CN1886512A - Expression of polypeptides in chloroplasts, and compositions and methods for expressing same - Google Patents

Abstract

Method of producing one or more polypeptides in a plant chloroplast, including methods of producing polypeptides that specifically associate in a plant chloroplast to generate a functional protein complex, are provided. An isolated polynucleotide that includes (or encodes) a first ribosome binding sequence (RBS) operatively linked to a second RBS, such that the first RBS directs translation of a polypeptide in a prokaryote and the second RBS directs translation of the polypeptide in a chloroplast, also is provided, as is a vector containing such a polynucleotide, particularly a chloroplast vector and a chloroplast/prokaryote shuttle vector. Also provided is a synthetic polynucleotide, which is chloroplast codon biased. A plant cell that is genetically modified to contain a polynucleotide or vector as described above, as well as transgenic plants containing or derived from such a genetically modified cell, are provided. Polypeptides encoded by a synthetic polynucleotide as described also are provided.

Description

The expression of polypeptide in chloroplast(id) and composition and the method that is used for express polypeptide

[0001] the present invention is undertaken by government-funded, and these subsidies are respectively: fund GM54659 is provided by the state-run commune hospital of the U.S.; Fund DE-FG03-93ER20116 is provided by USDOE; Fund NA06RG00142 is provided by the Ni Fuliya ocean fund university plan of adding of state-run ocean of the U.S. and air management office.United States Government may enjoy certain right to the present invention.

Technical field

[0002] relate generally to of the present invention is used for composition and the method at vegetable cell chloroplast(id) express polypeptide, more specifically, the polynucleotide with chloroplast(id) codon preference (chloroplast codon biased) of heterologous polypeptide relates to encode, and the carrier that can make heterologous polypeptide strongly expressed in bacterium and chloroplast(id), described heterologous protein comprises, for example, albumen composition, the antibody and the antibody chimeric body that form as specific connection by the polypeptide subunit.

Background technology

[0003] molecular biology and genetic engineering can produce great deal of bioactive substances, and these materials can be as the nutritional supplementation of healthy individual or as the treatment reagent for the treatment of the individuality with pathology disorder.Be produced such as, tethelin, and recombinant human growth hormone has been used to treatment and has suffered from the handicapped individuality of growth by genetic engineering method.Similarly, the monoclonal antibody with needed specific characteristics also is found the reagent that can be used as the various disorders of treatment, and described disorder comprises cancer, as lymphatic cancer and breast cancer.

[0004] using gene engineering, to come a major advantage of production biological species treatment reagent be that this method can guarantee to produce a large amount of needed protein.In many cases, can obtain the method for distinguishing of the biological substance that for example is used as treatment reagent of q.s, just have only the active substance that generates naturally by purifying from the biomass cells that produces these materials to realize.Therefore, before the genetic engineering invention, tethelin can only extract from the pituitary body of animal such as ox.Regular Insulin is another example of such biological species reagent, and before the genetic engineering invention, it can only separate the Regular Insulin that can obtain q.s and biologically active form from the pancreas of animal such as pig.

[0005] though genetic engineering provides a kind of method of producing the large number of biological material, particularly protein and nucleic acid, still there are some limitation in present available method.Such as, human albumen can be in bacterial cell great expression.The complicated more proteinic assembling but bacterium can not provide suitable environment, such as antibody, it is the protein of the biologically active that forms of a kind of connection of passing through four polypeptide chains.Therefore, even bacterium can be used for producing biological substance, but still may need some extra steps as under given conditions protein being carried out sex change and folding, thereby obtain biologically active substance.

[0006] recombinant protein also can be expressed in eukaryotic cell, and such as insect cell and mammalian cell, these cells can provide the protein that will express to be processed into the necessary environment of bioactive agents and the necessary factor.Contain a heavy chain and a light chain such as antibody, they constitute dimer, and then the dimer that further is made of heavy chain and light chain with another one is connected into and has active antibody.Such processing can be carried out in eukaryotic cell such as mammalian cell.Yet eukaryotic cell also may modifying protein, and glycosylation modified such as protein is carried out, like this, described albumen just contains glycosyl in its certain location.This posttranslational modification may provide the characteristic of bringing benefit, simultaneously also may bring some shortcomings and has limited the purposes of recombinant protein.For example; glycosyl group may have very strong antigenicity; after in it is injected into body; can cause the intensive immunne response and make the recombinant protein inactivation; even in some cases; may produce very bad influence, individuality is caused injury bigger when being injected into individuality at first than this medicine.

[0007] usually, the polynucleotide of the polypeptide that coding will obtain by recombinant DNA method is included in the carrier, and described carrier is a kind of nucleic acid molecule that helps the operation of polynucleotide.Carrier can be used for interested polynucleotide is imported in prokaryotic cell prokaryocyte such as bacterial cell or eukaryotic cell such as the mammalian cell.According to the difference of carrier place host cell, carrier also contains controlling element, such as the element that carrier can be increased in host cell.In addition, carrier also can be designed to and can shuttle back and forth between prokaryotic cell prokaryocyte and eukaryotic cell.Such shuttle vectors may be very useful, such as, they can allow to produce earlier a large amount of carrier (polynucleotide be also contained in) in bacterium, carrier is transferred in the mammalian cell more then, makes can be produced being suitable for correctly assembling under the condition of biological activity protein by its encoded polypeptides.

[0008] shuttle vectors although it is so has some advantages at the carrier of one or several particular cell types specifically than those, but they still can not avoid the potential problem brought by posttranslational modification such as glycosylation, and this situation usually takes place in eukaryotic cell.Therefore a kind of method that can produce biological activity protein easily of needs, and the albumen that is produced can not have unwanted feature, for example produces the intensive antigenicity when being applied to individuality as the human individual.The present invention has just satisfied such needs, but also extra advantage is provided.

Summary of the invention

[0009] the present invention is based in part on a definite fact, promptly makes it to reflect that by the nucleotide sequence of coded polypeptide is modified the codon of chloroplast(id) uses (codon usage), can make described heterologous polypeptide strong expression in plant.Therefore, the present invention relates to the synthetic polynucleotide, it comprises at least the first nucleotide sequence of coding at least the first polypeptide, and wherein at least one codon in first nucleotide sequence reflects that the chloroplast(id) codon uses preference.In a kind of specific embodiments, each codon in first nucleotide sequence reflects that all the chloroplast(id) codon uses preference.

[0010] the synthetic polynucleotide can comprise the single nucleotide sequence of the single polypeptide of encoding, perhaps may further include at least the second nucleotide sequence of coding second polypeptide, one or more codon in wherein said second nucleotide sequence can reflect that also the chloroplast(id) codon uses preference.If two of synthetic polymerized nucleoside acid encodings or more a plurality of polypeptide, these coding nucleotide sequences can effectively be connected (operatively linked), the wall scroll polynucleotide can be transcribed out like this, and encoded polypeptides can be expressed respectively, perhaps can further effectively connect, the fusion rotein that contains first polypeptide and second polypeptide so simultaneously just can be expressed.In a kind of specific embodiments, first nucleotide sequence effectively is connected by a trinucleotide sequence with second nucleotide sequence, the described trinucleotide sequence one section connection peptides of for example can encoding.Like this, just can give expression to fusion rotein by described synthetic polynucleotide, it contains first polypeptide and second polypeptide that links together by connection peptides.

[0011] can be any interested polypeptide by synthetic polynucleotide encoded polypeptides among the present invention, and polypeptide expressed in plastid not under the normal circumstances normally, polypeptide expressed in chloroplast(id) not particularly.Such as, encoded polypeptides can be immunoglobulin (Ig) (Ig) family member's one or more of chains, for example, and Ig variable region, Ig constant region, Ig heavy chain, Ig light chain, perhaps its combination; Or TXi Baoshouti (TCR) α chain, TCR β chain, perhaps its combination; Or the acceptor of any solubility is such as the soluble form of TXi Baoshouti or such acceptor and for example fusion rotein of IG heavy chain formation.In a kind of specific embodiments, synthetic polymerized nucleoside acid encoding Ig family member fusion rotein, such as a single-chain antibody, it comprises the complete heavy chain that effectively is connected with a variable region of light chain.An example of such fusion rotein is strand anti-herpes simplex virus (HSV) antibody, its aminoacid sequence is shown in SEQ ID NO:16, it can be by the synthetic polymerized nucleoside acid encoding with the nucleotide sequence shown in the SEQ ID NO:15, and described nucleotide sequence has the chloroplast(id) codon and uses preference.In the another one example, fusion rotein by the synthetic polymerized nucleoside acid encoding with chloroplast(id) codon use preference is the anti-HSV antibody of a strand Fv segment, its aminoacid sequence is shown in SEQ IDNO:43, and the nucleotide sequence of this section aminoacid sequence of encoding is shown in SEQ ID NO:42.In another one example again, fusion rotein by the synthetic polymerized nucleoside acid encoding with chloroplast(id) codon use preference is HSV8-lsc (big strand) antibody, its aminoacid sequence is shown in SEQ ID NO:48, and the nucleotide sequence of this section aminoacid sequence of encoding is shown in SEQID NO:48.

[0012] among the present invention by synthetic polynucleotide encoded polypeptides also can be the report polypeptide (reporter polypeptide), such as a kind of luciferase polypeptide.An example of such luciferase reporting polypeptide is to contain by the effective bacterial luciferase A subunit that connects of connection peptides and the luciferase fusion rotein of bacterial luciferase B subunit, the aminoacid sequence of this fusion rotein is shown in SEQ ID NO:46, and the chloroplast(id) codon that has of this section aminoacid sequence of encoding uses the nucleotide sequence of preference shown in SEQ ID NO:45.Therefore, the invention provides the luciferase fusion polypeptide of the aminoacid sequence shown in a kind of SEQ of having ID NO:46.The synthetic polynucleotide with chloroplast(id) codon preference of coding report polypeptide, polynucleotide (SEQ IDNO:45) such as the coding bacterium luxAB fusion polypeptide (SEQ ID NO:46) that illustrates is very useful, such as, can be used as the instrument of identifying chloroplast(id) promotor, 5 ' non-translational region (5 ' UTRs), 3 ' UTR, proteolytic enzyme defective strain system and analogue, a kind of method that heterologous polypeptide is expressed of further improving in chloroplast(id) so just is provided.

[0013] the invention still further relates to and a kind ofly produce the method for heterologous polypeptide in the plastid by importing the synthetic polynucleotide, described synthetic polynucleotide comprises at least the first nucleotide sequence of coding at least the first polypeptide, wherein at least one codon has chloroplast(id) codon use preference in described first nucleotide sequence, and described importing is to carry out under the condition that allows described at least the first polypeptide to express in described plastid.This synthetic polynucleotide can effectively be connected with the nucleotide sequence of one section coding at least one ribosome binding sequence (RBS)---the RBS sequence that particularly guides described polypeptide to translate in plastid---.

[0014] the employed synthetic polynucleotide of the method according to this invention can be any synthetic polynucleotide, and it comprises that at least one codon has at least the first nucleotide sequence that the chloroplast(id) codon uses preference.So, described synthetic polynucleotide may further include at least the second nucleotide sequence of coding second polypeptide, wherein said first nucleotide sequence can but be not effectively to be connected with described second nucleotide sequence, and wherein said second polypeptide can but and nonessential be allogenic with chloroplast(id).If two (perhaps more a plurality of) polypeptide of described synthetic polymerized nucleoside acid encoding, the so described polypeptide that is encoded can be expressed as independently different polypeptide, or is expressed as the fusion rotein that contains first and second (perhaps more) polypeptide.

[0015] in a kind of specific embodiments, the fusion rotein of being expressed by the synthetic polynucleotide according to method of the present invention comprises first polypeptide and second polypeptide that connects by connection peptides.An example of such method is to express the single-chain antibody that comprises interconnective IgA heavy chain and variable region of light chain, this fusion rotein has the aminoacid sequence shown in the SEQ ID NO:16, and the chloroplast(id) codon that has of this section aminoacid sequence of encoding uses the nucleotide sequence of preference shown in SEQ IDNO:15, wherein the single-chain antibody of being expressed still possess antigen-binding specificity (also referring to, have the anti-HSV antibody of the strand Fv segment (by SEQ ID NO:42 coding) of the aminoacid sequence shown in the SEQ ID NO:43 and have the HSV8-lsc antibody (encoding) of the aminoacid sequence shown in the SEQ ID NO:48 by SEQ ID NO:48.

[0016] method of the present invention is to express a report polypeptide in this further example, the luciferase fusion rotein that particularly comprises the luciferase A subunit that effectively is connected with luciferase B subunit, described fusion rotein has the aminoacid sequence shown in the SEQ ID NO:46, and the nucleotide sequence of this section aminoacid sequence of encoding is shown in SEQ ID NO:45, and the expression of wherein said allos luciferase in chloroplast(id) can be in vivo or in vitro detection.

[0017] method of the present invention can be put into practice in any plastid, comprises the chloroplast(id) of plant.The plant that contains chloroplast(id) can be any plant that contains chloroplast(id) naturally, comprises algae (microalgae or bulk kelp class) and higher plant.Described method can further include the method that the heterologous polypeptide of expressing is separated from the vegetable cell (perhaps isolating chloroplast(id)) that contains described polypeptide.Therefore, the invention provides the heterologous polypeptide that utilizes method of the present invention to produce.

[0018] the invention further relates to the method that a kind of detection contains the vegetable cell of plastid.Such method can be implemented like this, for example, synthetic polynucleotide of the present invention is imported in the plastid of vegetable cell, as import in the chloroplast(id), wherein said polymerized nucleoside acid encoding report polypeptide, such importing allows described report polypeptide to express in chloroplast(id), detects described report polypeptide expression then.Described report polypeptide can be any desired polypeptide, and its example is to express the luciferase fusion rotein with the aminoacid sequence shown in the SEQ ID NO:46.

[0019] the present invention also relates to a kind of proteic method that in plastid, produces.Such method can be implemented like this, for example, at least the first recombinant nucleic acid molecules is imported in the plastid, wherein said first recombinant nucleic acid molecules comprises first nucleotide sequence, at least one allos polynucleotide of this first nucleotide sequence coded at least one ribosome binding sequence (RBS) and at least a polypeptide of coding that effectively is connected with it, wherein said RBS can instruct the translation of described polypeptide in plastid under the condition that allows described at least a expression of polypeptides, can produce described polypeptide thus in described plastid.Described plastid can be any plastid, comprises for example a kind of chloroplast(id).

[0020] according to present method, one or more codon in first polynucleotide can have the chloroplast(id) codon and use preference.In a kind of specific embodiments, the polypeptide that is encoded is an antibody, perhaps the subunit of antibody.In another embodiment, the first polymerized nucleoside acid encoding, first polypeptide and second polypeptide, for example, first polypeptide comprises Ig heavy chain or its variable region, second polypeptide comprises Ig light chain or its variable region.An example by the expressed such antibody that comes out of method of the present invention is the anti-tetanus toxin antibody with the aminoacid sequence shown in the SEQ ID NO:14, its nucleotide sequence coded by shown in the SEQ ID NO:13.In another specific embodiments, first polynucleotide has the chloroplast(id) codon and uses preference.The example of such antibody of being expressed by method of the present invention is to have SEQID NO:16, the anti-HSV antibody of the aminoacid sequence shown among SEQ ID NO:43 and the SEQ ID NO:48, these antibody can be respectively SEQ ID NO:15 by sequence, and SEQ IDNO:42 and SEQ ID NO:47's is nucleotide sequence coded.

[0021] in the another one specific embodiments, the first polymerized nucleoside acid encoding, first polypeptide and at least one second polypeptide, wherein said first polypeptide and second (or more a plurality of) polypeptide can but might not be a kind of subunit of albumen composition, described albumen composition for example is a heterodimer, heterotrimer etc.In the another one specific embodiments, described method may further include and import at least the second recombinant nucleic acid molecules in plastid.Second recombinant nucleic acid molecules like this can comprise first nucleotide sequence, the at least the second allos polynucleotide of described first nucleotide sequence coded at least the one RBS and coding at least the second polypeptide that effectively is connected with it, a wherein said RBS can instruct described the polypeptide translation in plastid, particularly chloroplast(id).Preferably, described first recombinant nucleic acid molecules and second recombinant nucleic acid molecules coexpression in plastid.

[0022] the method according to this invention, first recombinant nucleic acid molecules can be included within the carrier.In a kind of specific embodiments, described carrier is the chloroplast(id) carrier, and it contains the chloroplast genomic dna nucleotide sequence that can carry out homologous recombination with chloroplast genomic dna, and the carrier that contains described first recombinant nucleic acid molecules is directed in the chloroplast(id).Such carrier can further comprise procaryotic replication orgin.

[0023] method of the present invention may further include isolated polypeptide from plastid.Therefore, the present invention also provides the isolated polypeptide that obtains by such method, for example, a kind of in chloroplast(id) the isolated antibody of heterogenous expression.

[0024] the invention further relates to the method that in plant chloroplast, produces one or more polypeptide, comprise that generation can connect the method for the polypeptide that forms protein complexes specifically.Similarly, method of the present invention provides a kind of means that produce the functional protein mixture, such as, comprise first heavy chain and light chain and second heavy chain of associating with it and the bivalent antibody of light chain.Method of the present invention can be implemented like this, for example, imports first recombinant nucleic acid molecules in chloroplast(id), and it comprises first polynucleotide of at least a polypeptide of encoding; Effectively be connected with second polynucleotide, described second polynucleotide comprises the nucleotide sequence of the nucleotide sequence of coding first ribosome binding sequence (RBS) and coding the 2nd RBS that effectively is connected with it, wherein a RBS can instruct the translation of polypeptide in prokaryotic organism, the 2nd RBS can instruct the translation of polypeptide in chloroplast(id), such method is to carry out under the condition that allows described at least a expression of polypeptides, so just can express described polypeptide in chloroplast(id).These methods among the present invention can be carried out in any plant (vegetable cell) that contains chloroplast(id), comprise one-celled plants and algae, and metaphyte and algae.

[0025] in a kind of specific embodiments, the used in the method for the invention first polymerized nucleoside acid encoding, first polypeptide and at least one second polypeptide, such as, one first polypeptide and one second polypeptide; Perhaps one first polypeptide, one second polypeptide and one the 3rd polypeptide; Or the like, any or whole polypeptide can be same or different.In the another one specific embodiments, one or more codon of described first polynucleotide has the chloroplast(id) codon and uses preference.

[0026] as disclosed here, plant chloroplast such as the chloroplast(id) of little algae Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) in polypeptide expressed correctly assemble, and can unite with one or more other polypeptide of in chloroplast(id), expressing and form the functional protein mixture.Therefore, in the another one specific embodiments, the first useful in the method for the invention polynucleotide one or more polypeptide subunits of can encoding, these subunits can be united and formed the functional protein mixture.Described albumen composition can be dimer, tripolymer, the tetramer or similar polymer, and these subunits can be identical, also can be different, or the combination of the two.Such as, when described albumen composition was a dimer, it can be homodimer or heterodimer.When described albumen composition was a tripolymer, it can be homotrimer, heterotrimer or the tripolymer be made up of two identical polypeptide and a different polypeptide.

[0027] method of the present invention is particularly useful for producing the functional protein mixture, described albumen composition for example contains the antibody of two heavy chains and two light chains usually when spontaneous generation, perhaps cell surface receptor such as TXi Baoshouti, growth hormone receptor, hormone acceptor, g protein coupled receptor, wherein g protein coupled receptor can be united with G albumen, and similar mixture.Using method of the present invention produces albumen such as antibody in chloroplast(id) a advantage is can be by glycosylation after polypeptide is expressed in chloroplast(id), therefore, its antigenicity is compared with the antibody antigen that obtains in animal or express in the tenuigenin of eukaryotic cells greatly and is reduced.As disclosed here, the method that produces the functional protein mixture in chloroplast(id) can be implemented like this: use first recombinant nucleic acid molecules, and according to definition, two or more subunits of the wherein said first polymerized nucleoside acid encoding mixture; Perhaps use first recombinant nucleic acid molecules and second recombinant nucleic acid molecules, the former is according to definition, a polypeptide subunit of coding mixture, and the latter has the characteristic same with first recombinant nucleic acid molecules, the another one polypeptide subunit of code for said proteins mixture.

[0028] therefore, method of the present invention can be used first recombinant nucleic acid molecules and implement, the wherein said first polymerized nucleoside acid encoding, first polypeptide and second polypeptide, the former is heavy chain immunoglobulin (H) or its variable region, the latter is light chain immunoglobulin (L) or its variable region.If necessary, the nucleotide sequence of one section inherent ribosome entry site(RES) of coding can be placed between the nucleotide sequence of coding H and L, can promote the expression of second (downstream) coded polypeptide like this.At the H of coding with after the L chain is translated in chloroplast(id), a H chain can be united with a L chain and formed a univalent antibody (that is, a H:L mixture), and two H:L mixtures can further be united and formed a bivalent antibody.

[0029] method of the present invention also can be implemented like this, in plant chloroplast, import first recombinant nucleic acid molecules, the first polymerized nucleoside acid encoding wherein, such as, H chain or its variable region, further in chloroplast(id), import second recombinant nucleic acid molecules, it comprises first polynucleotide of coding L chain or its variable region, and described first polynucleotide effectively is connected with second polynucleotide, described second polynucleotide comprises the nucleotide sequence of coding the 2nd RBS that encodes the nucleotide sequence of a RBS and effectively connect with it, a wherein said RBS can instruct the translation of polypeptide in prokaryotic organism, and the 2nd RBS can instruct the translation of polypeptide in chloroplast(id), the polypeptide that is encoded coexpression basically in chloroplast(id).Wherein heavy chain (H) and light chain (L) can be united H:L complex body of formation, and wherein the H:L complex body can further be united bivalent antibody of generation.

[0030] when putting into practice method of the present invention, first recombinant nucleic acid molecules can be included in the middle of the carrier.And if present method is also used second (perhaps more) other recombinant nucleic acid molecules, second recombinant nucleic acid molecules also can be included in the middle of the carrier so, this carrier can but might not be identical with the carrier that contains first recombinant nucleic acid molecules.As selection, vegetable cell can be by genetic modification, so that the chloroplast(id) of plant contains the recombinant nucleic acid molecules of a subunit of the proteins encoded mixture of a stable integration, like this, method of the present invention can comprise, imports to such as the carrier that will contain second recombinant nucleic acid molecules in the chloroplast(id) of described plant, one or more subunit of the described second recombinant nucleic acid molecules proteins encoded mixture, like this, after polypeptide was expressed, a functional albumen composition had just produced.

[0031] used carrier can be any carrier that is used for importing polynucleotide in chloroplast(id) among the present invention.Especially, described carrier can comprise the nucleotide sequence of chloroplast genomic dna, can mediate homologous recombination with chloroplast genomic dna by it.Such chloroplast(id) carrier can comprise any additional nucleotide sequence that helps the application or the operation of carrier, such as, one or more transcriptional regulatory element, and perhaps selected marker, perhaps cloning site, perhaps similar elements comprises their combination.In a kind of specific embodiments, carrier can be a chloroplast(id) carrier, it comprises plant chloroplast gene transcription promotor and 5 ' non-translational region (5 ' UTR), it can also comprise, perhaps by comprising a foregoing RBS and the 2nd RBS that effectively connects with it after modifying.In the another one specific embodiments, carrier can be a chloroplast(id) carrier, it comprises procaryotic replication origin (ori), such as the ori of intestinal bacteria (E.coli), therefore provides the shuttle vectors that can shuttle back and forth and operate in prokaryotic host cell and chloroplast(id).Shuttle vectors of the present invention can contain any interested polynucleotide, comprise synthetic polynucleotide with chloroplast(id) codon preference, such as synthetic polynucleotide with the sequence shown in the SEQ ID NO:45, its a kind of bacterium luxAB fusion rotein (SEQ ID NO:46) of encoding.The advantage that the shuttle vectors of such expression SEQ ID NO:46 provides is, for the expression in bacterium, can check controlling element or other interested sequences, the carrier that contains those elements with needed expression characterization then can be shuttled back and forth, shuttled back and forth the expression that is enhanced of the heterologous polypeptide that can obtain to be encoded therein in chloroplast(id) together with effectively connect with it same or other synthetic polynucleotide or other polynucleotides.

[0032] method of the present invention may further include the expressed polypeptide of from chloroplast(id) separation or the step of albumen composition.Therefore, the present invention also provides isolated polypeptide or the albumen composition that obtains according to method disclosed herein.Such as, the invention provides the isolated antibody of in plant chloroplast, expressing and therefrom obtaining.The polypeptide composition that an advantage of isolated antibody of the present invention is described antibody does not have glycosylation, and therefore its antigenicity can reduce when antibody is bestowed individuality.And, this antibody of the present invention is compared with abiogenous antibody, the effect activity (effector activities) that reduction can be arranged, for example complement is in conjunction with active (complementfixation activity), therefore provides to be used for antibody that individuality is diagnosed.

[0033] the invention still further relates to the isolating ribonucleoside acid sequence that comprises first ribosome binding sequence (RBS) and the 2nd RBS that effectively is connected with it, a wherein said RBS and the 2nd RBS are spaced apart by 5 to 25 Nucleotide, wherein, when described ribonucleoside acid sequence effectively is connected with the polynucleotide of a coded polypeptide, a described RBS can instruct the translation of described polypeptide in prokaryotic organism, and the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).Isolating ribonucleoside acid sequence of the present invention generally is about 11 to 50 ribonucleotide length, can be about 15 to 40 ribonucleotide length, or about 20 to 30 ribonucleotides, can be used as an independently unit, perhaps can effectively be connected with an allogenic RNA molecule.

[0034] in ribonucleoside acid sequence of the present invention, effectively a RBS and the 2nd RBS that connects is generally spaced apart by about 5 to 25 Nucleotide, usually is about 10 to 20 Nucleotide, such as being about 15 Nucleotide.The one RBS and the 2nd RBS can be made up of about 3 to 9 Nucleotide independently of one another, usually be about 4 to 7 Nucleotide, and can contain the sequence of any Shine-Delgamo of having (SD) sequence signature, such as the sequence that comprises 5 '-GGAG-3 ', the part of the anti-SD sequence of this sequence and 16S rRNA is complementary.The 2nd RBS that instructs polypeptide to translate in chloroplast(id) can be included among the 5 ' UTR of a chloroplast gene, described chloroplast gene can be a kind of solubility chloroplast protein of coding or the protein-bonded chloroplast gene of chloroplast membranes, wherein said 5 ' UTR may further include transcriptional regulatory element, comprises promotor.

[0035] ribonucleoside acid sequence of the present invention may further include the initial AUG codon that effectively is connected with described first and second RBS.Initial AUG codon like this may further include the adjacent nucleotide of Kozak sequence, and such as ACCAUGG, this sequence can promote the translation of polypeptide in cell.Ribonucleoside acid sequence of the present invention can also effectively be connected with the polybribonucleotide of coded polypeptide, described polybribonucleotide sequence can contain an initial AUG codon of endogenous or can contain an initial AUG codon by after modifying, perhaps also can lack initial AUG codon, and this codon can be a component of ribonucleoside acid sequence of the present invention.

[0036] isolating ribonucleoside acid sequence of the present invention can be synthetic by chemical process, perhaps can adopt Enzymology method to generate, such as utilizing DNA dependent form RNA polymerase or RNA dependent form RNA polymerase to generate respectively from a deoxyribonucleotide (DNA) or ribonucleotide (RNA) template.Such dna profiling can chemosynthesis or can be separated from abiogenous dna molecular, perhaps based on abiogenous dna sequence dna, through possessing required characteristic after certain modification, dna sequence dna such as one section prokaryotic gene, it has the nucleotide sequence that coding is positioned at the RBS of about 5 to 15 Nucleotide in initial ATG codon upstream, but also can contain the 2nd RBS by further modification, the 2nd RBS is positioned at a RBS upstream and separates with a RBS, and such the 2nd RBS can instruct the translation of polypeptide in chloroplast(id).

[0037] therefore, the invention still further relates to the coding polynucleotide of a RBS and the 2nd RBS that effectively connects with it thus defined.Described polynucleotide can be DNA or RNA, can be strand or two strands.Polynucleotide of the present invention can comprise initial ATG codon, and it effectively is connected with the nucleotide sequence of coding the one RBS and the 2nd RBS.In addition, polynucleotide of the present invention can comprise cloning site, the position of described cloning site allow can coded polypeptide effable polynucleotide effectively be connected on a RBS and the 2nd RBS, described like this polypeptide can be expressed in chloroplast(id) or prokaryotic host cell.Described cloning site can be any nucleotide sequence that effable polynucleotide inserted or be connected to first and second RBS of being convenient to, the translation of coded polypeptide just can start from a RBS and the 2nd RBS under suitable condition like this, and described cloning site is one or more restriction enzyme enzyme recognition site or recombinase recognition site or their combination for example.

[0038] encode the as defined herein polynucleotide of first and second RBS can effectively be connected with an effable polynucleotide, and the described effable polynucleotide at least a polypeptide of can encoding comprises the peptide moiety of peptide or polypeptide.Equally, described effable polynucleotide can encode a kind of first polypeptide and a kind of or more kinds of extra polypeptide, these polypeptide can be same or different.Such as, described effable polynucleotide can encode one first polypeptide and one second polypeptide, these polypeptide can be different.Further, like this first polypeptide and second polypeptide can be used as expressing fusion protein, perhaps can be used as independently expression of polypeptides, if latter event, the nucleotide sequence of an inherent ribosome entry site(RES) of coding can but might not effectively be connected between the encoding sequence of the encoding sequence of described first polypeptide and described second polypeptide, can describedly be convenient to the translation of described second polypeptide like this.

[0039] both sides of polynucleotide of the present invention can also have first cloning site and second cloning site, and a sequence box that can insert or be connected to second polynucleotide easily so just is provided.First and second cloning sites of this side can be identical or different, and one of them or both can be independently as one in a series of cloning sites, i.e. multiple clone site.

[0040] in a kind of specific embodiments, polynucleotide of the present invention comprises the nucleotide sequence of coding the 2nd RBS, nucleotide sequence and an initial ATG codon of coding the one RBS, these sequences all are that effectively connection and direction are 5 ' to 3 '; And/or with such polynucleotide complementary nucleotide sequence.In the another one specific embodiments, polynucleotide of the present invention comprises the nucleotide sequence of coding the 2nd RBS, the nucleotide sequence of coding the one RBS, with an initial ATG codon, with at least one cloning site, these sequences all are that effectively connection and direction are 5 ' to 3 '; And/or with such polynucleotide complementary nucleotide sequence.In another one specific embodiments again, polynucleotide of the present invention comprises the nucleotide sequence of coding the 2nd RBS, the nucleotide sequence of coding the one RBS, be positioned at the cloning site of about 3 to 10 nucleotide positions of 3 ' side of the nucleotide sequence of coding the one RBS with at least one, these sequences all are effectively to connect and direction is 5 ' to 3 '; And/or with such polynucleotide complementary nucleotide sequence.

[0041] the invention still further relates to a kind of carrier, it comprises RBS that coding effectively connects as defined herein and the polynucleotide of the 2nd RBS, with the nucleotide sequence of one section chloroplast gene group thymus nucleic acid (DNA), it can with chloroplast genomic dna generation homologous recombination.Though such chloroplast genomic dna nucleotide sequence generally is must not be the nucleotide sequence of one section silence, its certain chloroplast gene of not encoding, and want sufficiently long so that carrier can with corresponding nucleotide sequences generation homologous recombination on the chloroplast gene group.

[0042] carrier of the present invention can also comprise one or more the extra nucleotide sequence that can give the carrier desired characteristic, comprise, such as, help the sequence of carrier operation.So, carrier can comprise, such as, one or more cloning site, and multiple clone site for example, its location makes the allos polynucleotide can be inserted in the middle of the carrier and with a RBS effectively to be connected with the 2nd RBS.Described carrier can also comprise prokaryotic organism replication origin (ori), such as the ori of colibacillary ori or clay, provides the shuttle vectors that can shuttle back and forth as required thus in prokaryotic host cell or plant chloroplast.Therefore, in a kind of specific embodiments, the invention provides chloroplast(id)/prokaryotic organism shuttle vectors, wherein said shuttle vectors comprises 1) nucleotide sequence of chloroplast genomic dna, it can with chloroplast genomic dna generation homologous recombination; 2) procaryotic initial point; 3) RBS who effectively is connected with the 2nd RBS, wherein said first (perhaps second) RBS can instruct the translation of effable polynucleotide in chloroplast(id) of effective connection, and second (perhaps first) RBS can instruct the translation of effable polynucleotide in prokaryotic organism of effective connection; With 4) effective effable polynucleotide that is connected, perhaps its location can make the allos polynucleotide insert the cloning site that also effectively is connected on first and second RBS.

[0043] carrier of the present invention can be a kind of cyclisation carrier, perhaps also can be the linear carrier with first end and second end.Linear carrier among the present invention can be at one end or two ends have one or more cloning site, the means of the described carrier of cyclisation so just are provided or described carrier is connected to method on second polynucleotide, described second polynucleotide can be second carrier identical or different with described carrier of the present invention.Described cloning site can comprise restriction enzyme enzyme recognition site (perhaps its cleaved products), recombinase site, the combination in perhaps such site.

[0044] described carrier may further include one or more and expresses controlling elements, such as transcriptional regulatory element, extra translation element and similar elements.In a kind of specific embodiments, described carrier contains the initial ATG codon that effectively is connected with the sequence of coding the one RBS and the 2nd RBS, the polynucleotide of coded polypeptide can be adjacent to effectively be connected with the ATG codon like this, after transcribing, can produce can be in prokaryotic organism and the RNA that translates in chloroplast(id).Therefore, described carrier can also contain cloning site, and its location allows at least one section allos polynucleotide effectively to be connected with such ATG codon.Carrier of the present invention also can contain the nucleotide sequence of coding first polypeptide that effectively is connected with a RBS and the 2nd RBS, wherein said coding nucleotide sequence contains one or more cloning site through after modifying, comprise, such as near the upstream of ATG codon, near the downstream of ATG codon, and/or near the C-terminal of the polypeptide that is encoded or its.Such carrier provides a kind of nucleotides sequence that inserts one section coding second polypeptide easily to list in wherein means, or realize by the nucleotide sequence that substitutes described first polypeptide of coding, or effectively connect at the N-terminal or the C-terminal of the described polypeptide that is encoded, the fusion rotein that comprises first polypeptide and second polypeptide like this can be expressed.

[0045] the invention still further relates to a kind of cell that contains polynucleotide of the present invention or carrier.Described cell can be the host cell at carrier of the present invention, can be prokaryote or eukaryotic cells, comprises, such as bacterial cell such as Bacillus coli cells; Vegetable cell such as algae or unifacial leaf or dicotyledons; Insect cell; Perhaps vertebrate cells such as mammalian cell.If described cell is a vegetable cell, so described polynucleotide or carrier can be included in the plastid of vegetable cell, particularly in the chloroplast(id), and can but might not be incorporated in the genome of described plastid.

[0046] usually, polynucleotide of the present invention can be included in the carrier, and effectively be connected with effable polynucleotide, therefore the cell that contains described polynucleotide just provides an expression system, and it allows the translation by one or more polypeptide of described effable polymerized nucleoside acid encoding.So, the codon that described effable polynucleotide can have described plastid uses preference, particularly the chloroplast(id) codon uses preference, and described effable polynucleotide coding at least the first polypeptide is such as coding first polypeptide and second polypeptide.In a kind of specific embodiments, described effable polynucleotide encoding antibody.In another specific embodiments, described effable polynucleotide has the chloroplast(id) codon and uses preference, for example, described effable polynucleotide has SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:42, the nucleotide sequence shown in SEQ ID NO:45 or the SEQ ID NO:47.

[0047] the invention further relates to a kind of transgenic plant, it comprises and contains polynucleotide of the present invention, and has been incorporated into the vegetable cell in the chloroplast genomic dna.Therefore, the invention provides the vegetable cell device or cell or the tissue that obtain from such transgenic plant, such as isolated chloroplast(id) from described transgenic plant, or from described transgenic plant isolated leaf or flower, isolated fruit or rhizome from described transgenic plant, perhaps transplanting of described transgenic plant, the perhaps seed that produces by described transgenic plant.In addition, the invention provides, perhaps the cDNA or the chloroplast genomic dna storehouse of preparation in vegetable cell that obtains by described transgenic plant or the tissue from transgenic plant of the present invention.Transgenic plant of the present invention can be the plants of any kind, comprise, and such as algae, can be little algae or bulk kelp; Monocotyledons; Perhaps dicotyledons comprises ornamental plant such as angiosperm (such as grain class plant, leguminous plants, oleaginous seed plant, perhaps palohierror).

[0048] the invention further relates to a kind of composition, comprising contain the vegetable material that obtains in the vegetable cell that is incorporated into the polynucleotide of the present invention the plant chloroplast genomic dna from transgenic plant of the present invention or by genetic modification.Preferably, in the vegetable cell that described transgenic plant or genetic modification are crossed, an encode RBS of described effective connection and the polynucleotide of the 2nd RBS effectively is connected with effable polynucleotide, described effable polynucleotide can but might not possess the chloroplast(id) codon and use preference.Like this, described vegetable material, can be organoid, cell, perhaps one or more tissues that obtain by transgenic plant, for example, chloroplast(id), perhaps leaf or flower, fruit or rhizome, perhaps by the seed of transgenic plant generation, they provide the source by one or more polypeptide of described effable polymerized nucleoside acid encoding.For example, if described effable polynucleotide encoding antibody, perhaps its antigen binding fragment is disconnected, so described vegetable material, just and described composition the source of described antibody is provided.

[0049] composition of the present invention can be formulated into the form that is suitable for bestowing live body, and described live body is vertebrates or other Mammalss for example, can be domestic animal or pet, perhaps can be the people.Correspondingly, according to by a peptide species of described effable polymerized nucleoside acid encoding or the difference of multiple polypeptides, the composition that comprises vegetable material as disclosed herein can be used as nutritional additive, treatment reagent and similar products like.Such as, if described effable polynucleotide encoding antibody, perhaps its antigen binding fragment is disconnected, so described composition can be used for for example being exposed to the passive immunization of the individuality of simplexvirus or tetanus toxin.Equally, the invention provides and be used to improve the medicine of a kind of pathological state such as herpesvirus infection.

[0050] the invention still further relates to the isolating polynucleotide of coding fluorescence albumen or its mutant or its varient, the codon of wherein said polynucleotide has the chloroplast(id) codon and uses preference.Described polynucleotide can be dna sequence dna or RNA sequence, can be strand or two strands, can be at one end or two ends contain the linear polynucleotide of cloning site.Described polynucleotide also can effectively be connected with the polynucleotide of coding the one RBS and the 2nd RBS, and wherein these two RBS sequences are separated by about 5 to 25 Nucleotide, and like this, described fluorescin can be translated in prokaryotic organism and chloroplast(id) easily.

[0051] one or more codons of coding fluorescin of the present invention can have preference, for example, contain VITAMIN B4 or thymus pyrimidine the 3rd position, can promote the translation of described fluorescin in chloroplast(id) like this.For example, described fluorescin can be green fluorescent protein (GFP), as the fluorescin that is produced by Aequorea jellyfish.The example of such polynucleotide of the present invention is that encoding sequence is the polynucleotide of the polypeptide of SEQ ID NO:2, such as, the polynucleotide shown in the SEQ ID NO:1.Therefore, the present invention also provides by the such polymerized nucleoside acid encoding and the fluorescin of expression, for example, has the fluorescin of the aminoacid sequence shown in the SEQ ID NO:2.

[0052] the invention further relates to recombinant nucleic acid molecules, it comprises first polynucleotide and second polynucleotide, encode at least a polypeptide and contain one or more and have the codon that the chloroplast(id) codon uses preference of described first polynucleotide, described second polynucleotide comprises the nucleotide sequence of the encode nucleotide sequence of a RBS and coding the 2nd RBS that effectively is connected with it, a wherein said RBS can instruct the translation of described polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).The described first polynucleotide single polypeptide of can encoding, perhaps can encode two or peptide more the more, if latter event, described polypeptide can be expressed respectively or as expressing fusion protein.If two of described first polymerized nucleoside acid encodings or peptide more the more, the nucleotide sequence between each encoding sequence can but the inherent ribosome entry site(RES) of might not encoding, the translation of the location in this site is convenient to second (perhaps other) polypeptide.Recombinant nucleic acid molecules of the present invention may further include the 3rd polynucleotide, and it can effectively be connected with described first and second polynucleotides, it can but one or more polypeptide of might not encoding.

[0053] the invention still further relates to the method for preparing chloroplast(id)/prokaryotic organism shuttle expression carrier.This method can be implemented like this, such as, in the nucleotide sequence that is enough to one section chloroplast genomic dna of chloroplast genomic dna generation homologous recombination, import following sequence: the nucleotide sequence that comprises the prokaryotic organism replication origin; The encode nucleotide sequence of a RBS; The encode nucleotide sequence of the 2nd RBS, a wherein said RBS and the 2nd RBS are by about 5 to 25 Nucleotide separately; And cloning site, wherein said cloning site is positioned and effectively is connected to a described RBS and the 2nd RBS with the polynucleotide that allows coded polypeptide, like this, a described RBS can instruct the translation of described polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).Preparation chloroplast(id)/prokaryotic organism shuttle expression carrier can also be realized by the nucleotide sequence of one section chloroplast gene group of genetic modification thymus nucleic acid (DNA), described this section nucleotide sequence can with chloroplast genomic dna generation homologous recombination, it contains the prokaryotic organism replication origin through after modifying, RBS that coding is separated by about 5 to 25 Nucleotide and the nucleotide sequence of the 2nd RBS, and cloning site, wherein said cloning site is positioned and effectively is connected to a described RBS and the 2nd RBS with the polynucleotide that allows coded polypeptide, like this, a described RBS can instruct the translation of described polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).Therefore, the present invention also provides aforesaid prepared according to the methods of the invention chloroplast(id)/prokaryotic organism shuttle vectors.

[0054] the invention further relates to the reorganization polynucleotide, it comprises first nucleotide sequence of coding chloroplast(id) RBS and second nucleotide sequence of the coded polypeptide that effectively is connected with it, and wherein said first nucleotide sequence is relative allogenic with described second nucleotide sequence.Such reorganization polynucleotide may further include the 3rd (perhaps more) nucleotide sequences of coding second (perhaps other) polypeptide of effective connection, and the reorganization polynucleotide of coding bicistronic mRNA (perhaps polycistron) polybribonucleotide sequence is provided thus.The general distance of the nucleotide sequence of the RBS that coding effectively links to each other about 20 to 40 nucleotide positions of initial ATG codon (upstream), initiator codon then effectively is connected with the described nucleotide sequence of coding said polypeptide.In a kind of specific embodiments, described first nucleotide sequence comprises the initial ATG codon of 3 ' about 20 to 40 nucleotide positions of the sequence of range coding RBS.In the another one specific embodiments, internal ribosome binding sequence effectively is connected in two sections of coded polypeptide or more between the polynucleotide sequence, described these nucleotide sequences can be the same or different.

[0055] the invention still further relates to a kind of carrier, it comprises the nucleotide sequence of the coding RBS that is positioned at about 20 to 40 nucleotide positions of cloning site 5 ' end.Described cloning site can be that any nucleotide sequence of being convenient to inserts or be connected to nucleotide sequence in the carrier, such as, one or more restriction enzyme enzyme recognition site, one or more recombinase recognition site, or the combination in these sites.Preferably, described cloning site is a multiple clone site, comprising a series of restriction enzyme enzyme recognition sites or recombinase recognition site, or the combination of at least one restriction enzyme enzyme recognition site and at least one recombinase recognition site.Described carrier can further comprise a 5 ' end and an adjacent with it initial ATG codon or a part wherein that is positioned at described cloning site, so just provides translation initiation site for the encoding sequence that lacks initial ATG codon.In addition, described carrier can comprise 3 ' non-translational region of chloroplast gene, and it is positioned at 3 ' end of described cloning site.

Description of drawings

[0056] Fig. 1 provides the contrast of GFPct (SEQ ID NO:1) and GFPncb (SEQ ID NO:3) coding region.The aminoacid sequence of GFPct (SEQ ID NO:2) marks below nucleotide sequence.The codon that changes comes out with the square frame mark, the codon shade mark that is significantly changed on codon uses.The codon of optimizing is defined as the codon (Nakamura etc., Nucl.Acids Res.27:292,1999) of per 1000 codons use above 10 times in Chlamydomonas reinhardtii (C.reinhardtii) the chloroplast gene group.Asterisk (*) indicates two amino acid changes between GFPct and the GFPncb, respectively on the 2nd and the 65th amino acids.

[0057] Fig. 2 provides the GFPct of pET expression and the sign of GFPncb.GFPct that is expressed by plasmid pET19b in intestinal bacteria and GFPncb albumen are by Ni agarose affinity chromatography purifying (embodiment 1).Contain the thick lysate of the proteic E.colli cell of GFPct or GFPncb (20 μ l) and directly go up on the SDS-PAGE of sample to 12% without boiling water bath, electrophoresis is taken electrophoresis equipment apart after finishing, and allows glue stay between the sheet glass.Fluorescence gel is observed by specified exciting (ex) and emission (em) filter.5 micrograms are separated on 12% SDS-PAGE with GFPncb albumen by the GFPct that affinity purification obtains, and use coomassie brilliant blue staining.100ng passes through GFPct that affinity purification obtains and GFPncb albumen through after 12% the SDS-PAGE, carries out western blotting again, with anti-GFP one anti-the detection.Excitation spectrum is to obtain from GFPct (4 μ g) and GFPncb (20 μ g) albumen through affinity purification.Write down at the relative intensity of fluorescence of excitation wavelength from 350 to 550nm the time, wherein emission wavelength is fixed on 510nm.The emission spectrum of GFPncb (long and short dash line) and GFPct (black line) as shown in the figure; What dotted line was represented is at the emission peak of two samples at the 510nm place.

[0058] Fig. 3 provides and has been used at the GFPct of the expression of Chlamydomonas reinhardtii chloroplast(id) and the collection of illustrative plates of GFPncb reporter gene.

What [0059] Fig. 3 A showed is with rbcL 5 ' UTR (Banm HI/Nde I; Also can be referring to SEQID NO:5) with GFPct (SEQ ID NO:1) or GFPncb (SEQ ID NO:3) coding region (Nde I/Xba I) and rbcL 3 ' UTR (Xba I/Bam HI; Also see also SEQ IDNO:10) the relevant limit site of demarcating.Each pulsating size is all indicated with base logarithm (bp).

[0060] integration site that Fig. 3 B shows is GFPct under rbcL 5 ' and 3 ' UTR are controlled and GFPncb gene integration in the Chlamydomonas reinhardtii chloroplast gene group.Eco RI to the Xho I segment of the chloroplast DNA of Chlamydomonas reinhardtii such as 5.7kb indicates.What double-headed arrow was indicated is the corresponding zone that DNA is hybridized probe used in (Southern blot) analysis.

What [0061] Fig. 4 showed is the linear order of the psbA 5 ' UTR (SEQ ID NOS:35 to 40) of sudden change, and its corresponding position is to arrive-36 positions with respect to initiator codon+3 of wild-type 5 ' UTR (SEQ ID NOS:34).5 ' UTR is placed in the upstream of D1cDNA, and this cDNA is the intronless copy of wild-type psbA gene.The change of primary sequence indicates with underscore, and initiator codon indicates with square frame.5 ' the end of mRNA represented in asterisk, and they are (referring to the Mayfield, Trends Plant Sci.4:190-195,1998, this piece document is put in order into as a reference at this) that produced by the processing incident of shearing 5 ' UTR in vivo.

[0062] Fig. 5 A to 5C provides the restriction map that is used for the HSV8-lsc gene of expressing at the Chlamydomonas reinhardtii chloroplast(id).HSV8-lsc nucleotide sequence (SEQ ID NO:47) and aminoacid sequence (SEQ ID NO:48) thereof provide in sequence table.

What [0063] Fig. 5 A and 5B showed is to describe following pulsating relevant limit site: rbcL5 ' UTR (Bam HI/Nde I), HSV8 coding region and flag mark (NdeI/Xba I) and rbcL 3 ' UTR (Xba I/Bam HI; Fig. 5 A), and atpA 5 ' UTR (Bam HI/NdeI), HSV8 coding region and flag mark (NdeI/Xba I) and rbcL 3 ' UTR (Xba I/BamHI; Fig. 5 B) relevant limit site.

[0064] Fig. 5 C provides a restriction map, its shown with the HSV8-lsc gene integration in plasmid p322 so that be integrated into integration site in the Chlamydomonas reinhardtii chloroplast gene group.The DNA of p322 plasmid contains the segment from Eco RI to Xho I of one section 5.7kb, this segment is corresponding in the chloroplast gene group of Chlamydomonas reinhardtii 44,877 to 50,577 position (referring to URL address, World Wide Web " biology.duke.edu/chlamy_genome/chloro.html ").The zone of double-headed arrow indication is corresponding to probe used in the Southem hybridization analysis.Black surround indication (from left to right) be respectively the exon 5 of psbA, the 23S ribosomal gene of 5S ribosomal gene and small portion.

[0065] Fig. 6 provides the sign of the HSV8-lsc that is incorporated into the HSV8 viral protein, and this obtains by ELISA.The HSV8-lsc that obtains by affinity purification from genetically modified Chlamydomonas reinhardtii strain system (10-6-3 and 16-3) screens with HSV albumen in elisa assay, and HSV albumen is from being prepared the cell of virus infection.100,80,70,60,30,20,10 or 5 microlitres use the viral protein bag of constant basis by incubation in titer plate by the HSV8-lsc albumen of Flag purifying.Proteic concentration is 13ng/ μ l in the extracting solution of these affinity purifications, and wherein about 10% is HSV8-lsc, and this identifies by Coomassie brilliant blue dyeing.The albumen of isopyknic wild-type Chlamydomonas reinhardtii is as negative control (concentration is 1 μ g/ μ l).

[0066] Fig. 7 provides the sequence alignment of luxAB (SEQ ID NO:44) and luxCt (amino-acid residue 2 to 695 of sequence SEQ ID NO:46) coding region.The aminoacid sequence that has the codon of revising is represented with the amino acid of square frame and shade mark.The codon of optimizing is defined as using in per 1000 codons in the Chlamydomonas reinhardtii chloroplast gene group and surpasses 10 times codon (Nakamura etc., 1999).These two proteinic aminoacid sequence differences indicate by the amino acid with square frame and shade mark, and cause obtaining the plain enzyme of active fluoro and reformed two amino acid mark above the amino acid of change with * *.

[0067] Fig. 8 A and 8B provide the collection of illustrative plates of the luxCt gene of expressing in the Chlamydomonas reinhardtii chloroplast(id).

[0068] Fig. 8 A has illustrated and has described atpA 5 ' UTR (Bam HI/Nde I), the relevant limit site of luxCt coding region (NdeI/Xba I) and rbcL 3 ' UTR (Xba I/Bam HI).

[0069] Fig. 8 B provides a collection of illustrative plates that shows the homology zone between plasmid p322 and the Chlamydomonas reinhardtii chloroplast gene group, and chimeric luxCt gene is inserted in this homology zone exactly.The Chlamydomonas reinhardtii chloroplast DNA is that the Eco RI with 5.7kb represents that to the segment of Xho I this segment is to be positioned in the reverse repeat region of chloroplast gene group.The zone that double-headed arrow is represented is corresponding to used probe in Southern and the Northern hybridization analysis.What black surround was from left to right indicated respectively is the exon 5 of psbA, 5S ribosome-RNA(rRNA) and 23S ribosomal RNA gene.

Detailed Description Of The Invention

[0070] the invention provides in plastid, expressive function polypeptide in plant chloroplast particularly, the composition and the method that comprise the functional protein mixture, and promote the composition that polynucleotide is expressed in chloroplast(id) and prokaryotic organism at the polypeptide that shuttles back and forth between plant chloroplast and the prokaryotic organism and allow to be encoded.In a kind of specific embodiments, method of the present invention is come illustration by expressive function antibody, described antibody comprises correctly the assembling and the single-chain antibody of conjugated antigen specifically, and expresses and can to unite the antibody and the antigen binding fragment thereof that form the homodimer of conjugated antigen specifically specifically disconnected as strand.

[0071] a method according to the present present invention, the polynucleotide of encoding said antibody effectively is connected with one 5 ' end non-translational region (5 ' UTR), and described non-translational region comprises the ribosome binding sequence (RBS) that instructs described antibody to translate in chloroplast(id).In the another one specific embodiments, a RBS of the polynucleotide of encoding said antibody and the translation of guidance in prokaryotic cell prokaryocyte, and the 2nd RBS of the translation of guidance in chloroplast(id) effectively connects.In another one specific embodiments again, the polynucleotide of encoding said antibody has the chloroplast(id) codon and uses preference.

[0072] according to another method of the present invention, with synthetic polynucleotide transfered cell, described synthetic polynucleotide comprises at least the first nucleotide sequence of coding at least the first polypeptide, at least one codon has chloroplast(id) codon use preference in wherein said first nucleotide sequence, and the polypeptide that is encoded can be expressed in cell.In a kind of specific embodiments, each codon all has chloroplast(id) codon use preference in described first nucleotide sequence, in the another one specific embodiments, described synthetic polynucleotide contains at least one second nucleotide sequence, described second nucleotide sequence can but might not effectively be connected with first nucleotide sequence, and described second nucleotide sequence coded at least a second polypeptide, the expression of wherein said polynucleotide can but might not generate the fusion rotein that comprises described first and second (perhaps more) polypeptide.Therefore, the invention provides the synthetic polynucleotide, it comprises at least the first nucleotide sequence of coding at least the first polypeptide, and at least one codon has chloroplast(id) codon use preference in wherein said first nucleotide sequence.Noun used herein " synthetic polynucleotide " means the nucleic acid molecule through modifying, wherein not having the chloroplast(id) codon in the polypeptide uses the codon of preference to be changed to having the codon (referring to table 1, as follows) of chloroplast(id) codon use preference.As disclosed in this, strong by the such expression of synthetic polynucleotide encoded polypeptides in chloroplast(id).

[0073] in other specific embodiments, provides the composition of putting into practice method of the present invention.Comprise the ability that can make functional polypeptide strong expression in plant chloroplast by advantage provided by the invention, wherein polypeptide also can be by glycosylation, therefore antigenicity can reduce when being applied to individuality, can also produce a large amount of functional polypeptides in addition under the situation that does not need fermentation equipment and associated expense.

[0074] method of the present invention provides the means of expressing one or more polypeptide in plant chloroplast, and described like this polypeptide can be assembled and produce the functional protein mixture.As disclosed herein, polypeptide expressed not only can correctly be assembled in the chloroplast(id), and when these polypeptide comprised the subunit of an albumen composition, these polypeptide can be united the systematic function albumen composition specifically.Here the term of using " albumen composition " means by the specificity of two polypeptide at least unites the composition that forms, and described polypeptide can be the same or different.The polypeptide that can unite the function that plays albumen composition specifically is known, comprises enzyme, somatomedin and hormone receptor, and analogue.

[0075] term of using here " specificity associating " or " specificity interaction " or " specificity in conjunction with " mean two or more the more peptide be formed on metastable complex body under the physiological condition.Here the term of using can be meant various interactions, comprises, for example the interaction of the first polypeptide subunit and the second polypeptide subunit can form the functional protein mixture by this interaction, and the interaction between antibody and its antigen.Specific interaction can be by being at least about 1 * 10 ^-6The dissociation constant of M characterizes, and generally is about at least 1 * 10 ^-7M usually is about at least 1 * 10 ^-8Be about at least 1 * 10 under the M, particular case ^-9M or 1 * 10 ^-10M or bigger.Specific interaction is stable under physiological condition generally, described physiological condition comprises condition in the active somatic cell or the condition in the subcellular compartment, described live body comprises plant or animal, wherein animal can be vertebrates or invertebrates, and the condition of in cell cultures, using, such as the condition that is used to keep certain biological cell or tissue.Whether interactional specifically method is known to detect two molecules, comprises for example equilibrium dialysis, surperficial plastid resonance, gel retardation assay, and similar method.

[0076] method of the present invention can be used to produce functional polypeptide, comprises the functional protein mixture, and this purposes is by this illustration that is created in of functional antibodies.Here widely used term " antibody " means the polypeptide or the albumen composition of the epitope of conjugated antigen specifically.Usually, antibody contains at least one antigen binding domains, and this structural domain is to unite formation by variable region of heavy chain structural domain and variable region of light chain structural domain, particularly hypervariable region.The antibody that method in according to the present invention generates can be based on abiogenous antibody, such as bivalent antibody, it (for example contains two antigen binding domainss that can form by first heavy chain and variable region of light chain and second heavy chain and variable region of light chain, IgG or IgA homotype), perhaps pass through two antigen binding domains (V that first variable region of heavy chain and second variable region of heavy chain form _HHAntibody; Referring to, such as U.S. Patent number 6,005,079), or based on non-abiogenous antibody, comprise such as, single-chain antibody, chimeric antibody, bifunctional antibody, (humanized) antibody of humanization, and the antigen binding fragment of antibody is disconnected, such as the Fab segment, and the Fd segment, Fv segment, and analogue.

[0077] in a kind of specific embodiments, method of the present invention comprises that with coding heavy chain comes illustration with the polynucleotide of the single-chain antibody of the light chain that effectively is connected with it, and wherein said antibodies specific ground is in conjunction with tetanus toxin (referring to SEQ ID NO:13 and 14).In another specific embodiments, present method is to comprise that with coding heavy chain comes illustration with the polynucleotide of the single-chain antibody of the light chain that effectively is connected with it, wherein said antibodies specific ground is in conjunction with herpes simplex virus type 1 and 2 types, and wherein the polynucleotide of encoding said antibody has chloroplast(id) codon use preference (referring to SEQ ID NO:15 and 16; SEQ ID NO:42 and 43; SEQ IDNO:47 and 48; Also see also embodiment 3).

[0078] polynucleotide that is used for putting into practice method of the present invention can separate from the cell that produces interested antibody, such as, from the B cell of immune body or from the individuality of contacted specific antigen, also can use known polynucleotide synthetic method de novo synthesis, can also produce by recombination method, perhaps can be for example the combinatorial library of polynucleotide by screening coding variable heavy chain and variable light chain obtain (referring to Huse etc., Science246:1275-1281 (1989), this piece document is put in order into for your guidance at this), and described polynucleotide can have the use of chloroplast(id) codon preference, (referring to embodiment 1 and table 1) if necessary.Preparation coding is for example chimeric, these methods and other method of the polynucleotide of peopleization, CDR transplanting, strand and bifunctional antibody are (Winter and the Harris that is widely known by the people to those skilled in the art, Immunol.Today 14:243-246,1993; Ward etc., Nature 341:544-546,1989; Harlow and Lane, Antibodies:A laboratorymanual (Cold Spring Harbor Laboratory Press, 1988); Hilyard etc., ProteinEngineermg:A practical approach (IRL Press 1992); Borrabeck, AntibodyEngineering, second edition (Oxford University Press 1995); All put in order into for your guidance for every piece) at this.

[0079] gives an example, the polynucleotide of the monoclonal antibody of coding peopleization can obtain like this, the nucleotide sequence of the complementarity determining region of coding rat immune globulin heavy chain and variable region of light chain is transferred in the human varied texture domain gene, in the corresponding frame area of mouse, replaced human residue then.The general technology that is used to clone the rat immune globulin variable domains be known (referring to, such as, Orlandi etc., Proc.Natl.Acad.Sci., USA 86:3833,1989, this piece document is put in order into for your guidance at this), preparation peopleization monoclonal antibody method also be known (referring to, such as, Jones etc., Nature 321:522,1986; Riechmann etc., Nature332:323,1988; Verhoeyen etc., Science 239:1534,1988; Carter etc., Proc.Natl.Acad.Sci., USA 89:4285,1992; Sandhu, Crit.Rev.Biotechnol.12:437,1992; With Singer etc., J.Immunol.150:2844,1993; Every piece of document is all put in order into as a reference at this).

[0080] method of the present invention can also use coding pulsating polynucleotide of isolated human antibodies from combination immunoglobulin (Ig) library put into practice (referring to, such as, Barbas etc., Methods:A Companion to Methods in Immunology 2:119,1991; Winter etc., Ann.Rev.Immunol.12:433,1994; Every piece of document is all put in order into for your guidance at this).The cloning vector and the expression vector that are used to produce the human immunoglobulin phage library can be by for example obtaining from Stratagene Cloning Systems (adding the Ni Fuliya state, the La Jolla).

[0081] coding human monoclonal antibody's polynucleotide can also for example obtain from transgenic mouse, and described transgenic mouse has passed through genetically engineered, can produce the antigen reactive specific human antibody-like of response.In this technology, the human heavy chain and the element of light chain gene seat are directed in the strain system of mouse, and described strain system comes from endogenous heavy chain and light chain gene seat by target destructive embryonic stem cell line.This transgenic mouse can synthesize the specificity human body antibody at the human antigen, and this mouse can be used for producing the hybridoma of secretion human body antibody, therefrom can obtain to be used to put into practice the polynucleotide of method of the present invention.The method that obtains human antibodies from transgenic mouse was described in following document, such as Green etc., and Nature Genet.7:13,1994; Lonberg etc., Nature 368:856,1994; With Taylor etc., Intl.Immunol.6:579,1994; Every piece of document is all put in order into for your guidance at this, and such transgenic mouse is (the Abgenix company that can obtain by the commercial channel; Add the Ni Fuliya state, Fo Limeng).

[0082] described polynucleotide also can be the disconnected polynucleotide of antigen binding fragment of encoding antibody.Antigen binding fragment is disconnected, comprises, and such as Fv, Fab, Fab ', Fd, and F (ab ') ₂Segment, these all are known in the art, and at first all are that protease hydrolysis by antibody identifies.For example, antibody fragment can obtain by traditional stomach en-and the whole antibody of papain hydrolysis.The antibody fragment that obtains with the stomach en-enzymolysis produces the segment of a 5S, is called as F (ab ') ₂Segment.This segment can be further with the also original reagent shearing of a kind of sulfydryl, and the sulfydryl that produces for the cutting that is connected by disulfide linkage can also selectively use blocking groups, so just produces the Fab ' monovalent fragments of a 3.5S.As selection, can carry out enzyme cutting with stomach en-, directly produce two Fab ' monovalent fragments and Fc segment (referring to, such as, Goldenberg, U.S. Patent number 4,036,945 and U.S. Patent number 4,331,647, each is all put in order into for your guidance at this; Nisonhoff etc., Arch.Biochem.Biophys.89:230.1960; Porter, Biochem.J.73:119,1959; Edelman etc., Meth.Enzymol.1:422 (Academic Press 1967); Coligan etc. are at Curr.Protocols Immunol.1992, referring to 2.8.1-2.8.10 and 2.10.1-2.10.4; Every piece of document is all put in order into for your guidance at this).

[0083] another form of antibody fragment is the peptide of the single complementary determining region of coding (CDR).The CDR peptide can be by making up the interested antibody of coding the polynucleotide of CDR obtain, such as by the polymerase chain reaction by the RNA of the cell that produces antibody synthesize described variable region (referring to, such as, Larrick etc., Methods:A Companion to Methods inEnzymology 2:106,1991, this piece document is put in order into for your guidance at this).The polynucleotide that such antibody fragment of encoding---comprises these pulsating subunits and is connected for example connection peptides of variable region of heavy chain and variable region of light chain---can prepare by chemical synthesis process or conventional recombinant DNA method, and such preparation can be initial from the polynucleotide of the coding total length heavy chain that obtains by foregoing method and light chain.

[0084] present method is based in part on such fact, and promptly ribosome binding sequence (RBS) can cause this section encoding sequence to be translated consumingly in plant chloroplast (referring to following content with respect to the correct location of encoding sequence; Also referring to embodiment 2), those have been notified when generating naturally in vivo and unite the polypeptide (for example antibody) that forms albumen composition specifically and also can correctly unite (referring to embodiment 3) in chloroplast(id).An advantage expressing such polypeptide in chloroplast(id) is that the polypeptide of expressing so can not pass through some cellular compartments as the polypeptide of being expressed by nuclear gene, so also just some posttranslational modification can not take place such as glycosylation.Equally, polypeptide or the albumen composition that produces by method of the present invention can have the lower antigenicity of expressing of same polypeptide from the polynucleotide that imports to eukaryotic cells nuclear.

[0085] method of the present invention provides the means that produce functional polypeptide and albumen composition, and the former is single-chain antibody for example, and the latter is bivalent antibody for example, comprise such as first heavy chain and light chain and with second heavy chain and the light chain of its associating.As disclosed in this, method of the present invention can be implemented like this, such as, in chloroplast(id), import recombinant nucleic acid molecules, wherein said recombinant nucleic acid molecules comprises at least one polypeptide of coding (promptly, 1,2,3,4 or more) first polynucleotide and second polynucleotide that effectively is connected with it, described second polynucleotide comprises the nucleotide sequence of the encode nucleotide sequence of a RBS and coding the 2nd RBS that effectively is connected with it, the described at least a polypeptide expression of the conditions permit of described importing.Such condition comprises and allows or help the condition that described recombinant nucleic acid molecules enters chloroplast(id), preferably, is with the condition of described recombinant nucleic acid molecule integrates in the chloroplast gene group.Such method is included in this method of enumerating, and in other method of routine known in the art.

[0086] term that here uses " effectively connects " and means two or more a plurality of molecule and located relative to each other, they can be used as a single unit and treat like this, and the function that shows is the characteristic that possesses one of them or two molecules or their combination.For example, the polynucleotide of coded polypeptide can effectively be connected with transcriptional regulatory element or translational control element, in this case, described element is just regulated and control the polymerized nucleoside acid sequence that normally links to each other with them as them for the regulating and controlling effect of described polynucleotide in cell.One first polynucleotide encoding sequence can effectively be connected with one second (perhaps more) encoding sequence, such chimeric polyeptides just can from this encoding sequence that effectively connects, express (referring to, such as, SEQ ID NO:30, it has shown coding GFP and has had polynucleotide that the chloroplast(id) codon uses preference (promptly, SEQ ID NO:1) be inserted into the site in the PsbD gene, a kind of like this fluorescent fusion protein that merges the PsbD gene product that GFP is arranged that comprised has just produced).Described chimeric polyeptides can be a fusion polypeptide, two (perhaps more) peptides that wherein are encoded are translated into single polypeptide, promptly link to each other by the peptide bond covalency, for example, comprise (by connection peptides, if necessary) the effective variable region of heavy chain that connects and the single-chain antibody of variable region of light chain; Perhaps can translate into two independently polypeptide, after translation, can unite mutually specifically then and form stable albumen composition, for example, the heavy chain of antibody and light chain, it forms quaternary structure, obtain functional univalent antibody, this univalent antibody can further be united the functional bivalent antibody of formation.The example of synthetic polynucleotide of such fusion rotein of encoding comprises SEQ ID NO:45, its coding bacteriofluorescein enzyme fusion proteins, and SEQ ID NO:15,42 and 47, the anti-HSV antibody of they coding strands.

[0087] term that here is widely used " polynucleotide " or " nucleotide sequence " or " nucleic acid molecule " mean the sequence that is coupled together by the phosphide key by two or more deoxyribonucleotides or ribonucleotide.Like this, these terms comprise RNA and DNA, can be the parts of a gene or gene, also comprise cDNA, synthetic polydeoxyribonucleotide sequence, perhaps similar molecule, and can be strand or two strands, also can be the DNA/RNA hybrid molecule.Further, here these terms of using comprise abiogenous nucleic acid molecule, they can separate from cell, also comprise the synthetic polynucleotide, and they can prepare such as the method by chemosynthesis or Enzymology method such as polymerase chain reaction (PCR).It should be noted that, here used different terms are the convenience in order to discuss only, such as in order to distinguish the different components of a composition, by way of exception be at this term of using " synthetic polynucleotide ", it is specially to refer to have the polynucleotide that the chloroplast(id) codon uses preference through after modifying.

[0088] usually, the Nucleotide of forming polynucleotide comprises abiogenous deoxyribonucleotide, such as the VITAMIN B4 that is connected on the 2 ' ribodesose, cytosine(Cyt), guanine or thymus pyrimidine, perhaps ribonucleotide is such as the VITAMIN B4 that is connected on the ribose, cytosine(Cyt), guanine or uridylic.Yet according to the difference of purposes, polynucleotide can also contain nucleotide analog, and these analogues comprise non-abiogenous synthetic Nucleotide or the abiogenous Nucleotide through modifying.Nucleotide analog is known in this area, and can obtain from commercial channels (such as Ambion company; Austin TX), the polynucleotide that contains such nucleotide analog also is (Lin etc., Nucl.Acids Res.22:5220-5234,1994 like this; Jellinek etc., Biochemistry 34:11363-11372,1995; Pagratis etc., NatureBiotechnol.15:68-73,1997, every piece of document is all put in order into for your guidance at this).The covalent linkage that connects Nucleotide in the polynucleotide generally all is the phosphide key.Yet, the purpose that is employed according to polynucleotide, described covalent linkage also can be a kind of in other a variety of keys, comprise thioester bond, sulfo-phosphide key, the similar key of peptide or any other known in the art can connect the key that Nucleotide generates the synthetic polynucleotide (referring to, such as, Tam etc., Nucl.Acids Res.22:977-986,1994; Ecker and Crooker, Biotechnology 13:351360,1995, every piece of document is all put in order into for your guidance at this).

[0089] containing the polynucleotide of abiogenous Nucleotide and phosphide key can chemosynthesis, perhaps can utilize suitable polynucleotide to prepare by recombinant DNA method as template.In contrast to this, the polynucleotide that contains the covalent linkage of nucleotide analog or non-phosphide key generally need prepare by chemosynthesis, although the enzyme such as the T7 polysaccharase also can be incorporated into the nucleotide analog of some type in the polynucleotide, and therefore can pass through the such polynucleotide of recombinant methods with the suitable template of cause.

[0090] term used herein " recombinant nucleic acid molecules " means the polynucleotide of operating by artificial intervention.Recombinant nucleic acid molecules can comprise two or more nucleotide sequences, and they are connected to be formed on does not have found product in the natural cell.Especially, described two or more nucleotide sequences can effectively connect, and the fusion polypeptide of can for example encoding, and perhaps can comprise coding nucleotide sequence and controlling element, especially, described controlling element refers to a RBS and the 2nd RBS of effectively connection.Recombinant nucleic acid molecules also can be based on abiogenous polynucleotide, but after operation, they and abiogenous polynucleotide are different, for example wherein there is one or more Nucleotide to change, normal first codon that occurs has had chloroplast(id) codon use preference in this polynucleotide like this, perhaps interested sequence is directed in the described polynucleotide, for example restriction enzyme enzyme recognition site or splice site, promotor, the dna replication dna initial point, perhaps similar sequence.

[0091] as disclosed in this, RBS is positioned at initiator codon and can translates (referring to embodiment 2) consumingly such as the feasible encoding sequence that originates in this AUG codon of about 20 to 40 Nucleotide in the upstream (5 ') of AUG codon.Like this, the RBS that is positioned about 20 to 40 nucleotide positions in AUG codon upstream can be considered to " effectively be connected " with described AUG codon.Further, what known is, the RBS that is positioned at about 5 to 15 nucleotide positions of upstream from start codon can instruct the translation of encoding sequence in prokaryotic organism, and as disclosed herein, such RBS can effectively be connected with the 2nd RBS that is positioned at about 20 to 40 nucleotide positions of upstream from start codon, and the translational control element that obtains like this can instruct the translation of encoding sequence in prokaryotic organism and chloroplast(id).Like this, first and second RBS are separated by about 5 to 25 Nucleotide, and they also are considered to effectively connect.It should be noted that, term used herein " first ", " second ", " 3rd " or the like is meant a RBS or polynucleotide or polypeptide, or analogue, the use of these terms is the convenience in order to discuss just, unless specialize, otherwise do not infer any order or the importance or the similar meaning.Similarly, when for example speaking of a RBS of the translation that can instruct in prokaryotic organism and can instruct the 2nd RBS of the translation in chloroplast(id), " first " used herein, " second " (perhaps similar title) are just for the ease of distinguishing these two (perhaps more a plurality of) elements.

[0092] have the ability of " instruct translation " for RBS, this is meant that when it effectively was connected with common encoding sequence with start codon initiation, described RBS can combine with rrna, translates so just can begin from described initiator codon.Term used herein " initiator codon " is meant as the ribonucleoside acid sequence of first codon of encoding sequence or coding deoxyribonucleotide sequence.Usually, initiator codon is one " initial AUG codon " (in RNA) or one " initial ATG codon " (in DNA), and its methionine(Met) of encoding although other codon also can be used as initiator codon, comprises for example CUG.

[0093] codon that can make one or more codon of coding polynucleotide show chloroplast(id) uses preference (embodiment 1).Most of amino acid are by two or more a plurality of different (degeneracy) codon coding, and what known is that some codon of different biological utilisations has priority with respect to other codon.This preferred codon uses and is also adopted " use of chloroplast(id) codon " that this just mentions by chloroplast(id) here.What table 1 (following) showed is the chloroplast(id) codon use of Chlamydomonas reinhardtii.

[0094] when term " was tended to (biased) " and is used to describe a codon, it was meant that the sequence of a codon in the polynucleotide is changed, and made this codon become the preferred codon (referring to table 1) that uses in chloroplast(id).Being tended to polynucleotide that the codon of chloroplast(id) uses can de novo synthesis, perhaps can use conventional recombinant DNA technology and carry out genetic modification and obtain, make them have the chloroplast(id) codon to use preference (referring to embodiment 1) such as change one or more codon by the site-specific nature mutation method.As disclosed herein, the codon preference of chloroplast(id) is some difference of possibility between different plants, compares with the chloroplast(id) of tobacco such as the chloroplast(id) of algae.Usually,---for example comprise when preparation synthetic polynucleotide disclosed herein---that according to purpose of the present invention selected chloroplast(id) codon preference will reflect that the codon of certain plant chloroplast uses, comprise a kind of like this codon preference, promptly A/T is partial in the 3rd of a codon the position, for example, the 3rd position has and surpasses about 66% AT preference, especially, has and surpasses about 70% AT preference.Like this, according to purpose of the present invention and the chloroplast(id) codon of preference got rid of for example biased good at Nicotiana tabacus (tobacco) the observed the 3rd, its on the 3rd of codon, have 34.56% GC preference (referring to, for example, World Wide Web URL " kazusa.or.jp/codon/ " and wherein " chloroplast(id) " link).In a kind of specific embodiments, it is the preference that algae chloroplast(id) codon uses that described chloroplast(id) codon uses preference, such as Chlamydomonas reinhardtii, has 74.6% AT preference on the 3rd of the codon of its chloroplast(id).

Table 1

Chloroplast(id) codon in the Chlamydomonas reinhardtii uses

UUU?34.1 ^*(348 ^**)?UCU?19.4(198) UAU?23.7(242) UGU?8.5(87)

UUC?14.2(145) UCC?4.9(50) UAC?10.4(106) UGC?2.6(27)

UUA?72.8(742) UCA?20.4(208) UAA?2.7(28) UGA?0.1(1)

UUG?5.6(57) UCG?5.2(53) UAG?0.7(7) UGG?13.7(140)

CUU?14.8(151) CCU?14.9(152) CAU?11.1(113) CGU?25.5(260)

CUC?1.0(10) CCC?5.4(55) CAC?8.4(86) CGC?5.1(52)

CUA?6.8(69) CCA?19.3(197) CAA?34.8(355) CGA?3.8(39)

CUG?7.2(73) CCG?3.0(31) CAG?5.4(55) CGG?0.5(5)

AUU?44.6(455) ACU?23.3(237) AAU?44.0(449) AGU?16.9(172)

AUC?9.7(99) ACC?7.8(80) AAC?19.7(201) AGC?6.7(68)

AUA?8.2(84) ACA?29.3(299) AAA?61.5(627) AGA?5.0(51)

AUG?23.3(238) ACG?4.2(43) AAG?11.0(112) AGG?1.5(15)

GUU?27.5(280) GCU?30.6(312) GAU?23.8(243) GGU?40.0(408)

GUC?4.6(47) GCC?11.1(113) GAC?11.6(118) GGC?8.7(89)

GUA?26.4(269) GCA?19.9(203) GAA?40.3(411) GGA?9.6(98)

GUG?7.1(72) GCG?4.3(44) GAG?6.9(70) GGG?4.3(44)

^*The frequency of utilization of certain codon in per 1000 codons.

^*Observed number of times in 36 chloroplast(id) encoding sequences (10193 codons).

[0095] the method for the present invention polynucleotide that can use one first polypeptide of coding and at least one second polypeptide is implemented.Like this, described polynucleotide can encoding ratio as one first polypeptide and one second polypeptide; One first polypeptide, one second polypeptide and one the 3rd polypeptide; Or the like.Further, any one or all polypeptide of being encoded can be the same or different.As disclosed herein, the polypeptide expressed assembling forms functional polypeptide and albumen composition (referring to embodiment 1 and 3) in the chloroplast(id) of little algae Chlamydomonas reinhardtii.Like this, method of the present invention provides a kind of means of producing the functional protein mixture, described albumen composition comprises for example dimer, the tripolymer and the tetramer, and the subunit of wherein said mixture can identical or different (for example being respectively homodimer or heterodimer).The method of expressive function polypeptide and albumen composition is come illustration by the production of antibody and the production of reporter protein in chloroplast(id), and described reporter protein comprises green fluorescent protein and luciferase (luxAB fusion rotein; Referring to embodiment 1 and 4; Also can be respectively referring to SEQ ID NOS:1 and 45), antibody can use the polynucleotide of preference express (referring to embodiment 3, also can referring to SEQ ID NOS:15,42 and 47) from having the chloroplast(id) codon.As illustrative at this, chloroplast(id) carries out transfection with recombinant nucleic acid molecules, described recombinant nucleic acid molecules comprises the polynucleotide of the single-chain antibody of encoding, described single-chain antibody has a complete heavy chain that is connected on the variable region of light chain, and having generated the homodimer that comprises two single-chain antibodies thus, described two single-chain antibodies are by the specificity interaction gang between its heavy chain structural domain.These results provide the proof heterologous polypeptide can unite the evidence that forms quaternary structure specifically in chloroplast(id), and proved that heteromultimers can produce by method of the present invention, single recombinant nucleic acid molecules by each different polypeptide of the described heteromultimers of importing coding in chloroplast(id), perhaps import two or more polynucleotides, encode (perhaps a plurality of) subunit of described heteromultimers of wherein each.

[0096] method of the present invention can be used one first recombinant nucleic acid molecules and implements, described recombinant nucleic acid molecules comprises that coding instructs the nucleotide sequence of the RBS of the translation in chloroplast(id), preferably, this section nucleotide sequence is the further RBS of the translation of guidance in prokaryotic organism that effectively connect of coding also, and described nucleotide sequence effectively is connected with at least one polynucleotide of the first polypeptide of encoding at least.For example, described recombinant nucleic acid molecules can comprise coding heavy chain immunoglobulin (H) or its variable region (V _H) polynucleotide, further coding second polypeptide, described second polypeptide is light chain immunoglobulin (L) or its variable region (V _L).If necessary, the nucleotide sequence of the inherent ribosome entry site(RES) of encoding can be positioned to encode between the nucleotide sequence of H chain and L chain, helps second (downstream) polypeptide expression that is encoded like this.After H chain that is encoded and L chain were translated out in chloroplast(id), a H chain can be united a formation univalent antibody (such as a H:L complex body) with a L chain, and two H:L complex bodys can further be united bivalent antibody of formation.

[0097] method of the present invention also can be put into practice like this, imports first recombinant nucleic acid molecules in plant chloroplast, and described first recombinant nucleic acid molecules comprises coding for example H chain or V _HThe polynucleotide of chain further imports second recombinant nucleic acid molecules to described chloroplast(id) again, and described second recombinant nucleic acid molecules comprises L chain of coding or V _LThe polynucleotide of chain, wherein each recombinant nucleic acid molecules all comprises the nucleotide sequence of the encode nucleotide sequence of a RBS and coding the 2nd RBS that effectively is connected with it, a wherein said RBS can instruct the translation of polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of polypeptide in chloroplast(id), and the nucleotide sequence of these two RBS that wherein encode effectively is connected with described coding polymerized nucleoside acid sequence.When the vegetable cell that contains described chloroplast(id) was exposed to the condition that allows the polypeptide coexpression that is encoded, H chain and L chain can be united and formed the H:L complex body, and the H:L complex body can further be united the generation bivalent antibody then.

[0098] recombinant nucleic acid molecules that includes the polynucleotide of coded polypeptide can further comprise peptide-labeled thing such as His-6 mark or similar mark, described mark effectively is connected with described encoding sequence, helps to identify the expression of described polypeptide in cell.For example the polyhistidine mark peptide of His-6 can be with divalent cation such as nickel ion, cobalt ion or similar ion detection.The peptide-labeled of other comprise, for example, the FLAG epi-position, it can detect by anti-FLAG antibody (referring to, such as, Hopp etc., BioTechnology 6:1204 (1988); U.S. Patent number 5,011,912, every piece of document are all put in order into for your guidance at this); The c-myc epi-position, it can detect with the antibody at this epi-position specifically; Vitamin H, it can or affinely usually detect with streptavidin; And glutathione s-transferase, it can detect with gsh.Such mark can provide some extra advantages, and for example, they help with it the effectively separation of the polypeptide of connection, if wish to obtain the polypeptide of purifying basically.

[0099] recombinant nucleic acid molecules of using in the method for the present invention can be included in the carrier.Further, if this method is used second (perhaps more) recombinant nucleic acid molecules, so described second recombinant nucleic acid molecules also can be included in the carrier, this carrier can but might not be identical with the carrier that contains first recombinant nucleic acid molecules.Described carrier can be any carrier that is used for importing to chloroplast(id) polynucleotide, preferably, described carrier contain one section chloroplast genomic dna nucleotide sequence so that with chloroplast gene group generation homologous recombination, include in the chloroplast genomic dna 400 to 1500 or the nucleotide sequence of more a plurality of continuous nucleotides such as one section.The chloroplast(id) carrier and select on the chloroplast gene group some zone as the method for carrier be known (referring to, such as, Bock, J.Mol.Biol.312:425-438,2001; Also can be referring to Staub and Maliga, Plant cell 4:39-45,1992; Kavanagh etc., Genetics 152:1111-1122,1999, every piece of document is all put in order into for your guidance at this).

[0100] the whole chloroplast gene group of Chlamydomonas reinhardtii is to obtain by the public database on the World Wide Web, its URL address is " biology.duke.edu/chlamy_genome/chloro.html " (referring to " checking the complete genome group with text file format " link and " chloroplast gene picture group spectrum " link), here each piece document is all put in order into (J.Maul for your guidance at this, J.W.Lilly, and D.B.Stern, do not deliver the result; Revise on January 28th, 2002; Will deliver with GenBank number of registration AF396929).Usually, the nucleotide sequence of selecteed chloroplast genomic dna is not the part of gene, comprise and be not regulating and controlling sequence or encoding sequence, especially, if because homologous recombination causes gene destroyed, thus can be to the influence of damaging property of chloroplast(id), such as influencing duplicating of chloroplast gene group, perhaps can the vegetable cell that contain chloroplast(id) be caused a devastating effect, so, the nucleotide sequence of selecteed chloroplast genomic dna can not be the part of such gene.In view of this consideration, announced that the website of Chlamydomonas reinhardtii chloroplast gene group sequence also provides the coding region of demonstration chloroplast gene group and the collection of illustrative plates of non-coding region, therefore helped to pick out the sequence that can be used for making up carrier of the present invention.For example, it approximately is that Eco (EcoRI) site of 143.1kb extends to the clone in the Xho that the position approximately is 148.5kb (Xho I) site (referring to the World Wide Web from the position that the chloroplast(id) carrier p322 that uses in this disclosed experiment is one, the URL address is " biology.duke.edu/chlamy genome/chloro.html ", click " chloroplast gene picture group spectrum " link, and then click " 140-150kb " link; Also can directly enter by World Wide Web URL " biology.duke.edu/chlamy/chloro/chloro140.html "; Also referring to embodiment 1).

[0101] described carrier can also comprise some the extra application that help carrier or the nucleotide sequence of operation, such as, one or more transcriptional regulatory element, the sequence of coding selected marker, one or more cloning site, and similar sequence.In a specific embodiments, described chloroplast(id) carrier contains procaryotic replication origin (ori), such as, colibacillary ori, thus the shuttle vectors that can shift and operate between prokaryotic host cell and chloroplast(id) is provided.

[0102] method of the present invention illustrates by using little algae Chlamydomonas reinhardtii.The method according to this invention, using little algae to come the advantage of express polypeptide or albumen composition is can cultivate in a large number by this little algae, comprises coml channel (Cyanotech company; Kailua-Kona HI), thereby can produce and isolate a large amount of needed products, if necessary.Yet any plant can be expressed the ability that for example functional Mammals polypeptide comprises albumen composition and also be allowed such plant to plant in a large number in chloroplast(id), therefore also can produce a large amount of polypeptide easily.Therefore, method of the present invention can be put into practice with any plant that contains chloroplast(id), comprise, such as, bulk kelp such as marine algae and sea grass, and be grown in plant in the soil, such as cereal (corn (Zea mays)), Chinese cabbage is (such as B.napus, B.rapa, B.juncea), particularly those are used to produce the kind of vegetables oil, clover (Medicago sativa), paddy rice (Oryza sativa), rye (Secale cereale), Chinese sorghum (Sorghum bicolor, Sorghum vulgare), Zea mays (such as, pearl broomcorn millet (Pennisetum glaucum), millet broomcorn millet (Panicum miliaceum), foxtail (Setaria italica), finger broomcorn millet (Eleusinecoracana)), Sunflower Receptacle (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotianatabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocosnucifera), pineapple (Ananas comosus), mandarin tree (Citrus spp.), cocoa tree (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea ultilane), Fructus Fici (Ficus casica), piscidia (Psidiumguajava), mango (Mangifera indica), olive wood (Olea europaea), papaya (Carica papaya), cashew nut (Anacardium occidentale), Hawaii drupe (Macadamia integrifolia), almond fruit (Prunus amygdalus), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), oat, duckweed (Lemna), barley, tomato (Lycopersicon esculentum), lettuce (such as Lactuca sativa), mung bean (Phaseolus vulgaris), lima bean (Phaseolus limensis), pea (Lathvrusspp.), and melon such as cucumber (C.sativus), muskmelon (C.cantalupensis), Moschus melon (C.melo).Ornamental plant is also includable; such as rhododendron (Rhododendron spp.); Flower of Largeleaf Hydrangea (Macrophylla hydrangea), lotus (Hibiscusrosasanensis), rose (Rosa spp.); turmeric (Tulipa spp.); flower of Chinese Narcissus (Narcissusspp.), petunia (Petunia hybrida), carnation (Dianthus caryophyllus); poinsettia (Euphorbia pulcherrima), and chrysanthemum.The ornamental plant that other can be used to put into practice method of the present invention comprises Flower of Garden Balsam, Flower of Evans Begonia, Flos Pelargonii, violet, primrose, vervain, Vinca, Flower of Aztec Marigold, the plant of primula, Sao Paulo flower, wrinkled giant hyssop, amaranth, Antihirrhinum, Herba Ranunculi Japonici, chrysanthemum, clover, Cosmo flower, cowpea, Garden Dahlia, Flos Daturae, Herba delphinii grandiflori, Herba Leibnitziae, gladiolus, gloxinia, belladonna lily, mesembrianthemum, salpiglossis, and zinnia.The conifer trees also can be used for putting into practice the present invention's method, such as comprising, pine tree such as torch pine (Pinus taeda), Ai Shi pine (Pinus elliotii), yellow oregon pine (Pinusponderosa), balk pine (Pinus contorta), and pine (Pinus radiata), Pseudotsuga menziesii (Mirbel) Franco (Pseudotsuga menziesii); The West Pinaceae evergreen tree (Tsuga ultilane); Dragon spruce (Picea glauca); U.S. Chinese fir (Sequoia sempervirens); Fir such as silver-colored China fir (Abies amabilis) and rubber China fir (Abies balsamea); And Chinese fir such as Western Chinese larch (Thuja plicata) and Alaska Douglas fir (Chamaecyparis nootkatensis).

[0103] leguminous plants that can be used to put into practice method of the present invention comprises bean and pea.Bean comprises guar-bean, the thorn locust bean, and trigonella, soybean, the garden beans, cowpea, mung bean, broad bean, French beans, garbanzo, or the like.Leguminous plants includes, but are not limited to, and Semen arachidis hypogaeae is such as peanut, and is vetch such as the coronule flower, villose vetch, red bean, mung bean, and garbanzo, lupinus is such as lupine, trifolium, Phaseolus is such as Kidney bean and lima bean, and Pisum is such as vetch, and the draft sweet-scented osmanthus belongs to such as cloves, Medicago is such as clover, and Nelumbo is such as trefoil; Lentil, such as root of Szemao crotalaria, and false indigo.The preferred forage grass and the meadow straw bag that use are in the method for the invention drawn together clover, rural area grass, Festuca Arundinacea, English ryegrass, this spy of crawling property grass, and white bent.Can be used for other plant of the present invention and comprise acacia, vanilla, arithoke, chrysanthemum south leaf mustard, black berry, rape, cilantro, Mediterranean Sea is tangerine early, witloof, eucalyptus, fennel, natsudaidai, hami melon, yam bean, Kiwifruit, lemon, bitter orange, mushroom, nut, gumbo, orange, parsley, persimmon, plantain, pomegranate, white poplar, pine, purple leaf witloof, the south pine, sweetgum, tangerine, triticale, grapevine, Chinese yam, apple, pear tree, warm cypress, cherry, apricot, muskmelon, hemp, buckwheat, grape, raspberry, lamb's-quarters, blueberry, nectarine, peach, plum, strawberry, watermelon, eggplant, pepper, Cauliflower, Chinese cabbage belongs to, such as cabbage, Chinese cabbage, avocado seedling, onion, Radix Dauci Sativae, leek, beet, broad bean, celery, radish, pumpkin, witloof, cucurbit, garlic, string bean, spinach, bottle gourd, turnip, avocado, romaine lettuce, Semen arachidis hypogaeae and green cucumber.

[0104] method of the present invention can obtain to contain through containing plant (that is Pignus pignoris body (transplastomes), of the chloroplast(id) of the polynucleotide that is stabilized integration after the genetic modification; Referring to, such as, Hager and Bock, Appl.Microbiol.Biotechnol.54:302-310,2000, this piece document is put in order into for your guidance at this; Also can be referring to, Bock, as above, 2001).The described polynucleotide that is integrated for example can comprise the coding polynucleotide that effectively is connected with first and second RBS in this definition.Therefore, the present invention further provides transgenosis (Pignus pignoris body) plant, it contains one or more chloroplast(id) that contains the polynucleotide of one or more heterologous polypeptides of encoding, and described polypeptide comprises that those can unite the polypeptide that forms the functional protein mixture specifically.The transgenic plant that contain the Pignus pignoris body are incorporated into transgenic plant in the nuclear gene group with those with polynucleotide and compare and have some advantages.Such as, in most of crop plants, chloroplast(id) is strictly by the ovum matrocliny; Then lack in the pollen (sperm) chloroplast(id) (referring to, such as, Hager and Bock, as above, 2000).Like this, the transgenic plant that contain the Pignus pignoris body just can not with other plant generation crossing pollination, comprise may be near transgenic plant eciophyte, the growth that has so just reduced transgenic plant may threaten to the ecology that environment brings.

[0105] widely used here term " plant " is meant a kind of particularly eukaryote of chloroplast(id) of plastid that contains, and comprise the such biology that is in each developmental stage, perhaps be meant the part of plant, comprise that plant transplants, vegetable cell, plant cell, plant organ, plant seed, plantling.Vegetable cell is structure and the physiology unit of plant, comprises protoplastis and cell walls.Vegetable cell can be the form of isolating individual cells or the form of cultured cells, perhaps can be the more part of high-grade organization unit, such as plant tissue, and plant organ, perhaps plant.Therefore, vegetable cell can be a protoplastis, can generate the cell of conjugant, or can be regenerated as cell or the cell mass of a whole strain plant.Like this, the seed that contains a plurality of cells and the whole plant of renewable one-tenth can be regarded as and be used for vegetable cell of the present invention.Plant tissue or plant organ can be seeds, protoplastis, and callus perhaps is organized into any other vegetable cell group of a structure or functional unit.Useful especially part comprises and can gather in part and the part useful to the breeding of filial generation plant in the plant, such as flower, and pollen, cotyledon, stem tuber, leaf, stem, fruit, seed, root, and similar part.The part that plant is used to breed comprises that fruit is transplanted such as, seed, cotyledon, stem tuber, rhizome, and similar part.

[0106] transgenic plant can be by the transformed plant cells regeneration that contains through the chloroplast(id) of genetic modification.Here the term of Shi Yonging " regeneration " is meant from the sub-fraction of vegetable cell, a group vegetable cell, protoplastis, seed or plant and goes out a whole plant such as callus or tissue growth.It is different between different plant species to be derived from protoplasts regenerated.Such as, can prepare the suspended substance of protoplastis, in some species, can be by the forming of described protoplastis suspended substance inducing embryo, and then to ripe, go down to posterity.Substratum generally contains the growth and the necessary various component of regenerating, and comprises such as hormone such as plant growth hormones and phytokinin; And amino acid is such as L-glutamic acid and proline(Pro), and these components depend on specific floristics.Effectively depend on substratum, genotype, and the history of cultivating regeneration section.Yet if these variable Be Controlled, regeneration is repeatably so.

[0107] callus from plant, explant, certain part of organ or plant can take place in regeneration.Can in organ or plant part regeneration, implement to transform.(referring to, Meth.Enzymol. the 118th volume; Klee etc., Ann.Rev.Plant Physiol.38:467,1987, these documents are put in order into for your guidance at this).For example, utilize leaf dish-transform-regenerate this method, the leaf dish is cultivated on selective medium, then about two to around in young shoot formation.Young shoot in the growth is taken from callus, and is transplanted to and is suitable for inducing in the selective medium of root.The seedling that goes out root is transplanted in the soil after root occurs as early as possible.Such seedling can be transplanted as required once more, up to reaching the ripening stage.

[0108] in the crop that nourishes and generates, sophisticated transgenic plant are by transplanting or tissue culture technique produces a plurality of identical plant and realizes breeding.Required transgenosis (transgenotes) is selected, and new mutation can be obtained and can be used for nourishing and generating of commercial purpose.In germplasm breeding crop, sophisticated transgenic plant can selfing produce the inbreeding plant of isozygotying.The inbreeding plant that obtains like this can produce the seed that contains the allos polynucleotide that is imported into, and they can be obtained expressing the plant by described polynucleotide encoded polypeptides by cultivation.Like this, the present invention further provides the seed that produces by the transgenic plant that obtain by method of the present invention.

[0109] if desired, of the present invention contain the transgenic plant that can express the chloroplast(id) of different heterologous polypeptides through genetic modification can the phase mutual cross, provide a kind of acquisition to contain the means of two or more a plurality of different genetically modified transgenic plant thus.The method that plant mating and selection have the hybrid plant of desired characteristic or other interested characteristics is known in the art.

[0110] method of producing heterologous polypeptide or albumen composition in chloroplast(id) or transgenic plant of the present invention may further include from the vegetable cell chloroplast(id) and separates by the step of polypeptide expressed or albumen composition.Term used herein " isolating " or " purifying basically " are meant polypeptide or polynucleotide with the relative protein of contact with it of having removed under state of nature, nucleic acid, and lipid, the form of carbohydrate or other material exists.Usually, isolated polypeptide (perhaps polynucleotide) has been formed about at least 20% of sample, usually having formed at least 50% of sample, is the about at least 80% of sample under the particular case, more particularly is about at least 90% or 95% or more of sample under the situation.

[0111] term used herein " allos " is said so on a kind of comparative sense and is referred to that one section nucleotide sequence (perhaps polypeptide) is from non-source with reference to the source, perhaps be connected on second nucleotide sequence that does not under normal circumstances interrelate (perhaps polypeptide), perhaps through making after modifying that the form after modifying is improper relevant with the reference material.For example, the polymerized nucleoside acid sequence of encoding antibody is allogenic with respect to the nucleotide sequence of plant chloroplast, the component that for example contains the recombinant nucleic acid molecules of first nucleotide sequence that effectively is connected with second nucleotide sequence also is allogenic, and the sudden change polynucleotide that is directed in the chloroplast(id) also is allogenic when described sudden change polynucleotide is not under normal circumstances found in chloroplast(id).

[0112] polypeptide or albumen composition can utilize any method that is suitable for specific polypeptide or albumen composition to separate from chloroplast(id), these methods for example comprise, salt fractionation method and chromatographic process are as utilizing the affinity chromatography method of can be specifically carrying out in conjunction with the part or the acceptor of polypeptide or albumen composition.Be used for determining that the polypeptide of the method according to this invention generation or the method that albumen composition has been isolating form are the methods of having known, such as carrying out electrophoresis, identify that specific molecule is relatively independent band, identify that perhaps specific mixture is a series of bands.Therefore, the present invention also provides isolated polypeptide or the albumen composition that the method according to this invention produces.

[0113] the present invention also provides and may be used singly or in combin to obtain the composition of the strong expression of heterologous polypeptide in chloroplast(id).In a kind of specific embodiments, the invention provides the nucleotide sequence that comprises (perhaps coding) RBS and the 2nd RBS, a wherein said RBS and the 2nd RBS spatially are separated, such RBS instructs the translation in prokaryotic cell prokaryocyte, and another one RBS can instruct the translation in plant chloroplast.In one aspect, described nucleotide sequence can also comprise the initiator codon that (perhaps coding) and a described RBS and the 2nd RBS effectively are connected, such as initiator codon AUG (perhaps ATG), perhaps can comprise cloning site, its location allows one section encoding sequence effectively to be connected with the 2nd RBS with a described RBS.Aspect another one, described nucleotide sequence is comprised in the carrier, and described carrier preferably contains the nucleotide sequence of one section chloroplast genomic dna, make can with chloroplast gene group generation locus specificity homologous recombination.Aspect another one again, described carrier is a shuttle vectors, and it may further include the prokaryotic organism replication origin.

[0114] in the another one specific embodiments, utilize codon to select to make the coding polynucleotide to be partial to the chloroplast(id) codon and use, thereby provide a kind of in chloroplast(id) the means of one or more polypeptide that are encoded of strong expression.Utilize codon to select to optimize the expression of polypeptide in chloroplast(id), this comes illustration (GFP by expressing jellyfish (Aequeoria victoria) green fluorescent protein; Example 1).Like this, the present invention also provides the polynucleotide of coding GFP, wherein said polynucleotide had can be in chloroplast(id) the codon of optimization expression.As disclosed in this, variation polymerized nucleoside acid encoding can be with a certain amount of GFP that expresses, and this makes it to comprise the genetic expression that detects in the chloroplast(id) as the reagent that detects plant chloroplast.The codon optimized generally useful property for express polypeptide of chloroplast(id) further proves (embodiment 4) by the synthetic polynucleotide for preparing the plain enzyme of coding fluorescence, this proteic expression can be in vivo or is external detected, can also prove (embodiment 3) by the polynucleotide of encoding antibody.Further, composition that is illustrated as an example and method proved functional fusion rotein can be in chloroplast(id) strong expression, comprise single-chain antibody and report polypeptide (referring to embodiment 3 and 4).

[0115] chloroplast(id) of higher plant and algae probably originates from the endosymbiosis integration of photosynthetic prokaryotic organism to the eukaryote host.Many genes move on to from chloroplast transgenic the host cell nuclear (Gray, Curr.Opin.Gen.Devel.9:678-687,1999) in the process of integrating.Like this, normal light compositing function need be examined the fellowship of proteins encoded and plastid proteins encoded in the chloroplast(id), and the cooperation of genetic expression between these two genomes.Being expressed in of plant center coding and chloroplast(id) encoding gene to cooperation very sensitively in the response of growth and environmental factor.

[0116] in chloroplast(id), the regulation and control of genetic expression generally be occur in transcribe after, and usually occur in the translation initiation stage.This regulation and control depend on the translating equipment of chloroplast(id), and the regulatory factor of nuclear coding is (referring to Barkan and Goldschmidt-Clermont, Biochemie82:559-572,2000; Zerges, Biochemie 82:583-601,2000; Bruick and Mayfield, as above, 1999).The chloroplast(id) translating equipment is more similar to the translating equipment in the bacterium; Chloroplast(id) contains the rrna of 70S; Its mRNA lacks 5 ' cap and does not generally contain 3 ' end polyadenylic acid tail (Harris etc., Microbiol.Rev.58:700-754,1994); And the translation of chloroplast(id) and bacterium can be suppressed by selectivity reagent such as paraxin.

[0117] in bacterium, the RNA element that mediates correct translation initiation comprises initiator codon, RBS, the specific interval of RBS and initiator codon, the translation enhancement sequences, the deflection of second codon, but and the secondary structure (Gold that influences the RNA contact, Ann.Rev.Biochem.57:199-233,1988).In chloroplast(id), the correct selection of ribosomal combination and translation initiation site be by, be that part is passed through at least, (referring to Bruick and the Mayfield, as above, 1999) that cis acting RNA element is finished.Similar to bacterium, the chloroplast(id) initiator codon also can influence the efficient of translation initiation, but does not determine position (Chen etc., the PlantCell 7:1295-1305 of initiation site, 1995), this shows the extra determinative of selection needs of translation initiation site in the chloroplast(id).

[0118] several RNA elements of mediation translational control are identified (Alexander etc., Nucl.Acids Res.26:2265-2272,1998 in 5 ' the end non-translational region of chloroplast(id) mRNA; Hirose and Sugiura, EMBO J.15:1687-1695,1996; Mayfield etc., J.Cell Biol.127:1537-1545,1994; Sakamoto etc., Plant J.6:503-512,1994; Zerges etc., as above, 1997, every piece of document is all put in order into for your guidance at this).These elements may with nuclear coding factor interaction, and general and known prokaryotic organism regulating and controlling sequence does not have similarity (McCarthy and Brimacombe, Trends Genet.10:402-407,1994).

[0119] feature of Bao Shou prokaryotic organism RBS element is Shine-Dalgamo (SD) sequence, and this section sequence contains 3 to 9 Nucleotide, wherein generally has the 3 ' end of about 4,5 or 6 Nucleotide and 16S rRNA complementary.Early stage at translation initiation, the ribosomal subunit of 30S combines with mRNA at SD sequence place by means of the anti-SD sequence of complementary in the 16S rRNA.Because the SD sequence of prokaryotic organism mRNA is positioned at the position of 5 to 15 Nucleotide of upstream from start codon, the 30S ribosomal subunit is positioned like this, and correct initiator codon just enters rrna P site.

[0120] many chloroplast(id) mRNA contain element (Bonham-Smith and Bourque, Nucl.Acids Res.17:2057-2080,1989 that are similar to prokaryotic organism RBS element; Ruf and Kossel, FEBS Lett.240:41-44,1988, every piece of document is all put in order into for your guidance at this).Yet the function effectiveness of these RBS sequences in chloroplast(id) translation is still unclear, this be because the location of these elements than usually in prokaryotic organism observed position to be positioned at the upstream of initiator codon more.In some researchs, the change of the RBS that infers in 5 ' the end non-translational region among the report chloroplast(id) mRNA can influence translation (Betts and Spremulli, J.Biol.Chem.269:26456-26465,1994; Hirose etc., FEBS Lett.430:257-260,1998; Hirose and Sugiura, as above, 1996; Mayfield etc., as above, 1994) change of possible RBS element is to influence very little (Fargo etc., Mol.Gen.Genet.257:271-282,1998 of translation, and in other chloroplast(id) mRNA; Koo and Spremulli, J.Biol.Chem.269:7494-7500,1994; Rochaix, Plant Mol.Biol.32:327-341,1996; Sakamoto etc., as above, 1994).Become complicated for these results' explanation owing to chloroplast(id) RBS element lacks conservative property, but also because the sudden change that produces in order to study these RBS sequences of inferring may change in 5 ' the end non-translational region other important sequence.

[0121] the functional effect here revealed (embodiment 2) of RBS element in the chloroplast(id) translation.The anti-SD sequence of chloroplast(id) 16S rRNA is positioned at the 3 ' end of 16S rRNA, its sequence is 3 '-CUUCCUCCAC-5 ' (SEQ ID NO:29), it is suddenlyd change, remove the possible base pairing that itself and chloroplast(id) mRNA go up the SD sequence, this has seriously weakened the proteic translation of several conformity membranes (embodiment 2) of Chlamydomonas reinhardtii chloroplast(id) coding.The rrna that has the anti-SD sudden change of 16SrRNA still can be translated, because the synthetic influence that is not subjected to these sudden changes to a great extent of solubility chloroplast protein.

[0122] possible SD element in 5 ' the end non-translational region of the proteic chloroplast(id) psbA of the II of encoded light system reactive center D1 mRNA is analyzed, discovery is similar to the existence of procaryotic single RBS element, and described element is positioned at 5 ' end (upstream) 27 Nucleotide places of initiator codon AUG.This RBS is arranged in upstream from start codon position too far away so that rrna 30S subunit can not be as contacting with initiator codon with the RBS element simultaneously on bacterium.When reorientating this RBS element when making it nearer apart from initiator codon, it is initial that it but no longer supports to translate in the chloroplast(id), but can make this transcripton translate (embodiment 2) in intestinal bacteria.Because complex body can form at this RBS element place before initial, so it has the feature of the true recognition site of rrna 30S subunit.Yet this RBS element is not having can not correctly to limit translation initiation site under the extra factor participation, and the described factor comprises translation activator (Danon and Mayfield, 1991 of nuclear coding; Yohn etc., 1998a; Yohn etc., 1998b).This result shows, among the mRNA of psbA between RBS and the initiator codon extra distance be used to hold extra translation factor, its illustration is that RBS element and light regulation and control trans-acting factor are united and played the function that promotes translation initiation in the chloroplast(id).

[0123] therefore, the invention provides a kind of isolating ribonucleoside acid sequence, it comprises a RBS who effectively is connected with the 2nd RBS.As disclosed herein, first and second RBS of effective connection are so generally separated by about 5 to 25 Nucleotide, like this, when this section ribonucleoside acid sequence effectively is connected with the polynucleotide of coded polypeptide, a described RBS can instruct the translation of described polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).When a RBS sequence being positioned at about at least 19 Nucleotide in initiator codon AUG upstream (5 ' end), this RBS is activated in the translation process of chloroplast(id), the activity that has described here comprises the polysomal formation of permission, if the position of RBS from AUG more recently, will cause active reduce (referring to the Fig. 4) of its translation in chloroplast(id).As shown in Figure 4, the position (that is ,-27 positions of AUG codon upstream after) of the RBS of psbA mRNA (SD sequence) from-27.The disappearance that causes the distance of RBS and AUG codon to be less than about 19 Nucleotide causes in the chloroplast(id) the active and polymeric formation of translation obviously to reduce, but find that but translation activity in bacterium has increase (referring to embodiment 2, also shown in the bacterium translation was active when the position of RBS distance A UG codon surpassed about 15 Nucleotide can reduce).

[0124] the general length of isolating ribonucleoside acid sequence of the present invention is about 11 to 50 Nucleotide, can be about 15 to 40 Nucleotide, perhaps about 20 to 30 Nucleotide.It generally is about 3 to 9 Nucleotide that such length allows, two SD sequences that usually are about 4 to 7 Nucleotide are separated (generally by about 10 to 20 Nucleotide, being separated by about 15 Nucleotide in particular) by about 5 to 25 Nucleotide.For example, ribonucleoside acid sequence of the present invention can comprise the RBS that 4 Nucleotide are long, such as GGAG, it is opened such as the GGAG branch by the 2nd RBS of 5 Nucleotide and about 4 Nucleotide length, and it is the ribonucleoside acid sequence of 13 Nucleotide that a segment length so just is provided.The one RBS and the 2nd RBS can have the sequence of any SD of having sequence signature respectively independently.As disclosed herein, be used for instructing plant chloroplast translation RBS need with the anti-SD sequence of 3 ' end of 16S rRNA (3 '-CUUCCUCCAC-5 '; SEQ ID NO:29) at least three specifically, are four, five, and perhaps six, perhaps more Nucleotide complementation, especially, with 8 Nucleotide complementations in centre of anti-SD sequence.For example, comprise GGAG, GGAGG, perhaps the RBS sequence of ACGAGA (indicating with italic with SEQ ID NO:29 complementary Nucleotide) can instruct its translation in plant chloroplast when effectively being connected with one section coded polypeptide.

[0125] being used to the RBS for preparing composition of the present invention or put into practice method of the present invention can chemosynthesis, perhaps can separate from abiogenous nucleic acid molecule.For example, instruct the RBS of the translation in the chloroplast(id) generally to be present in 5 ' end non-translational region of chloroplast gene, so it can be separated from a chloroplast gene.In addition, comprise into normally more relevant extra nucleotide sequence and also have some benefits with the SD sequence of described gene.For example, 5 ' end non-translational region may comprise transcriptional regulatory element such as promotor, thereby helps to make up the recombinant nucleic acid molecules that can transcribe in plant chloroplast and translate.In addition, as disclosed herein, the putting in order of 5 ' extra UTR sequence that comes from the chloroplast gene of relevant D1 (psbA) chloroplast protein of coding film causes the expression (embodiment 3) of film heterologous polypeptide in chloroplast(id).Like this, the ribonucleotide that contains the RBS that instructs the translation in the chloroplast(id) of the present invention can further comprise 5 ' end non-translational region of a chloroplast gene, for example, 5 ' end non-translational region of the chloroplast gene of a soluble proteins of coding, 5 ' the end non-translational region of a film of perhaps encoding in conjunction with the gene of chloroplast protein.5 ' end non-translational region is like this known in this area, comprise those 5 ' end non-translational regions by the genes encoding of coding soluble proteins, such as, 5 ' the end non-translational region (SEQ ID NO:5) of 5 ' the end non-translational region (SEQ ID NO:4) of AtpA or RbcL, and those are by 5 ' end non-translational region of the chloroplast gene coding of coding embrane-associated protein, such as 5 ' the end non-translational region (SEQ ID NO:6) of PsbD, perhaps 5 ' of PsbA end non-translational region (SEQ ID NO:7).In addition, 5 ' the end non-translational region (SEQ ID NO:8) of 16S rRNA can be used to for example instruct with it effectively transcribing of the allos polynucleotide of connection, and can modify with the complementary sequence place of anti-SD sequence, make the RBS that so generates can instruct especially effectively by the translation of polynucleotide encoded polypeptides in plant chloroplast.

[0126] ribonucleoside acid sequence of the present invention may further include initiator codon, such as initiator codon AUG, and effectively is connected with first and second RBS.Such initiator codon AUG may further include the adjacent nucleotide of Kozak sequence, such as, ACCAUGG or GCCAUGG or CC (A/G) CCAUGG or similar sequence are (referring to Kozak, J.Mol.Biol.196:947-950,1987, this piece document is put in order into for your guidance at this), such sequence helps the translation of coded polypeptide in cell.In addition, ribonucleoside acid sequence of the present invention can effectively be connected with the polynucleotide of coded polypeptide, wherein said polynucleotide comprises initiator codon, described codon can be but might not be endogenic initiator codon, perhaps can contain an initiator codon by after modifying.

[0127] isolating ribonucleoside acid sequence of the present invention can chemosynthesis, perhaps can utilize Enzymology method to generate, such as, utilize DNA dependent form RNA polymerase or RNA dependent form RNA polymerase to generate respectively by DNA or RNA template.Encoding the dna profiling of ribonucleotide of the present invention can chemosynthesis, can separate from abiogenous dna molecular, perhaps can get by deriving in the abiogenous dna sequence dna that have desired characteristic through modifying.For example, contain the nucleotide sequence of the RBS that encodes under the dna sequence dna normal circumstances of prokaryotic gene, this section sequence is at about 5 to 15 the Nucleotide places of upstream from start codon.Such nucleotide sequence can be separated, and modify by the DNA recombination method of routine and to make it to contain the 2nd RBS that is positioned at appropriate location, endogenous protokaryon RBS upstream (5 ').Therefore, the invention provides the polynucleotide of first and second RBS of the effective connection that is coded in this definition.

[0128] polynucleotide of the RBS that effectively is connected with the 2nd RBS of coding can be DNA or RNA, can be strand or two strands, a wherein said RBS can instruct the translation in the prokaryotic organism, and described the 2nd RBS can instruct the translation in chloroplast(id).The initiator codon that described polynucleotide can also comprise and the nucleotide sequence of coding a described RBS and the 2nd RBS effectively is connected, ATG for example, initiator codon is positioned at the position of about 3 to 15 Nucleotide in a described RBS downstream (3 ' holds), be included in described downstream about 4,5,6,7,8,9,10,11, the position of 12,13 or 14 Nucleotide.Polynucleotide of the present invention can also comprise cloning site, the location of described cloning site allows the effable polynucleotide of coded polypeptide effectively to be connected with described first and second RBS, and effectively be connected with the ATG codon, if there is such codon, like this, described polypeptide just can be expressed in chloroplast(id) or prokaryotic host cell.

[0129] term of here extensively using " cloning site " is meant any Nucleotide or nucleotide sequence that helps first polynucleotide to be connected with second polynucleotide.Usually, cloning site comprises one or more restriction enzyme enzyme recognition site, multiple clone site for example, perhaps one or more recombinase recognition sites, for example loxP site or att site, the combination in perhaps such site.The cloning site that is provided is for the ease of inserting or attended operation, described connection can be effective connection of first and second polynucleotides, first polynucleotide such as effective first and second RBS that connect of coding effectively is connected with second polynucleotide of the interested polypeptide of coding, and described polypeptide will be translated in prokaryotic organism or in the chloroplast(id) or in the two.

[0130] polynucleotide at coding first and second RBS of this definition can effectively be connected with effable polynucleotide, and the described effable polynucleotide at least a polypeptide of encoding comprises the peptide moiety of peptide or polypeptide.Like this, described effable polynucleotide one first polypeptide of can only encoding, perhaps can encode two or more a plurality of polypeptide, these polypeptide can be identical with first polypeptide or different.For example, described effable polynucleotide can encode one first polypeptide and one second polypeptide, these two polypeptide are different, especially, first polypeptide and second polypeptide can be united specifically and formed functional heterodimer such as antibody; Enzyme; Cell surface receptor is such as TXi Baoshouti, growth factor receptors, cannabis acceptor; Perhaps analogue.First and second (perhaps other) polypeptide like this can be expressed as a fusion rotein, such as comprising the H chain that links together and the single-chain antibody of L chain, perhaps can be expressed as the polypeptide that independently separates, these polypeptide can but might not have and unite the ability that forms the functional protein mixture specifically.If be isolating unit with these expression of polypeptides, at the nucleotide sequence of ribosome entry site(RES) (IRES), help the translation of second (perhaps downstream) polypeptide like this in can between the sequence of the sequence of described first polypeptide of coding and described second polypeptide of encoding, encoding on effective the connection so.

[0131] polynucleotide of first and second RBS that effectively connect at the coding of this definition can be linear nucleotide sequence, and can have first cloning site at an end, have second cloning site at the another one end, the sequence box that can insert or connect second polynucleotide easily so just is provided.The first and second terminal cloning sites can be the same or different, and one of them or both can comprise multiple clone site independently.Described polynucleotide may further include any other interested nucleotide sequence, such as the initiator codon ATG that effectively connects with it.

[0132] as defined herein, the present invention further provides the carrier of the polynucleotide that contains effective first and second RBS that connect of coding.Described carrier can be any carrier that can be used for one section polynucleotide is imported to protokaryon or eukaryotic cell, comprises cloning vector or expression vector.In a kind of specific embodiments, described carrier comprises the nucleotide sequence of one section chloroplast genomic dna, the reticent nucleotide sequence of any chloroplast gene of particularly not encoding, had it to guarantee and chloroplast genomic dna between homologous recombination.Such chloroplast(id) carrier is known in this area, comprises that for example p322 is (referring to embodiment 1; Also can be referring to, Kindle etc., Proc.Natl.Acad.Sci., USA 88:1721-1725,1991, this piece document is put in order into for your guidance at this; Hager and Bock, as above, 2000; Bock, as above, 2001).

[0133] carrier of the present invention can also comprise one or more extra nucleotide sequence that can bring desired characteristic for carrier, comprise such as, the cloning site that helps the operation of carrier, the controlling element of transcribing of the nucleotide sequence that instructs carrier to duplicate or wherein comprised, the sequence of coding selected marker, and similar sequence.Like this, described carrier can comprise for example one or more cloning site such as multiple clone site, the location of described cloning site can but be not to make the allos polynucleotide can be inserted in the described carrier, and effectively be connected with described first and second RBS.Described carrier can also comprise procaryotic replication origin (ori), such as the ori of colibacillary ori or clay, and so just can be so that described carrier shifts in prokaryotic host cell and in the plant chloroplast as required.

[0134] widely used here term " controlling element " means the location of transcribing of regulation and control polynucleotide or translation or polypeptide and the nucleotide sequence that effectively connects with it.Except RBS, expression control sequenc can also be a promotor, enhanser, transcription terminator, initiator codon, the splicing signal that is used to excise intron and keeps the proper reading frame frame, terminator codon, amber codon or He Shi codon, IRES, perhaps polypeptide is directed to the sequence of specific position, for example cellular compartment turns into and uses signal, and it can be used for polypeptide being directed to tenuigenin, nucleus, plasma membrane, endoplasmic reticulum, mitochondrial membrane or matrix, chloroplast membranes or inner chamber, golgi body pond, middle anti-district, perhaps lysosome or endosome.Cellular compartment turns into structural domain to be known in this area, the peptide that comprises the 1st to 81 amino-acid residue that for example contains human II type film anchorin galactosyltransferase, perhaps contain cytochrome c oxidase IV subunit precursor sequence the 1st to 12 amino-acid residue peptide (also can referring to, Hancock etc., EMBO J.10:4033-4039,1991; Buss etc., Mol.Cell.Biol.8:3960-3963,1988; U.S. Patent number 5,776,689, every piece of document are all put in order into for your guidance at this).In using the polypeptide that method of the present invention produces, add cellular compartment and turn into and use structural domain, the polypeptide that comprises albumen composition is directed in the cellular compartment specific in individual as required, and use there.

[0135] but carrier of the present invention or other recombinant nucleic acid molecules can comprise the nucleotide sequence of coding report polypeptide or other selective markers.Term " report " or " selected marker " are meant the polynucleotide (polypeptide that perhaps is encoded) that can give phenotype that can be detected.Generally, report the polypeptide that sub-coding can be detected, such as, a kind of green fluorescent protein or a kind of enzyme such as luciferase, when they with will produce the signal (Giacomin that can be detected by naked eyes or arrive by suitable instrument detecting after suitable reagent (being respectively the light or the fluorescein of specific wavelength) contacts, Plant Sci.116:59-72,1996; Scikantha, J.Bacteriol.178:121,1996; Gerdes, FEBS Lett.389:44-47,1996; Also can referring to, Jefferson, EMBO J.6:3901-3907,1997, f1-glucuronidase).Selected marker generally is a kind of molecule, when it exists in cell or expresses, certain selective advantage (perhaps inferior position) is provided for the cell that contains this mark, possess the ability of growth such as the cell that contains this mark under the condition that a kind of reagent exists, the cell that does not contain this mark then can be killed by this reagent.

[0136] selected marker can provide prokaryotic cell prokaryocyte or vegetable cell or the means of the two that to express this mark, like this, this selected marker just can be used as carrier of the present invention a part (referring to, for example, Bock, as above, 2001).The example of selected marker comprises that those give the mark of metabolic antagonist resistance, such as, Tetrahydrofolate dehydrogenase, it can give the resistance to methotrexate (Reiss, Plant Physiol. (Life Sci.Adv.) 13:143-149,1994); Neomycin phosphotransferase, it can give the resistance to the aminoglycoside Xin Meisu, the resistance of kalamycin resistance and paromycin (Herrera-Estrella, EMBO J.2:987-995,1983), hygro, it can give the resistance to Totomycin (Marsh, Gene 32:481-485,1984), trpB, it allows cell to utilize indoles to substitute tryptophane; HisD, it allows cell to utilize histidinol alternate sets propylhomoserin (Hartman, Proc.Natl.Acad.Sci.USA 85:8047,1988); Seminose 6 phosphoric acid isomerases, it allows cell to utilize seminose (WO 94/20627); Ornithine decarboxylase, it can be given the ornithine decarboxylase inhibitor, the resistance (DMFO of 2-(difluoromethyl)-DL-ornithine; McConlogue, 1987, at Current Communication inMolecular Biology, Cold Spring Habor Laboratory ed.); And the desaminase that comes from terreus (Aspergillus terreus), it can give the resistance to blasticidin S (Tamura, Biosci.Biotechnol.Biochem.59:2336-2338,1995).The other selected marker comprises the mark that those can conferring herbicide resistance, and such as the phosphinothricin acyl transferase gene, it can give the resistance to phosphinothricin (White etc., Nucl.Acids Res.18:1062,1990; Spencer etc., Theor.Appl.Genet.79:625-631,1990), the mutant of EPSPV synthase, it can conferring glyphosate resistance (Hinchee etc., BioTechnology 91:915-922,1998), the mutant of acetolactate synthestase, it can give imidazolone or sulphur urea resistance (Lee etc., EMBO J.7:1241-1248,1988), mutant psbA, it can give the resistance (Smeda etc. to atrazine, Plant Physiol.103:911-917,1993), perhaps the mutant of proporphyrinogen oxidase (referring to U.S. Patent number 5,767,373), perhaps other can be given the mark of weedicide such as the resistance of careless fourth phosphine.Selected marker comprises that those make eukaryotic cell have Tetrahydrofolate dehydrogenase or neomycin resistance, make prokaryotic organism have tsiklomitsin such as intestinal bacteria, penicillin resistance; And make plant have bleomycin, gentamicin, glyphosate, Totomycin, kantlex, methotrexate, phleomycin, phosphinothricin, spectinomycin, Streptomycin sulphate, the polynucleotide of sulfonamide and sulphur urea resistance (referring to, such as Maliga etc., Methods in Plant Molecular Biology, Cold Spring Habor Laboratory Press, 1995,39 pages).Because composition of the present invention or method can make polypeptide express in chloroplast(id), if can give the polypeptide of the certain selective advantage of vegetable cell so effectively is connected with the nucleotide sequence of the interior location of one section Codocyte motif, just can be with described polypeptide transporte to cells matter, nucleus, perhaps other subcellular organelles, and these places just in time be for example the toxic effect that causes of the selected marker place of working (referring to, such as, Von Heijne etc., Plant Mol.Biol.Rep.9:104,1991; Clark etc., J.Biol.Chem.264:17544,1989; Della Cioppa etc., Plant Physiol.84:965,1987; Romer etc., Biochem.Biophys.Res.Comm.196:1414,1993; Shah etc., Science 233:478,1986; Archer etc., J.Bioenerg Biomemb.22:789,1990; Scandalios, Prog.Clin.Biol.Res.344:515,1990; Weisbeek etc., J.Cell Sci.Suppl.11:199,1989; Bruce, Trends Cell Biol.10:440,2000).

[0137] the shuttle vectors of the present invention ability of shuttling back and forth in prokaryotic organism makes more convenient to operate for carrier.For example, contain described carrier and suppose that the reaction mixture of the interested polynucleotide that has inserted can be transformed into prokaryotic host cell such as in the intestinal bacteria, adopt conventional method amplification and collection then, it is tested contain the carrier that inserts segment or interested construction with evaluation.If necessary, described carrier can also further be operated, such as the polynucleotide that is inserted into being carried out site-specific nature sudden change, and then amplification and pick out the carrier that contains interested sudden change polynucleotide.Described shuttle vectors can be directed in the vegetable cell chloroplast(id) then, and interested there polypeptide can be expressed, and if necessary, can also isolate these polypeptide by method of the present invention.

[0138] polynucleotide of the present invention or recombinant nucleic acid molecules can be included in the middle of the carrier, described carrier comprises carrier of the present invention, and described polynucleotide or recombinant nucleic acid molecules can be directed in the plant chloroplast by any method known in the art.Here the term of using " importing " is meant to be transferred to polynucleotide in the cell, and described cell comprises prokaryotic cell prokaryocyte or vegetable cell, particularly imports in the plastid of vegetable cell.Polynucleotide can be directed in the cell by the whole bag of tricks, and these methods are known in this area, and the selection of these methods partly depends on specific host cell.For example, polynucleotide can be directed in the vegetable cell by direct gene transfer method, such as electroporation, the little conversion of launching mediation (biology launches) that perhaps utilizes particle gun to carry out, perhaps " granulated glass sphere method " (referring to, such as, Kindle etc., as above, 1991), the perhaps conversion of pollen-mediated, liposome-mediated conversion, the conversion that the prematurity embryo of utilization damage or enzyme liberating carries out, perhaps utilize the conversion that embryo's generation callus of damage or enzyme liberating carries out (referring to, Potrykus, Ann.Rev.Plant.Physiol.Plant Mol.Biol.42:205-225,1991, this piece document is put in order into for your guidance at this).

[0139] plastid transform be a kind of routine and the method that polynucleotide is imported the vegetable cell chloroplast(id) known (referring to, U.S. Patent number 5,451,513,5,545,817 and 5,545,818; WO 95/16783; McBride etc., Proc.Natl.Acad.Sci.USA 91:7301-7305,1994, every piece of document is put in order into for your guidance at this).Chloroplast(id) transforms to relate to and utilizes biological example to launch (biolistic) or protoplast transformation method (for example conversion of calcium chloride or PEG mediation), has the required nucleotide sequence in some zone of chloroplast DNA to import to suitable destination organization both sides.The chloroplast genomic dna nucleotide sequence of 1 to 1.5kb both sides can make between carrier and the described chloroplast gene group homologous recombination takes place, and can substitute or modify the specific region of plastom (plastome).Adopt this method, can give can be as the selected marker (Svab etc., Proc.Natl.Acad.Sci.USA 87:8526-8530,1990 that transform to the chloroplast(id) 16S rRNA and the rpsl2 point mutation of the resistance of spectinomycin and Streptomycin sulphate; Staub and Maliga, as above, 1992), the result can obtain stable homogeneity transformant (homoplasmic transformants), and successful frequency approximately is to have one in the target blade that bombards of 100 quilts.To be present in cloning site between these marks be carrier construction---comprising carrier of the present invention---provides nucleotide sequence (Staub and Maliga, EMBOJ.12:601-606,1993) easily.Selected marker with dominance, coding spectinomycin detoxification enzyme aminoglycoside-3 '-VITAMIN B4 transferring enzyme substitutes recessive rRNA or ribosomal protein antibiotics resistance gene, can obtain transformation efficiency (Svab and the Maliga of obviously increase, Proc.Natl.Acad.Sci.USA 90:913-917,1993).Usually need about 15 to 20 cell division cycle can reach the homogeneity state after the conversion.In plastid is expressed, gene is inserted in all thousands of ring-type plastom copies of each vegetable cell by homologous recombination, owing to compare with the gene of nuclear expression, it can utilize huge copy number, therefore makes expression level can easily surpass 10% of plant total soluble protein.

[140] directly gene transfer method also can be used for polynucleotide of the present invention is imported to plant chloroplast (1985, this piece document is put in order into for your guidance at this for Fromm etc., Proc.Natl.Acad.Sci.USA82:5824) such as electroporation.The electricimpulse of high field intensity can reversibly make cytolemma penetrate, thereby allows the importing of polynucleotide.Again formed cell walls by the plant protoplast of electroporation, division also forms the plant callus.Microinjection can carry out according to the method for describing among the Gene Transfer To Plants (NY 1995 for Springer Verlag, Berlin) that Potrykus and Spangenberg showed.The transformed plant cells that contains the polynucleotide of importing can identify that described phenotype for example is the expression of reporter gene or selected marker by detecting the phenotype of being brought by the polynucleotide that imports.

[0141] also can use being converted of little projectile (microprojectile) mediation and polynucleotide be imported in the vegetable cell chloroplast(id) (1987, this piece document is put in order into for your guidance at this for Klein etc., Nature 327:70-73).This method utilizes little projectile such as gold or tungsten, on it by calcium chloride, spermidine or polyethylene glycol precipitation method and be coated with required polynucleotide.Described little projectile particle is squeezed at a high speed in the plant tissue, and the equipment of using can be BIOLISTIC PD-1000 particle gun (BioRad for example; The California, He Kulesi).The method of utilizing biological catapult technique to transform is (Wan, Plant Physiol.104:37-48,1984 of having known; Vasil, BioTechnology 11:1553-1558,1993; Christou, Trends in Plant Science 1:423-431,1996).The conversion of little projectile mediation for example can be used for, and obtains various transgenic plant species, comprises cotton, tobacco, corn, hybridization white poplar or papaya.Important cereal crop are such as wheat, and oat, barley, Chinese sorghum and paddy rice also can adopt the transmission of little projectile mediation to transform (Duan etc., Nature Biotech.14:494-498,1996; Shimamoto, Curr.Opin.Biotech.5:158-162,1994).Most of dicotyledonss can adopt aforesaid method to transform.Monocotyledons also can transform, such as the above-mentioned biological catapult technique of employing, and protoplast transformation, the electroporation of the cell that part is penetrating imports DNA with glass fibre, the granulated glass sphere stirring means (Klindle etc., as above, 1991), and similar method.

[0142] the present invention also provides the carrier of the nucleotide sequence that comprises the coding RBS that is positioned at about 20 to 40 nucleotide positions of cloning site 5 ' end.Described cloning site can be that nucleotide sequence in the carrier is inserted or be connected to any heterologous nucleotide sequence that helps, one or more restriction enzyme enzyme recognition site for example, one or more recombinase recognition site, the perhaps combination in these sites.Preferably, described cloning site is a multiple clone site, and it comprises a series of restriction enzyme enzyme recognition sites or recombinase recognition site, perhaps the combination of at least one restriction enzyme enzyme recognition site and at least one recombinase recognition site.Described carrier can further contain near initiator codon or its part that is positioned at the described cloning site 5 ' end, thereby provides a translation initiation site (perhaps hidden initiation site) for the encoding sequence that lacks initiator codon ATG or cause only containing the part initiator codon owing to the cutting of for example restriction enzyme.Described carrier can also contain the chloroplast gene 3 ' UTR that is positioned at described cloning site 3 ' end, such as the 3 ' UTR (SEQ ID NO:9) of PsbA, the 3 ' UTR of RbcL (SEQID NO:10), the 3 ' UTR of AtpA (SEQ ID NO:11), tRNA ^ARG3 ' UTR (SEQ ID NO:12), perhaps 3 ' of PsbD UTR is (referring to SEQ ID NO:30, from the position 1553; The insertion site that has also shown the GFP construction of coding PsbD-GFP fusion rotein).

[0143] also provides a kind of method for preparing chloroplast(id)/prokaryotic organism shuttle expression carrier.Shuttle vectors of the present invention can be by preparing like this, for example, imports following sequence in the nucleotide sequence that is enough to one section chloroplast genomic dna of chloroplast genomic dna generation homologous recombination: the nucleotide sequence that comprises the prokaryotic organism replication origin; The encode nucleotide sequence of a RBS; The encode nucleotide sequence of the 2nd RBS, a wherein said RBS and the 2nd RBS are by about 5 to 25 Nucleotide separately; And cloning site, wherein said cloning site is positioned and effectively is connected to a described RBS and the 2nd RBS with the polynucleotide that allows coded polypeptide, like this, a described RBS can instruct the translation of described polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).Preparation chloroplast(id)/prokaryotic organism shuttle expression carrier can also be realized by the nucleotide sequence of one section chloroplast genomic dna of genetic modification, described this section nucleotide sequence can with chloroplast genomic dna generation homologous recombination, it contains the prokaryotic organism replication origin through after modifying, RBS that coding is separated by about 5 to 25 Nucleotide and the nucleotide sequence of the 2nd RBS, and cloning site, wherein said cloning site is positioned and effectively is connected to a described RBS and the 2nd RBS with the polynucleotide that allows coded polypeptide, like this, a described RBS can instruct the translation of described polypeptide in prokaryotic organism, and described the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).Therefore, the present invention also provides aforesaid prepared according to the methods of the invention chloroplast(id)/prokaryotic organism shuttle vectors.

[0144] the present invention also provides a kind of recombinant nucleic acid molecules, it comprises first nucleotide sequence of the coding chloroplast(id) RBS that effectively is connected with second nucleotide sequence of coded polypeptide, wherein, described first nucleotide sequence is relative allogenic with described second nucleotide sequence.Effectively the RBS that connects generally is positioned at the position of initiator codon 5 ' end (upstream) about 20 to 40 Nucleotide, and described initiator codon effectively is connected with the polynucleotide of coded polypeptide.In a kind of specific embodiments, described first nucleotide sequence comprises the ATG codon of about 20 to 40 nucleotide positions of 3 ' end of the nucleotide sequence that is positioned at coding RBS.Recombinant nucleic acid molecules of the present invention may further include other controlling element or interested coding polynucleotide, as illustrative or known in the art at this.

[0145] reporter gene in the chloroplast(id) of higher plant by successful Application, and high levels of recombinant proteins is expressed report is also arranged.In addition, reporter gene also is employed in the chloroplast(id) of Chlamydomonas reinhardtii, but all is to have only a spot of protein to be expressed in most cases.In many living species, reporter gene can strengthen the ability of gene expression greatly.In the chloroplast(id) of higher plant, beta-glucuronidase (uidA, Staub and Maliga, EMBO J.12:601-606,1993), neomycin phosphotransferase (nptII, Carrer etc., Mol.Gen.Genet.241:49-56,1993), adenosine-3-VITAMIN B4 transferring enzyme (aadA, Svab and Maliga, Proc.Natl.Acad.Sci.USA 90:913-917,1993), and the GFP (Sidorov etc. among the Aequoreavictoria, Plant J.19:209-216,1999) all be used as reporter gene (Heifetz, Biochemie 82:655-666,2000).In these genes each all has the characteristic that can make it report of expressing as chloroplast gene, has analytically accessibility such as them, and susceptibility perhaps has the ability of in situ detection genetic expression.Based on these research, other heterologous protein also can be expressed in the chloroplast(id) of higher plant, such as the Cry toxin of Bacillus thuringiensis (Bacillus thuringiensis), it can make plant have resistance (Kota etc., Proc.Natl.Acad.Sci. to herbivorous insect, USA 96:1840-1845,1999), perhaps human growth hormone (Staub etc., Nat.Biotechnol.18:333-338,2000), this is a kind of biological species medicine.

[0146] there have been several reporter genes in the chloroplast(id) of eucaryon green alga Chlamydomonas reinhardtii, to express, although the degree of successful expression is different.These genes comprise aadA (Goldschmidt-Clermont, Nucl.Acids Res.19:4083-4089 1991; Zerges and Rochaix, Mol.Cell Biol.14:5268-5277,1994), uidA (Sakamoto etc., Proc.Natl.Acad.Sci., USA 90:477-501,1993; Ishikura etc., J.Biosci.Bioeng.87:307-314 1999), sea pansy (Renilla) luciferase (Minko etc., Mol.Gen.Genet.262:421-425,1999) and come from the aminoglycoside phosphotransferase of Acinetobacter baumannii (Acinetobacter baumanii), aphA6 (Bateman and Purton, Mol.Gen.Genet.263:404-410,2000).The amount of the recombinant protein that produces only has report (Ishikura etc., as above, 1999) for the uidA gene, and based on protein imprinted analysis and determination of activity, its expression amount is very low.In order to improve the expression of heterologous polypeptide in chloroplast(id), the present invention has studied at the influence of the codon preference of Chlamydomonas reinhardtii chloroplast gene group (Nakamura etc., as above, 1999) to expressing.

[0147] because the inborn degeneracy of genetic code, 6 kinds of nucleotide triplets same seed amino acid of encoding nearly sometimes, and isoacceptor is usually encoded by multigene family.For example, in all nuclear all known nematodes of tRNA gene (Caenorhabditis elegans), 31 tRNA are arranged _UCC ^GlyEncoding gene (Duret, Trends Genet.16:287-289,2000).A result of this degeneracy is exactly that many species show tangible codon preference, and wherein the frequency of utilization of some codon substantially exceeds other codons.The influence that codon preference is expressed for heterologous protein has had detailed description in prokaryotic organism and eukaryote, even virogene all shows certain codon preference, thereby influences their temporary transient and tissue-specific expression.Generally speaking, the use of codon is relevant with the level of isoacceptor.Like this, the proteic gene that efficiently expresses of coding utilize often the extra high codon of its isoaccepting tRNA level (Duret, as above, 2000; Kanaya etc., Gene 238:143-155,1999).

[0148] the chloroplast gene group of Chlamydomonas reinhardtii shows the intensive codon and uses preference, uses VITAMIN B4 or uridylic (perhaps thymus pyrimidine) (Nakamura etc., as above, 1999) at the 3rd optimum seeking site.Use the polynucleotide of preference to study the chloroplast(id) codon by de novo synthesis coding GFP and the chloroplast(id) codon that most of albumen had with Chlamydomonas reinhardtii chloroplast(id) coding and use effect (embodiment 1) when recombinant polypeptide is expressed in the Chlamydomonas reinhardtii chloroplast(id).With the GFP sequence box (GFPct that is in the codon that has optimization under Chlamydomonas reinhardtii RbcL 5 ' UTR and 3 ' UTR (being respectively SEQ ID NOS:5 and the 10) control; SEQ IDNO:1) transform the Chlamydomonas reinhardtii chloroplast(id), monitor the wherein accumulation of GFP, and with the GFP sequence box (GFPncb that not have optimization; SEQ ID NO:3) accumulation of GFP is compared in the Chlamydomonas reinhardtii of Zhuan Huaing.As disclosed herein, accumulation volume with GFP in the Chlamydomonas reinhardtii chloroplast(id) of GFPct sequence box conversion approximately is with more than 80 times in the strain system of GFPncb conversion, and this expression is enough strong, so that can report the difference (embodiment 1) of the albumen that causes based on the subtle change of envrionment conditions on synthetic.Similar result also obtains on luciferase, wherein a kind of coding comprises bacterial luciferase A subunit and the expression of the synthetic polynucleotide with chloroplast(id) codon preference (SEQ ID NO:45) of the fusion luciferase protein (SEQ ID NO:46) of the bacterial luciferase B subunit that merges with it by connection peptides has caused the intensive of luciferase to be expressed, but also other advantage is provided, can detected in vivo (referring to embodiment 4) as the expression of luciferase.

[0149] therefore, the invention provides the isolating synthetic polynucleotide of coding fluorescence albumen or its mutant or varient, the codon of wherein said polynucleotide has the chloroplast(id) codon and uses preference.Described synthetic polynucleotide can be DNA or RNA, can be strand or two strands, can be at one end or two ends contain the linear polynucleotide of cloning site.Described polynucleotide can be included in the carrier, and RBS that can also be separated by about 5 to 25 Nucleotide with coding and the polynucleotide of the 2nd RBS effectively are connected, and fluorescin can be translated in prokaryotic organism and chloroplast(id) easily like this.

[0150] table 1 shows the preferred codon that uses in the algae chloroplast gene.The term here " use of chloroplast(id) codon " is exactly to be used to refer to such codon, and but it is when being used with respect to those same amino acid of coding but seldom for the degenerate codon of finding in the chloroplast gene.When term " preference " is to make when being used for using with reference to the chloroplast(id) codon, be meant polynucleotide is operated, one or more Nucleotide on its one or more codon is changed, and the result obtains the preferred codon that uses in chloroplast(id).The chloroplast(id) codon preference here illustrates by the algae chloroplast(id) codon preference that is listed in the table 1.The chloroplast(id) codon preference can but be not to select based on the specified plant that will express the synthetic polynucleotide therein.Above-mentioned operation can be that the method by for example rite-directed mutagenesis changes codon, the primer that just utilizes and will reformed Nucleotide mispairing carries out PCR, make amplified production have the chloroplast(id) codon and use preference, above-mentioned operation or can be de novo synthesis polymerized nucleoside acid sequence makes such variation (perhaps preference) import in the sequence by synthesis step.

[0151] except utilizing the chloroplast(id) codon preference as the means that effective polypeptide translation is provided, it should be noted that, also have method for distinguishing can make the efficient translation of polypeptide in chloroplast(id), for example chloroplast gene group (for example chloroplast gene group of Chlamydomonas reinhardtii) is carried out engineeredly, make it to express and can't help the tRNA that described chloroplast gene group expresses under the normal circumstances.Such through engineering approaches algae is expressed one or more allos tRNA molecule, and this just can provide an advantage, can avoid making amendment to importing in the chloroplast gene group and by each polynucleotide of its expression; The algae that chloroplast gene group with genetic modification so just can be provided is such as Chlamydomonas reinhardtii, and can the method according to this invention it be used for effective translation of polypeptide.Codon in the gene that efficiently expresses use and the tRNA abundance between dependency known (Franklin etc., Plant J.30:733-744,2002; Dong etc., J.Mol.Biol.260:649-663,1996; Duret, Trends Genet.16:287-289,2000; Goldman etc., J.Mol.Biol.245:467-473,1995; Komar etc., Biol.Chem.379:1295-1300,1998, every piece of document is all put in order into for your guidance at this).For example, in intestinal bacteria, the bacterial strain that the tRNA that underuses is expressed is processed again can be made the gene that utilizes these codons strengthen to express (referring to, Novy etc., inNovations 12:1-3,2001, this piece document is put in order into for your guidance at this).When utilizing external source tRNA gene, can use rite-directed mutagenesis and prepare synthetic tRNA gene, such tRNA gene can be directed in the chloroplast(id) to replenish the chloroplast gene group such as tRNA gene rare in the Chlamydomonas reinhardtii chloroplast gene group or that be not used.

[0152] one or more codons of coding fluorescin of the present invention can have preference, for example contain VITAMIN B4 or thymus pyrimidine on the 3rd position, help the translation of fluorescin in chloroplast(id) like this.As disclosed herein, the polynucleotide of coding jellyfish (Aequorea victoria) GFP can contain the encoding sequence that 121 synonym change by one section of de novo synthesis makes it to have skewed popularity, comprising 66 54 variations (embodiment 1) that the codons that change and cause being of little use use to change towards chloroplast(id) representing the appropriateness used towards the chloroplast(id) codon to change.Like this, the polynucleotide of the GFP of the coding modified shown in SEQ ID NO:1 (SEQ ID NO:2) provides an example of polynucleotide of the present invention, coding SEQ ID NO:2 but polynucleotide with codon with preference still less then provides extra example.The present invention also provides the GFP of the modified with the aminoacid sequence shown in SEQ ID NO:2.

[0153] GFP be a kind of known in the art from the northwest Pacific jellyfish, Portuguese man-of-war, sea pansy, isolated albumen (Ward etc., Photochem.Photobiol.35:803-808,1982 among Renilla reiniformis and the Phialidium gregarium, Levine etc., Comp.Biochem.Physiol.72B:77-85,1982, every piece of document is all put in order into for your guidance at this).Similarly, red fluorescent protein is also known, can from coral polyp Discosoma, separate (Matz etc., Nature Biotechnol.17:969-973,1999, this piece document is put in order into for your guidance at this).In addition,, can obtain the various fluorescins relevant with jellyfish GFP by the aminoacid sequence of abiogenous GFP in the jellyfish is made amendment, they have useful excite and emission spectrum (referring to Prasher etc., Gene 111:229-233,1992; Heim etc., Proc.Natl.Acad.Sci., USA 91:12501-12504,1994; U.S. Patent number 6,319,669; International patent application no PCT/US95/14692, every piece of document put in order into for your guidance at this).Like this, the nucleotide sequence that it should be noted that the such fluorescin of coding also can make it to have the chloroplast(id) codon and use preference, thereby provides extra example for fluorescin of the present invention.

[0154] attempts the present invention to be described, but be not restriction the present invention by the following examples.

Embodiment 1

Carry out the optimization of encoding sequence in order in chloroplast(id), to express

[0155] this embodiment proved the nucleotide sequence of encoding green fluorescent protein with chloroplast(id) codon preference can be in the algae chloroplast(id) express efficiently (also can referring to, Franklin etc., Plant J.30:733-744,2002, this piece document is put in order into for your guidance at this).

Chlamydomonas reinhardtii strain system transforms and growth conditions

[0156] all conversions are to carry out on the 137c (mt+) in the Chlamydomonas reinhardtii strain all.Cell is cultured to logarithmic phase late period (about 7 days) (Gorman and Levine in the TAP of the floxuridine that contains 40mM substratum, Proc.Natl.Acad.Sci., USA 54:1665-1669,1965, this piece document is put in order into for your guidance at this), culture condition is that temperature is 23 ℃, and illumination is constant to be 450 luxs (Lux), at speed setting is to cultivate on the rotary shaker of 100rpm.4000 * g, 4 ℃ obtain 50 milliliters cell after centrifugal 5 minutes.Supernatant is removed, and then cell is resuspended in 4 milliliters the TAP substratum, transforms (Cohen etc., as above, 1998) so that use partickle bombardment method (particlebombardment) to carry out chloroplast(id) subsequently.All conversions all are to carry out under the selective action of spectinomycin (150 μ g/ml), wherein the resistance to spectinomycin is (Chlamydomonas Stock Center, the Duke University) that realizes by the spectinomycin resistance ribosomal gene on the cotransformation p228 plasmid.

[0157] cultivation that is used for expressing the Chlamydomonas reinhardtii transformant of GFP is carried out (Gorman and Levine at the TAP substratum, as above, 1965), culture condition is that temperature is 23 ℃, illumination is constant to be 5000 luxs, be to cultivate on the rotary shaker of 100rpm at speed setting, unless specialize, otherwise all be this culture condition.Before cell harvesting, culture will be 1 * 10 in density ⁷Kept at least 48 hours under the situation that individual cell is every milliliter.

Plasmid construction

[0158] all DNA and RNA operation is all basically according to Sambrook etc., as above, and 1989 and Cohen etc., as above, the carrying out described in 1998.The coding region of GFP gene is by PCR increase from the plasmid that contains original GFP sequence (GFPncb) (1998, this piece document is put in order into for your guidance at this for Tsien, Ann.Rev.Biochem.67:509-544).The PCR primer design can be so that be close to one 5 ' end Nde I site and one 3 ' end XbaI site in the outside of described coding region, and this helps ensuing clone.5 ' the sequence of holding at GFPncb is 5 '-CATATGAGTAAAGGAGAAGAAC-3 ' (SEQ ID NO:17); 3 ' end primer sequence at GFPct is 5 '-TCTAGATTATTTGTATAGTTCATCC-3 ' (SEQID NO:18).The coding region of GFPct is according to Stemmer etc., Gene 164:49-53, and the description in 1995 (this piece document is put in order for your guidance at this) obtains by de novo synthesis from each primer length is the primer storehouse of 40 Nucleotide.The primer of 5 ' end and 3 ' end contains Nde I and Xba I restriction site respectively.

[0159] operational manual that provides according to manufacturers to plasmid pCR2.1 TOPO (Invitrogen company), obtains plasmid pCrGFPct and pCrGFPncb with the PCR product cloning that contains GFPct and GFPncb gene of the 717bp that obtains respectively.3 ' the UTR of rbcL utilizes the 1.6kb Hind III segment of being cloned into the Chlamydomonas reinhardtii chloroplast genomic dna among the plasmid pUC19 as template, obtains by the PCR reaction.Sequence corresponding to the PCR primer of the 5 ' end of rbcL 3 ' UTR and the part of pUC19 multiple clone site is 5 '-TCTAGAGTCGACCTGCAG-3 ' (SEQ ID NO:19), comprising Xba I site.The sequence of PCR primer corresponding to the 3 ' end of rbcL 3 ' UTR is 5 '-GGATCCGTCGACGTATG-3 ' (SEQ ID NO:20), comprising the Bam HI restriction site that is provided with for subsequently clone.The product cloning of the 433bp that obtains in plasmid pCR2.1TOPO, is obtained the p3rbcL plasmid.

[0160] 5 ' of rbcL UTR is that template obtains by PCR with the Chlamydomonas reinhardtii genomic dna.With the rbcL gene 5 ' end complementary PCR primer sequence that originates in position-189 with respect to translation initiation site be 5 '-GAATTCATATACCTAAAGGCCCTTTCTATGC-3 ' (SEQ IDNO:21), wherein comprise Eco RI restriction site.With 3 ' the end complementary PCR primer of 5 ' UTR of rbcL gene from translation initiation site, its sequence is 5 '-CATATGTATAAATAAATGTAACTTC-3 ' (SEQ ID NO:22), wherein comprises Nde I restriction site.The PCR product cloning of the 24lbp that obtains in plasmid pCR2.1TOPO, is obtained the p5rbcL plasmid.

[0161] plasmid p5rbcL is cut by Bam HI and Nde I enzyme, the segment that obtains is connected on same pCrGFPct that cuts through Bam HI and Nde I enzyme or the pCrGFPncb produces plasmid p5CrGFPct and p5CrGFPncb respectively.Finally, p5CrGFPct and p5CrGFPncb are cut by Bam HI and Xba I enzyme again, and the segment of the 958bp that obtains is connected on the same p3rbcL after cutting with Bam HI and Xba I enzyme, obtain p53rGFPct and p53rGFPncb.

[0162] p53rGFPct and p53rGFPncb use Nde I and Bam HI enzyme to cut, and the segment of 1.2kb is connected among the pET19b (Novagen), and producing respectively can be at the plasmid pETGFPct and the pETGFPncb of expression in escherichia coli.P53rGFPct and p53rGFPncb then cut with Bam HI enzyme, the segment of 1.43kb is connected to Chlamydomonas reinhardtii chloroplast(id) conversion carrier p322 (Chlamydomonas Genetics Center, Duke University), form pExGFPct and pExGFPncb plasmid.

[0163] the p322 carrier is based on the nucleotide sequence structure of Chlamydomonas reinhardtii chloroplast genomic dna, this section sequence be Eco (Eco RI) sites of 143073 beginnings from the position to the position 148561 Xho (Xho I) sites that begin (referring to the World Wide Web, URL " biology.duke.edu/chlamy_genome/chloro.html " clicks " watching whole genome with text file format "; Also can link referring to " chloroplast gene picture group spectrum ", and then click " 140-150kb " link, watching the position approximately is the Eco site of 143.1kb and the Xho site that the position approximately is 148.5kb).This section Eco/Xho chloroplast gene group sequence is inserted into the pBS plasmid of cutting with Eco RI/Xho I enzyme (Stratagene company, La Jolla, California).Bam HI site on the p322 corresponding on the chloroplast genomic dna sequence since the site of 146522 positions.

Southern and Northern hybridization

[0164] Southern hybridization and DNA ³²The P mark is according to Sambrook etc., and as above, the method for describing in 1989 is carried out.The radioactive probe of using in Southern hybridization comprises the p322Bam HI/Pst I segment (surveying 5 ' p322) of 2.2kb, the p322 BamHI/Xho I segment of 2.0kb (surveying 3 ' p322) and come from p53rGFPct (surveying GFPct) and the Nde I/Xba I segment of the 717bp of p53rGFPncb (detection GFPncb).Latter two probe also is used for detecting in Northern hybridization the mRNA of GFPct and GFPncb.Other radioactive probe of using in the Northern hybridization analysis comprises psbA and rbcL cDNA.Southern and Northern hybridization all are to adopt the Packard Cyclone Storage Phosphor System that has been equipped with the OPTIQUANT software package to observe.

Protein expression, western blotting and fluorescence gel

[0165] according to the operational manual of manufacturers, it is among the BL21 that plasmid pETGFPct and pETGFPncb are transformed into e. coli strains, induces the GFPct or the proteic expression of GFPncb of band 6His mark with IPTG.The proteic purifying of His mark is undertaken by nickel-agarose affinity chromatography (Qiagen).The method that western blotting is described according to (as above, 1998) such as Cohen utilizes the anti-mouse two anti-(Sigma) of a kind of mouse-anti GFP one anti-(Clontech) and a kind of alkali phosphatase enzyme mark to carry out.The operation of fluorescence gel is identical with the gel operation that is used for Coomassie brilliant blue dyeing and protein transduction film, but protein need not boil before last sample.GFP in the gel is the Berthold Night Owl CCD camera looks of LB 981 by model, and this camera has been equipped with the emission filter that excites filter and 535nm (Chroma company) of 485nm.Image generates by WinLight software.

The formation of GFPct and GFPncb excitation spectrum

[0166] excitation spectrum is to utilize the GFPct of protein affinity purification and GFPncb albumen to produce on the Perkin Elmer fluorophotometer of model for LS50.Recombinant protein was diluted to 50mM NaH before reading on the photometer ₂PO ₄, 300mM NaCl, the 250mM imidazoles is in the solution of pH 8.0.Scanning wavelength in 510nm place record emissive porwer, thereby obtains excitation spectrum from 350 to 550nm.

De novo synthesis has the GFP gene of Chlamydomonas reinhardtii chloroplast(id) codon preference

[0167] for develop can be in the Chlamydomonas reinhardtii chloroplast(id) reporter gene of strong expression, synthesized the green fluorescence protein gene that its codon uses the codon that can reflect Chlamydomonas reinhardtii chloroplast gene group through optimizing to use.Designed two amino acid at original GFP (GFPncb) coding region and changed, to improve proteic fluorescence and expression characterization.In these amino acid changes first changes does not think the spectral quality that can have influence on GFP, and it is that the Serine on amino acid position 2 is become L-Ala, and initiator codon is in the more suitable sequence environment.The Serine that second change is amino acid position 65 becomes Threonine, this changes can to strengthen with respect to original GFP and excites amplitude (about 6 times) at the 485nm place, that reduces the 395nm place simultaneously excites amplitude (Heim etc., Nature 373:663-664,1995, this piece document is put in order into for your guidance at this).This change is introduced in the GFPct encoding sequence to improve the fluoroscopic examination when using visible light.As what show in Fig. 1, the GFPncb gene is compared with wild-type GFP gene, also has an amino acid change, Q80R.This change is before selected clone, is imported in the pcr amplification process of original GFP gene.This Q80R sudden change is a kind of change common when adopting the original GFP encoding sequence of pcr amplification (Tsien, as above, 1998), and it is to the not influence of proteinic function.Like this, for consistence, this change also is introduced in the GFPct gene.

The GFPct of expression in escherichia coli and the sign of GFPncb

[0168], is used to check by the Bacillus coli cells lysate of the cell preparation that has transformed pETGFPct or pETGFPncb in order to determine whether GFPct gene and GFPncb gene can produce functional GFP albumen.The nickel affinity chromatogram of intestinal bacteria lysate has obtained having the albumen of correct GFP molecular weight.With the albumen that the SDS-PAGE separating Escherichia coli produces, directly fluorometric analysis shows that these two kinds of albumen all can send fluorescence under blue light illumination, and the fine difference of their photoluminescent properties of demonstrating also changes consistent with the amino acid that imports.S65T variation among the GFPct causes albumen to strengthen greatly in the fluorescence level at 485nm place (with respect to the consumption of GFPncb albumen in detection, the proteic consumption of the GFPct of expression in escherichia coli have only the former 1/5), and its fluorescence when 395nm excites reduces (referring to Fig. 2) greatly.The western blot analysis that utilizes the mouse polyclonal antibody of antigen beginning GFP to carry out shows that GFPct and GFPncb send similar signal.The proteic spectral quality of GFPct is had a mind to the enhanced situation with respect to GFPncb albumen under, seem very important of this result.Like this, fluoroscopic examination meeting based on exciting under the visible light (485nm) helps the proteic detection of GFPct more, simultaneously immune labeled then as broad as long, this just permission to GFPct albumen and GFPncb albumen the accumulation in the Chlamydomonas reinhardtii chloroplast(id) directly compare.

The Southern of GFPct and GFPncb transformant and Northern hybridization

[0169] proved that GFPct and GFPncb encoding sequence can produce after functional GFP albumen, just transformed the Chlamydomonas reinhardtii chloroplast(id) with pExGFPct and pExGFPncb.In addition, these cells also carry out cotransformation with the selected marker plasmid p228 that can give the spectinomycin resistance.In former generation,, transformant screened by PCR and Southern hybridization analysis, then the male transformant goes out homogenous cell system (homoplasmiclines) by extra number wheel screening and separating again, and all chloroplast genes all contain the GFP gene of importing in such clone.

[0170] two homogeneity GFPct transformant 18.3 and 21.2 and two GFPncb homogeneity transformants 5.8 and 12.1 be selected to come out to do further analysis (referring to Fig. 3 A, demonstration be to indicate GFPct and the GFPncb construction that relevant restriction site is arranged).The probe that utilization marks on gene mapping determines that the Eco/Xho zone of the 7.1kb of plasmid pExGFPct and pExGFPncb correctly has been incorporated into (Fig. 3 B) in the chloroplast gene group.The genomic dna that obtains from wild-type and GFPct transformant and GFPncb transformant is cut through Eco RI and Xho I enzyme, separates on agar gel, then carries out the Southern hybridization analysis.Because the 5 ' UTR of rbcL contains an Eco RI restriction site (Fig. 3 A), compares with wild-type DNA so cut transformant DNA with Eco RI with Xho I enzyme, should be able to produce segment one section littler and 5 ' end p322 or 3 ' end probe hybridization.

[0171] the Southern hybridization analysis of the GFPct of Chlamydomonas reinhardtii chloroplast(id) and GFPncb transformant has proved that transgenic cell line is a homogeneity.The DNA of Chlamydomonas reinhardtii cuts with Eco RI and Xho I enzyme simultaneously, and filter membrane is hybridized with radioactive probe more then.5 ' p322's ³²P label probe and 3 ' p322's ³²The P label probe respectively with GFPct and GFPncb transformant in the Eco RI segment hybridization of 3.7kb and 3.3kb.Yet as what expected, these same probes are the Eco RI/Xho I segment hybridization of 5.7kb in being with the wild-type Chlamydomonas reinhardtii strain that does not have to transform.Southern blotting technique is washed off, surveys with GFPct and the specific probe of GFPncb again then.In transformant 5.8 and 12.1, detect the EcoRI/Xho I segment (Fig. 4, intermediate plate) of 3.3kb with the specific probe of GFPncb, and the segment of similar size arrives with the specific probe in detecting of GFPct in transformant 18.3 and 21.2 also.And detect less than any signal in wild-type Chlamydomonas reinhardtii DNA with these two kinds of probes.

The accumulation of GFP mRNA in transgenic line

[0172] the Northern hybridization analysis of total RNA is used to determine whether GFPct and GFPncb gene transcribe in the chloroplast(id) of transgene Chlamydomonas reinhardtii.The total RNA of 10 micrograms is from wild-type, and transgenic cell line 5.8,12.1 is separated in 18.3,21.2, and separates on the sex change agar gel, is imprinted onto on the nylon membrane then.Two parts of same filter membranes with ³²The psbA of P mark or rbcL cDNA probe hybridization, the level of the psbA of accumulation and rbcL mRNA is all similar in each strain system, this the proof equivalent RNA by last sample in each swimming lane, and chloroplast(id) transcribe with being accumulated in these transgenic lines of mRNA all be normal.

[0173] these filter membranes are surveyed with GFPct and GFPncb specific probe after the wash-out more again.Strain is 5.8 and 12.1 accumulation GFPncb mRNA, and strain is 18.3 and 21.2 accumulation GFPct mRNA then.As prediction, there is not the GFP signal in wild-type cell, to be observed.All four kinds of cDNA probes are marked as has almost equal specific activity, the GFPct that obtains is similar with the GFPncb signal, and two kinds of filter membranes that the GFP probe is surveyed need the time shutter of longer (approximately being 4 times) can reach and the similar signal of rbcL probe.These results show that the accumulating level of GFP mRNA approximately is 1/4th of endogenous rbcL mRNA.

Analyze the accumulation of GFP in the transgene Chlamydomonas reinhardtii chloroplast(id)

[0174] in order to measure GFPct and the accumulating level of GFPncb albumen in transgenic cell line, measures GFP by fluorometric analysis and western blot analysis.Relatively expressing the Chlamydomonas reinhardtii transgenic line 21.2 of GFPct and the strain of expression GFPncb is the accumulation of GFP in 5.8 and 12.1.It is 1 * 10 that cell (5000 lux) under continuous illumination grows into density ⁷Every milliliter in individual cell, this growth conditions are the known conditions that can accumulate GFP to greatest extent.The solubility total protein is carried out SDS-PAGE analyze, then the antiserum(antisera) with anti-GFP carries out western blot analysis.For GFPncb transgenic line 5.8 and 12.1, with sample on the 20 microgram solubility total proteins, and for GFPct transgenic line 21.2, with 250ng (1/80) sample to the solubility total protein of 20 micrograms (1/1).

[0175] 6 microgram solubility total protein (tsp) separates by SDS-PAGE, and the gel that obtains is used for Coomassie brilliant blue dyeing, fluorescence imaging, perhaps western blot analysis.The albumen that gel after the Coomassie brilliant blue dyeing (the 6 microgram solubility total proteins of separating from specified Chlamydomonas reinhardtii strain system are by on the SDS-PAGE gel of last sample to 12%) shows isodose by last sample in each swimming lane.(situation of protein Preparation and Coomassie brilliant blue dyeing gel is similar, but sample did not boil before last sample for fluorescence gel; Albumen separates by SDS-PAGE)---excitation wavelength is arranged on 485nm, and emission wavelength is arranged on 535nm; 485nm excites, imaging during the 535nm emission---and show and have only GFPct transformant 18.3 and 21.2 that signal is just arranged.When excitation wavelength is 366nm, all there is not fluorescent signal for any GFP transformant.Show that GFPct and GFPncb albumen expresses in the chloroplast(id) of transgene Chlamydomonas reinhardtii strain system.

[0176] western blot analysis that same sample is carried out has provided and the similar result of fluorometric analysis, in the GFPncb transformant, do not detect GFP, and in GFPct strain system, good signal is arranged (in western blot analysis, the GFP albumen of chloroplast expression is transferred on the nitrocellulose filter, and detects with anti-GFP antiserum(antisera)).Carry out burette test to confirm the difference of GFP accumulation between GFPct and the GFPncb transformant more exactly.The 20 microgram tsp that come from GFPncb transformant 5.8 and 12.1 are separated with the tsp that comes from GFPct transformant 21.2.For GFPct strain system, protein concentration changes to 250ng from 20 micrograms.Comparison shows that between the sample, the accumulating level of GFPct approximately is in any GFPncb transformant observed about 80 times in transformant 21.2.

Report that the GFP that utilization is optimized at chloroplast(id) expresses as chloroplast gene

[0177] studied the effect of accumulation of different growth conditionss to GFPct in the transgenic line, GFPct gene for confirmation can be used as report that chloroplast gene is expressed.Before harvested cell, the GFPct transgenic line 21.2 of Chlamydomonas reinhardtii maintains 1 * 10 under the constant illumination condition ⁶The density that individual cell is every milliliter, the intensity of described illumination are 5000 luxs (high illumination) or 450 luxs (low light shines).Handle sample for each part, get 1 microgram tsp and carry out western blot analysis.Studied the effect of accumulation of light intensity to GPFct in the Chlamydomonas reinhardtii.Before results, Chlamydomonas reinhardtii transgenic line 21.2 is maintained at 1 * 10 under the constant light of specifying light intensity is shone ⁶Every milliliter in individual cell or 1 * 10 ⁷The concentration that individual cell is every milliliter reaches 48 hours.Solubility total protein (1 microgram) is gone up sample and is gone up separation to 12%SDS-PAGE, resists with anti-GFP one and carries out western blot analysis.

[0178] cell is maintained at 1 * 10 under shining with the constant light of 5000 luxs ⁶During concentration that individual cell is every milliliter, the amount of the GFPct of accumulation approximately be low light according under be maintained at 1 * 10 ⁶About 10% of expression amount during concentration that individual cell is every milliliter in the cell.Shaking bottle with the 3rd, to keep cell concn under the constant illumination condition of 5000 luxs be 1 * 10 ⁷Every milliliter in individual cell, GFP can build up to high level once more, because high cell density has played the effect that reduces intensity of illumination in culture, in fact is exactly the environment of having created the low light photograph.These results prove that the GFPct gene can be used to report because the small albumen synthetic difference that environmental change caused has proved that the GFPct gene can be used as report that chloroplast gene is expressed.

[0179] had several heterologous genes to be used as report of chloroplast(id) genetic expression in the Chlamydomonas reinhardtii, but their practicality is all owing to low-level protein expression is restricted.The low expression level of these heterologous proteins in the Chlamydomonas reinhardtii chloroplast(id) has several possible reasons.For example, the promotor that is used for ordering about these genetic transcriptions may cause low-level transcribing.Perhaps, the congenital instability of the mRNA of some of them reporter gene, result cause low-level mRNA accumulation.Another possibility is that these chimeric mRNA lack the needed RNA element of translation.Chlamydomonas reinhardtii chloroplast gene intensive codon preference also might hinder the translation of allos mRNA.

[0180], the analysis revealed of transgene Chlamydomonas reinhardtii chloroplast(id) there are enough allos mRNA accumulation to support high-caliber protein synthesis though the stability of the activity of promotor and mRNA can influence the expression of gene in chloroplast(id) to a great extent.In addition, in most cases, 5 ' UTR of Chlamydomonas reinhardtii and 3 ' UTR can be used in the structure of mosaic gene, and this makes the mRNA of these reporter genes unlikely lack crucial RNA element.As disclosed herein, the change codon uses a kind of means that improve the accumulation of heterologous protein in the Chlamydomonas reinhardtii chloroplast(id) that can be used as.The method that this change codon uses can illustrate by the green fluorescent protein (GFP) of jellyfish.

[0181] the GFP coding region of GFP is matched so that the codon of albumen coded sequence uses with Chlamydomonas reinhardtii chloroplast gene group by engineered.Place 5 ' the end non-translational region and 3 ' of Chlamydomonas reinhardtii Chloroplast rbcL to hold under the control of non-translational region the expression of this GFPct gene and original GFP gene (GFPncb).GFPncb gene and GFPct gene can be transcribed in the Chlamydomonas reinhardtii chloroplast(id), and being on close level of mRNA accumulation.

[0182] accumulation volume of expressing GFP in the transgenic line of GFPct approximately is 80 times that express in the strain system of GFPncb.Under the growth conditions of optimizing, the strain that produces GFPct is that the GFP of accumulation in 21.2 accounts for about 0.5% of solubility total protein greatly.The feasible expression that can analyze GFP by the fluoroscopic examination of total protein of cell of the protein expression of this level.Report was arranged in the past, and in the Chlamydomonas reinhardtii chloroplast(id), uidA (GUS) shows low-level protein expression at the 5 ' UTR of rbcL and the expression under 3 ' the UTR control, accounts for 0.01% of soluble proteins greatly; Suitable (the Ishikura etc. of the GUS accumulation of this level with the GFP accumulating level that obtains from the GFPncb gene that has used identical rbcL controlling elements, as above, 1999, this piece document has also been reported the low-level relatively accumulation of rbcL-GUS mRNA) (be similar to the low-level of rbcL GFP mRNA, as disclosed herein).

[0183] compare with the GFPncb gene, one has 123 codons variations in the GFPct gene, changes (referring to top content) comprising the codons that 121 synonym change and two make amino acid that replacement take place.During these 121 synonym change, there are 66 variations representing gentleness to change to more optimal codon use.In remaining codon, 54 codon quilt codon replacements commonly used that variation causes being of little use.This codon optimized being evenly distributed in basically in the whole GFP gene, wherein have 15 variations be positioned at coding region first three/one, 20 variation be positioned in the middle of 1/3rd and 18 variations be positioned at back 1/3rd.

[0184] for the analysis of the gene of in the Chlamydomonas reinhardtii chloroplast(id), expressing of former report, these genes comprise the Renilla luciferase (Minko etc., as above, 1999), and uidA (Sakamoto etc., as above, 1993), and aadA (Goldschmidt-Clermont, as above, 1991) and aph A6 encoding sequence (Bateman and Purton, as above, 2000), showing in above each gene has 61 respectively, 252,121 and 65 not preferred codons.If the number of these not preferred codons in these reporter genes recently calculated with the percentage that they account for total codon number, is respectively 20%, 42%, 46% and 25%.This can compare with the situation of GFPncb gene, and 23% the not preferred codon that accounts for the codon sum is arranged in the GFPncb gene.These results show that these other expression of report in the Chlamydomonas reinhardtii chloroplast(id) can be strengthened greatly by changing the codon use.

[0185] because the based composition of GFP sequence has been changed significantly, so studied the influence of these changes for the mRNA structure at the mRNA of GFPct and GFPncb.This analysis has determined that the enhancing of the translation of GFPct mRNA is because the difference that codon uses, rather than the mRNA secondary structure may hinder GFPncb navigate on the rrna influence.Preceding 250 Nucleotide of the mRNA of GFPct and GFPncb fold program mfold (Zucker etc. with RNA, at RNA Biochemistry and Biotechnology 11-43 (ed.Barciszewski and Clark, NATO ASI Series, Kluwer Acad.Publ.1999; Matthews etc., J.Mol.Biol.288:911-940,1999) study.Do not predict any tangible secondary structure difference between these two genes, wherein the free energy of the suitableeest structure of GFPct sequence is-42kcal, the GFPncb sequence then is similarly-and 38kcal.

[0186] result disclosed herein proves that the optimizing codon use can promote the translation and the expression of polypeptide, and such as the GFPct gene of optimizing, it can be as report of Chlamydomonas reinhardtii chloroplast gene expression.Proved codon optimized can being used in the expression of Chlamydomonas reinhardtii acquisition high levels of recombinant proteins, this shows the codon optimized translation efficiency of other heterologous polypeptide in plant chloroplast that generally can improve.Compare relative low-level GFPmRNA accumulation with endogenic rbcL mRNA and show that promoter activity and the mRNA stability of optimizing GFPct may provide a kind of signal with GFPct to be strengthened to the method for level higher or that need more.Like this, the GFPct gene provides one can be used for optimizing easily GFP comprises the transcribing of Chlamydomonas reinhardtii chloroplast(id), mRNA stability and translation at plant chloroplast instrument.

Embodiment 2

The sign of plant chloroplast ribosome binding sequence (RBS)

[0187] present embodiment has illustrated evaluation and the sign that instructs the ribosome binding sequence of translation in chloroplast(id).

The structure of mutant and sign

[0188] utilize following oligonucleotide that psbA 5 ' UTR is carried out pcr amplification, produce the locus specificity sudden change:

5-GAAGCTTGAATTTATAAATTAAAATATTTTTACAATATTTTACCCAGAAATTAAAAC-3’(RBS-Alt；SEQ?ID?NO：23)；

5’-TGTCATATGTTAATTTTTTTAAAGTTTTTCTCCGTAAAATATTG-3’(RBS-23；SEQ?ID?NO：24)；

5’-TGTCATATGTTAATTTTTTTAAAGTCTCCGTAAAATATTG-3’(RBS-19，SEQ?ID?NO：25)；

5’-TGTCATATGTTAATTTTTTTTCTCCGTAAAATATTG-3’(RBS-15；SEQ?ID?NO：26)；

5 '-GTCATATGTTAATTTCTCCG-3 ' (RBS-11; SEQ ID NO:27); With

5’-TGTCATATGTTAATCCTCCTAAAGTTTTAATTTCTCCG-3’(RBS-Add；SEQ?ID?NO：28)。

[0189] conversion of plasmid construction and Chlamydomonas reinhardtii is to carry out according to the description of (as above, 1994) such as Mayfield.16S-1470/71 and 16S-1467/68 mutant are to adopt QUICK-CHANG sudden change test kit (Qiagen) to make up.Mutant characterizes by northern hybridization or western blot analysis.RNA extracts, the northern hybridization analysis, protein separation, western blot analysis, and in the body under the condition to albumen with ( ¹⁴C)-pulse labelling that acetate carries out all is to carry out according to the description of (as above, 1998) such as Cohen.

[0190] analyze for " the toe marking (toeprinting) ", the ribosomal subunit of 30S separates according to the method that Harris (Microbiol.Rev.58:700-754,1989, this piece document is put in order into for your guidance at this) describes, and has done some little changes.Wild-type Chlamydomonas reinhardtii cell (2137a) is resuspended in TKMD damping fluid (25mM Tris-HCl (pH 7.8), 25mM KCl, 25mMMgOAc, 5mM DTT), coming smudge cells, pressure by a broken instrument of high pressure cell (French press) then is 5000psi.Cellular exudate is 40000 * g in Beckman JA-20 rotary head again, and 4 ℃ centrifugal 30 minutes.With 200 A ₂₆₀The supernatant of unit is put in the middle of the TKMD damping fluid with 10-30% sucrose linear gradient, also contains 100mM KCl in the damping fluid, separates with a step of the ribosomal subunit that is used for 30S and 50S.Described gradient is 22500rpm in the BeckmanSW28.1 rotary head, and 2 ℃ centrifugal 20 hours.Handle described gradient with optical scanner that can read 260nm place absorbance and Fraction Collector.30S and 50S fraction are collected respectively together, then with the dilution proportion of the high salt TKMD that contains 800mM KCl with 1: 1, and then in Beckman TLA-100 rotary head 200000 * g, 4 ℃ are centrifugal 20 hours.These are deposited in the TKMD damping fluid that contains 100mM KCl resuspendedly, freezing in liquid nitrogen then again, leave-70 ℃ of refrigerations in.The degree of the crossed contamination between 30S and the 50S subunit detects (Cohen etc., as above, 1998) with the analysis of RNA dot blot.

[0191] formation of initial recombination body by as the extension inhibition described such as Hartz (J.Mol.Biol.218:83-97,1988, this piece document is put in order into for your guidance at this) analyze, done some little changes.Reaction mixture at 10 microlitres has the 5 ' end that contains 0.6pmol ³²The annealing mixture (referring to embodiment 2) of the synthetic psbA D1-HA transcript of the oligonucleotide of P mark and 0.2pmol.Extend and suppress from adding 3.75mM dNTPs and 8 * 10 ^-5To 2 * 10 ^-3The 30S ribosomal subunit that the high eluting salt of μ M is crossed begins.Be reflected at after 37 ℃ of incubations 5 minutes uncharged intestinal bacteria tRNA (tRNA _f ^MetThe Luo Shi diagnostic reagent) join in the reaction system, final concentration is 5 μ M.Again AMV ThermoScript II (0.5 unit) is added, be reflected at 37 ℃ of incubations 15 minutes again.Be reflected on 8% the sequencing gel and analyze.Sequencing reaction carries out according to described above, and the final concentration of the dNTPs of use is 200 μ M, and does not have rrna or tRNA to exist.

Gel retardation assay (gel shift assays)

[0192] about 1 microgram is by the albumen (Cohen etc. of heparin-agarose purifying, 1998) and the PRIME RNase inhibitor (5Prime → 3Prime of 0.4 unit, Inc.) at cumulative volume be dialysis buffer liquid (the 20mM Tris-HCl (pH 7.5) of 8 microlitres, 100mM KOAc, 0.2mM EDTA (pH 8.0), 2mM DTT, 20% glycerine, 4mM MgCl ₂) in incubation 10 minutes under the room temperature.The in-vitro transcription of adding 0.04pmol ( ³²P) the psbA RNA of mark, 20 microgram wheatgerm tRNA (Sigma), and after the total RNA of 3 microgram FuD7 (the Chlamydomonas reinhardtii strain system that lacks psbA mRNA), reacted at room temperature incubation 10 minutes, the zone that described psbARNA covers is to+171 positions with respect to translation initiation codon-90.In some reaction, the unlabelled psbA RNA of the in-vitro transcription that does not have mark of 10pmol is added in the reaction system as the rival.The RNA/ protein complexes separates on 5% non-denaturing polyacrylamide gel.

Shine-Delgarno ribosome binding sequence among the chloroplast(id) 30S ribosomal subunit identification psbA 5 ' UTR

[0193] in order to identify that chloroplast(id) mRNA translates needed RNA element, the psbA gene variant that contains locus specificity sudden change among the 5 ' UTR is directed in the chloroplast(id) of psbA defective Chlamydomonas reinhardtii strain system (Mayfield etc., as above, 1994).The potential that the possible RBS that is positioned 27 Nucleotide of upstream from start codon discerns the antisense SD sequence on the chloroplast(id) 16SrRNA based on it among the psbA 5 ' UTR is identified out.The disappearance of this section sequence (RBS-del) causes the failure of psbA mRNA and rrna bonded, thereby causes can not synthesizing fully corresponding D 1 albumen (Mayfield etc., as above, 1994).Though this result shows this element and may play the function of RBS, but this disappearance also might influence ribosomal combination by a series of other mechanism, comprise the binding site (Yohn etc. that directly or indirectly destroy the trans-acting factor that is incorporated into 5 ' contiguous UTR of described RBS, Proc.Natl.Acad.Sci., USA95:2238-2243,1998a; Yohn etc., J.Cell Biol.142:435-442,1998b; Danon and Mayfield, EMBO J.10:3993-4001,1991, every piece of document all is hereby incorporated by; Also can be referring to, Fargo etc., as above, 1998).

[0194] the SD sequence in the RBS element by with the translation (Voorma of complementary sequence (anti-SD sequence) the pairing promotion protokaryon transcripton of the 3 ' end of the 16S rRNA of 30S small subunit ribosome, at Translational Control (ed.Hershey etc., Cold Spring Harbor LaboratoryPress 1996), this piece document is hereby incorporated by).This interaction joins in the protokaryon transcripton at the external 30S ribosomal subunit that utilizes purifying and is measured (Hartz etc., as above, 1991).Bonded 30S subunit has stoped the extension of the downstream oligonucleotide primer on the mRNA, and the result forms ribosomal " the toe marking (toeprint) ".

[0195] whether can discern RBS in 5 ' the end non-translational region of psbA mRNA for the subunit of determining 30S, the ribosomal subunit of 30S extracts from Chlamydomonas reinhardtii.With 5 ' end of the regional complementarity in psbA mRNA initiator codon downstream ( ³²P) the external synthetic psbA transcripton annealing of the oligonucleotide primer of mark and purifying.Will ³²The Chlamydomonas reinhardtii 30S ribosomal subunit of the purifying that the oligonucleotide of P mark/RNA mixture and concentration increase progressively, and colibacillary fMet tRNA (referring to embodiment 2) incubation.Because the combination of ribosomal subunit can cause occurring in the primer extension process stopping site (pause sites).Carry out sequencing reaction to determine the ribosomal position of bonded.In containing the ribosomal reaction of 30S, the stop of toe marking reaction occurs in Shine-Delgamo sequence 3 ' end 12 Nucleotide (RBS stop) and initiator codon 3 ' end 12 Nucleotide (AUG stop).When chloroplast(id) 30S ribosomal subunit with corresponding to psbA mRNA 5 ' end the sub-incubation of rna transcription the time, observe the primer extension toe marking.These stops occur in about 12 Nucleotide of SD sequence and initiator codon downstream of supposition, this result with two sequences of 30S ribosomal subunit and this in conjunction with consistent.Intestinal bacteria 30S subunit can be incorporated on the psbA mRNA that comes from barley, its result has also disclosed the toe marking corresponding to potential SD sequence, the position class of this SD sequence is similar to position (Kim and the Mullet of the SD sequence of the psbA mRNA that comes from Chlamydomonas reinhardtii, Plant Mol.Biol.25:437-448,1994, this piece document is incorporated herein for your guidance).These results show that the RBS element of supposition has the characteristic of a functional r BS element.Therefore, these external physicochemical datas are supported the explanation of hereditary evidence in the body that early-stage Study obtain, and just the RBS element among the psbA mRNA is positioned at initiator codon 5 ' end (upstream) 27 base places (Mayfield etc., as above, 1994).

The sudden change of anti-SD sequence suppresses the translation of a series of chloroplast(id) mRNA among the 16S rRNA

[0196] in order to prove chloroplast ribosome by interacting identifying information with the SD sequence, made up two homogeneity Chlamydomonas reinhardtii strain systems, wherein the anti-SD sequence of chloroplast(id) 16S rRNA is suddenlyd change.Be positioned at 16S rRNA 3 ' end anti-SD sequence in Nucleotide become GGUCC (1467 and 1468 Nucleotide of 16S rRNA) or become CCUGG (1470 and 1471 Nucleotide of 16S rRNA from CCUCC from CCUCC, also can be) referring to, SEQ IDNO:29.All survive when these mutant are cultivated in can supporting not have the perfect medium of photosynthetic growth, and do not show the anomalad that any variation of significantly synthesizing owing to the chloroplast(id) energy causes.16S-1467/68 mutant strain system can be on minimal medium the growth to reduce, 16S-1470/71 mutant strain system then cannot grow on minimal medium, and these show in these mutant that photosynthetic function reduces respectively and removed.

[0197] accumulation of chloroplast(id) encoded protein is checked by western blot analysis in these strain systems.From wild-type (wt) Chlamydomonas reinhardtii strain system or mutant 16S-1467/68 and 16S-1470/71, prepare the total protein (measuring) of isodose by Coomassie brilliant blue dyeing, separate by SDS-PAGE again, trace is to nitrocellulose filter then, follow with specific at D1, D2, ATPase, perhaps the proteic rabbit polyclonal antiserum(antisera) of Lsu is handled.The sudden change of the anti-SD sequence among the 16SrRNA has influenced the accumulation of some chloroplast protein.The D1 albumen of psbA coding is not accumulation in the 16S-1470/71 mutant, and has only 20% of accumulating level in the wild-type in the 16S-1467/68 mutant.The D2 albumen of psbD coding shows similar situation, and the accumulating level in the 16S-1470/71 mutant is less than 10% of wild-type, and in the 16S-1467/68 mutant nearly 25%.Being accumulated in of chloroplast(id) ATPase also weakened in the 16S-1470/71 mutant (have only wild-type accumulating level 50%) is although level that accumulates in the 16S-1467/68 mutant and wild-type are approaching.On the contrary, the accumulation in the big subunit of the ribulose bis phosphoric acid carboxylase (Lsu) of the solubility of chloroplast(id) coding what a kind of 16S mutating strain series in office all is not affected substantially.

[0198] failure that accumulates in mutating strain series of D1 albumen shows that it is that to optimize translation needed that the RBS element of supposition and the Shine-Delgamo between the 16SrRNA interact.The failure of D2 protein accumulation may be because the translation of psbD mRNA needs and the same anti-SD sequence of psbAmRNA in these strains system, or since the D1 subunit lose cause D2 albumen synthetic after instability.For example, the Chlamydomonas reinhardtii nuclear transmutation body that can not synthesize the single subunit of PSII can not accumulate other core polypeptides of the PSII that is encoded by chloroplast(id), although these albumen also with the same speed of wild-type in synthetic (Erickson etc., EMBO J 5:1745-1754,1986).

[0199] in order to check the translation speed of single chloroplast protein, wild-type strain system has the strain system of the 16S rRNA of sudden change, and the Chlamydomonas reinhardtii strain system that lacks the psbA gene, all use ( ¹⁴C)-the acetate pulse labelling.Nearly all membranin in the 16S-1470/71 mutant comprises D1 as a result, D2, and P5 and P6 albumen all do not have synthetic.Relevant total protein of the film of isodose or solubility total protein (Cohen etc., Meth.Enzymol.297:192-208,1998, this piece document is put in order into for your guidance at this; Also can be referring to embodiment 2) from by ( ¹⁴C) prepare in the wild-type of acetate pulse labelling and the strain of the mutant Chlamydomonas reinhardtii system, proteic amount is to measure with Coomassie brilliant blue dyeing, and then all albumen are separated on SDS-PAGE. ¹⁴The albumen of C mark is by the radioautograph imaging.The sudden change of the anti-SD sequence of 16S rRNA has reduced the synthesis rate of several chloroplast(id) encoded protein.This result shows that the decline of D2 protein accumulation is not that shortage owing to the D1 protein accumulation causes, but the translation of psbD gene is to need anti-SD sequences.The translation of the mRNA of ATPase equally also reduces in this strain system, though the degree that reduces is less than other membranins.The smaller slightly influence of degree of susceptibility is observed in the 16S-1467/68 mutant, and this is consistent with observed protein accumulation level.The relevant albumen of some films in 16S-1470/71 strain system with wild-type in the same level continue to translate.Diametrically opposite with membranin is that the translation of solubility chloroplast protein does not almost change in 16S rRNA mutant.Soluble proteins still has function with the chloroplast ribosome that the synthetic explanation of the speed in the wild-type has the change of anti-SD element among the 16S rRNA, can support translation.These results show that the expression regulation of soluble proteins and membranin is differently regulated and control by the mechanism of a kind of RBS of depending in the chloroplast(id).

The proteic expression of D1 of psbA coding needs the existence of SD sequence among the RBS and specific space requirement

[0200] checking of Chlamydomonas reinhardtii strain system is further adopted in the effect of RBS in the translation of psbA mRNA, and wherein RBS changes into CCAG (RBS-Alt) by GGAG.Each strain system all is grown under successive illumination in complete (TAP) substratum (referring to embodiment 2), the membranin of equivalent (measuring by Coomassie brilliant blue dyeing) separates by SDS-PAGE, trace is used at the proteic rabbit polyclonal antiserum(antisera) of D1 again and is handled to nitrocellulose filter then.Because the proteic incomplete sex change of D1 produces a plurality of bands by the bonded chloroplast(id).The RBS-Alt sudden change has destroyed potential SD base pairing between psbA mRNA and 16S rRNA 3 ' end, but does not destroy other relative positions (referring to Fig. 4) in the 5 ' UTR.The same as the result of the RBS-del that shows previously (Mayfield etc., as above, 1994), D1 albumen is also not accumulation in RBS-Alt.This result proves that the GGAG sequence is essential for the expression of psbA, as same real RBS.

[0201] if when thinking that interval in RBS and initiator codon is greater than 15 Nucleotide, the ribosomal subunit of 30S can not contact (Chen etc. with these two sections sequences simultaneously, Nucl.AcidsRes.22:4953-4957,1994), should just can not instruct translation to begin with the psbA initiator codon at a distance of the SD sequence of the supposition of 27 Nucleotide among the psbA mRNA so at correct initiator codon place.For the relative position of the RBS that checks psbA mRNA is how to influence expression, a series of deletion mutantions are directed to and make the distance more and more nearlyer (Fig. 4) of RBS element distances initiator codon in 5 ' the end non-translational region.Along with RBS is little by little shifted near to initiator codon, the accumulation of D1 albumen in the Chlamydomonas reinhardtii cell begins to descend.Those make and approach the disappearance of the optimum position of protokaryon RBS element in the position of described RBS (RBS-15 RBS-11), causes not having the proteic accumulation of D1 in the chloroplast(id) of Chlamydomonas reinhardtii.Further, under the condition that the RBS of wild-type psbA sequence exists, add the accumulation that extra protokaryon RBS element (SD-add) does not promote D1 at 7 nucleotide positions of upstream from start codon.

The accumulation failure of D1 albumen in the psbA mutating strain series is not that the stability of mRNA is lost and caused

[0202] though in the supposition SD sequence of the 5 ' UTR of its psbA, exists the strain system of sudden change can not accumulate D1 can not to discern with rrna and explain to also have other to explain existence.Such as, make the unsettled sudden change of transcripton usually reduce the level of mRNA accumulation, this also can cause translation skill to reduce or the protein accumulation level reduces.PsbA mRNA accumulation in the Chlamydomonas reinhardtii strain system of the rite-directed mutagenesis that contains influential RBS sequence.Come from total RNA storehouse or RNA storehouse that rrna is relevant in the level of psbA mRNA detect by the radiolabeled probe of specificity at psbA or 16S rRNA (guaranteeing same applied sample amount).Relative psbAmRNA level is proofreaied and correct by the difference of 16S rRNA, and then carries out normalization method by wild-type.

[0203] though in 5 ' the end non-translational region of psbA the sudden change of SD sequence cause the steady-state level of the psbAmRNA that accumulates to reduce, the level relatively of the mRNA that accumulates is not consistent with the proteic accumulating level of observed D1.Such as, the proteic accumulating level of D1 is just than higher in the RBS-23 mutant, and the level of psbA mRNA has descended 50%.RBS-15 and RBS-11 strain system can not accumulate any D1 albumen, or can not under minimum growth conditions, grow, still, nonetheless, the level of the psbA mRNA of their accumulation is similar with the RBS-19 mutant but, and the latter can accumulate D1 albumen.In fact, accumulation is equivalent to only 10% of wild-type psbA mRNA level, such as observed situation in RBS-del and RBS-Alt mutant, just enough can observe the D1 protein accumulation (Mayfield etc. of wild-type level in other psbA mutant, as above, 1994).Like this, the viewed influence that causes owing to those sudden changes can not be attributed to the variation of mRNA stability/accumulation.

[0204] if the sudden change/disappearance of 5 ' the end non-translational region of psbA causes structural modification and causes the transcripton of generation to be translated, also losing of D1 protein accumulation may be taken place.Whether can discern the SD sequence in order to determine rrna, even if exist the sudden change of the relative position that changes SD sequence and initiator codon, by cell extract centrifugal relevant RNA of the rrna that comes from various mutant strains that makes in the sucrose damping fluid is separated with free mRNA.In containing the strain system of RBS that change or disappearance, the psbA mRNA level relevant with rrna reduces greatly.Yet, each strain system of containing the RBS element all have significant amounts (greater than the wild-type level 50%) psbA mRNA and rrna form connection, even can not accumulate during the proteic strain of D1 is at some.Can not accumulate D1 albumen and show, the relevant RNA of rrna is mainly by forming with monosome rather than polysome bonded RNA in RBS-15 and the RBS-11 mutant strain.

[0205] in order to prove that further making the SD sequence is not the translation that by mistake stops on the 70S rrna near the sudden change of initiator codon more, made up mosaic gene, the coding region of the bacterial luciferase that wherein contains is placed in the back of wild-type or mutant psbA 5 ' UTR.These mosaic genes are transformed in the intestinal bacteria, and the translation of luciferase mRNA detects by luminescence activity.The expression pattern of luciferase is opposite with the expression pattern of observed D1 in Chlamydomonas reinhardtii in intestinal bacteria.Make psbA SD sequence promote translation in the bacterium again near the sudden change of initiator codon more.The coding region that comes from the bacterial luciferase gene (lux AB) of Vibrio harveyi (Vibrio harveyi) is fused to wild-type (wt) or mutant psbA 5 ' UTR, is connected in the plasmid that contains wild-type psbA promotor and 3 ' UTR.These plasmids are transformed in e. coli bl21 (DE3) the strain system again, then under the condition that luciferase substrate n-capraldehyde exists, use pick up camera and monitor translation (Welsh and the Kay of luciferase by photon counting, Curr.Opin.Biotech.5:617-622,1997, this piece document is hereby incorporated by).Each strain system comes out with respect to the per-cent of optimum expression (RBS-11) is determined.In bacterium, translate luciferase efficiently, but when the orientation distance upstream from start codon of RBS during above 19 Nucleotide, translation efficiency is very low by the construction that contains apart from the RBS of 11 to 15 Nucleotide of upstream from start codon.This result presents a contrast with report about the 5 ' UTR of the atpB mRNA of Chlamydomonas reinhardtii, and the latter is in the news and can orders about the translation of carrying out similar level in bacterium or chloroplast(id) (Fargo etc., as above, 1998).

[0206] sequence in 5 ' of the psbA of Chlamydomonas reinhardtii and the psbD transcripton end non-translational region may influence mRNA processing.PsbA 5 ' UTR is sheared at 4 nucleotide position places, RBS sequence upstream in vivo, and this ripening process and rrna be in conjunction with being associated, and depends on the existing of RBS sequence (Bruick and Mayfield, as above, 1998).The factor that the analysis of psbA 5 ' end provides extra evidence to confirm that psbA RBS sequence from mutant is participated in the early stage assembling of rrna is discerned.5 ' the end non-translational region of the primer extension analysis of chloroplast(id) psbA mutant proof psbA all passes through the course of processing in each contains the strain system of RBS sequence, but does not observe (referring to Fig. 4 in RBS-Alt and RBS-del mutant; Also can referring to, Bruick, Graduate Thesis, Scipps institute, 1998).These results show that the RBS element is by the identification of the ribosomal subunit in the chloroplast(id) in RBS-15 and the RBS-11 strain system, and still this identification because itself, is not enough to instruct the correct translation at the initiator codon place initial.

The deletion mutantion of psbA 5 ' end non-translational region does not stop the combination of the trans-acting translation factor of nuclear coding

[0207] nuclear encoded protein mixture is discerned psbA 5 ' end non-translational region specifically, and can greatly strengthen by stimulating translation initiation D1 proteic synthetic (Danon and Mayfield, as above, 1991; Yohn etc., as above, 1998a; Yohn etc., as above, 1998b).For whether the sudden change of determining psbA 5 ' end non-translational region can influence the ability of this complex body in conjunction with mRNA, use external gel retardation assay and measure RNA bonded avidity in each mutant.Carry out the bonded gel retardation assay of psbA specific complex and psbA 5 ' UTR.The RNA segment of radiolabeled 5 ' end corresponding to wild-type psbA is transcribed out external, reacts with albumen with the heparin-agarose purifying then together.The RNA/ protein-interacting causes RNA to block on non-sex change PAGE glue.The cold competition RNA of 250 times of amounts of exceeding also is added in the same reaction.Be used to compete albumen composition and combining corresponding to the excessive unlabelled RNA of the psbA 5 ' UTR that comes from each mutant corresponding to the labeled rna of wild-type psbA5 ' UTR.5 ' the end non-translational region of the psbA of each mutant is all discerned by albumen composition under conditions in vitro, has only the RNA of RBS-11 not have can perfect competition to fall combining of wild type rna and albumen composition.This result shows that the most translation in these mutant is lost and is not to be caused by the forfeitures of particular combination sequence at these translation activators.

[0208] the transcribing with translating mechanism of procaryotic chloroplast(id) of originating from endosymbiosis is similar to bacterium usually.The chloroplast(id) promotor contains some elements that are similar to bacterium, and the plastid promotor can drive transcribing in intestinal bacteria.The rrna of chloroplast(id) significantly with bacterium in rrna kinship is arranged, and the ribosome-RNA(rRNA) of chloroplast(id) and ribosomal protein show with bacterium in the conservative property (Harris etc., as above, 1994) of counterpart height.Chloroplast(id) mRNA is also the same with procaryotic mRNA, and end does not have cap structure, generally also few polyadenylation, and can contain polycistronic message.Translating equipment in chloroplast(id) keeps in its some prokaryotic organism features, the accumulation in elapsed time, and many modulating properties have begun restricted and nucleus.The similar procaryotic component of chloroplast(id) is how to integrate with being derived from nuclear trans-acting regulatory factor, still knows little so far.

[0209] owing to the prokaryotic organism characteristic of chloroplast(id) translating equipment, Shine-Delgamo (SD) interacts and is construed in early days is the possible regulator of chloroplast(id) translation.Yet, in most of the cases can certified SD sequence all be positioned at relative upstream from start codon position too far away, down to the RBS element of not thinking to guard.In conjunction with Study on Variation, wherein the sudden change of the bacteroid conservative SD sequence of class does not cause the forfeiture of translating, and the importance that SD interacts in the chloroplast(id) translation has been disengaged (Fargo etc., 1998; Koo and Spremulli, 1994; Rochaix, 1996; Sakamoto etc., 1994).

[0210] interact generally for the influence of chloroplast(id) translation in order to study SD, particularly for the influence of the translation of psbA mRNA, anti-SD sequence is suddenlyd change among the chloroplast(id) 16S rRNA, thereby destroys the possible base pairing of SD sequence with supposition.The such rrna of result still keeps the ability of synthesizing soluble chloroplast protein, and this shows that these 16S sudden changes generally do not suppress ribosomal activity or function.Yet, the overwhelming majority, but be not that the synthetic sudden change owing to anti-SD zone among the 16S rRNA of the relevant chloroplast protein of whole film has been subjected to seriously influencing.These results have shown the importance of anti-SD zone in the chloroplast(id) translation, and show that this element may be a component of translational control in the plastid.

[0211] for this studies the SD interaction on one side from mRNA, a series of sudden changes are directed in the possible SD sequence that is positioned at 27 nucleotide positions of upstream from start codon among the psbA mRNA, the research work in early stage hints important element (Bruick and Mayfield in processing that this possible SD sequence is psbA mRNA and the translation process, as above, 1999; Mayfield etc., as above, 1994).As disclosed herein, the sudden change of the SD element of psbA has destroyed the ribosomal associating of mRNA/, and broken translation and the proteic accumulation of D1 that has encircled psbA.The comprehensive 16S mutation analysis and the toe marking are analyzed, and these results prove that the translation that Shine-Delgarno interacts for psbA mRNA needs, and also are like this for other a series of chloroplast(id) mRNA.

[0212] consider among the psbA mRNA uncommon interval between the SD element and initiator codon, position effect is studied for the influence of SD function in the chloroplast(id).The location of the SD element of a series of psbA of making more near with bacterium in the deletion mutantion of conservative position of components cause the translation of D1 in chloroplast(id) to reduce accordingly, but but can promote the translation of these transcriptons in bacterium.This result shows that chloroplast(id) adopts different mechanism to determine initiator codon with bacterium after SD interacts.These results have also proved among the psbA mRNA SD element not in the conservative position of procaryotic SD element, and can be interpreted as among what other plastids mRNA in the not influence of translation for them of the disappearance of the possible SD element of the conservative position of bacterium.

[0213] because information stability, rrna associates and translation usually closely links together, and has caused the difficulty of primary effect of the sudden change of 5 ' the end non-translational region of identifying mRNA like this.Shown that 5 ' of psbD holds class RBS element (the AUGAG sequence: PRB2) can influence the proteic synthetic (Nickelsen etc. of D2 in the chloroplast(id) as an information stability element that is positioned at about 30 nucleotide positions of upstream from start codon in the non-translational region, Plant Cell 11:957-970,1999).Based on the SD relative positions with PRB2 element and psbA of losing of psbD translation in the 16S mutant, the PRB2 element is likely the SD element of psbD mRNA.Lack the reduction of psbD mRNA stability in the mutant of this element; the same with those observed various sudden changes that influence psbA SD; reflect that probably rrna can not associate; and this association originally can protect mRNA exempt to be degraded (Wagner etc., J.Bacteriol.176:1683-1688.1994).

[0214] contrast of membranin and soluble proteins translation shows that it may be a component that makes a difference of translational control in the chloroplast(id) that SD interacts in the 16S mutant.The membranin synthetic be studies show that translation skill and the wild-type of at least two kinds of embrane-associated proteins in the 16S mutant is suitable.The translation difference of membranin shows that chloroplast(id) mRNA may adopt slightly different sequence as the SD element in two kinds of 16S mutant, and has two types rrna in the hint chloroplast(id) and exist.

[0215] location of RBS element shows and exists a kind of new early stage initiation complex that impels to move to the mechanism of initiator codon from RBS in the chloroplast(id) among the psbA mRNA.Secondary structure can shorten the distance between the localized RBS element of atypia in some former nuclear information.Yet the RBS element of psbA and the Nucleotide between the initiator codon can be changed significantly but the translation of psbA can not lost, and predict that this zone is not have influence to structure comparatively speaking.Observed a kind of scanning (scanning) mechanism also is considered to be applicable to chloroplast(id) mRNA in Eukaryotic translation initiation process, but needs the energy derive of ATP as helicase activity, and this feature also is not described in the chloroplast(id) translation.In addition, chloroplast(id) mRNA may utilize protein factor to move the initiator codon place at RBS place bonded 30S subunit to act on it.The specific proteins factor of 5 ' the end non-translational region of a kind of psbA of being incorporated into mRNA with known and the interactional eukaryotic protein of translation initiation factor have homology (Yohn etc., as above, 1998a).Like this translation initiation complex may be taken to correct initiator codon place with the similar albumen of eukaryotic protein, bring into play its function like this with regard to can be used as chloroplast(id) translational control son.Interval extra between RBS element and the initiator codon can provide the position of laying for these protein factors, can not stop the external combination of these factors just as most mutant of research here.Similarly remote SD sequence is also identified at 5 ' the end non-translational region of the psbA of higher plant, and this shows that such SD element is the feature of plant chloroplast mRNA.

Embodiment 3

Expressing antibodies in chloroplast(id)

What [0216] present embodiment had proved the coding single-chain antibody passes through the expression of the codon optimized polynucleotide of chloroplast(id) in chloroplast(id), and this single-chain antibody is assembled into dimer.

[0217] will encode can be specifically in conjunction with the single-chain antibody (HSV8 of hsv (HSV) 1 type and 2 types; SEQ ID NO:16) polynucleotide (SEQ ID NO:15) is transformed into (referring to embodiment 1) in the Chlamydomonas reinhardtii chloroplast(id) by pExGFP plant chloroplast carrier, and different is to use the polynucleotide (SEQ ID NO:15) of coding HSV8 to replace the GFP encoding sequence.The solubility total protein sample that comes from two transformants (10.6 and 11.3) is collected under the situation of reductive agent dithiothreitol dithio (DTT) having and do not have, on 10% SDS-PAGE, adopt the Laemmli Laemmli buffer system Laemmli to separate then, transfer to again that (Cohen etc., 1998) are used for western blot analysis on the nitrocellulose filter.It is peptide-labeled that described HSV8 antibody comprises a FLAG who effectively connects, and can adopt the peptide-labeled antibody of anti-FLAG (M2 monoclonal antibody; Sigma) and the coupled antibody (Sigma) of anti-mouse alkaline phosphatase detect.

[0218] the HSV8 single-chain antibody of expressing in described two different transformants is moved to the position (approximately 65kDa) at the molecular weight place of expectation.Significantly, separating the HSV8 antibody that obtains under the condition that does not have DTT to exist is to move as a dimer.These results prove that albumen composition can assemble such as the antibody dimer in plant chloroplast.Equally, the synthetic polynucleotide (SEQ ID NO:42) of the strand Fv segment of the anti-HSV antibody of the coding of chloroplast(id) codon preference (SEQ ID NO:43) is fabricated out, and express in Chlamydomonas reinhardtii, the result obtains the anti-HSV Fv of functional strand antibody.

[0219] when combinatorial antibody library had solved the problem of utilizing a large amount of immunity spectrums, effectively producing these compound molecules still was a problem.Here, the effective expression of unique big strand (lsc) antibody is unicellular organism, and green alga is realized in the chloroplast(id) of Chlamydomonas reinhardtii.By synthetic lsc gene, and drive the expression of mosaic gene, thereby realize high-caliber protein accumulation by utilizing one in two cover Chlamydomonas reinhardtii chloroplast(id) promotors and 5 ' end and 3 ' the end RNA element to overlap with chloroplast(id) codon preference.The lsc antibody of the glycoprotein D of this Direct Recognition hsv is produced by the form of algae with solubility, and is assembled into more high-grade complex body under the condition in vivo.Except the mediation of the formation by disulfide linkage dimerization, described antibody without any can detected posttranslational modification.Further, these results prove that the particular growth control that the accumulation of described antibody can be used to cultivate described algae regulate and control, but also can 5 ' of antibody gene expression is held and 3 ' end member spare is regulated and control by selecting to be used to drive.These results prove the practicality of algae as a kind of platform of express recombinant protein, and have described a kind of novel single-chain antibody that whole heavy chain protein comprises the Fc structural domain that contains.

[0220] had a large amount of heterologous protein expression systems to can be used for producing recombinant protein at present, and each system all provide different advantages (1) on the expense of the convenience of protein output and operation and operation.Monoclonal antibody (mAbs) is mainly produced by cultivate the transgene mammal cell in fermentation equipment.Because high fund input and the inborn complicacy of mammalian expression system, the throughput of monoclonal antibody will sink into the condition that supply falls short of demand (2) at the five-year.

[0221] has defective owing to cultivate the manufacture order clonal antibody, just need set up the high production monoclonal antibody method of other input-output ratio to adapt to the proteic developing steps of present treatment by mammalian cell.Though yeast and bacterial expression system on nutrient media components more economically, several shortcomings are arranged, comprise producing correct folding functional molecular effectively, and complicated more proteinic low yield.Except traditional fermentation, there have been several study group to begin to expand terrestrial plant and come production monoclonal antibody (3,4,5).In such system, itself has become bio-reactor plant, and antibody then is deposited in leaf or the seed tissue.Though plant can provide unprecedented economic scale in the biotechnology industry (such as, people can plant several thousand acres corn), this method also has several geneogenous shortcomings.At first, the needed time span of recombinant protein of (milligram is to restraining) may reach 3 years to obtaining that consumption is arranged from initial transformation event, such as for the such species of corn.Second consideration is round the expression of human treatment's medicine in alimentary crop, this probably since (passing through pollen) towards periphery the gene flow of crop (gene flow) (6) take place, as the situation (7) that between transgenic corns of expressing Bacillus thuringiensis insecticidal proteins and local landrace, takes place.These considerations make the open plantation of the transgenosis food plant (as corn, paddy rice and soybean) that controlling organization may forbidden list intelligent class medicine.

[0222] chloroplast(id) being carried out the engineered expression treatment that makes it still is fewer (8) with proteic trial, though the quite high-caliber expression of recombinant protein has realized (9-12) in this organoid in some instances.Be used for about generation express recombinant protein the transgenosis algae report just still less, although green alga is used to understand mechanism from light regulation and control and nutrition regulation genetic expression to the assembling of light compositing device and all respects of function (13) as a kind of model animals.Disclosed as embodiment 1, make the GFP reporter gene reflect that the codon of the codon preference of Chlamydomonas reinhardtii chloroplast gene group use to optimize about 80 times of the accumulation that has increased GFP, account for 0.5% (also can referring to 14) of soluble proteins.

[0223] as disclosed herein, human monoclonal antibody and segment thereof can be expressed in the chloroplast(id) of transgenosis algae.Big single-chain antibody gene has Chlamydomonas reinhardtii chloroplast(id) codon preference after being transformed, and utilizes the promotor of Chlamydomonas reinhardtii chloroplast(id) atpA or rbcL gene and 5 ' end non-translational region to drive expression.This antibody Direct Recognition herpes simplex virus glycoprotein D (15), and comprise variable region of light chain and the entire I gA heavy chain that merges by flexible peptide linker.This lsc antibody accumulates as soluble proteins in the transgenosis chloroplast(id), and determines that by elisa assay it can be in conjunction with protein herpesvirus.This big single-chain antibody can be assembled into more high-grade structure (dimer) in vivo, and does not contain tangible posttranslational modification, except the disulfide linkage relevant with dimerization.These results have proved the practicality of algae as complicated Recombinant Protein Expression platform.

Method

Chlamydomonas reinhardtii strain system transforms and growth conditions

[224] all conversion is to carry out on the 137c (mt+) according to (14) as previously mentioned in the Chlamydomonas reinhardtii strain all.The cultivation of Chlamydomonas reinhardtii transformant that is used for expressing HSV8-LSC is in TAP substratum (19), and culture temperature is 23 ℃, carries out under the certain condition of illumination and cell density.

Plasmid construction

[0225] all DNA and RNA operation all is basically according to document (20; 21; Also can be referring to, Mayfield etc., Proc.Natl.Acad.Sci., USA 100:438-442,2003, this piece document be incorporated herein for your guidance) describe and carry out.The coding region of HSV8-lsc gene (SEQ IDNO:47) is a method de novo synthesis of describing (14) according to the method for document (22) and front.According to the operational manual of manufacturers, with the PCR product cloning of the 1893bp that obtains in plasmid pCR2.1 TOPO (Invitrogen company).The promotor of atpA and rbcL and 5 ' end non-translational region, and 3 ' the end non-translational region of rbcL generates (14) by PCR.

Southern and northern hybridization analysis

[0226] Southern hybridization and as the DNA's of probe ³²The P mark is to carry out according to the method for document description (20).Be used in the p322 Bam HI/Pst I segment (surveying the 5 ' end of p322) that radiolabeled probe in the Southern hybridization comprises 2.2kb, the p322 BamHI/Xho I segment of 2.0kb (surveying the 3 ' end of p322) and come from the Nde I/Xba I segment of the 1926bp of HSV8-lsc.The radioactive probe that is used for the northern hybridization analysis in addition comprises psbAcDNA.Southern hybridization and northern hybridization all are to adopt the Packard Cyclone Storage Phosphor System that has been equipped with Optiquant software to come detected result.

Protein expression, western blotting and ELISA

[0227] in order to carry out western blot analysis, from Chlamydomonas reinhardtii, isolates albumen according to the method that document (14) is described.The Chlamydomonas reinhardtii HSV8-lsc of Flag protein affinity purification is containing adequate proteins enzyme inhibitors cocktail agent sheet (complete protease inhibitor cocktail tablets, Roche is the TRIS buffer salt solution (TBS of the phenylmethylsulfonyl fluoride (PMSF) of 1mM with final concentration Inc.); 25mM TRIS pH 7.4,150mM NaCl) separates in.According to the operational manual of manufacturers, extract is with anti-Flag M2 sepharose 4B (Sigma) purifying.Elisa assay carries out in 96 hole titer plate (Costar), and the volume of 100 microlitres is with the HSV albumen bag quilt of 100 microlitres.

[0228] sample that is used for ELISA seals damping fluid by phosphate buffered saline buffer (PBS with the dilution of sealing damping fluid; 137mM NaCl, 2.7mM KCl, 1.8mM K ₂PO ₄, 10mMNa ₂HPO ₄, pH 7.4) and 5% degreasing anhydrous milk form.Incubation is 8 hours under 4 ℃ and the condition of rocking.Use Tween 20 rinsings 96 orifice plates 3 times of PBS and 0.5% then, and then at 4 ℃ with anti-Flag antibody incubation 8 hours.Rinsing 96 orifice plates use for three times the coupled sheep anti-mouse antibody of alkaline phosphatase (Santa Cruz biotech company) 4 ℃ of incubations 8 hours then once more.Use Tween 20 rinsings 96 orifice plates three times of PBS and 0.5% once more, use 100 microlitre p-nitrophenyl phosphoric acid ester (pNPP, Sigma) colour developing then.Reaction stops by the NaOH that adds 50 microlitre 3N.

[0229] protein concentration utilizes BioRad analysis of protein reagent to measure.Western blotting is the method for describing according to document (23), utilizes the coupled sheep anti mouse of mouse-anti Flag one anti-(Sigma) and alkaline phosphatase two anti-(Santa Cruz biotech companies) to carry out.

The result

Utilization has polynucleotide big single-chain antibody gene of de novo synthesis in the Chlamydomonas reinhardtii chloroplast(id) of codon preference

[0230], utilizes to reflect that through optimization the codon of the preference of the Chlamydomonas reinhardtii chloroplast(id) mRNA that is translated in a large number comes the synthetic single-chain antibodies gene in order to realize recombinant antibodies at the strong expression in Chlamydomonas reinhardtii.The antibody of this through engineering approaches is to come from the human antibodies library that is illustrated on the phage, and by being identified out (15) with the effect of hsv protein.The antibody of this HSV8 of being called as was proved to be in the past can be in conjunction with viral surface antigen glycoprotein D (16), and the Fab of this antibody or IgG1 form in vitro and in vivo can be as effective neutralizing antibody (15,16).

[0231] since simple scfv antibody can in bacterium or Yeast system, synthesize, therefore attempt in chloroplast(id) synthetic complicated more, but still the antibody that can translate by single mRNA.Article one, single-chain antibody contains whole heavy chain and the variable region of light chain that merges by flexible peptide linker through design.The proteic one-level aminoacid sequence of big strand (lsc) of this uniqueness is shown in SEQ IDNO:48, and this section sequence is encoded by sequence SEQ ID NO:47.

Make up the chimeric big single-chain antibody gene of Chlamydomonas reinhardtii chloroplast(id)

[0232] in order to obtain the transgenosis chloroplast(id) of expressing recombinant antibody, synthetic one section mosaic gene, this fragment gene contains atpA or rbcL promotor and 5 ' end non-translational region, and be fused on the codon optimized HSV8-lsc coding region, then also have 3 ' the end non-translational region (being respectively Fig. 5 A and 5B) of rbcL.Integrator gene is finished by homologous recombination in the chloroplast gene group, and this needs the sequence homology (17) between conversion carrier and the chloroplast gene group.The Chlamydomonas reinhardtii chloroplast(id) transforms carrier p322 (14) and is utilized.Showed that as Fig. 5 B described chimeric antibody gene is connected to the Bam HI site of p322, thereby produced plasmid p322/atpA-HSV8 and p322/rbcL-HSV8.These p322/HSV8 constructions and plasmid p228 by partickle bombardment (17) by cotransformation in the chloroplast(id) of Chlamydomonas reinhardtii, p228 contains the one section 16S ribosomal gene that can give the spectinomycin resistance.

The Southern hybridization analysis of HSV8-lsc transgenosis chloroplast(id)

[0233] initial conversion (primary transformants) is containing selected come out on the substratum of spectinomycin, and filters out the transformant that is integrated with the HSV8 gene by the Southern hybridization analysis.The HSV8 positive transformant is selected to isolate homogenous cell system through extra number wheel, and wherein the copy of all chloroplast gene groups all contains the HSV8-lsc gene of importing.Two homogeneity transformant are selected comes out, and one is 10-6-3, and it contains the atpA promotor of controlling HSV8-lsc, and another one is 20-4-4, and it contains the rbcL promotor of controlling HSV8-lsc.Cut with Eco RI and Xho I enzyme from the genomic dna that wild-type and these two HSV8-lsc transformants obtain, on agar gel, separate then, carry out the Southern hybridization analysis again.The preparation method of Chlamydomonas reinhardtii DNA carries out according to the method for describing among the embodiment 3, cuts with Eco RI and Xho I enzyme, and filter membrane is hybridized with radioactive probe more then, and the position of probe indicates with double-headed arrow in Fig. 5 C.

[0234] uses ³²The hybridization of the Nde I/Xba I segment of the HSV8 coding region of P mark all identifies the band of 6.0kb in atpA-HSV8 and rbcL-HSV8 transgenic line, but do not identify in the swimming lane at wild-type place as expecting any can detected band.When same Hybond membrane with ³²When the segment of Eco RI between PstI of the 1.5kb of the 5 ' end of the p322 of P mark hybridized, in the wild-type sample, there is the segment of a 5.7kb to be displayed, in two transgenic lines, then identified out greater than the segment of 6.0kb slightly.Hold with 3 ' of p322 ³²The hybridization of the Bam HI/Xho I segment of P mark then identifies the segment of 2.5kb and 2.0kb respectively at 10-6-3 and 20-4-4, what then manifest once more in wild-type strain system is the band of 5.7kb.These results prove that the HSV8 gene correctly has been incorporated into the p322 silent site of chloroplast gene group, and the copy of all chloroplast gene groups all contains the HSV8 gene.The accumulation of HSV8-lsc mRNA in transgenic line

[0235] in transgene Chlamydomonas reinhardtii strain system, detected the HSV8-lscmRNA of chloroplast expression.The strain system (wt) that never transforms, isolating total RNA after separating on the sex change sepharose is transferred on the nylon membrane in the strain system (20-4-4) that strain system (10-6-3) that atpA HSV8-lsc transforms and rbcL transform.These films with Methylene blue dyeing or with psbA cDNA probe, perhaps with the specific probe hybridization of hsv8.Whether the northern hybridization analysis of total RNA is used for detecting the HSV8 gene and transcribes in the transgene Chlamydomonas reinhardtii chloroplast(id).The total RNA of 10 micrograms after separating on the sex change agarose gel from wild-type and two transgenic cell lines is transferred on the nylon membrane.The same filter membrane of double dyes with Methylene blue and uses ³²The psbA cDNA probe of P mark or the specific probe hybridization of HSV8.Ribosome-RNA(rRNA) is similar with the accumulating level in the various transgenic lines in wild-type with psbA mRNA, and the amount that this proof goes up the RNA of sample is the same, and genetically modified importing does not change the accumulation of endogenous mRNA.Hybridize with the specific probe of HSV8, show in 10-6-3 and the 20-4-4 strain system to have accumulated big or small correct HSV8-lsc mRNA, and as expectation, swimming lane does not detect the HSV8 signal at the wild-type place.

Analyze the accumulation of HSV8-lsc albumen in the transgene Chlamydomonas reinhardtii chloroplast(id)

[0236] utilize anti-flag antibody to measure the level of HSV8-lsc antibody, to determine whether HSV8-lsc albumen accumulates in transgenic cell line by western blot analysis.20 micrograms come from the total protein of the e. coli strains system of expressing HSV8-lsc from the pET carrier, 20 micrograms come from the total protein of wild-type and two transgenosis Lay mattress strain systems, separate by SDS-PAGE, carry out western blot analysis with Coomassie brilliant blue dyeing or with anti-Flag antiserum(antisera) then.In order to express in bacterium, the Nde I/Bam HI segment of codon optimized HSV8-lsc gene is connected in the pET carrier, and by adding the IPTG abduction delivering.The proteic amount of sample all was the same on the coomassie stained gel showed in each swimming lane, but also showed that the accumulation of total protein in the transgenic line also is normal.Utilize anti-Flag antibody that same sample is carried out western blot analysis, show the strong signal that correct molecular weight is arranged in HSV8-lsc transgenic line and intestinal bacteria, but as expectation, do not have signal at Chlamydomonas reinhardtii wild-type place swimming lane.

The sign of the HSV8-lsc antibody of in intestinal bacteria and chloroplast(id), expressing

[0237] for whether the HSV8-lsc that determines to accumulate in the Chlamydomonas reinhardtii chloroplast(id) has function, the HSV8-lsc albumen of the albumen of chloroplast expression and bacterial expression is characterized together.Genetically modified bacterium of HSV8-lsc and algae are resuspended in TBS solution, use the ultrasonication cell then.Soluble proteins and insolubility albumen are separated by centrifugal.The albumen that takes out same amount from soluble component and insolubility component separates with SDS-PAGE, and HSV8-lsc albumen manifests by western blot analysis then.The HSV8-lsc that produces in the bacterium proteic about 60% is distributed in the insoluble component, and the albumen that produces in the chloroplast(id) almost all is found in soluble component.

[0238] in order to determine whether the antibody of expressing in the chloroplast(id) contains posttranslational modification, and these antibody detect with SDS-PAGE and western blot analysis on reductibility and irreducibility gel.The soluble proteins that comes from Chlamydomonas reinhardtii transgenic line 10.6.3 before separating with SDS-PAGE with the (+Bme) processing or handle of B mercaptoethanol without reductibility reagent (not having Bme).Albumen is transferred on the nitrocellulose filter, and then with anti-flag antibody test.Under non-reduced condition, any disulfide linkage that forms between two heavy chains of described antibody all remains unchanged, and migrates and make antibody can be used as bigger molecule.The HSV8-lsc of chloroplast expression moves with a bigger albumen under non-reduced condition, and molecular weight approximately is 140kDa, and this size is that a dimer has.After the Bme processing, disulfide linkage is reduced, and the result makes HSV8-lsc albumen monomer molecule amount---68kDa---with prediction in electrophoresis of chloroplast(id) move.

[0239] in order to determine whether that other posttranslational modifications are present in the albumen of chloroplast expression, the albumen of bacterium and chloroplast expression characterizes by mass spectrum.The mass spectrum that comes from the peptide section of expressed proteins in intestinal bacteria and the chloroplast(id) is a spectral pattern much at one, and this explanation is without any extra modification for chloroplast protein.

[0240] HSV8-lsc of chloroplast expression is verified to confirm that the HSV8-lsc that accumulates has function in the transgenosis chloroplast(id) in conjunction with the proteic ability of HSV8.HSV8-lsc obtains by anti-flag affinity resin purifying from the transgenosis chloroplast(id).As shown in Figure 6, in elisa assay, the antibody that chloroplast(id) produces is discerned HSV8 albumen in powerful mode.

The adjusting of the accumulation of HSV8-lsc in transgenic alga

[0241] different growth conditionss are detected to determine whether the expression of HSV8-lsc in the Chlamydomonas reinhardtii chloroplast(id) can be modulated for antibody effect of accumulation in two transgenic line 10-6-3 and 20-4-4.The cultivation of each strain system maintains 10 ⁶Perhaps 10 ⁷Every milliliter in individual cell is grown under circulation (5000 lux) condition of 12/12 illumination-dark, perhaps growth under continuous illumination (5000 lux) illumination condition.Cell is by centrifugal collection, and 20 microgram soluble proteinss separate in SDS-PAGE then, and HSV8-lsc manifests with the western blotting that anti-Flag antibody carries out then.

[0242] accumulation of HSV8-lsc depends on growth conditions significantly and changes.In strain is among the 20-4-4, and the protein expression under rbcL promotor/5 ' UTR control shows tangible antibody accumulation and increases in the latter stage of dark period or after just entering photoperiod, and irrelevant with cell density.Comparatively speaking, be among the 10-6-3 in strain, atpA promotor/5 ' UTR is 10 ⁶At the flat substantially invariable HSV8-lsc protein expression of illumination-dark cycle middle finger water guide, be 10 under the cell density that individual cell is every milliliter yet work as cell culture density ⁷In the time of every milliliter in individual cell, after entering photoperiod, can show the obvious increase of lsc accumulation.When being grown in continuous illumination condition following time, it is 10 that two strains tie up to cell concn ⁶It is 10 that protein accumulation during every milliliter in individual cell will be higher than cell concn ⁷Protein accumulation during every milliliter in individual cell.These results prove that the accumulation of HSV8-lsc in the Chlamydomonas reinhardtii chloroplast(id) can be optimised, and this depends on the illumination condition that is used for culturing cell, cell residing cyclostage during cell harvesting, and be used to drive expression promoter/UTR.

[0243] a kind of human monoclonal antibody is expressed in chlorophta chlorophyll body.The expression of high levels of recombinant proteins is the codon use that makes it to reflect most of chloroplast proteins by the codon of optimizing antibody coding sequence, and realize with the expression that promotor and the 5 ' UTR of chloroplast(id) atpA or rbcL drives mosaic gene.This big strand (lsc) antibody contains whole heavy chain of the IgA that merges by one section flexible peptide linker and variable region of light chain, and accumulates with completely soluble albumen form in chloroplast(id).This antibody is used for discerning the glycoprotein D of hsv, and this antibody of being expressed by algae and the combination between the bleb albumen are measured by ELISA.This lsc antibody contains the Fc part of heavy chain, relates to the formation of intermolecular disulfide bond under this part normal circumstances, thereby causes the dimerization of antibody.The antibody of described chloroplast expression is assembled into more high-grade mixture, and this mixture is responsive for the reduction by the Bme mediation, and this antibody that shows chloroplast expression forms dimer in vivo.The formation of disulfide linkage was described (8) in the recombinant protein of expressing in chloroplast(id) in the human growth hormone that tobacco chloroplast is expressed, owing to have protein disulfide bond isomerase in the algae chloroplast(id), so the formation disulfide linkage also is among expecting somewhat (18) in the recombinant protein of expressing in the chloroplast(id).The N that this lsc antibody also contains supposition connects glycosylation site.The chloroplast(id) encoded protein has not been considered to glycosylated generation, and in fact based on mass spectroscopy, there is no evidence that the antibody of chloroplast expression has glycosylation yet.

[0244] transgenic line of Chan Shenging shows the antibody accumulation of different levels, and this depends on and is used for the kinesin expression promoter, and the illumination condition of cell density and these strain systems of cultivation.The reason of these wide fluctuations of antibody accumulation may be caused by various factors, comprises stability and the translation ability of chimeric mRNA, the renewal of antibody protein (turnover).These results prove that the accumulation of antibody can be subjected to the favourable influence of growth conditions, but also show that high-level antibody accumulation (surpass soluble proteins 1%) can realize in algae, as long as the growth conditions that is suitable for specific promotor and UTR combination by optimization just can be realized simply.

[0245] recombinant protein can be produced in various protein expression systems.Complicated treatment albumen such as monoclonal antibody (mAbs), mainly is to produce in the transgene mammal cell of cultivating.The cost of manufacture order clonal antibody on average approximately is 150 dollars of every gram starting material in the mammalian cell of cultivating, and the monoclonal antibody production cost estimates it is 0.05 dollar of every gram starting material (1) in botanical system.The cost of manufacture order clonal antibody is considered to have more competitive edge than production cost in terrestrial plant in algae systems, and the price of the substratum of algae is quite rationally (0.002 dollar every liter) just.In addition, algae can cultured continuously and their growth medium can circulate.

[0246] the manufacture order clonal antibody also has a series of special benefits to make algae become recombinant protein and produces the ideal candidate except impayable cost advantage in algae.At first, the transgenosis algae can obtain soon, only needs several weeks from producing initial conversion to scaling up to this process of production volume.The second, algae chloroplast gene group and nuclear gene group can be by genetic transformations, and this just provides possibility for produce a series of different transgene proteins in species, and this needs in the time will producing the polyprotein mixture such as secretory antibody for this.In addition, algae has the ability of cultivating in very economical mode on various scales, from several milliliters to 500000 liters.These advantages and green alga are included into the fact of GRAS catalogue (being regarded as security catalog usually), make Chlamydomonas reinhardtii become an attractive especially selection that substitutes other system expression recombinant protein.At last, though the production of this example lay special stress on antibody in algae, this system can be competent at the production of any recombinant protein in fact.

Citing document

Below every piece of article all be hereby incorporated by.

1.Dove，(2002)Nature?Biotechnol.20，777-779

2.Motmans and Bouche, Antibodies:The Next Generation (2000) Reportto Auerbach Grayson ﹠amp; Company, Inc.

3.Hiatt etc., (1989) Nature 342,76-78

4.Ma etc., (1994) Eur.J.Immunol.24,131-138

5.Ma etc., (1995) Science 268,716-719

6.Ellstrand，(2001)Plant?Physiol.125，1543-1545.

7.Quist and Chapela, (2001) Nature 414,541-543.

8.Staub etc., (2000) Nature Biotechnol.18,333-338.

9.Kota etc., (1999) Proc.Natl.Acad.Sci.USA 96,1840-1845.

10.Sidrov etc., (1999) Plant J.19,209-216.

11.Ruf etc., (2001) Nature Biotechnol.19,870-875.

12.Heifetz，(2000)Biochemie?82，655-666

13.Harris，(1989)The?Chlamudomonas?Sourcebook?Academic?Press，Inc.

14.Franklin etc., (2002) Plant J.30,733-734.

15.Burioni etc., (1994) Proc.Natl.Acad.Sci.USA.91,355-359.

16.De Logu etc., (1998) J.Clin.Microbiol.36,3198-3204.

17.Boynton etc., (1988) Science 240,1534-1538

18.Kim and Mayfield, (1997) Science 278,1954-1957

19.Gorman etc., (1965) Proc.Natl.Acad.Sci.USA 54,1665-1669.

20.Sambrook etc., (1989) Moecular Cloning.A Laboratory Manual ColdSpring Harbor Laboratory Press.

21.Cohen etc., (1998) .Meth.Enzymol.297,192-208.

22.Stemmer etc., (1995) Gene 164,49-53.

Embodiment 4

By bacterium luxAB genetic expression luciferase fusion rotein with chloroplast(id) codon preference

[0247] present embodiment has confirmed the strong expression of luciferase fusion rotein in chloroplast(id) by the synthetic polymerized nucleoside acid encoding with chloroplast(id) codon preference.

[0248] luciferase reporter gene successfully has been used for detecting the genetic expression in the viable cell in various biologies, but does not also have in chloroplast(id) by the successful Application mistake.Described in embodiment 1, green fluorescent protein (gfp) is expressed from the polynucleotide with chloroplast(id) codon preference, and can be as the reporter gene of genetic expression in the chloroplast(id).Because gfp can send high-intensity spontaneous fluorescence, and in chloroplast(id) high-caliber relatively expression and gfp protein accumulation for visual be essential, luciferase reporting albumen by polymerized nucleoside acid encoding with chloroplast(id) codon preference, by synthetic two bacteriofluorescein enzyme subunits, luxAB, become a fusion rotein, and be developed with Chlamydomonas reinhardtii chloroplast(id) codon preference.As disclosed herein, the chloroplast(id) luciferase gene, luxCt is expressing in the chloroplast(id) at Chlamydomonas reinhardtii under the control of the 3 ' UTR of atpA promotor and 5 ' UTR and rbcL.This luxCt is responsive report that chloroplast gene is expressed, and can allow to measure the active of luciferase or measure its activity with photometer under conditions in vitro under the condition in vivo with the CCD camera.Further, in the luxCt protein accumulation of measuring by western blot analysis and the body and luminous being directly proportional that records under the conditions in vitro.These results prove the practicality of luxCt gene as the general and sensitive reporter gene of chloroplast(id) genetic expression in the active somatic cell.

[0249] reporter gene has strengthened the ability of gene expression in large number of biological greatly.In the chloroplast(id) of higher plant, β-Pu Taotanggansuanmei (uidA, Staub and Maliga, 1993), neomycin phosphotransferase (nptII, Carrer etc., 1993), adenosine-3-VITAMIN B4 transferring enzyme (aadA, Svab and Maliga, 1993) with from green fluorescent protein (Sidorov etc., 1999 of jellyfish; Reed etc., 2001) all be used as reporter gene (Heifetz, 2000).Several reporter genes also the eucaryon green alga, are expressed in the chloroplast(id) of Chlamydomonas reinhardtii, and these genes comprise aadA (Goldschmidt-Clermont, 1991; Zerges and Rochaix, 1994), uidA (Sakamoto etc., 1993, Ishikura etc., 1999), aphA6 (Bateman and Purton, 2000) and renilla luciferase (Minko etc., 1999).Unfortunately, the protein accumulation level that these early stage reporter gene expression boxes are produced is very low, make they can not be well as report of quantitative analysis genetic expression.

[0250] as disclosed in embodiment 1, the expression of high-caliber reporter gene makes and is used for obtaining (also can referring to, Franklin etc., 2002) by the codon of optimizing the green fluorescent protein reporter gene.Relatively transformed the proteic accumulation of GFP in the strain system of cgfp gene of unoptimizable gfp gene and optimization, found that the proteic accumulation of GFP that is come by the cgfp gene of Chlamydomonas reinhardtii chloroplast(id) has 80 times increase.These results prove, it is that codon by the Chlamydomonas reinhardtii chloroplast gene uses preference to cause that the expression of former reporter gene in the Chlamydomonas reinhardtii chloroplast(id) can not reach high-caliber reason.

[0251] in order to expand the result who obtains with gfp, and in order to obtain report observed under a kind of condition in vivo, the bacterial luciferase gene with Chlamydomonas reinhardtii chloroplast(id) codon preference is synthesized out.The lux gene of this de novo synthesis is based on the bacterium luxAB gene synthetic (Baldwin etc., 1984, Johnson etc., 1986) of Vibrio harveyi (Vibrioharveyi).The encoding sequence of luciferase makes after being synthesized luciferase A and B subunit by flexible peptide linker (Kirchener etc. are expressed in the single encoded zone that A and B subunit link together as one, 1989, Olsson etc., 1989, Almashanu etc., 1990).This with regard to chloroplast(id) and optimised luciferase gene (luxCt) is placed in the expression cassette of 3 ' the end non-translational region that contains atpA promotor and 5 ' end non-translational region and rbcL.Containing in the transgenic cell line of described luxCt gene has luxCt mRNA and LUXCt protein accumulation, and this is (referring to the following content) judged by RNA hybridization and western blot analysis respectively.When cell was handled with the substrate capraldehyde of bacterial luciferase, the luminous of transgenic cell line of expressing luxCt was easy to observe by the CCD camera, then do not show any luminous in same test in the wild-type cell.The expression of the luxCt that measures by western blot analysis and be directly proportional by luminescence analysis of using the CCD camera and the expression of using the luxCt of external photometer assay determination.The activity of luciferase can be measured on several orders of magnitude in the transgenic line, and this proof luxCt has susceptibility and practicality as report of genetic expression in the active somatic cell chloroplast(id).

Method

Chlamydomonas reinhardtii strain system transforms and growth conditions

[0252] transforming is to be 137c (mt+) with the Chlamydomonas reinhardtii strain, and perhaps the strain of psbA defective type is that cc744 (REF) carries out.Cell in the TAP of the floxuridine that contains 40mM substratum (Gorman and Levine, 1965), at rotating speed be on the shaking table of 100rpm in 23 ℃, the constant light of 4000 luxs (Gao Guang) according in grow into back logarithmic phase (about 7 days).Through 4000 * g, collect 50 milliliters cell after centrifugal 5 minutes for 4 ℃.Supernatant is discarded, and cell is resuspended in 4 milliliters of TAP substratum, carries out ensuing chloroplast(id) by the partickle bombardment method of stating (Cohen etc., 1998) and transforms.All transform all and select to carry out down at spectinomycin (150 μ g/ml), and wherein the spectinomycin resistance is spectinomycin resistance ribosomal gene (Chlamydomonas Stock Center, the Duke University) realization by cotransformation plasmid p228.In order to express luxCt, the cultivation of Chlamydomonas reinhardtii transformant is according to carrying out in 23 ℃ in the middle constant light of TAP substratum (Gorman and Levine, 1965).

Plasmid construction

[0253] DNA and RNA operation mainly is to carry out according to the method for description such as Sambrook etc. (1989) and Cohen (1998).The coding region of luxCt gene is that the primer storehouse that the method according to (1995) such as Stemmer all is 40 Nucleotide by each length obtains by de novo synthesis.5 ' and 3 ' of employed primer end contains Nde I and XbaI site respectively in such assembling.According to the operational manual of manufacturers,, produce plasmid pluxCt with the PCR product cloning that contains the luxCt gene (Invitrogen company) in pCR2.1 TOPO plasmid of the 2094bp that obtains.3 ' the end non-translational region of the promotor of atpA and 5 ' end non-translational region and rbcL is the method generation according to document description (Mayfield etc., 2002).Chloroplast(id) transforms plasmid p322 and makes up according to document description (Franklin etc., 2002).

Southern hybridization and northern hybridization analysis

[0254] Southern hybridization and the DNA that uses as probe ³²The P mark is according to document (Sambrook etc., 1989; With Cohen etc., 1998) method of describing is carried out.The radioactive probe that is used for Southern hybridization comprises the Bam HI/Xho I segment (surveying the 3 ' end of p322) of the 2.0kb of the Nde I/Xba I segment of the 2kb of luxCt coding region (surveying luxCt) and p322.0.9kb Eco RI/Xba I luxCt probe in the northern hybridization analysis, be used to detect luxCt mRNA.Other radioactive probe that uses in the northern hybridization analysis comprises rbcL cDNA.Southern hybridization and northern hybridization are to detect by the Packard Cyclone Storage Phosphor System that has been equipped with Optiquant software.

Protein expression, western blot analysis and luminescence analysis

[0255] plasmid pluxAB and pluxCt are transformed in the e. coli bl21 strain system, incubated overnight in the cell liquid medium within.In order to carry out western blot analysis, albumen extraction separation from intestinal bacteria or Chlamydomonas reinhardtii is come out, the damping fluid of employing contains 750mM TrisCl, pH 8.0,15% sucrose (wt/vol), 100mM Bme, 1mM PMSF.Sample is in 13000 * g, 4 ℃ centrifugal 30 minutes, the supernatant that obtains is used for western blot analysis.Used Chlamydomonas reinhardtii albumen is at 50mM Na in external luminescence analysis ₂HPO ₄, pH 7.0,50mM Bme prepares in the damping fluid of 400mM sucrose, thick then lysate is in 13000 * g, 4 ℃ centrifugal 30 minutes, the supernatant that obtains is used for luminescence analysis.96 hole microtitration plate analysis are to revise from bacteriofluorescein enzyme method (Langridge and Szalay, 1994) to obtain.The Chlamydomonas reinhardtii soluble proteins extracts damping fluid with luciferase and is diluted to each sample of 100 microlitres, contains the 50mM Tris-Cl of 500 μ M NADH, the damping fluid of pH 8.0, and the 50mM Na that contains the 0.025U diaphorase to wherein adding 125 microlitres ₂HPO ₄, 50mM Bme, 1% bovine serum albumin damping fluid.Add 130 microlitre solution in the mixture that obtains toward above again, wherein contain 200mM three (methylol) methylglycine that 125 microlitres contain 100 μ M FMN, pH 7.0 solution and 5 microlitres contain the 50mM Na of 0.1% capraldehyde in ultrasonic 10 seconds ₂HPO ₄, pH 7.4 solution.(relative light units is at FMN for the photon survey of unit rlu) with relative light unit ^-/ capraldehyde adds and begins after 5 seconds, and measurement is to carry out with the LJL BiosystemsAnalyst AD photometer (fluorescence intensity meter) that has been equipped with Criterion Host software.Protein concentration is measured with BioRad analysis of protein reagent.

[0256] Western blot is the method for describing according to (1998) such as Cohen, adopts the goat-anti rabbit two of anti-luxAB one of rabbit anti-(REF) and alkali phosphatase enzyme mark to resist (Sigma) to carry out.Cloning luminous is to be the Berthold Night Owl CCD camera imaging of LB 981 by model, and camera has been equipped with the emission filter of 700nm to stop chloroplast(id) fluorescence (Chroma company).30 seconds to 5 minutes time shutter enough can manifest the luminous of luciferase in most cases.Image adopts WinLight software to generate.

The result

De novo synthesis has the luxAB gene of Chlamydomonas reinhardtii chloroplast(id) codon preference

[0257] report son in order to develop a kind of sensitive of monitoring genetic expression in the chloroplast(id), (embodiment 1 to utilize the codon that reflects the codon use of the gene of great expression in the Chlamydomonas reinhardtii chloroplast(id) through optimization to synthesize luciferase gene; Franklin etc., 2002).This luciferase gene, luxCt (Fig. 7) is based on (luxAB of the bacterial luciferase AB gene design of Vibrio harveyi; Baldwin etc., 1984).For the expression in chloroplast(id), two subunits of luxAB are connected to one section single encoded sequence, wherein by the terminator codon of removing on the A subunit it is linked to each other in correct reading frame with the B subunit, A subunit and B subunit generate single fusion rotein (Fig. 7) by one section flexible peptide sequence.Vibrio harveyi luxAB sequence obtains from the GenBank database, design a series of oligonucleotides based on this aminoacid sequence, use to reflect that those codons at the gene of Chlamydomonas reinhardtii chloroplast(id) camber expression use but changed codon.Described gene is according to the method assembling of (1995) such as Stemmer.The PCR product is cloned in the escherichia coli plasmid, and the synthetic gene is checked order, and some sequence errors are proofreaied and correct by point mutation.A Nde I site is designed in the place of initiator codon, and Xba I site then is placed in the place followed by the terminator codon downstream, and this prepares for ensuing clone.The gene that obtains, luxCt is cloned in the escherichia coli expression box, and the expression of luciferase is analyzed by the luminescence imaging that utilizes the CCD camera.Surprisingly, in the bacterium that contains the luxCt gene, be detected, though in the bacterium that has transformed bacterium luxAB gene, can detect high-intensity luminous (Kondo etc., 1993) without any luminous.

Whether [0258] have sudden change by mistake to be imported in the luxCt gene when being cloned into escherichia coli vector in order to confirm, luxCt gene that contains in colibacillus expression plasmid and bacterium luxAB gene are all checked order.Compare in the luxCt gene, not detecting mistake with required sequence, but from the sequence difference that is used in bacterium is expressed the luxAB sequence (Kondo etc., 1993) of plasmid of luxAB, identifying the luxAB sequence of reporting in a large amount of and the GenBank database (Acc.No.E12410).Coming from the luxAB albumen of several different bacterial species and the sequence alignment between the synthetic luxCt albumen and identify a large amount of differences on the aminoacid sequence, is on conserved amino acid but have only one in these differences.Therefore, the practical site specific mutant recovers the L-glutamic acid on 204 amino acid positions, and 205 adjacent locational leucines.There is not other aminoacid sequence to be changed, because none is conservative in these a series of luxAB albumen of being investigated in these other the amino acid.

The luxCt antigen-4 fusion protein gene produces functional luciferase in bacterium

[0259] in order to determine whether synthetic luxCt gene can produce functional luciferase, in having transformed the Bacillus coli cells that contains luxAB or luxCt expression of gene plasmid, measure luminous.The thick lysate that utilization comes from the Bacillus coli cells of expressing luxAB gene or luxCt gene carries out western blot analysis; 20 microlitres are used to SDS-PAGE, transfer on the nitrocellulose filter then.Resistive connection with anti-luxAB is incorporated on these films again, and then anti-rabbit two resistive connections of using alkaline phosphatase coupled close, and then albumen dyes by alkaline phosphatase activities and manifests.The alpha of luxAB (A) and beta (B) subunit are identified, and the single fusion rotein (FP) of luxCt is identified.In addition, being expressed in incubated overnight on the agarose substratum and the intestinal bacteria with the capraldehyde vapour cure of luciferase measured.Do not have Bacillus coli cells that transforms or the cell of expressing luxAB or luxCt gene to take pictures (taking pictures), perhaps observe (luminous) by the luminescence imaging that utilizes the CCD camera with reflected light.When Bacillus coli cells is handled with capraldehyde and with the CCD camera imaging time, two strains systems of containing luciferase are all luminous, not have the intestinal bacteria of conversion then not have optical signal as expecting.Western blot analysis carries out with the proteic polyclonal antibody of the original luxAB of identification, the result shows the signal of proteic A of bacterial luciferase and B subunit in luxAB strain system, and in luxCt strain system, what identify is single band corresponding to described fusion rotein.The level of the A albumen of luxAB and B protein accumulation will be higher than the single fusion rotein of luxCt in bacterium, and the accumulation of these proteic luminous signals and luciferase protein is proportional relation roughly, and 2 parts of luxAB1 signals are corresponding to 1 part of luxCt signal.

The Southern hybridization analysis of the structure of luxCt expression cassette and luxCt transformant

[0260] proved that the luxCt encoding sequence produces after functional luciferase, transformed the chloroplast(id) of Chlamydomonas reinhardtii with the luxCt expression cassette.For luciferase is expressed in chloroplast(id), be fabricated out according to expression cassette shown in Figure 8.The luxCt encoding sequence is connected to the downstream of atpA promotor and 5 ' end non-translational region, the upstream (Fig. 8 A) of 3 ' the end non-translational region of rbcL.This chimeric atpA/luxCt gene and then be connected to chloroplast(id) and transform carrier p322 and go up unique Bam HI site forms plasmid p322-atpA/luxCt (Fig. 8 B).

[0261] transform wild-type Chlamydomonas reinhardtii cell with p322-atpA/luxCt plasmid and selected marker plasmid p228, wherein plasmid p228 can make cell have the spectinomycin resistance.Initial conversion is carried out luminescence analysis with the CCD camera, and to filter out existing of luxCt gene, the male transformant is further verified by the Southern hybridization analysis.Transformant through extra number wheel screening, is therefrom isolated homogenous cell system again, and the chloroplast gene group of all copies all contains the luxCt gene of importing in such clone.

[0262] two homogeneity luxCt transformant, 10.6 and 11.5, selectedly come out further to analyze.What Fig. 8 showed is the luxCt construction, and relevant restriction site is labeled out.It is the Nde I-Xba I segment of utilizing luxCt that the 8.7kb Eco/Xho zone of plasmid p322-atpA/luxCt correctly is incorporated in the chloroplast gene group, perhaps the Bam HI-Xho I segment of plasmid p322 is confirmed, these segments all are labeled out in Fig. 8.Chlamydomonas reinhardtii chloroplast transgenic beggar to luxCt has carried out the Southern hybridization analysis.The DNA preparation of Chlamydomonas reinhardtii is cut with Eco RI and Xho I enzyme as described in Example 4 then simultaneously, carries out the Southern hybridization analysis again.Filter membrane and the radioactive probe hybridization that is indicated as Fig. 8 B.Described two transformants all contain luxCt hybridization band, and wild-type strain system does not have signal with luxCt coding region probe hybridization the time.Identify two bands in transgenic cell line, this is because the luxCt gene contains an Eco RI site in the position therebetween.Hybridize with the Bam HI-Xho I segment that comes from the p322 plasmid, identify single band in wild-type, then identify the band that varies in size with it in two transformants, these are all identical with predicting the outcome before.The molecular weight size of each band of wild-type and transformant clone all is the correct size of being predicted.These results prove that described two transgenic lines are homogeneities.

The accumulation of the mRNA of luxCt in transgenic line

[0263] the northern hybridization analysis of using total RNA is confirmed luxCt gene transcribing in the transgene Chlamydomonas reinhardtii chloroplast(id).The total RNA of 10 micrograms that extracts from wild-type and described two transgenic lines separates at the sex change sepharose respectively, transfers on the nylon membrane then.Two parts of same filter membranes dye or usefulness with Methylene blue ³²The luxCt probe of P mark or rbcLcDNA probe hybridization.The level of the rRNA (painted band) of accumulation and rbcLmRNA is all similar in each strain system, and this illustrates that the RNA of sample on each swimming lane is an equivalent, and chloroplast(id) transcribe with being accumulated in the transgenic cell line of mRNA be normal.The hybridization of filter membrane and luxCt specific probe shows the luxCtmRNA that has all accumulated the prediction size in two transgenic cell lines, does not then equally observe the luxCt signal in wild-type cell as expected.

The analysis that luxCt albumen accumulates in the transgene Chlamydomonas reinhardtii chloroplast(id)

[0264] uses western blot analysis and confirm whether luxCt albumen accumulates in transgenic cell line.The 20 microgram solubility total proteins (tsp) that come from wild-type and described two transgenic cell lines separate by SDS-PAGE, dye then or with Coomassie brilliant blue, perhaps carry out western blot analysis.Show that through the coomassie stained gel protein content of going up sample in each swimming lane is the same, and accumulated a series of in the transgenic cell line and the similar albumen of wild-type.The western blot analysis of same sample identifies the single band corresponding to described fusion rotein in described two luxCt transgenosis swimming lanes.At wild-type Chlamydomonas reinhardtii swimming lane, as expectation, do not observe signal.

With report of luxCt as chloroplast(id) genetic expression in the viable cell

[0265] in order to determine the practicality of luxCt gene, the wild-type and the transgenic cell of growing carried out fluorescence measurement on agar plate as report of chloroplast(id) genetic expression in the live body Chlamydomonas reinhardtii cell.Cell is painted on the solid medium, maintains under the continuous illumination condition (1000 lux) 7 days.The substrate capraldehyde of luxAB is coated onto on the lid of Pi Shi flat board, and flat board is placed on below the CCD camera then.Transgenic cell ties up under the ambient lighting and seems very similar to wild-type cell.After 5 minutes dark adaptations (adaptation) removal chloroplast(id) fluorescence, use the CCD camera imaging, the result shows bright luminous signal in described two transgenic cell lines, then do not have signal to take place for wild-type strain system.Even coming from the signal of luxCt transgenic cell line is enough to show very little single clone under the condition in vivo.

[0266] except transforming the luxCt expression cassette in wild-type (137c) cell, described expression cassette also is transformed into (cc744 in the Chlamydomonas reinhardtii strain system of psbA defective type, ChlamydomonasGenetics Center, http://www.botany.duke.edu/chlamy/).The same with the situation of front, the initial conversion body screens by the luminescence analysis that utilizes the CCD camera to carry out, and positive transgenic cell line is again through obtaining homogenous cell system after the extra number wheel screening.To be higher than 137c/luxCt strain system far away from the luminous of cc744/luxCt strain system.In order to determine that whether this enhanced is luminous directly relevant with the luciferase accumulation that increases, and measured proteic accumulation of luxCt and uciferase activity in 137c and the cc744 transgenic cell line.Wild-type and luxCt transgenic cell line luxCt137c and luxCtcc744 grow on the agar culture plate, handle with capraldehyde then.Cell or under reflected light, take a picture (photo, photograph), perhaps by the CCD camera manifest (luminous, luminescence).Albumen extracts from cell, carries out western blot analysis (with anti-luxAB antibody) then or analyses quantitatively (photometer) with the luminosity score.When cell was grown on the solid TAP substratum under illumination, western blot analysis disclosed, than 137c clone, and nearly luxAB protein accumulation more than 10 times in the cc744 clone.The luminescence analysis of CCD camera discloses, and cc744 clone has similar signal to increase than 137c clone.The quantitative announcement cc744-luxCt clone of the uciferase activity that the employing photometer carries out is than the nearly uciferase activity more than 11 times of 137c-luxCt clone.These results prove that the luxCt gene is powerful report that chloroplast gene is expressed, and the lux activity measurement under the condition is consistent with the luciferase accumulation that records by western blot analysis and external luminescence analysis in the body.

[0267] several heterologous genes have been used as report that chloroplast gene is expressed, but their practicality is all owing to the susceptibility difference or can't visually in vivo be restricted.Luciferase has been used as reporter gene (Greer and Szalay, 2002 in many biologies; Langeridge etc., 1994; Kondo etc., 1993; Kay, 1993), this is because high-caliber susceptibility of luciferase and luciferase can be visual easily in active somatic cell, and has only slight influence for organism.Present embodiment has been showed the structure of the luciferase reporter gene that is used for chloroplast expression, two subunits of wherein synthetic bacterial luciferase luxAB are as one fusion rotein, and make its codon that reflects the gene that efficiently expresses in Chlamydomonas reinhardtii chloroplast(id) use by the codon of optimizing this synthetic luciferase gene.

[0268] present embodiment has been expanded the result of embodiment 1, the gfp of optimised codon has proved that codon uses and has greatly influenced the expression (Franklin etc., 2002) of heterologous protein in the Chlamydomonas reinhardtii chloroplast(id) embodiment 1 by synthetic have with regard to chloroplast(id).This cgfp runs up to 0.5% of solubility total protein in genetically modified chloroplast(id), and can manifest by the fluorometric analysis of chloroplast(id) extract.Yet, even the accumulation of high-caliber relatively GFP also is not enough to see in vivo this report gene.The gfp gene that adopts a kind of plastosome to optimize, Komine etc. have been reported the gfp (Komine etc., 2002) that uses in the fluorescence microscope transgene Chlamydomonas reinhardtii chloroplast(id).However, for this transgenic cell line, the proteic accumulating level of GFP still is very low, and the output of the fluorescence of mGFP strain system looks and is not higher than the background fluorescence that the strain that not have to transform is.

[0269] gfp that optimizes based on chloroplast(id) is in the success that promotes aspect the protein accumulation, still make it the visual fact in chloroplast(id) under the condition in vivo even add high-caliber relatively GFP, codon with chloroplast(id) optimization has synthesized a kind of luciferase gene, thereby obtains significant report of sensitive.The optimised luxCt gene of this codon places under the control of 3 ' UTR of the 5 ' promotor of Chlamydomonas reinhardtii chloroplast(id) atpA and UTR and rbcL, and this expression of gene has illustrated mRNA and the accumulation of albumen in the transgene Chlamydomonas reinhardtii chloroplast(id) of luxCt.Further, the transgenic line of expressing luxCt has accumulated the luciferase of q.s, make it can with the CCD camera easily in vivo by luminescence analysis by visual.Proportional by the luxCt protein accumulation that western blot analysis is measured with the uciferase activity that passes through luminescence analysis of CCD camera or external photometer assay determination.

[0270] Chlamydomonas reinhardtii is called as " green yeast ", and this is a title that fame follows merit, and the splendid hereditary property of this biology makes it can be used to disclose a large amount of cell processes, and foremost is exactly the biology generation of flagellum and light compositing device.Yet what obviously lack at present is exactly a kind of means that make things convenient for of analyzing gene expression, the particularly genetic expression in chloroplast(id).This result has proved the practicality of the luxCt gene of described optimization as report of chloroplast(id) genetic expression under the condition in the body.This result has proved that also luxCt can monitor even the genetic expression in very little cell clone report that any high throughput analysis that makes luxCt become the chloroplast gene expression can be selected as sensitive report.

Citing document

Every piece of following article all is hereby incorporated by.

Almashanu etc., J.Biolumin.Chemilumin.5:89-97,1990.

Bateman and Purton, Mol.Gen Genet.263:404-410,2000.

Baldwin etc., Biochemistry 23:3663-7,1984.

Carrer etc., Mol.Gen.Genet.241:49-56,1993.

Cohen etc., Meth.Enzymol.297,192-208,1998.

Franklin etc., Plant J.30:733-44,2002.

Goldschmidt-Clermont，Nucl.Acids?Res.19：4083-4089，1991.

Greer and Szalay, Luminescence 17:43-47,2002.

Gorman and Levine, Proc.Natl.Acad.Sci.USA 54:1665-1669,1965.

Heifetz，Biochemie?82：655-666，2000.

Ishikura etc., J.Biosci.Bioeng.87:307-314,1999.

Johnson etc., J.Biol.Chem.261:4805-11,1986.

Kirchner etc., Gene 81:349-54,1989.

Komine etc., Proc.Natl.Acad.Sci.USA 19:4085-90,2000.

Kondo etc., Proc.Natl.Acad.Sci.USA 90:5672-5676,1993.

Langridge etc., J.Biolumin.Chemilumin.9:185-200,1994.

Minko etc., Mol.Gen.Genet.262:421-425,1999.

Nakamura etc., Nucl.Acids Res.27:292,1999.

Olsson etc., Gene 81:335-47,1989.

Reed etc., Plant J.27:257-265,2001.

Sambrook etc., Molecular Cloning:A Laboratory Manual (Cold SpringHarbor Laboratory Press 1989).

Sakamoto etc., Proc.Natl.Acad.Sci.USA 90:497-501,1993.

Sidrov etc., Plant J.25:209-216,1999.

Staub and Maliga, EMBO J.12:601-606,1993.

Stemmer etc., Gene.164:49-53,1995.

Svab and Maliga, Proc.Natl.Acad.Sci.USA 913-917,1993.

Zerges and Rochaix, Mol.Cell.Biol.14:5268-5277,1994.

Though the present invention is described with reference to the foregoing description, it will be appreciated that [0271] modification or variation also all comprise within the spirit and scope of the present invention.Therefore, the present invention is only limited by the claims.

Sequence table

＜110〉The Scripps Research Inst.

＜120〉expression of polypeptide in chloroplast(id) and composition and the method that is used for express polypeptide

<130>CPUSZ42310

<150>US?60/375,129

<151>2002-04-23

<150>US?60/434,957

<151>2002-12-19

<160>48

<170>PatentIn?version?3.1

<210>1

<211>717

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the codon optimized green fluorescent protein of chloroplast(id)

<400>1

atggctaaag?gtgaagaatt?attcacaggt?gttgtaccta?ttttagtaga?attagacggt 60

gatgtaaacg?gtcacaaatt?ttcagtttct?ggtgaaggtg?aaggtgacgc?aacttatggt 120

aaattaacac?ttaaattcat?ttgtactaca?ggtaaattac?cagtaccttg?gccaacttta 180

gttacaactt?ttacatacgg?tgtacaatgt?ttcagtcgtt?accctgatca?catgaaacaa 240

catgactttt?tcaaatctgc?tatgccagaa?ggttatgttc?aagaacgtac?tatttttttc 300

aaagatgacg?gtaattataa?aacacgtgct?gaagtaaaat?ttgaaggtga?tactttagtt 360

aaccgtattg?aattaaaagg?tattgacttc?aaagaagatg?gtaatatttt?aggtcacaaa 420

cttgaatata?actacaattc?acataacgta?tatattatgg?cagacaaaca?aaaaaatggt 480

attaaagtaa?actttaaaat?tcgtcataat?atcgaggatg?gttctgtaca?attagctgac 540

cactatcaac?aaaacacacc?aattggtgat?ggtcctgttt?tacttccaga?caatcattat 600

ttaagtactc?aatctgcttt?atcaaaagat?cctaacgaaa?aacgtgacca?catggtatta 660

cttgaatttg?ttacagcagc?tggtattact?cacggtatgg?atgaattata?caaataa 717

<210>2

<211>238

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the codon optimized green fluorescent protein of chloroplast(id)

<400>2

Met?Ala?Lys?Gly?Glu?Glu?Leu?Phe?Thr?Gly?Val?Val?Pro?Ile?Leu?Val

1 5 10 15

Glu?Leu?Asp?Gly?Asp?Val?Asn?Gly?His?Lys?Phe?Ser?Val?Ser?Gly?Glu

20 25 30

Gly?Glu?Gly?Asp?Ala?Thr?Tyr?Gly?Lys?Leu?Thr?Leu?Lys?Phe?Ile?Cys

35 40 45

Thr?Thr?Gly?Lys?Leu?Pro?Val?Pro?Trp?Pro?Thr?Leu?Val?Thr?Thr?Phe

50 55 60

Thr?Tyr?Gly?Val?Gln?Cys?Phe?Ser?Arg?Tyr?Pro?Asp?His?Met?Lys?Gln

65 70 75 80

His?Asp?Phe?Phe?Lys?Ser?Ala?Met?Pro?Glu?Gly?Tyr?Val?Gln?Glu?Arg

85 90 95

Thr?Ile?Phe?Phe?Lys?Asp?Asp?Gly?Asn?Tyr?Lys?Thr?Arg?Ala?Glu?Val

100 105 110

Lys?Phe?Glu?Gly?Asp?Thr?Leu?Val?Asn?Arg?Ile?Glu?Leu?Lys?Gly?Ile

115 120 125

Asp?Phe?Lys?Glu?Asp?Gly?Asn?Ile?Leu?Gly?His?Lys?Leu?Glu?Tyr?Asn

130 135 140

Tyr?Asn?Ser?His?Asn?Val?Tyr?Ile?Met?Ala?Asp?lys?Gln?Lys?Asn?Gly

145 150 155 160

Ile?Lys?Val?Asn?Phe?Lys?Ile?Arg?His?Asn?Ile?Glu?Asp?Gly?Ser?Val

165 170 175

Gln?Leu?Ala?Asp?His?Tyr?Gln?Gln?Asn?Thr?Pro?Ile?Gly?Asp?Gly?Pro

180 185 190

Val?Leu?Leu?Pro?Asp?Asn?His?Tyr?Leu?Ser?Thr?Gln?Ser?Ala?Leu?Ser

195 200 205

Lys?Asp?Pro?Asn?Glu?Lys?Arg?Asp?His?Met?Val?Leu?Leu?Glu?Phe?Val

210 215 220

Thr?Ala?Ala?Gly?Ile?Thr?His?Gly?Met?Asp?Glu?Leu?Tyr?Lys

225 230 235

<210>3

<211>717

<212>DNA

＜213〉ocean jellyfish (Aequorea victoria)

<400>3

atgagtaaag?gagaagaact?tttcactgga?gttgtcccaa?ttcttgttga?attagatggt 60

gatgttaatg?ggcacaaatt?ttctgtcagt?ggagagggtg?aaggtgatgc?aacatacgga 120

aaacttaccc?ttaaatttat?ttgcactact?ggaaaactac?ctgttccatg?gccaacactt 180

gtcactactt?tctcttatgg?tgttcaatgc?ttttcaagat?acccagatca?tatgaaacgg 240

catgactttt?tcaagagtgc?catgcccgaa?ggttatgtac?aggaaagaac?tatatttttc 300

aaagatgacg?ggaactacaa?gacacgtgct?gaagtcaagt?ttgaaggtga?tacccttgtt 360

aatagaatcg?agttaaaagg?tattgatttt?aaagaagatg?gaaacattct?tggacacaaa 420

ttggaataca?actataactc?acacaatgta?tacatcatgg?cagacaaaca?aaagaatgga 480

atcaaagtta?acttcaaaat?tagacacaac?attgaagatg?gaagcgttca?actagcagac 540

cattatcaac?aaaatactcc?aattggcgat?ggccctgtcc?ttttaccaga?caaccattac 600

ctgtccacac?aatctgccct?ttcgaaagat?cccaacgaaa?agagagacca?catggtcctt 660

cttgagtttg?taacagctgc?tgggattaca?catggcatgg?atgaactata?caaataa 717

<210>4

<211>544

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>4

ggatcccatt?tttataactg?gtctcaaaat?acctataaac?ccattgttct?tctcttttag 60

ctctaagaac?aatcaattta?taaatatatt?tattattatg?ctataatata?aatactatat 120

aaatacattt?acctttttat?aaatacattt?accttttttt?taatttgcat?gattttaatg 180

cttatgctat?cttttttatt?tagtccataa?aacctttaaa?ggaccttttc?ttatgggata 240

tttatatttt?cctaacaaag?caatcggcgt?cataaacttt?agttgcttac?gacgcctgtg 300

gacgtccccc?ccttcccctt?acgggcaagt?aaacttaggg?attttaatgc?aataaataaa 360

tttgtcctct?tcgggcaaat?gaattttagt?atttaaatat?gacaagggtg?aaccattact 420

tttgttaaca?agtgatctta?ccactcacta?tttttgttga?attttaaact?tatttaaaat 480

tctcgagaaa?gattttaaaa?ataaactttt?ttaatctttt?atttattttt?tcttttttca 540

tatg 544

<210>5

<211>241

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>5

ggatccacta?gtaacggccg?ccagtgtgct?ggaattcggc?ttccgaattc?atatacctaa 60

aggccctttc?tatgctcgac?tgataagaca?agtacataaa?tttgctagtt?tacattattt 120

tttatttcta?aatatataat?atatttaaat?gtatttaaaa?tttttcaaca?atttttaaat 180

tatatttccg?gacagattat?tttaggatcg?tcaaaagaag?ttacatttat?ttatacatat 240

g 241

<210>6

<211>468

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>6

ggatccctag?taacggccgc?cagtgtgctg?gaatttgagt?atatgaaatt?aaatggatat 60

ttggtacatt?taattccaca?aaaatgtcca?atacttaaaa?tacaaaatta?aaagtattag 120

ttgtaaactt?gactaacatt?ttaaatttta?aattttttcc?taattatata?ttttacttgc 180

aaaatttata?aaaattttat?gcatttttat?atcataataa?taaaaccttt?attcatggtt 240

tataatataa?taattgtgat?gactatgcac?aaagcagttc?tagtcccata?tatataacta 300

tatataaccc?gtttaaagat?ttatttaaaa?atatgtgtgt?aaaaaatgct?tatttttaat 360

tttattttat?ataagttata?atattaaata?cacaatgatt?aaaattaaat?aataataaat 420

ttaacgtaac?gatgagttgt?ttttttattt?tggagataca?cgcatatg 468

<210>7

<211>373

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>7

ggatccgtcg?actggtaccg?ccactgcctg?cttcctcctt?cggagtatgt?aaaccccttc 60

gggcaactaa?agtttatcgc?agtatataaa?tataggcagt?tggcaggcaa?ctgccactga 120

cgtcctattt?taatactccg?aaggaggcag?ttggcaggca?actgccactg?acgtcccgta 180

agggtaaggg?gacgtccact?ggcgtcccgt?aaggggaagg?ggacgtaggt?acataaatgt 240

gctaggtaac?taacgtttga?ttttttgtgg?tataatatat?gtaccatgct?tttaatagaa 300

gcttgaattt?ataaattaaa?atatttttac?aatattttac?gagaaattaa?aactttaaaa 360

aaattaacat?atg 373

<210>8

<211>223

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>8

ggatccgttg?gcaggcaaca?aatttattta?ttgtcccgta?aggggaaggg?ggaaacaatt 60

attattttac?tgcggagcag?cttgttattg?aagttttatt?aaaaaaaaaa?taaaaatttg 120

acaaaaaaaa?taaaaaagtt?aaattaaaaa?cactgggaat?gttctacatc?ataaaaatca 180

aaagggttta?aaatcccgac?aaaatttaaa?ctttaaacat?atg 223

<210>9

<211>397

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>9

tctagactta?gcttcaacta?actctagctc?aaacaactaa?ttttttttta?aactaaaata 60

aatctggtta?accatacctg?gtttatttta?gtttagttta?tacacacttt?tcatatatat 120

atacttaata?gctaccatag?gcagttggca?ggacgtcccc?ttacgggaca?aatgtattta 180

ttgttgcctg?ccaactgcct?aatataaata?ttagtggacg?tccccttccc?cttacgggca 240

agtaaactta?gggattttaa?tgctccgtta?ggaggcaaat?aaattttagt?ggcagttgcc 300

tcgcctatcg?gctaacaagt?tccttcggag?tatataaata?tcctgccaac?tgccgatatt 360

tatatactag?gcagtggcgg?taccactcga?cggatcc 397

<210>10

<211>434

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>10

ctagagtcga?cctgcaggca?tgcaagcttg?tactcaagct?cggaacgaag?gtcgtgcctt 60

gctcggaagg?tggcgacgta?attcgttcag?cttgtaaatg?gtctcccaga?acttgctgct 120

gcatgtgaag?tttggaaaga?aattaaattc?gaatttgata?ctattgacaa?actttaattt 180

ttatttttca?tgatgtttat?gtgaatagca?taaacatcgt?ttttattttt?atggtgttta 240

ggttaaatac?ctaaacatca?ttttacattt?ttaaaattaa?gttctaaagt?tatcttttgt 300

ttaaatttgc?ctgtctttat?aaattacgat?gtgccagaaa?aataaaatct?tagcttttta 360

ttatagaatt?tatctttatg?tattatattt?tataagttat?aataaaagaa?atagtaacat 420

acgtcgacgg?atcc 434

<210>11

<211>411

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>11

tctagatttt?aattaagtag?gaactcggta?tatgctcttt?tggggtctta?ttagctagta 60

ttagttaact?aacaaaagat?caatatttta?gtttgtttta?tatattttat?tacttaagta 120

gtaaggattt?gcatttagca?atcttaaata?cttaagtaat?aatctataaa?taaaatatat 180

tttcgcttta?aaacttataa?aaattatttg?ctcgttataa?gcctaaaaaa?acgtaggatc 240

tctacgagat?attacattgt?ttttttcttt?aattggcttt?aatattactt?tgtatatata 300

aaccaaagta?cttgttaata?gttattaaat?tatattaact?atacagtaca?aagaaatttt 360

ttgctaaaaa?aagtatgtta?acattaaaaa?tttttgttta?tacagggatc?c 411

<210>12

<211>266

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>12

tctagattat?aatacattaa?aattgtaacg?cctttacaag?acagtataaa?atgggaatta 60

attaattagg?agggtcactt?tcagccactc?gttttttaaa?taggtaagta?acctttttaa 120

gagaacgtaa?gagattgtgg?attacgttct?caagagacat?aactcaaaat?actagtaggt 180

ttgagcttga?cttcaagctt?taacctccgt?cagcgataaa?acctattttg?agcgcatttt 240

aatatatttg?ggacgccagt?ggatcc 266

<210>13

<211>792

<212>DNA

＜213〉artificial sequence

<220>

＜223〉chloroplast(id) codon optimized specifically at the antibody of tetanus toxin

<220>

<221>CDS

<222>(1)..(789)

<223>

<400>13

atg?ctc?gag?cag?tct?ggg?gct?gag?gtg?aag?aag?cct?ggg?tcc?tcg?gtg 48

Met?Leu?Glu?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro?Gly?Ser?Ser?Val

1 5 10 15

aag?gtc?tcc?tgc?agg?gct?tct?gga?ggc?acc?ttc?aac?aat?tat?gcc?atc 96

Lys?Val?Ser?Cys?Arg?Ala?Ser?Gly?Gly?Thr?Phe?Asn?Asn?Tyr?Ala?Ile

20 25 30

agc?tgg?gtg?cga?cag?gcc?cct?gga?caa?ggg?ctt?gag?tgg?atg?gga?ggg 144

Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met?Gly?Gly

35 40 45

atc?ttc?cct?ttc?cgt?aat?aca?gca?aag?tac?gca?caa?cac?ttc?cag?ggc 192

Ile?Phe?Pro?Phe?Arg?Asn?Thr?Ala?Lys?Tyr?Ala?Gln?His?Phe?Gln?Gly

50 55 60

agg?gtc?acc?att?acc?gcg?gac?gaa?tcc?acg?ggc?aca?gcc?tac?atg?gag 240

Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Gly?Thr?Ala?Tyr?Met?Glu

65 70 75 80

ctg?agc?agc?ctg?aga?tct?gag?gac?acg?gcc?ata?tat?tat?tgt?gcg?aga 288

Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Ile?Tyr?Tyr?Cys?Ala?Arg

85 90 95

ggg?gat?acg?att?ttt?gga?gtg?acc?atg?gga?tac?tac?gct?atg?gac?gtc 336

Gly?Asp?Thr?Ile?Phe?Gly?Val?Thr?Met?Gly?Tyr?Tyr?Ala?Met?Asp?Val

100 105 110

tgg?ggc?caa?ggg?acc?acc?gtc?acc?gtc?tcc?tct?ggt?ggc?ggt?ggc?tcg 384

Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser

115 120 125

ggc?ggt?ggt?ggg?tcg?ggt?ggc?ggc?gga?tct?gag?ctc?gtt?ctc?acg?cag 432

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Glu?Leu?Val?Leu?Thr?Gln

130 135 140

tct?cca?ggc?acc?ctg?tct?ttg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc 480

Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser

145 150 155 160

tgc?agg?gcc?agt?cac?agt?gtt?agc?agg?gcc?tac?tta?gcc?tgg?tac?cag 528

Cys?Arg?Ala?Ser?His?Ser?Val?Ser?Arg?Ala?Tyr?Leu?Ala?Trp?Tyr?Gln

165 170 175

cag?aaa?cct?ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?aca?tcc?agc 576

Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Thr?Ser?Ser

180 185 190

agg?gcc?act?ggc?atc?cca?gac?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca 624

Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr

195 200 205

gac?ttc?act?ctc?acc?atc?agc?aga?ctg?gag?cct?gaa?gat?ttt?gca?gtg 672

Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu?Pro?Glu?Asp?Phe?Ala?Val

210 215 220

tac?tac?tgt?cag?cag?tat?ggt?ggc?tca?ccg?tgg?ttc?ggc?caa?ggg?acc 720

Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Gly?Ser?Pro?Trp?Phe?Gly?Gln?Gly?Thr

225 230 235 240

aag?gtg?gaa?ctc?aaa?cga?act?agt?ggc?cag?gcc?ggc?cag?tac?ccg?tac 768

Lys?Val?Glu?Leu?Lys?Arg?Thr?Ser?Gly?Gln?Ala?Gly?Gln?Tyr?Pro?Tyr

245 250 255

gac?gtt?ccg?gac?tac?gct?tct?taa 792

Asp?Val?Pro?Asp?Tyr?Ala?Ser

260

<210>14

<211>263

<212>PRT

＜213〉artificial sequence

<220>

＜223〉chloroplast(id) codon optimized specifically at the antibody of tetanus toxin

<400>14

Met?Leu?Glu?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro?Gly?Ser?Ser?Val

1 5 10 15

Lys?Val?Ser?Cys?Arg?Ala?Ser?Gly?Gly?Thr?Phe?Asn?Asn?Tyr?Ala?Ile

20 25 30

Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met?Gly?Gly

35 40 45

Ile?Phe?Pro?Phe?Arg?Asn?Thr?Ala?Lys?Tyr?Ala?Gln?His?Phe?Gln?Gly

50 55 60

Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Gly?Thr?Ala?Tyr?Met?Glu

65 70 75 80

Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Ile?Tyr?Tyr?Cys?Ala?Arg

85 90 95

Gly?Asp?Thr?Ile?Phe?Gly?Val?Thr?Met?Gly?Tyr?Tyr?Ala?Met?Asp?Val

100 105 110

Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser

115 120 125

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Glu?Leu?Val?Leu?Thr?Gln

130 135 140

Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser

145 150 155 160

Cys?Arg?Ala?Ser?His?Ser?Val?Ser?Arg?Ala?Tyr?Leu?Ala?Trp?Tyr?Gln

165 170 175

Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Thr?Ser?Ser

180 185 190

Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr

195 200 205

Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu?Pro?Glu?Asp?Phe?Ala?Val

210 215 220

Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Gly?Ser?Pro?Trp?Phe?Gly?Gln?Gly?Thr

225 230 235 240

Lys?Val?Glu?Leu?Lys?Arg?Thr?Ser?Gly?Gln?Ala?Gly?Gln?Tyr?Pro?Tyr

245 250 255

Asp?Val?Pro?Asp?Tyr?Ala?Ser

260

<210>15

<211>1926

<212>DNA

＜213〉artificial sequence

<220>

＜223〉chloroplast(id) codon optimized specifically at the antibody of hsv

<220>

<221>CDS

<222>(1)..(1917)

<223>

<400>15

cat?atg?gct?gct?cac?cac?cac?cac?cac?cac?gtt?gct?caa?gct?gct?tca 48

His?Met?Ala?Ala?His?His?His?His?His?His?Val?Ala?Gln?Ala?Ala?Ser

1 5 10 15

tca?gaa?tta?acg?caa?tca?cca?ggt?acc?tta?tca?tta?tca?cca?ggt?gaa 96

Ser?Glu?Leu?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly?Glu

20 25 30

cgt?gct?acc?tta?tca?tgt?cgt?gct?tca?caa?tca?gtt?tca?tca?gct?tac 144

Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ala?Tyr

35 40 45

tta?gct?tgg?tac?caa?caa?aaa?cca?ggt?caa?gct?cca?cgt?tta?tta?att 192

Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile

50 55 60

tac?ggt?gct?tca?tca?cgt?gct?act?ggt?att?cca?gat?cgt?ttc?tca?ggt 240

Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly

65 70 75 80

tca?ggt?tca?ggt?aca?gat?ttc?act?tta?acc?att?tca?cgt?tta?gaa?cca 288

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu?Pro

85 90 95

gaa?gat?ttc?gct?gtt?tac?tac?tgt?caa?caa?tac?ggt?cgt?tca?cca?act 336

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Arg?Ser?Pro?Thr

100 105 110

ttc?ggt?ggt?ggt?acc?aaa?gtt?gaa?att?aaa?cgt?act?tca?tca?ggt?ggt 384

Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Ser?Ser?Gly?Gly

115 120 125

ggt?ggt?tca?ggt?ggt?ggt?ggt?ggt?ggt?tca?tca?cgt?tca?tca?tta?gaa 432

Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Ser?Arg?Ser?Ser?Leu?Glu

130 135 140

caa?tca?ggt?gct?gaa?gtt?aaa?aaa?cca?ggt?tca?tca?gtt?aaa?gtt?tca 480

Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro?Gly?Ser?Ser?Val?Lys?Val?Ser

145 150 155 160

tgt?aaa?gct?tca?ggt?ggt?tca?ttc?tca?tca?tac?gct?att?aac?tgg?gtt 528

Cys?Lys?Ala?Ser?Gly?Gly?Ser?Phe?ger?Ser?Tyr?Ala?Ile?Asn?Trp?Val

165 170 175

cgt?caa?gct?caa?ggt?caa?ggt?tta?gaa?tgg?atg?ggt?ggt?tta?atg?cca 576

Arg?Gln?Ala?Gln?Gly?Gln?Gly?Leu?Glu?Trp?Met?Gly?Gly?Leu?Met?Pro

180 185 190

att?ttc?ggt?aca?aca?aac?tac?gct?caa?aaa?ttc?caa?gat?cgt?tta?acg 624

Ile?Phe?Gly?Thr?Thr?Asn?Tyr?Ala?Gln?Lys?Phe?Gln?Asp?Arg?Leu?Thr

195 200 205

att?acc?gct?gat?gtt?tca?acg?tca?aca?gct?tac?atg?caa?tta?tca?ggt 672

Ile?Thr?Ala?Asp?Val?Ser?Thr?Ser?Thr?Ala?Tyr?Met?Gln?Leu?Ser?Gly

210 215 220

tta?aca?tac?gaa?gat?acg?gct?atg?tac?tac?tgt?gct?cgt?gtt?gct?tac 720

Leu?Thr?Tyr?Glu?Asp?Thr?Ala?Met?Tyr?Tyr?Cys?Ala?Arg?Val?Ala?Tyr

225 230 235 240

atg?tta?gaa?cca?acc?gtt?act?gct?ggt?ggt?tta?gat?gtt?tgg?ggt?aaa 768

Met?Leu?Glu?Pro?Thr?Val?Thr?Ala?Gly?Gly?Leu?Asp?Val?Trp?Gly?Lys

245 250 255

ggt?acc?acg?gtt?acc?gtt?tca?cca?gct?tca?cca?acc?tca?cca?aaa?gtt 816

Gly?Thr?Thr?Val?Thr?Val?Ser?Pro?Ala?Ser?Pro?Thr?Ser?Pro?Lys?Val

260 265 270

ttc?cca?tta?tca?tta?tgt?tca?acc?caa?cca?gat?ggt?aac?gtt?gtt?att 864

Phe?Pro?Leu?Ser?Leu?Cys?Ser?Thr?Gln?Pro?Asp?Gly?Asn?Val?Val?Ile

275 280 285

gct?tgt?tta?gtt?caa?ggt?ttc?ttc?cca?caa?gaa?cca?tta?tca?gtt?acc 912

Ala?Cys?Leu?Val?Gln?Gly?Phe?Phe?Pro?Gln?Glu?Pro?Leu?Ser?Val?Thr

290 295 300

tgg?tca?gaa?tca?ggt?caa?ggt?gtt?acc?gct?cgt?aac?ttc?cca?cca?tca 960

Trp?Ser?Glu?Ser?Gly?Gln?Gly?Val?Thr?Ala?Arg?Asn?Phe?Pro?Pro?Ser

305 310 315 320

caa?gat?gct?tca?ggt?gat?tta?tac?acc?acg?tca?tca?caa?tta?acc?tta 1008

Gln?Asp?Ala?Ser?Gly?Asp?Leu?Tyr?Thr?Thr?Ser?Ser?Gln?Leu?Thr?Leu

325 330 335

cca?gct?aca?caa?tgt?tta?gct?ggt?aaa?tca?gtt?aca?tgt?cac?gtt?aaa 1056

Pro?Ala?Thr?Gln?Cys?Leu?Ala?Gly?Lys?Ser?Val?Thr?Cys?His?Val?Lys

340 345 350

cac?tac?acg?aac?cca?tca?caa?gat?gtt?act?gtt?cca?tgt?cca?gtt?cca 1104

His?Tyr?Thr?Asn?Pro?Ser?Gln?Asp?Val?Thr?Val?Pro?Cys?Pro?Val?Pro

355 360 365

tca?act?cca?cca?acc?cca?tca?cca?tca?act?cca?cca?acc?cca?tca?cca 1152

Ser?Thr?Pro?Pro?Thr?Pro?Ser?Pro?Ser?Thr?Pro?Pro?Thr?Pro?Ser?Pro

370 375 380

tca?tgt?tgt?cac?cca?cgt?tta?tca?tta?cac?cgt?cca?gct?tta?gaa?gat 1200

Ser?Cys?Cys?His?Pro?Arg?Leu?Ser?Leu?His?Arg?Pro?Ala?Leu?Glu?Asp

385 390 395 400

tta?tta?tta?ggt?tca?gaa?gct?aac?tta?acg?tgt?aca?tta?acc?ggt?tta 1248

Leu?Leu?Leu?Gly?Ser?Glu?Ala?Asn?Leu?Thr?Cys?Thr?Leu?Thr?Gly?Leu

405 410 415

cgt?gat?gct?tca?ggt?gtt?acc?ttc?acc?tgg?acg?cca?tca?tca?ggt?aaa 1296

Arg?Asp?Ala?Ser?Gly?Val?Thr?Phe?Thr?Trp?Thr?Pro?Ser?Ser?Gly?Lys

420 425 430

tca?gct?gtt?caa?ggt?cca?cca?gaa?cgt?gat?tta?tgt?ggt?tgt?tac?tca 1344

Ser?Ala?Val?Gln?Gly?Pro?Pro?Glu?Arg?Asp?Leu?Cys?Gly?Cys?Tyr?Ser

435 440 445

gtt?tca?tca?gtt?tta?cca?ggt?tgt?gct?gaa?cca?tgg?aac?cac?ggt?aaa 1392

Val?Ser?Ser?Val?Leu?Pro?Gly?Cys?Ala?Glu?Pro?Trp?Asn?His?Gly?Lys

450 455 460

acc?ttc?act?tgt?act?gct?gct?tac?cca?gaa?tca?aaa?acc?cca?tta?acc 1440

Thr?Phe?Thr?Cys?Thr?Ala?Ala?Tyr?Pro?Glu?Ser?Lys?Thr?Pro?Leu?Thr

465 470 475 480

gct?acc?tta?tca?aaa?tca?ggt?aac?aca?ttc?cgt?cca?gaa?gtt?cac?tta 1488

Ala?Thr?Leu?Ser?Lys?Ser?Gly?Asn?Thr?Phe?Arg?Pro?Glu?Val?His?Leu

485 490 495

tta?cca?cca?cca?tca?gaa?gaa?tta?gct?tta?aac?gaa?tta?gtt?acg?tta 1536

Leu?Pro?Pro?Pro?Ser?Glu?Glu?Leu?Ala?Leu?Asn?Glu?Leu?Val?Thr?Leu

500 505 510

acg?tgt?tta?gct?cgt?ggt?ttc?tca?cca?aaa?gat?gtt?tta?gtt?cgt?tgg 1584

Thr?Cys?Leu?Ala?Arg?Gly?Phe?Ser?Pro?Lys?Asp?Val?Leu?Val?Arg?Trp

515 520 525

tta?caa?ggt?tca?caa?gaa?tta?cca?cgt?gaa?aaa?tac?tta?act?tgg?gct 1632

Leu?Gln?Gly?Ser?Gln?Glu?Leu?Pro?Arg?Glu?Lys?Tyr?Leu?Thr?Trp?Ala

530 535 540

tca?cgt?caa?gaa?cca?tca?caa?ggt?acc?acc?acc?ttc?gct?gtt?acc?tca 1680

Ser?Arg?Gln?Glu?Pro?Ser?Gln?Gly?Thr?Thr?Thr?Phe?Ala?Val?Thr?Ser

545 550 555 560

att?tta?cgt?gtt?gct?gct?gaa?gat?tgg?aaa?aaa?ggt?gat?acc?ttc?tca 1728

Ile?Leu?Arg?Val?Ala?Ala?Glu?Asp?Trp?Lys?Lys?Gly?Asp?Thr?Phe?Ser

565 570 575

tgt?atg?gtt?ggt?cac?gaa?gct?tta?cca?tta?gct?ttc?aca?caa?aaa?acc 1776

Cys?Met?Val?Gly?His?Glu?Ala?Leu?Pro?Leu?Ala?Phe?Thr?Gln?Lys?Thr

580 585 590

att?gat?cgt?tta?gct?ggt?aaa?cca?acc?cac?gtt?aac?gtt?tca?gtt?gtt 1824

Ile?Asp?Arg?Leu?Ala?Gly?Lys?Pro?Thr?His?Val?Asn?Val?Ser?Val?Val

595 600 605

atg?gct?gaa?gtt?gat?ggt?acc?tgt?tac?gat?tat?aaa?gat?cac?gat?ggt 1872

Met?Ala?Glu?Val?Asp?Gly?Thr?Cys?Tyr?Asp?Tyr?Lys?Asp?His?Asp?Gly

610 615 620

gat?tac?aaa?gat?cac?gat?att?gat?tat?aaa?gat?gat?gat?gat?aaa 1917

Asp?Tyr?Lys?Asp?His?Asp?Ile?Asp?Tyr?Lys?Asp?Asp?Asp?Asp?Lys

625 630 635

taatctaga 1926

<210>16

<211>639

<212>PRT

＜213〉artificial sequence

<220>

＜223〉chloroplast(id) codon optimized specifically at the antibody of hsv

<400>16

His?Met?Ala?Ala?His?His?His?His?His?His?Val?Ala?Gln?Ala?Ala?Ser

1 5 10 15

Ser?Glu?Leu?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly?Glu

20 25 30

Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ala?Tyr

35 40 45

Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile

50 55 60

Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly

65 70 75 80

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu?Pro

85 90 95

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Arg?Ser?Pro?Thr

100 105 110

Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Ser?Ser?Gly?Gly

115 120 125

Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Ser?Arg?Ser?Ser?Leu?Glu

130 135 140

Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro?Gly?Ser?Ser?Val?Lys?Val?Ser

145 150 155 160

Cys?Lys?Ala?Ser?Gly?Gly?Ser?Phe?Ser?Ser?Tyr?Ala?Ile?Asn?Trp?Val

165 170 175

Arg?Gln?Ala?Gln?Gly?Gln?Gly?Leu?Glu?Trp?Met?Gly?Gly?Leu?Met?Pro

180 185 190

Ile?Phe?Gly?Thr?Thr?Asn?Tyr?Ala?Gln?Lys?Phe?Gln?Asp?Arg?Leu?Thr

195 200 205

Ile?Thr?Ala?Asp?Val?Ser?Thr?Ser?Thr?Ala?Tyr?Met?Gln?Leu?Ser?Gly

210 215 220

Leu?Thr?Tyr?Glu?Asp?Thr?Ala?Met?Tyr?Tyr?Cys?Ala?Arg?Val?Ala?Tyr

225 230 235 240

Met?Leu?Glu?Pro?Thr?Val?Thr?Ala?Gly?Gly?Leu?Asp?Val?Trp?Gly?Lys

245 250 255

Gly?Thr?Thr?Val?Thr?Val?Ser?Pro?Ala?Ser?Pro?Thr?Ser?Pro?Lys?Val

260 265 270

Phe?Pro?Leu?Ser?Leu?Cys?Ser?Thr?Gln?Pro?Asp?Gly?Asn?Val?Val?Ile

275 280 285

Ala?Cys?Leu?Val?Gln?Gly?Phe?Phe?Pro?Gln?Glu?Pro?Leu?Ser?Val?Thr

290 295 300

Trp?Ser?Glu?Ser?Gly?Gln?Gly?Val?Thr?Ala?Arg?Asn?Phe?Pro?Pro?Ser

305 310 315 320

Gln?Asp?Ala?Ser?Gly?Asp?Leu?Tyr?Thr?Thr?Ser?Ser?Gln?Leu?Thr?Leu

325 330 335

Pro?Ala?Thr?Gln?Cys?Leu?Ala?Gly?Lys?Ser?Val?Thr?Cys?His?Val?Lys

340 345 350

His?Tyr?Thr?Asn?Pro?Ser?Gln?Asp?Val?Thr?Val?Pro?Cys?Pro?Val?Pro

355 360 365

Ser?Thr?Pro?Pro?Thr?Pro?Ser?Pro?Ser?Thr?Pro?Pro?Thr?Pro?Ser?Pro

370 375 380

Ser?Cys?Cys?His?Pro?Arg?Leu?Ser?Leu?His?Arg?Pro?Ala?Leu?Glu?Asp

385 390 395 400

Leu?Leu?Leu?Gly?Ser?Glu?Ala?Asn?Leu?Thr?Cys?Thr?Leu?Thr?Gly?Leu

405 410 415

Arg?Asp?Ala?Ser?Gly?Val?Thr?Phe?Thr?Trp?Thr?Pro?Ser?Ser?Gly?Lys

420 425 430

Ser?Ala?Val?Gln?Gly?Pro?Pro?Glu?Arg?Asp?Leu?Cys?Gly?Cys?Tyr?Ser

435 440 445

Val?Ser?Ser?Val?Leu?Pro?Gly?Cys?Ala?Glu?Pro?Trp?Asn?His?Gly?Lys

450 455 460

Thr?Phe?Thr?Cys?Thr?Ala?Ala?Tyr?Pro?Glu?Ser?Lys?Thr?Pro?Leu?Thr

465 470 475 480

Ala?Thr?Leu?Ser?Lys?Ser?Gly?Asn?Thr?Phe?Arg?Pro?Glu?Val?His?Leu

485 490 495

Leu?Pro?Pro?Pro?Ser?Glu?Glu?Leu?Ala?Leu?Asn?Glu?Leu?Val?Thr?Leu

500 505 510

Thr?Cys?Leu?Ala?Arg?Gly?Phe?Ser?Pro?Lys?Asp?Val?Leu?Val?Arg?Trp

515 520 525

Leu?Gln?Gly?Ser?Gln?Glu?Leu?Pro?Arg?Glu?Lys?Tyr?Leu?Thr?Trp?Ala

530 535 540

Ser?Arg?Gln?Glu?Pro?Ser?Gln?Gly?Thr?Thr?Thr?Phe?Ala?Val?Thr?Ser

545 550 555 560

Ile?Leu?Arg?Val?Ala?Ala?Glu?Asp?Trp?Lys?Lys?Gly?Asp?Thr?Phe?Ser

565 570 575

Cys?Met?Val?Gly?His?Glu?Ala?Leu?Pro?Leu?Ala?Phe?Thr?Gln?Lys?Thr

580 585 590

Ile?Asp?Arg?Leu?Ala?Gly?Lys?Pro?Thr?His?Val?Asn?Val?Ser?Val?Val

595 600 605

Met?Ala?Glu?Val?Asp?Gly?Thr?Cys?Tyr?Asp?Tyr?Lys?Asp?His?Asp?Gly

610 615 620

Asp?Tyr?Lys?Asp?His?Asp?Ile?Asp?Tyr?Lys?Asp?Asp?Asp?Asp?Lys

625 630 635

<210>17

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>17

catatgagta?aaggagaaga?ac 22

<210>18

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>18

tctagattat?ttgtatagtt?catcc 25

<210>19

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>19

tctagagtcg?acctgcag 18

<210>20

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>20

ggatccgtcg?acgtatg 17

<210>21

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>21

gaattcatat?acctaaaggc?cctttctatg?c 31

<210>22

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>22

catatgtata?aataaatgta?acttc 25

<210>23

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>23

gaagcttgaa?tttataaatt?aaaatatttt?tacaatattt?tacccagaaa?ttaaaac 57

<210>24

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>24

tgtcatatgt?taattttttt?aaagtttttc?tccgtaaaat?attg 44

<210>25

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>25

tgtcatatgt?taattttttt?aaagtctccg?taaaatattg 40

<210>26

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>26

tgtcatatgt?taattttttt?tctccgtaaa?atattg 36

<210>27

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>27

gtcatatgtt?aatttctccg 20

<210>28

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉amplimer

<400>28

tgtcatatgt?taatcctcct?aaagttttaa?tttctccg 38

<210>29

<211>10

<212>RNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>29

caccuccuuc 10

<210>30

<211>2000

<212>DNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<220>

<221>CDS

<222>(497)..(1552)

<223>

<400>30

cgtcatagta?tatcaatatt?gtaacagatt?gacacccttt?aagtaaacat?tttttttgag 60

tcatatggag?tcatatgaaa?ttaaatggat?atttggtaca?tttaattcca?caaaaatgtc 120

caatacttaa?aatacaaaat?taaaagtatt?agttgtaaac?ttgactaaca?ttttaaattt 180

taaatttttt?cctaattata?tattttactt?gcaaaattta?taaaaatttt?atgcattttt 240

atatcataat?aataaaacct?ttattcatgg?tttataatat?aataattgtg?atgactatgc 300

acaaagcagt?tctagtccca?tatatataac?tatatataac?ccgtttaaag?atttatttaa 360

aaatatgtgt?gtaaaaaatg?cttattttta?attttatttt?atataagtta?taatattaaa 420

tacacaatga?ttaaaattaa?ataataataa?atttaacgta?acgatgagtt?gtttttttat 480

tttggagata?cacgca?atg?aca?att?gcg?atc?ggt?aca?tat?caa?gag?aaa?cgc 532

Met?Thr?Ile?Ala?Ile?Gly?Thr?Tyr?Gln?Glu?Lys?Arg

1 5 10

aca?tgg?ttc?gat?gac?gct?gat?gac?tgg?ctt?cgt?caa?gac?cgt?ttc?gta 580

Thr?Trp?Phe?Asp?Asp?Ala?Asp?Asp?Trp?Leu?Arg?Gln?Asp?Arg?Phe?Val

15 20 25

ttc?gta?ggt?tgg?tca?ggt?tta?tta?cta?ttc?cct?tgt?gct?tac?ttt?gca 628

Phe?Val?Gly?Trp?Ser?Gly?Leu?Leu?Leu?Phe?Pro?Cys?Ala?Tyr?Phe?Ala

30 35 40

tta?ggt?ggt?tgg?tta?act?ggt?act?act?ttc?gtt?act?tca?tgg?tat?acg 676

Leu?Gly?Gly?Trp?Leu?Thr?Gly?Thr?Thr?Phe?Val?Thr?Ser?Trp?Tyr?Thr

45 50 55 60

cat?ggt?tta?gct?act?tct?tac?tta?gaa?ggt?tgt?aac?ttc?tta?aca?gca 724

His?Gly?Leu?Ala?Thr?Ser?Tyr?Leu?Glu?Gly?Cys?Asn?Phe?Leu?Thr?Ala

65 70 75

gct?gtt?tct?aca?cct?gct?aac?agt?atg?gct?cac?tct?ctt?cta?ttt?gtt 772

Ala?Val?Ser?Thr?Pro?Ala?Asn?Ser?Met?Ala?His?Ser?Leu?Leu?Phe?Val

80 85 90

tgg?ggt?cca?gaa?gct?caa?ggt?gat?ttc?act?cgt?tgg?tgt?caa?ctt?ggt 820

Trp?Gly?Pro?Glu?Ala?Gln?Gly?Asp?Phe?Thr?Arg?Trp?Cys?Gln?Leu?Gly

95 100 105

ggt?tta?tgg?gca?ttc?gtt?gct?tta?cac?ggt?gca?ttt?ggt?tta?att?ggt 868

Gly?Leu?Trp?Ala?Phe?Val?Ala?Leu?His?Gly?Ala?Phe?Gly?Leu?Ile?Gly

110 115 120

ttc?atg?ctt?cgt?cag?ttt?gaa?att?gct?cgt?tca?gta?aac?tta?cgt?cca 916

Phe?Met?Leu?Arg?Gln?Phe?Glu?Ile?Ala?Arg?Ser?Val?Asn?Leu?Arg?Pro

125 130 135 140

tac?aac?gca?att?gct?ttc?tca?gca?cca?att?gct?gta?ttc?gtt?tca?gta 964

Tyr?Asn?Ala?Ile?Ala?Phe?Ser?Ala?Pro?Ile?Ala?Val?Phe?Val?Ser?Val

145 150 155

ttc?cta?att?tac?cca?tta?ggt?caa?tca?ggt?tgg?ttc?ttt?gca?cct?agt 1012

Phe?Leu?Ile?Tyr?Pro?Leu?Gly?Gln?Ser?Gly?Trp?Phe?Phe?Ala?Pro?Ser

160 165 170

ttc?ggt?gta?gct?gct?atc?ttc?cgt?ttc?att?tta?ttc?ttc?caa?ggt?ttc 1060

Phe?Gly?Val?Ala?Ala?Ile?Phe?Arg?Phe?Ile?Leu?Phe?Phe?Gln?Gly?Phe

175 180 185

cac?aac?tgg?aca?ctt?aac?cca?ttc?cac?atg?atg?ggt?gtt?gct?ggt?gtt 1108

His?Asn?Trp?Thr?Leu?Asn?Pro?Phe?His?Met?Met?Gly?Val?Ala?Gly?Val

190 195 200

tta?ggt?gct?gct?tta?tta?tgt?gct?att?cac?ggt?gct?act?gtt?gaa?aac 1156

Leu?Gly?Ala?Ala?Leu?Leu?Cys?Ala?Ile?His?Gly?Ala?Thr?Val?Glu?Asn

205 210 215 220

aca?tta?ttc?gaa?gac?ggt?gac?ggt?gct?aac?aca?ttc?cgt?gca?ttc?aac 1204

Thr?Leu?Phe?Glu?Asp?Gly?Asp?Gly?Ala?Asn?Thr?Phe?Arg?Ala?Phe?Asn

225 230 235

cct?aca?cag?gct?gaa?gaa?aca?tac?tct?atg?gtt?act?gct?aac?cgt?ttc 1252

Pro?Thr?Gln?Ala?Glu?Glu?Thr?Tyr?Ser?Met?Val?Thr?Ala?Asn?Arg?Phe

240 245 250

tgg?tca?caa?atc?ttc?ggt?gtt?gct?ttc?tct?aac?aaa?cgt?tgg?ctt?cac 1300

Trp?Ser?Gln?Ile?Phe?Gly?Val?Ala?Phe?Ser?Asn?Lys?Arg?Trp?Leu?His

255 260 265

ttc?ttc?atg?tta?tta?gtt?cca?gta?act?ggt?ctt?tgg?atg?agt?gct?att 1348

Phe?Phe?Met?Leu?Leu?Val?Pro?Val?Thr?Gly?Leu?Trp?Met?Ser?Ala?Ile

270 275 280

ggt?gtt?gta?ggt?tta?gct?cta?aac?tta?cgt?gct?tac?gac?ttc?gta?tca 1396

Gly?Val?Val?Gly?Leu?Ala?Leu?Asn?Leu?Arg?Ala?Tyr?Asp?Phe?Val?Ser

285 290 295 300

caa?gag?att?cgt?gct?gct?gaa?gac?cct?gaa?ttc?gaa?aca?ttc?tac?act 1444

Gln?Glu?Ile?Arg?Ala?Ala?Glu?Asp?Pro?Glu?Phe?Glu?Thr?Phe?Tyr?Thr

305 310 315

aaa?aac?att?ctt?ctt?aac?gaa?ggt?att?cgt?gct?tgg?atg?gct?gct?caa 1492

Lys?Asn?Ile?Leu?Leu?Asn?Glu?Gly?Ile?Arg?Ala?Trp?Met?Ala?Ala?Gln

320 325 330

gac?caa?cca?cac?gaa?cgt?tta?gta?ttc?cct?gaa?gaa?gta?tta?cca?cgt 1540

Asp?Gln?Pro?His?Glu?Arg?Leu?Val?Phe?Pro?Glu?Glu?Val?Leu?Pro?Arg

335 340 345

ggt?aac?gct?cta?taatatattt?ttatataaat?taccaatact?aattagtatt 1592

Gly?Asn?Ala?Leu

350

ggtaatttat?attactttat?tatttaaaag?aaaatgcccc?tttggggcta?aaaatcacat 1652

gagtgcttga?gccgtatgcg?aaaaaactcg?catgtacggt?tctttaggag?gatttaaaat 1712

attaaaaaat?aaaaaaacaa?atcctacctg?actaaaccag?gacatttttc?acgtactctg 1772

tcaaaaggtc?caaacacaac?aacttggatt?tggaaccttc?acgcagatgc?tcatgacttt 1832

gacagtcata?caagtgatct?agaagaaatt?tctagaaaag?tattcagtgc?acactttggt 1892

caattaggta?tcattttcat?ttggttaagt?gggtgcgaca?cgaagacgta?tatattttta 1952

tagtttaaaa?agatactttt?acactgtagt?tgaaaagtat?aagcactt 2000

<210>31

<211>352

<212>PRT

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>31

Met?Thr?Ile?Ala?Ile?Gly?Thr?Tyr?Gln?Glu?Lys?Arg?Thr?Trp?Phe?Asp

1 5 10 15

Asp?Ala?Asp?Asp?Trp?Leu?Arg?Gln?Asp?Arg?Phe?Val?Phe?Val?Gly?Trp

20 25 30

Ser?Gly?Leu?Leu?Leu?Phe?Pro?Cys?Ala?Tyr?Phe?Ala?Leu?Gly?Gly?Trp

35 40 45

Leu?Thr?Gly?Thr?Thr?Phe?Val?Thr?Ser?Trp?Tyr?Thr?His?Gly?Leu?Ala

50 55 60

Thr?Ser?Tyr?Leu?Glu?Gly?Cys?Asn?Phe?Leu?Thr?Ala?Ala?Val?Ser?Thr

65 70 75 80

Pro?Ala?Asn?Ser?Met?Ala?His?Ser?Leu?Leu?Phe?Val?Trp?Gly?Pro?Glu

85 90 95

Ala?Gln?Gly?Asp?Phe?Thr?Arg?Trp?Cys?Gln?Leu?Gly?Gly?Leu?Trp?Ala

100 105 110

Phe?Val?Ala?Leu?His?Gly?Ala?Phe?Gly?Leu?Ile?Gly?Phe?Met?Leu?Arg

115 120 125

Gln?Phe?Glu?Ile?Ala?Arg?Ser?Val?Asn?Leu?Arg?Pro?Tyr?Asn?Ala?Ile

130 135 140

Ala?Phe?Ser?Ala?Pro?Ile?Ala?Val?Phe?Val?Ser?Val?Phe?Leu?Ile?Tyr

145 150 155 160

Pro?Leu?Gly?Gln?Ser?Gly?Trp?Phe?Phe?Ala?Pro?Ser?Phe?Gly?Val?Ala

165 170 175

Ala?Ile?Phe?Arg?Phe?Ile?Leu?Phe?Phe?Gln?Gly?Phe?His?Asn?Trp?Thr

180 185 190

Leu?Asn?Pro?Phe?His?Met?Met?Gly?Val?Ala?Gly?Val?Leu?Gly?Ala?Ala

195 200 205

Leu?Leu?Cys?Ala?Ile?His?Gly?Ala?Thr?Val?Glu?Asn?Thr?Leu?Phe?Glu

210 215 220

Asp?Gly?Asp?Gly?Ala?Asn?Thr?Phe?Arg?Ala?Phe?Asn?Pro?Thr?Gln?Ala

225 230 235 240

Glu?Glu?Thr?Tyr?Ser?Met?Val?Thr?Ala?Asn?Arg?Phe?Trp?Ser?Gln?Ile

245 250 255

Phe?Gly?Val?Ala?Phe?Ser?Asn?Lys?Arg?Trp?Leu?His?Phe?Phe?Met?Leu

260 265 270

Leu?Val?Pro?Val?Thr?Gly?Leu?Trp?Met?Ser?Ala?Ile?Gly?Val?Val?Gly

275 280 285

Leu?Ala?Leu?Asn?Leu?Arg?Ala?Tyr?Asp?Phe?Val?Ser?Gln?Glu?Ile?Arg

290 295 300

Ala?Ala?Glu?Asp?Pro?Glu?Phe?Glu?Thr?Phe?Tyr?Thr?Lys?Asn?Ile?Leu

305 310 315 320

Leu?Asn?Glu?Gly?Ile?Arg?Ala?Trp?Met?Ala?Ala?Gln?Asp?Gln?Pro?His

325 330 335

Glu?Arg?Leu?Val?Phe?Pro?Glu?Glu?Val?Leu?Pro?Arg?Gly?Asn?Ala?Leu

340 345 350

<210>32

<211>45

<212>RNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>32

caauauuuua?cggagaaauu?aaaacuuuaa?aaaaauuaac?auaug 45

<210>33

<211>13

<212>RNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>33

gcucaccucc?uuc 13

<210>34

<211>38

<212>RNA

＜213〉Chlamydomonas reinhardtii (Chlamydomonas reinhardtii)

<400>34

uuacggagaa?auuaaaacuu?uaaaaaaauu?aacauaug 38

<210>35

<211>34

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>35

uuacaaauua?aaacuuuaaa?aaaauuaaca?uaug 34

<210>36

<211>38

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>36

uuacccagaa?auuaaaacuu?uaaaaaaauu?aacauaug 38

<210>37

<211>34

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>37

uuacggagaa?aaacuuuaaa?aaaauuaaca?uaug 34

<210>38

<211>30

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>38

uuacggagac?uuuaaaaaaa?uuaacauaug 30

<210>39

<211>26

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>39

uuacggagaa?aaaaaauuaa?cauaug 26

<210>40

<211>22

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>40

uuacggagaa?auuaaacaua?ug 22

<210>41

<211>38

<212>RNA

＜213〉artificial sequence

<220>

＜223〉mutant nucleotide sequence of SEQ ID NO:32

<400>41

uuacggagaa?auuaaaacuu?uaggaggauu?aacauaug 38

<210>42

<211>840

<212>DNA

＜213〉human (homo sapiens)

<400>42

catatggttg?ctcaagctgc?ttcatcagaa?ttaacgcaat?caccaggtac?cttatcatta 60

tcaccaggtg?aacgtgctac?cttatcatgt?cgtgcttcac?aatcagtttc?atcagcttac 120

ttagcttggt?accaacaaaa?accaggtcaa?gctccacgtt?tattaattta?cggtgcttca 180

tcacgtgcta?ctggtattcc?agatcgtttc?tcaggttcag?gttcaggtac?agatttcact 240

ttaaccattt?cacgtttaga?accagaagat?ttcgctgttt?actactgtca?acaatacggt 300

cgttcaccaa?ctttcggtgg?tggtaccaaa?gttgaaatta?aacgtacttc?atcaggtggt 360

ggtggttcag?gtggtggtgg?tggtggttca?tcacgttcat?cattagaaca?atcaggtgct 420

gaagttaaaa?aaccaggttc?atcagttaaa?gtttcatgta?aagcttcagg?tggttcattc 480

tcatcatacg?ctattaactg?ggttcgtcaa?gctcaaggtc?aaggtttaga?atggatgggt 540

ggtttaatgc?caattttcgg?tacaacaaac?tacgctcaaa?aattccaaga?tcgtttaacg 600

attaccgctg?atgtttcaac?gtcaacagct?tacatgcaat?tatcaggttt?aacatacgaa 660

gatacggcta?tgtactactg?tgctcgtgtt?gcttacatgt?tagaaccaac?cgttactgct 720

ggtggtttag?atgtttgggg?taaaggtacc?acggttaccg?tttcagatta?taaagatcac 780

gatggtgatt?acaaagatca?cgatattgat?tataaagatg?atgatgataa?ataatctaga 840

<210>43

<211>277

<212>PRT

＜213〉human (homo sapiens)

<400>43

His?Met?Val?Ala?Gln?Ala?Ala?Ser?Ser?Glu?Leu?Thr?Gln?Ser?Pro?Gly

1 5 10 15

Thr?Leu?Ser?Leu?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala

20 25 30

Ser?Gln?Ser?Val?Ser?Ser?Ala?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35 40 45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr

50 55 60

Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr

65 70 75 80

Leu?Thr?Ile?Ser?Arg?Leu?Glu?Pro?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85 90 95

Gln?Gln?Tyr?Gly?Arg?Ser?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu

100 105 110

Ile?Lys?Arg?Thr?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly

115 120 125

Gly?Ser?Ser?Arg?Ser?Ser?Leu?Glu?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys

130 135 140

Pro?Gly?Ser?Ser?Val?Lys?Val?Ser?Cys?Lys?Ala?Ser?Gly?Gly?Ser?Phe

145 150 155 160

Ser?Ser?Tyr?Ala?Ile?Asn?Trp?Val?Arg?Gln?Ala?Gln?Gly?Gln?Gly?Leu

165 170 175

Glu?Trp?Met?Gly?Gly?Leu?Met?Pro?Ile?Phe?Gly?Thr?Thr?Asn?Tyr?Ala

180 185 190

Gln?Lys?Phe?Gln?Asp?Arg?Leu?Thr?Ile?Thr?Ala?Asp?Val?Ser?Thr?Ser

195 200 205

Thr?Ala?Tyr?Met?Gln?Leu?Ser?Gly?Leu?Thr?Tyr?Glu?Asp?Thr?Ala?Met

210 215 220

Tyr?Tyr?Cys?Ala?Arg?Val?Ala?Tyr?Met?Leu?Glu?Pro?Thr?Val?Thr?Ala

225 230 235 240

Gly?Gly?Leu?Asp?Val?Trp?Gly?Lys?Gly?Thr?Thr?Val?Thr?Val?Ser?Asp

245 250 255

Tyr?Lys?Asp?His?Asp?Gly?Asp?Tyr?Lys?Asp?His?Asp?Ile?Asp?Tyr?Lys

260 265 270

Asp?Asp?Asp?Asp?Lys

275

<210>44

<211>681

<212>PRT

＜213〉Vibrio harveyi (Vibrio harveyi)

<400>44

Met?Lys?Phe?Gly?Asn?Phe?Leu?Leu?Thr?Tyr?Gln?Pro?Pro?Glu?Leu?Ser

1 5 10 15

Gln?Thr?Glu?Val?Met?Lys?Arg?Leu?Val?Asn?Leu?Gly?Lys?Ala?Ser?Glu

20 25 30

Gly?Cys?Gly?Phe?Asp?Thr?Val?Trp?Leu?Leu?Glu?His?His?Phe?Thr?Glu

35 40 45

Phe?Gly?Leu?Leu?Gly?Asn?Pro?Tyr?Val?Ala?Ala?Ala?His?Leu?Leu?Gly

50 55 60

Thr?Thr?Glu?Thr?Leu?Asn?Val?Gly?Thr?Ala?Ala?Ile?Val?Leu?Pro?Thr

65 70 75 80

Ala?His?Pro?Val?Arg?Gln?Ala?Glu?Asp?Val?Asn?Leu?Leu?Asp?Gln?Met

85 90 95

Ser?Lys?Gly?Arg?Phe?Arg?Phe?Gly?Ile?Cys?Arg?Gly?Leu?Tyr?Asp?Lys

100 105 110

Asp?Phe?Arg?Val?Phe?Gly?Thr?Asp?Met?Asp?Asn?Ser?Arg?Ala?Leu?Met

115 120 125

Asp?Cys?Trp?Tyr?Asp?Leu?Met?Lys?Glu?Gly?Phe?Asn?Glu?Gly?Tyr?Ile

130 135 140

Ala?Ala?Asp?Asn?Glu?His?Ile?Lys?Phe?Pro?Lys?Ile?Gln?Leu?Asn?Pro

145 150 155 160

Ser?Ala?Tyr?Thr?Gln?Gly?Gly?Ala?Pro?Val?Tyr?Val?Val?Ala?Glu?Ser

165 170 175

Ala?Ser?Thr?Thr?Glu?Trp?Ala?Ala?Glu?Arg?Gly?Leu?Pro?Met?Ile?Leu

180 185 190

Ser?Trp?Ile?Ile?Asn?Thr?His?Glu?Lys?Lys?Ala?Gln?Leu?Asp?Leu?Tyr

195 200 205

Asn?Glu?Val?Ala?Thr?Glu?His?Gly?Tyr?Asp?Val?Thr?Lys?Ile?Asp?His

210 215 220

Cys?Leu?Ser?Tyr?Ile?Thr?Ser?Val?Asp?His?Asp?Ser?Asn?Arg?Ala?Lys

225 230 235 240

Asp?Ile?Cys?Arg?Asn?Phe?Leu?Gly?His?Trp?Tyr?Asp?Ser?Tyr?Val?Asn

245 250 255

Ala?Thr?Lys?Ile?Phe?Asp?Asp?Ser?Asp?Gln?Thr?Lys?Gly?Tyr?Asp?Phe

260 265 270

Asn?Lys?Gly?Gln?Trp?Arg?Asp?Phe?Val?Leu?Lys?Gly?His?Lys?Asp?Thr

275 280 285

Asn?Arg?Arg?Ile?Asp?Tyr?Ser?Tyr?Glu?Ile?Asn?Pro?Val?Gly?Thr?Pro

290 295 300

Glu?Glu?Cys?Ile?Ala?Ile?Ile?Gln?Gln?Asp?Ile?Asp?Ala?Thr?Gly?Ile

305 310 315 320

Asp?Asn?Ile?Cys?Cys?Gly?Phe?Glu?Ala?Asn?Gly?Ser?Glu?Glu?Glu?Ile

325 330 335

Ile?Ala?Ser?Met?Lys?Leu?Phe?Gln?Ser?Asp?Val?Met?Pro?Tyr?Leu?Lys

340 345 350

Glu?Lys?Gln?Glx?Met?Lys?Phe?Gly?Leu?Phe?Phe?Leu?Asn?Phe?Met?Asn

355 360 365

Ser?Lys?Arg?Ser?Ser?Asp?Gln?Val?Ile?Glu?Glu?Ile?Leu?Asp?Thr?Ala

370 375 380

His?Tyr?Val?Asp?Gln?Leu?Lys?Phe?Asp?Thr?Leu?Ala?Val?Tyr?Glu?Asn

385 390 395 400

His?Phe?Ser?Asn?Asn?Gly?Val?Val?Gly?Ala?Pro?Leu?Thr?Val?Ala?Gly

405 410 415

Phe?Leu?Leu?Gly?Met?Thr?Lys?Asn?Ala?Lys?Val?Ala?Ser?Leu?Asn?His

420 425 430

Val?Ile?Thr?Thr?His?His?Pro?Val?Arg?Val?Ala?Glu?Glu?Ala?Cys?Leu

435 440 445

Leu?Asp?Gln?Met?Ser?Glu?Gly?Arg?Phe?Ala?Phe?Gly?Phe?Ser?Asp?Cys

450 455 460

Glu?Lys?Ser?Ala?Asp?Met?Arg?Phe?Phe?Asn?Arg?Pro?Thr?Asp?Ser?Gln

465 470 475 480

Phe?Gln?Leu?Phe?Ser?Glu?Cys?His?Lys?Ile?Ile?Asn?Asp?Ala?Phe?Thr

485 490 495

Thr?Gly?Tyr?Cys?His?Pro?Asn?Asn?Asp?Phe?Tyr?Ser?Phe?Pro?Lys?Ile

500 505 510

Ser?Val?Asn?Pro?His?Ala?Phe?Thr?Glu?Gly?Gly?Pro?Ala?Gln?Phe?Val

515 520 525

Asn?Ala?Thr?Ser?Lys?Glu?Val?Val?Glu?Trp?Ala?Ala?Lys?Leu?Gly?Leu

530 535 540

Pro?Leu?Val?Phe?Arg?Trp?Asp?Asp?Ser?Asn?Ala?Gln?Arg?Lys?Glu?Tyr

545 550 555 560

Ala?Gly?Leu?Tyr?His?Glu?Val?Ala?Gln?Ala?His?Gly?Val?Asp?Val?Ser

565 570 575

Gln?Val?Arg?His?Lys?Leu?Thr?Leu?Leu?Val?Asn?Gln?Asn?Val?Asp?Gly

580 585 590

Glu?Ala?Ala?Arg?Ala?Glu?Ala?Arg?Val?Tyr?Leu?Glu?Glu?Phe?Val?Arg

595 600 605

Glu?Ser?Tyr?Ser?Asn?Thr?Asp?Phe?Glu?Gln?Lys?Met?Gly?Glu?Leu?Leu

610 615 620

Ser?Glu?Asn?Ala?Ile?Gly?Thr?Tyr?Glu?Glu?Ser?Thr?Gln?Ala?Ala?Arg

625 630 635 640

Val?Ala?Ile?Glu?Cys?Cys?Gly?Ala?Ala?Asp?Leu?Leu?Met?Ser?Phe?Glu

645 650 655

Ser?Met?Glu?Asp?Lys?Ala?Gln?Gln?Arg?Ala?Val?Ile?Asp?Val?Val?Asn

660 665 670

Ala?Asn?Ile?Val?Lys?Tyr?His?Ser?Glx

675 680

<210>45

<211>2094

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the luxAB gene of the codon of use chloroplast(id) preference

<400>45

catatgaaat?ttggtaactt?ccttttaact?tatcaaccac?ctgaactatc?tcaaacagaa 60

gttatgaaac?gtttagttaa?tttaggtaaa?gcttctgaag?gttgtggttt?cgacacagtt 120

tggttattag?aacatcactt?tactgaattt?ggtttattag?gtaaccctta?tgttgctgct 180

gcacatctat?taggtgctac?agaaaaatta?aatgttggta?ctgctgctat?tgtattacct 240

actgctcacc?ctgttcgtca?agcagaagac?gtaaatttat?tagatcaaat?gtcaaaagga 300

cgttttcgtt?ttggtatttg?tcgtggttta?tacgacaaag?atttccgtgt?ttttggtaca 360

gacatggata?atagtcgtgc?tttaatggac?tgttggtatg?acttaatgaa?agaaggtttt 420

aacgaaggtt?atattgctgc?agataatgaa?catattaaat?tccctaaaat?tcaattaaat 480

ccatcagctt?acacacaagg?tggtgctcct?gtttatgttg?ttgctgaatc?agcatcaaca 540

acagaatggg?ctgctgaacg?tggtttacca?atgattctaa?gttggattat?taatactcac 600

gaaaaaaaag?cacaacttga?tctttataat?gaagttgcta?ctgaacacgg?ttacgatgta 660

actaaaattg?accattgttt?atcttatatt?acttcagttg?atcacgattc?aaacaaagct 720

aaagatattt?gtcgtaattt?tttaggtcat?tggtatgact?catacgtaaa?tgctacaaaa 780

atttttgatg?actctgatca?aacaaaaggt?tatgacttta?ataaaggtca?atggcgtgat 840

tttgttttaa?aaggtcacaa?agatactaac?cgtcgtattg?attatagtta?cgaaattaat 900

ccagtaggta?cacctgaaga?atgtatcgca?attattcaac?aagatatcga?tgctacaggt 960

attaataata?tttgttgtgg?ttttgaagct?aacggttctg?aagaagaaat?tatcgcttct 1020

atgaaattat?ttcaatctga?tgtaatgcca?tatcttaaag?aaaaacaatc?tggtggtgga 1080

ggttcttcag?gtggtggagg?cggtggttct?tcaatgaaat?ttggattatt?tttccttaat 1140

tttatgaatt?caaaacgttc?ttctgatcaa?gttattgaag?aaatgttaga?tactgcacat 1200

tatgtagatc?aattaaaatt?tgacacatta?gctgtttacg?aaaatcactt?ttcaaacaat 1260

ggtgtagttg?gtgctccatt?aacagtagct?ggttttttac?ttggtatgac?aaaaaacgct 1320

aaagtagctt?cattaaatca?tgttattact?acacaccatc?cagtacgtgt?agctgaagaa 1380

gcatgtttac?ttgatcaaat?gagtgaaggt?cgttttgttt?ttggttttag?tgattgtgaa 1440

aaaagtgctg?atatgcgttt?ttttaatcgt?ccaacagatt?ctcaatttca?attattcagt 1500

gaatgtcaca?aaattatcaa?tgatgcattt?actactggtt?attgtcatcc?aaataatgat 1560

ttttacagtt?ttcctaaaat?ttctgttaac?ccacacgctt?atactgaagg?tggtcctgca 1620

caatttgtaa?atgctacaag?taaagaagta?gttgaatggg?cagctaaatt?aggtcttcca 1680

cttgtattta?aatgggacga?ttcaaatgct?caacgtaaag?aatatgctgg?tttataccat 1740

gaagttgctc?aagcacacgg?tgttgatgtt?agtcaagttc?gtcataaatt?aacactatta 1800

gttaatcaaa?acgtagatgg?tgaagcagct?cgtgcagaag?ctcgtgttta?tttagaagaa 1860

tttgttcgtg?aatcttatcc?taatactgac?ttcgaacaaa?aaatggtaga?attattatca 1920

gaaaacgcta?ttggtactta?cgaagaaagt?actcaagcag?ctcgtgttgc?aattgaatgt 1980

tgtggtgctg?cagacttatt?aatgtctttt?gaatcaatgg?aagataaagc?tcacgaacgt 2040

gcagttattg?atgtagtaaa?tgctaacatt?gttaaatatc?attcataatc?taga 2094

<210>46

<211>698

<212>PRT

＜213〉artificial sequence

<220>

＜223〉LuxAB fusion rotein

<400>46

His?Met?Lys?Phe?Gly?Asn?Phe?Leu?Leu?Thr?Tyr?Gln?Pro?Pro?Glu?Leu

1 5 10 15

Ser?Gln?Thr?Glu?Val?Met?Lys?Arg?Leu?Val?Asn?Leu?Gly?Lys?Ala?Ser

20 25 30

Glu?Gly?Cys?Gly?Phe?Asp?Thr?Val?Trp?Leu?Leu?Glu?His?His?Phe?Thr

35 40 45

Glu?Phe?Gly?Leu?Leu?Gly?Asn?Pro?Tyr?Val?Ala?Ala?Ala?His?Leu?Leu

50 55 60

Gly?Ala?Thr?Glu?Lys?Leu?Asn?Val?Gly?Thr?Ala?Ala?Ile?Val?Leu?Pro

65 70 75 80

Thr?Ala?His?Pro?Val?Arg?Gln?Ala?Glu?Asp?Val?Asn?Leu?Leu?Asp?Gln

85 90 95

Met?Ser?Lys?Gly?Arg?Phe?Arg?Phe?Gly?Ile?Cys?Arg?Gly?Leu?Tyr?Asp

100 105 110

Lys?Asp?Phe?Arg?Val?Phe?Gly?Thr?Asp?Met?Asp?Asn?Ser?Arg?Ala?Leu

115 120 125

Met?Asp?Cys?Trp?Tyr?Asp?Leu?Met?Lys?Glu?Gly?Phe?Asn?Glu?Gly?Tyr

130 135 140

Ile?Ala?Ala?Asp?Asn?Glu?His?Ile?Lys?Phe?Pro?Lys?Ile?Gln?Leu?Asn

145 150 155 160

Pro?Ser?Ala?Tyr?Thr?Gln?Gly?Gly?Ala?Pro?Val?Tyr?Val?Val?Ala?Glu

165 170 175

Ser?Ala?Ser?Thr?Thr?Glu?Trp?Ala?Ala?Glu?Arg?Gly?Leu?Pro?Met?Ile

180 185 190

Leu?Ser?Trp?Ile?Ile?Asn?Thr?His?Glu?Lys?Lys?Ala?Gln?Leu?Asp?Leu

195 200 205

Tyr?Asn?Glu?Val?Ala?Thr?Glu?His?Gly?Tyr?Asp?Val?Thr?Lys?Ile?Asp

210 215 220

His?Cys?Leu?Ser?Tyr?Ile?Thr?Ser?Val?Asp?His?Asp?Ser?Asn?Lys?Ala

225 230 235 240

Lys?Asp?Ile?Cys?Arg?Asn?Phe?Leu?Gly?His?Trp?Tyr?Asp?Ser?Tyr?Val

245 250 255

Asn?Ala?Thr?Lys?Ile?Phe?Asp?Asp?Ser?Asp?Gln?Thr?Lys?Gly?Tyr?Asp

260 265 270

Phe?Asn?Lys?Gly?Gln?Trp?Arg?Asp?Phe?Val?Leu?Lys?Gly?His?Lys?Asp

275 280 285

Thr?Asn?Arg?Arg?Ile?Asp?Tyr?Ser?Tyr?Glu?Ile?Asn?Pro?Val?Gly?Thr

290 295 300

Pro?Glu?Glu?Cys?Ile?Ala?Ile?Ile?Gln?Gln?Asp?Ile?Asp?Ala?Thr?Gly

305 310 315 320

Ile?Asn?Asn?Ile?Cys?Cys?Gly?Phe?Glu?Ala?Asn?Gly?Ser?Glu?Glu?Glu

325 330 335

Ile?Ile?Ala?Ser?Met?Lys?Leu?Phe?Gln?Ser?Asp?Val?Met?Pro?Tyr?Leu

340 345 350

Lys?Glu?Lys?Gln?Ser?Gly?Gly?Gly?Gly?Ser?Ser?Gly?Gly?Gly?Gly?Gly

355 360 365

Gly?Ser?Ser?Met?Lys?Phe?Gly?Leu?Phe?Phe?Leu?Asn?Phe?Met?Asn?Ser

370 375 380

Lys?Arg?Ser?Ser?Asp?Gln?Val?Ile?Glu?Glu?Met?Leu?Asp?Thr?Ala?His

385 390 395 400

Tyr?Val?Asp?Gln?Leu?Lys?Phe?Asp?Thr?Leu?Ala?Val?Tyr?Glu?Asn?His

405 410 415

Phe?Ser?Asn?Asn?Gly?Val?Val?Gly?Ala?Pro?Leu?Thr?Val?Ala?Gly?Phe

420 425 430

Leu?Leu?Gly?Met?Thr?Lys?Asn?Ala?Lys?Val?Ala?Ser?Leu?Asn?His?Val

435 440 445

Ile?Thr?Thr?His?His?Pro?Val?Arg?Val?Ala?Glu?Glu?Ala?Cys?Leu?Leu

450 455 460

Asp?Gln?Met?Ser?Glu?Gly?Arg?Phe?Val?Phe?Gly?Phe?Ser?Asp?Cys?Glu

465 470 475 480

Lys?Ser?Ala?Asp?Met?Arg?Phe?Phe?Asn?Arg?Pro?Thr?Asp?Ser?Gln?Phe

485 490 495

Gln?Leu?Phe?Ser?Glu?Cys?His?Lys?Ile?Ile?Asn?Asp?Ala?Phe?Thr?Thr

500 505 510

Gly?Tyr?Cys?His?Pro?Asn?Asn?Asp?Phe?Tyr?Ser?Phe?Pro?Lys?Ile?Ser

515 520 525

Val?Asn?Pro?His?Ala?Tyr?Thr?Glu?Gly?Gly?Pro?Ala?Gln?Phe?Val?Asn

530 535 540

Ala?Thr?Ser?Lys?Glu?Val?Val?Glu?Trp?Ala?Ala?Lys?Leu?Gly?Leu?Pro

545 550 555 560

Leu?Val?Phe?Lys?Trp?Asp?Asp?Ser?Asn?Ala?Gln?Arg?Lys?Glu?Tyr?Ala

565 570 575

Gly?Leu?Tyr?His?Glu?Val?Ala?Gln?Ala?His?Gly?Val?Asp?Val?Ser?Gln

580 585 590

Val?Arg?His?Lys?Leu?Thr?Leu?Leu?Val?Asn?Gln?Asn?Val?Asp?Gly?Glu

595 600 605

Ala?Ala?Arg?Ala?Glu?Ala?Arg?Val?Tyr?Leu?Glu?Glu?Phe?Val?Arg?Glu

610 615 620

Ser?Tyr?Pro?Asn?Thr?Asp?Phe?Glu?Gln?Lys?Met?Val?Glu?Leu?Leu?Ser

625 630 635 640

Glu?Asn?Ala?Ile?Gly?Thr?Tyr?Glu?Glu?Ser?Thr?Gln?Ala?Ala?Arg?Val

645 650 655

Ala?Ile?Glu?Cys?Cys?Gly?Ala?Ala?Asp?Leu?Leu?Met?Ser?Phe?Glu?Ser

660 665 670

Met?Glu?Asp?Lys?Ala?His?Glu?Arg?Ala?Val?Ile?Asp?Val?Val?Asn?Ala

675 680 685

Asn?Ile?Val?Lys?Tyr?His?Ser?Glx?Ser?Arg

690 695

<210>47

<211>1893

<212>DNA

＜213〉artificial sequence

<220>

＜223〉single-chain antibody

<400>47

atggttgctc?aagctgcttc?atcagaatta?acgcaatcac?caggtacctt?atcattatca 60

ccaggtgaac?gtgctacctt?atcatgtcgt?gcttcacaat?cagtttcatc?agcttactta 120

gcttggtacc?aacaaaaacc?aggtcaagct?ccacgtttat?taatttacgg?tgcttcatca 180

cgtgctactg?gtattccaga?tcgtttctca?ggttcaggtt?caggtacaga?tttcacttta 240

accatttcac?gtttagaacc?agaagatttc?gctgtttact?actgtcaaca?atacggtcgt 300

tcaccaactt?tcggtggtgg?taccaaagtt?gaaattaaac?gtacttcatc?aggtggtggt 360

ggttcaggtg?gtggtggtgg?tggttcatca?cgttcatcat?tagaacaatc?aggtgctgaa 420

gttaaaaaac?caggttcatc?agttaaagtt?tcatgtaaag?cttcaggtgg?ttcattctca 480

tcatacgcta?ttaactgggt?tcgtcaagct?caaggtcaag?gtttagaatg?gatgggtggt 540

ttaatgccaa?ttttcggtac?aacaaactac?gctcaaaaat?tccaagatcg?tttaacgatt 600

accgctgatg?tttcaacgtc?aacagcttac?atgcaattat?caggtttaac?atacgaagat 660

acggctatgt?actactgtgc?tcgtgttgct?tacatgttag?aaccaaccgt?tactgctggt 720

ggtttagatg?tttggggtaa?aggtaccacg?gttaccgttt?caccagcttc?accaacctca 780

ccaaaagttt?tcccattatc?attatgttca?acccaaccag?atggtaacgt?tgttattgct 840

tgtttagttc?aaggtttctt?cccacaagaa?ccattatcag?ttacctggtc?agaatcaggt 900

caaggtgtta?ccgctcgtaa?cttcccacca?tcacaagatg?cttcaggtga?tttatacacc 960

acgtcatcac?aattaacctt?accagctaca?caatgtttag?ctggtaaatc?agttacatgt 1020

cacgttaaac?actacacgaa?cccatcacaa?gatgttactg?ttccatgtcc?agttccatca 1080

actccaccaa?ccccatcacc?atcaactcca?ccaaccccat?caccatcatg?ttgtcaccca 1140

cgtttatcat?tacaccgtcc?agctttagaa?gatttattat?taggttcaga?agctaactta 1200

acgtgtacat?taaccggttt?acgtgatgct?tcaggtgtta?ccttcacctg?gacgccatca 1260

tcaggtaaat?cagctgttca?aggtccacca?gaacgtgatt?tatgtggttg?ttactcagtt 1320

tcatcagttt?taccaggttg?tgctgaacca?tggaaccacg?gtaaaacctt?cacttgtact 1380

gctgcttacc?cagaatcaaa?aaccccatta?accgctacct?tatcaaaatc?aggtaacaca 1440

ttccgtccag?aagttcactt?attaccacca?ccatcagaag?aattagcttt?aaacgaatta 1500

gttacgttaa?cgtgtttagc?tcgtggtttc?tcaccaaaag?atgttttagt?tcgttggtta 1560

caaggttcac?aagaattacc?acgtgaaaaa?tacttaactt?gggcttcacg?tcaagaacca 1620

tcacaaggta?ccaccacctt?cgctgttacc?tcaattttac?gtgttgctgc?tgaagattgg 1680

aaaaaaggtg?ataccttctc?atgtatggtt?ggtcacgaag?ctttaccatt?agctttcaca 1740

caaaaaacca?ttgatcgttt?agctggtaaa?ccaacccacg?ttaacgtttc?agttgttatg 1800

gctgaagttg?atggtacctg?ttacgattat?aaagatcacg?atggtgatta?caaagatcac 1860

gatattgatt?ataaagatga?tgatgataaa?taa 1893

<210>48

<211>630

<212>PRT

＜213〉artificial sequence

<220>

＜223〉single-chain antibody

<400>48

Met?Val?Ala?Gln?Ala?Ala?Ser?Ser?Glu?Leu?Thr?Gln?Ser?Pro?Gly?Thr

1 5 10 15

Leu?Ser?Leu?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser

20 25 30

Gln?Ser?Val?Ser?Ser?Ala?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly

35 40 45

Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Ser?Arg?Ala?Thr?Gly

50 55 60

Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu

65 70 75 80

Thr?Ile?Ser?Arg?Leu?Glu?Pro?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln

85 90 95

Gln?Tyr?Gly?Arg?Ser?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile

100 105 110

Lys?Arg?Thr?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly

115 120 125

Ser?Ser?Arg?Ser?Ser?Leu?Glu?Gln?Ser?Gly?Ala?Glu?Val?Lys?Lys?Pro

130 135 140

Gly?Ser?Ser?Val?Lys?Val?Ser?Cys?Lys?Ala?Ser?Gly?Gly?Ser?Phe?Ser

145 150 155 160

Ser?Tyr?Ala?Ile?Asn?Trp?Val?Arg?Gln?Ala?Gln?Gly?Gln?Gly?Leu?Glu

165 170 175

Trp?Met?Gly?Gly?Leu?Met?Pro?Ile?Phe?Gly?Thr?Thr?Asn?Tyr?Ala?Gln

180 185 190

Lys?Phe?Gln?Asp?Arg?Leu?Thr?Ile?Thr?Ala?Asp?Val?Ser?Thr?Ser?Thr

195 200 205

Ala?Tyr?Met?Gln?Leu?Ser?Gly?Leu?Thr?Tyr?Glu?Asp?Thr?Ala?Met?Tyr

210 215 220

Tyr?Cys?Ala?Arg?Val?Ala?Tyr?Met?Leu?Glu?Pro?Thr?Val?Thr?Ala?Gly

225 230 235 240

Gly?Leu?Asp?Val?Trp?Gly?Lys?Gly?Thr?Thr?Val?Thr?Val?Ser?Pro?Ala

245 250 255

Ser?Pro?Thr?Ser?Pro?Lys?Val?Phe?Pro?Leu?Ser?Leu?Cys?Ser?Thr?Gln

260 265 270

Pro?Asp?Gly?Asn?Val?Val?Ile?Ala?Cys?Leu?Val?Gln?Gly?Phe?Phe?Pro

275 280 285

Gln?Glu?Pro?Leu?Ser?Val?Thr?Trp?Ser?Glu?Ser?Gly?Gln?Gly?Val?Thr

290 295 300

Ala?Arg?Asn?Phe?Pro?Pro?Ser?Gln?Asp?Ala?Ser?Gly?Asp?Leu?Tyr?Thr

305 310 315 320

Thr?Ser?Ser?Gln?Leu?Thr?Leu?Pro?Ala?Thr?Gln?Cys?Leu?Ala?Gly?Lys

325 330 335

Ser?Val?Thr?Cys?His?Val?Lys?His?Tyr?Thr?Asn?Pro?Ser?Gln?Asp?Val

340 345 350

Thr?Val?Pro?Cys?Pro?Val?Pro?Ser?Thr?Pro?Pro?Thr?Pro?Ser?Pro?Ser

355 360 365

Thr?Pro?Pro?Thr?Pro?Ser?Pro?Ser?Cys?Cys?His?Pro?Arg?Leu?Ser?Leu

370 375 380

His?Arg?Pro?Ala?Leu?Glu?Asp?Leu?Leu?Leu?Gly?Ser?Glu?Ala?Asn?Leu

385 390 395 400

Thr?Cys?Thr?Leu?Thr?Gly?Leu?Arg?Asp?Ala?Ser?Gly?Val?Thr?Phe?Thr

405 410 415

Trp?Thr?Pro?Ser?Ser?Gly?Lys?Ser?Ala?Val?Gln?Gly?Pro?Pro?Glu?Arg

420 425 430

Asp?Leu?Cys?Gly?Cys?Tyr?Ser?Val?Ser?Ser?Val?Leu?Pro?Gly?Cys?Ala

435 440 445

Glu?Pro?Trp?Asn?His?Gly?Lys?Thr?Phe?Thr?Cys?Thr?Ala?Ala?Tyr?Pro

450 455 460

Glu?Ser?Lys?Thr?Pro?Leu?Thr?Ala?Thr?Leu?Ser?Lys?Ser?Gly?Asn?Thr

465 470 475 480

Phe?Arg?Pro?Glu?Val?His?Leu?Leu?Pro?Pro?Pro?Ser?Glu?Glu?Leu?Ala

485 490 495

Leu?Asn?Glu?Leu?Val?Thr?Leu?Thr?Cys?Leu?Ala?Arg?Gly?Phe?Ser?Pro

500 505 510

Lys?Asp?Val?Leu?Val?Arg?Trp?Leu?Gln?Gly?Ser?Gln?Glu?Leu?Pro?Arg

515 520 525

Glu?Lys?Tyr?Leu?Thr?Trp?Ala?Ser?Arg?Gln?Glu?Pro?Ser?Gln?Gly?Thr

530 535 540

Thr?Thr?Phe?Ala?Val?Thr?Ser?Ile?Leu?Arg?Val?Ala?Ala?Glu?Asp?Trp

545 550 555 560

Lys?Lys?Gly?Asp?Thr?Phe?Ser?Cys?Met?Val?Gly?His?Glu?Ala?Leu?Pro

565 570 575

Leu?Ala?Phe?Thr?Gln?Lys?Thr?Ile?Asp?Arg?Leu?Ala?Gly?Lys?Pro?Thr

580 585 590

His?Val?Asn?Val?Ser?Val?Val?Met?Ala?Glu?Val?Asp?Gly?Thr?Cys?Tyr

595 600 605

Asp?Tyr?Lys?Asp?His?Asp?Gly?Asp?Tyr?Lys?Asp?His?Asp?Ile?Asp?Tyr

610 615 620

Lys?Asp?Asp?Asp?Asp?Lys

625 630

Claims (206)

1. method of producing polypeptide in plastid, described method comprise to plastid and import first recombinant nucleic acid molecules,

Wherein first recombinant nucleic acid molecules comprises

First polynucleotide, its at least a polypeptide of encoding,

Effective with it second polynucleotide that connects, it comprises first nucleotide sequence of coding first ribosome binding sequence (RBS), and second nucleotide sequence of coding the 2nd RBS that effectively connects with it,

Wherein a RBS can instruct the translation of polypeptide in prokaryotic organism, and the 2nd RBS can instruct the translation of polypeptide in plastid,

Under the condition that allows described at least a expression of polypeptides, thereby in plastid, produce described polypeptide.

2. the process of claim 1 wherein a kind of first polypeptide of the first polymerized nucleoside acid encoding and at least a second polypeptide.

3. the method for claim 2, wherein said first polypeptide and at least the second polypeptide constitute fusion rotein.

4. the method for claim 3, wherein fusion rotein comprises single-chain antibody.

5. the method for claim 4, wherein first polynucleotide comprises the nucleotide sequence of SEQ ID NO:13 or coding SEQ ID NO:14.

6. the process of claim 1 wherein that plastid is a chloroplast(id).

7. the method for claim 6, wherein the codon of first polynucleotide codon that tended to show chloroplast(id) uses.

8. the method for claim 7, the wherein first polymerized nucleoside acid encoding reporter protein or its mutant or varient.

9. the method for claim 8, wherein reporter protein is a kind of green fluorescent protein.

10. the method for claim 9, wherein first polynucleotide comprises SEQ ID NO:15, the nucleotide sequence of the nucleotide sequence of coding SEQ ID NO:1 or coding SEQ ID NO:2.

11. the method for claim 7, wherein a kind of first polypeptide of the first polynucleotide sequence encoding and at least a second polypeptide.

12. the method for claim 11, wherein said first polypeptide and at least the second polypeptide constitute fusion rotein.

13. the method for claim 11, it plays the subunit that described first polypeptide and second polypeptide comprise albumen composition.

14. the method for claim 13, wherein albumen composition is a heterodimer.

15. the method for claim 13, wherein albumen composition comprises reporter protein.

16. the method for claim 15, wherein reporter protein comprises luciferase or its mutant or varient.

17. the method for claim 16, wherein luciferase comprises the product of bacterium luxAB gene.

18. the method for claim 17, wherein first polynucleotide comprises the nucleotide sequence of SEQ ID NO:45 or coding SEQ ID NO:46.

19. the method for claim 11, wherein first polypeptide comprises heavy chain or its variable region of immunoglobulin (Ig), and second polypeptide comprises light chain or its variable region of immunoglobulin (Ig).

20. the method for claim 19, wherein first polypeptide and second polypeptide constitute fusion rotein, can produce single-chain antibody thus.

21. the method for claim 20, wherein first polynucleotide comprises SEQ ID NO:15; The nucleotide sequence of coding SEQ ID NO:16; SEQ ID NO:42; The nucleotide sequence of coding SEQ ID NO:43; SEQ ID NO:47; Perhaps the encode nucleotide sequence of SEQ ID NO:48.

22. the method for claim 1 further comprises importing at least a second recombinant nucleic acid molecules in plastid.

23. the method for claim 22, wherein plastid is a chloroplast(id).

24. the method for claim 23, wherein second recombinant nucleic acid molecules comprises first polynucleotide and second polynucleotide that effectively is connected with it, the first polynucleotide at least a polypeptide of encoding, second polynucleotide comprise effective connection coding first ribosome binding sequence (RBS) first nucleotide sequence and the coding the 2nd RBS second nucleotide sequence, wherein a RBS can instruct the translation of described polypeptide in prokaryotic organism, and the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).

25. the method for claim 24, wherein first recombinant nucleic acid molecules and second recombinant nucleic acid molecules coexpression in chloroplast(id).

26. the process of claim 1 wherein that first recombinant nucleic acid molecules is comprised in the carrier.

27. the method for claim 26, wherein plastid is a chloroplast(id).

28. the method for claim 27, wherein carrier is a kind of chloroplast(id) carrier, and this carrier contains one section nucleotide sequence of chloroplast gene group thymus nucleic acid (DNA), it can with chloroplast genomic dna generation homologous recombination.

29. the method for claim 28, wherein carrier further comprises procaryotic replication origin.

30. the method for claim 1 further comprises and isolate polypeptide from plastid.

31. the isolated polypeptide that obtains according to the method for claim 30.

32. the isolated polypeptide of claim 31 comprises SEQ ID NO:2, SEQ ID NO:16, SEQ ID NO:43, SEQ ID NO:46, perhaps SEQ ID NO:48.

33. an isolated ribonucleoside acid sequence comprises first ribosome binding sequence (RBS) and the 2nd RBS that effectively is connected with it, wherein a RBS and the 2nd RBS are separated by about 5 to 25 Nucleotide.

34. this ribonucleoside acid sequence of claim 33, wherein a RBS and the 2nd RBS are separated by about 10 to 20 Nucleotide.

35. this ribonucleoside acid sequence of claim 33, wherein a RBS and the 2nd RBS are separated by about 15 Nucleotide.

36. this ribonucleoside acid sequence of claim 33, wherein each among a RBS and the 2nd RBS all is to be made up of 3 to 9 Nucleotide independently.

37. this ribonucleoside acid sequence of claim 33, wherein each among a RBS and the 2nd RBS all is to be made up of 4 to 7 Nucleotide independently.

38. the ribonucleoside acid sequence of claim 33, wherein a RBS or the 2nd RBS or the two all contain GGAG.

39. the ribonucleoside acid sequence of claim 33, wherein the 2nd RBS further comprises a kind of 5 ' end non-translational region (5 ' UTR) of chloroplast gene.

40. the ribonucleoside acid sequence of claim 39, wherein 5 ' UTR is by listed nucleotide sequence coded of among the SEQ ID NO:4 to 8 any one.

41. the ribonucleoside acid sequence of claim 39, wherein chloroplast gene coding soluble proteins.

42. the ribonucleoside acid sequence of claim 33, it effectively is connected with initial AUG codon.

43. the ribonucleoside acid sequence of claim 42, wherein initial AUG codon further comprises the Kozak sequence.

44. the ribonucleoside acid sequence of claim 43, the initial AUG codon that wherein further comprises the Kozak sequence is ACCAUGG.

45. the ribonucleoside acid sequence of claim 33, it effectively is connected with the polynucleotide of coded polypeptide.

46. the ribonucleoside acid sequence of claim 45, wherein polynucleotide comprises initial AUG codon.

47. the ribonucleoside acid sequence of claim 33, it is made up of about 11 to 50 ribonucleotides.

48. the ribonucleoside acid sequence of claim 33, it is made up of about 15 to 40 ribonucleotides.

49. the ribonucleoside acid sequence of claim 33, it is made up of about 20 to 30 ribonucleotides.

50. the ribonucleotide of claim 33 further comprises the polynucleotide of coding one peptide species of effective connection, a RBS can instruct the translation of described polypeptide in prokaryotic organism thus, and the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).

51. the polynucleotide of the ribonucleoside acid sequence in the coding claim 33.

52. the polynucleotide of claim 51, it comprises the initial ATG codon that the nucleotides sequence that effectively is connected to coding the one RBS and the 2nd RBS lists.

53. the polynucleotide of claim 51, it comprises cloning site, and the location in described site makes effable polynucleotide can effectively be connected with the 2nd RBS with a RBS.

54. the polynucleotide of claim 53, wherein cloning site comprises at least one restriction enzyme enzyme recognition site, perhaps at least one recombinase recognition site, the perhaps combination of the two.

55. the polynucleotide of claim 51, its both sides have first cloning site and second cloning site respectively.

56. the polynucleotide of claim 55, wherein first cloning site is different with second cloning site.

57. the polynucleotide of claim 51, it effectively is connected with effable polynucleotide.

58. the polynucleotide of claim 57, wherein effable polynucleotide coding at least the first polypeptide.

59. the polynucleotide of claim 58, wherein effable polymerized nucleoside acid encoding first polypeptide and at least a second polypeptide.

60. the polynucleotide of claim 59, wherein effable polymerized nucleoside acid encoding first polypeptide and a kind of second polypeptide.

61. the polynucleotide of claim 60, wherein first polypeptide is different with second polypeptide.

62. the polynucleotide of claim 60, wherein first polypeptide and second polypeptide constitute fusion rotein.

63. the polynucleotide of claim 62, wherein effable polynucleotide is included in SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:42, SEQ ID NO:45, perhaps listed nucleotide sequence among the SEQ IDNO:47.

64. the polynucleotide of claim 60 further comprises the nucleotide sequence of the inherent ribosome entry site(RES) of encoding, it effectively is connected between the encoding sequence of the encoding sequence of first polypeptide and second polypeptide.

65. the polynucleotide of claim 51, it is a duplex molecule.

66. the polynucleotide of claim 65, it comprises the nucleotide sequence of the 2nd RBS that encodes, the nucleotide sequence of the RBS that encodes and initial ATG codon, and these sequences effectively connect with 5 ' to 3 ' direction; Or with described polynucleotide complementary nucleotide sequence.

67. the polynucleotide of claim 66 further comprises 3 ' at least one cloning site held that is positioned at initial ATG codon.

68. the polynucleotide of claim 65, it comprises the nucleotide sequence of the 2nd RBS that encodes, the encode nucleotide sequence of a RBS, with at least one cloning site of about 3 to 10 nucleotide positions of nucleotide sequence 3 ' end that are positioned at coding the one RBS, these sequences connect with 5 ' to 3 ' direction; Perhaps with described polynucleotide complementary nucleotide sequence.

69. the polynucleotide of claim 65, it has at least one cloning site in each side.

70. carrier comprises the polynucleotide of claim 51 and one section nucleotide sequence of chloroplast gene group thymus nucleic acid (DNA), wherein said nucleotide sequence can and chloroplast genomic dna between homologous recombination takes place.

71. the carrier of claim 70 further comprises cloning site, the location in described site allows at least a allos polynucleotide effectively to be connected with the 2nd RBS with a RBS.

72. the carrier of claim 70 further comprises procaryotic replication origin.

73. the carrier of claim 72, wherein replication origin is colibacillary replication origin.

74. the carrier of claim 70, wherein the nucleotide sequence of chloroplast genomic dna comprises first terminal and second end.

75. the carrier of claim 74, wherein first terminal or second terminal or the two all comprise at least one cloning site, perhaps it shears product.

76. the carrier of claim 70, it is the molecule of cyclisation.

77. the carrier of claim 70 further comprises the initial ATG codon that effectively is connected with a RBS and the 2nd RBS.

78. the carrier of claim 77 further comprises cloning site, its location allows at least a allos polynucleotide effectively to be connected with the ATG codon.

79. the carrier of claim 72 further comprises the effable polynucleotide that effectively is connected with a RBS and the 2nd RBS.

80. the carrier of claim 79, wherein effable polynucleotide comprises SEQ ID NO:1, the nucleotide sequence of coding SEQ ID NO:2, SEQ ID NO:45, the nucleotide sequence of coding SEQ ID NO:46, perhaps its combination.

81. cell contains the polynucleotide in the claim 51.

82. the cell of claim 81, it is a kind of vegetable cell.

83. the vegetable cell of claim 82, wherein said polynucleotide is in chloroplast(id).

84. the vegetable cell of claim 83, wherein said polynucleotide effectively is connected with effable polynucleotide.

85. the vegetable cell of claim 83, wherein effable polynucleotide coding at least the first polypeptide.

86. the vegetable cell of claim 85, wherein effable polymerized nucleoside acid encoding first polypeptide and at least a second polypeptide.

87. the vegetable cell of claim 85, wherein effable polymerized nucleoside acid encoding first polypeptide and a kind of second polypeptide.

88. the vegetable cell of claim 87, wherein first polypeptide is different with second polypeptide.

89. the vegetable cell of claim 87, wherein first polypeptide and second polypeptide constitute fusion rotein.

90. the vegetable cell of claim 84, wherein effable polynucleotide comprises SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47 or the represented nucleotide sequence of its combination.

91. transgenic plant comprise the vegetable cell in the claim 83.

92. the vegetable cell or the tissue that obtain by the transgenic plant of claim 81.

93. the grafting part of the transgenic plant of claim 92.

94. the seed that the transgenic plant of claim 92 produce.

95. by the transgenic plant of claim 91, the perhaps cDNA or the chloroplast genomic dna library of preparing by vegetable cell that comes from described transgenic plant or plant tissue.

96. the transgenic plant of claim 91, wherein said plant is to belong to algae.

97. the transgenic plant of claim 91, wherein said plant is an angiosperm.

98. the transgenic plant of claim 97, wherein said angiosperm is a cereal grass, bean, oleaginous seed plant, perhaps palohierror.

99. the transgenic plant of claim 91, wherein said plant is an ornamental plant.

100. composition comprises the vegetable material that obtains in the transgenic plant of Accessory Right requirement 91.

101. the composition of claim 100, wherein the polynucleotide in the chloroplast(id) effectively is connected with effable polynucleotide.

102. being tended to the codon of chloroplast(id), the composition of claim 101, wherein effable polynucleotide use.

103. the composition of claim 101, wherein effable polynucleotide encoding antibody, perhaps its antigen binding fragment is disconnected.

104. the composition of claim 103, it is to be suitable for being administered to individual form and to exist with a kind of.

105. the composition of claim 104, wherein said individuality is a Mammals.

106. the composition of claim 104, wherein said individuality is the people.

107. chloroplast(id)/prokaryotic organism shuttle vectors,

One section nucleotide sequence of chloroplast genomic dna, it can with chloroplast genomic dna generation homologous recombination;

The prokaryotic organism initial point; With

First ribosome binding sequence (RBS) that effectively is connected with the 2nd RBS, wherein a RBS can instruct the translation of effable polynucleotide in chloroplast(id) of effective connection, and the 2nd RBS can instruct the translation of effable polynucleotide in prokaryotic organism of effective connection.

108. the shuttle vectors of claim 107 further comprises cloning site, the location of wherein said cloning site makes the allos polynucleotide to be inserted in the carrier, and effectively is connected with the 2nd RBS with a RBS.

109. the shuttle vectors of claim 107 further comprises the effable polynucleotide of effective connection.

110. the shuttle vectors of claim 109, wherein effable polynucleotide comprise the polynucleotide with chloroplast(id) codon deflection.

The codon that the isolated polynucleotide of a kind of albumen or its mutant or varient 111. encode, the codon of wherein said polynucleotide are tended to show chloroplast(id) uses.

112. the polynucleotide of claim 111, it comprises the deoxyribonucleotide sequence.

113. being tended to, the polynucleotide of claim 111, wherein said codon contain VITAMIN B4 or thymus pyrimidine at the 3rd nucleotide position.

114. the polynucleotide of claim 111, its both sides have first cloning site and second cloning site respectively.

115. the polynucleotide of claim 111, wherein said albumen comprises fusion rotein.

116. the polynucleotide of claim 111, wherein said albumen is reporter protein.

117. the polynucleotide of claim 116, wherein reporter protein is green fluorescent protein or luciferase.

118. the polynucleotide of claim 117 comprises SEQ ID NO:1, the nucleotide sequence of coding SEQ IDNO:2, SEQ ID NO:45, the nucleotide sequence of the SEQ ID NO:46 that perhaps encodes.

119. the polynucleotide of claim 111, wherein said albumen comprise that the antigen binding fragment of antibody or antibody is disconnected.

120. the polynucleotide of claim 119 comprises SEQ ID NO:15, the nucleotide sequence of coding SEQ IDNO:16, SEQ ID NO:42, the nucleotide sequence of coding SEQ ID NO:43, SEQ ID NO:47, the nucleotide sequence of coding SEQ ID NO:48.

121. the polynucleotide of claim 111, its polynucleotide with coding first ribosome binding sequence (RBS) and the 2nd RBS effectively is connected, wherein a RBS and the 2nd RBS are separated by about 5 to 25 Nucleotide, wherein a RBS instructs the translation of fluorescin in prokaryotic organism, and the 2nd RBS instructs the translation of fluorescin in chloroplast(id).

122. polynucleotide encoded polypeptides by claim 111.

123. recombinant nucleic acid molecules comprises

Encode first polynucleotide of at least a polypeptide, wherein the codon of first polynucleotide codon that tended to show chloroplast(id) uses; With

Second polynucleotide, it comprises the nucleotide sequence of coding first ribosome binding sequence (RBS) and the nucleotide sequence of coding the 2nd RBS that effectively is connected, wherein a RBS can instruct the translation of described polypeptide in prokaryotic organism, and the 2nd RBS can instruct the translation of described polypeptide in chloroplast(id).

124. the recombinant nucleic acid molecules of claim 123, wherein first polynucleotide comprises first nucleotide sequence of first polypeptide of encoding, and this sequence then has second nucleotide sequence of coding second polypeptide that effectively connects with it.

125. the recombinant nucleic acid molecules of claim 124, the nucleotide sequence of the inherent ribosome entry site(RES) of wherein encoding effectively are connected to second nucleotides sequence of coding second polypeptide and list.

126. the recombinant nucleic acid molecules of claim 123 further comprises the 3rd polynucleotide that effectively is connected with first polynucleotide and second polynucleotide.

127. the recombinant nucleic acid molecules of claim 126, wherein the 3rd polynucleotide at least a polypeptide of encoding.

128. comprising to the nucleotide sequence that is enough to one section chloroplast gene group thymus nucleic acid (DNA) of chloroplast genomic dna generation homologous recombination, a method for preparing chloroplast(id)/prokaryotic organism shuttle expression carrier, described method import

The nucleotide sequence that includes the prokaryotic organism replication origin,

The encode nucleotide sequence of first ribosome binding sequence (RBS) and the nucleotide sequence of the 2nd RBS that encodes,

Wherein a RBS and the 2nd RBS are separated by about 5 to 25 Nucleotide, and

Cloning site, the location of wherein said cloning site allows the polynucleotide of coded polypeptide effectively to be connected on a RBS and the 2nd RBS, a RBS can instruct the translation of polypeptide in prokaryotic organism thus, and the 2nd RBS can instruct the translation of polypeptide in chloroplast(id).

129. chloroplast(id)/prokaryotic organism shuttle expression carrier that the method by claim 128 prepares.

130. method for preparing chloroplast(id)/prokaryotic organism shuttle expression carrier, described method comprises carries out genetic modification to being enough to the nucleotide sequence of one section chloroplast gene group thymus nucleic acid (DNA) of chloroplast genomic dna generation homologous recombination, contains making it

The prokaryotic organism replication origin,

The encode nucleotide sequence of first ribosome binding sequence (RBS) and the nucleotide sequence of the 2nd RBS that encodes, a RBS and the 2nd RBS are separated by about 5 to 25 Nucleotide, and

Cloning site, the location of wherein said cloning site allows the polynucleotide of coded polypeptide effectively to be connected on a RBS and the 2nd RBS, a RBS can instruct the translation of polypeptide in prokaryotic organism thus, and the 2nd RBS can instruct the translation of polypeptide in chloroplast(id).

131. chloroplast(id)/prokaryotic organism shuttle expression carrier that the method by claim 130 prepares.

132. reorganization polynucleotide, first nucleotide sequence that comprises coding chloroplast ribosome binding sequence (RBS), and second nucleotide sequence of the coded polypeptide that effectively connects with it, wherein first nucleotide sequence is relative allogenic with second nucleotide sequence.

133. the reorganization polynucleotide of claim 132, wherein chloroplast(id) RBS is positioned at the position of 20 to 40 Nucleotide of initial ATG codon 5 ' end, and initiator codon effectively is connected with the nucleotide sequence of coded polypeptide.

134. the reorganization polynucleotide of claim 132, wherein first nucleotide sequence comprises the ATG codon of about 20 to 40 nucleotide positions of 3 ' end that are positioned at RBS.

135. carrier, it comprises the nucleotide sequence that coding is positioned at the ribosome binding sequence (RBS) of about 20 to 40 nucleotide positions of cloning site 5 ' end.

136. the carrier of claim 135, wherein cloning site comprises at least one restriction enzyme enzyme recognition site or at least one recombinase recognition site, perhaps its combination.

137. the carrier of claim 135, wherein cloning site comprises multiple clone site, described multiple clone site is made up of a plurality of restriction enzyme enzyme recognition sites or a plurality of recombinase recognition site, perhaps is made up of the combination of at least one restriction enzyme enzyme recognition site and at least one recombinase recognition site.

138. the carrier of claim 134 further comprises initial ATG codon or its part, it is positioned at 5 ' end and adjacent with cloning site of cloning site.

139. the carrier of claim 135 further comprises chloroplast gene 3 ' the end non-translational region that is positioned at cloning site 3 ' end.

140. a method of producing polypeptide in plastid comprises importing at least the first recombinant nucleic acid molecules in plastid,

Described first recombinant nucleic acid molecules comprises first nucleotide sequence of at least one ribosome binding sequence of coding (RBS), and at least a allos polynucleotide of at least a polypeptide of coding that effectively connects with it,

Wherein said RBS instructs the translation of described polypeptide in plastid, under the condition that allows described at least a expression of polypeptides, produces polypeptide thus in plastid.

141. the method for claim 140, wherein plastid is a chloroplast(id).

142. the method for claim 141, wherein the codon of first polynucleotide is tended to show the codon use of chloroplast(id).

143. the method for claim 140, the wherein first polynucleotide encoding antibody, the perhaps subunit of antibody.

144. the method for claim 143, wherein antibodies specific ground is in conjunction with tetanus toxin or hsv.

145. the method for claim 140, the wherein first polymerized nucleoside acid encoding, first polypeptide, and second polypeptide that can select.

146. the method for claim 145, wherein first polynucleotide is tended to the codon use of chloroplast(id).

147. the method for claim 146, wherein first polypeptide comprises heavy chain immunoglobulin or its variable region, and second polypeptide comprises light chain immunoglobulin or its variable region.

148. the method for claim 147, wherein antibody is included in SEQ ID NO:16, SEQ IDNO:43, perhaps listed aminoacid sequence among the SEQ ID NO:48.

149. the method for claim 147, wherein first polynucleotide is included in SEQ IDNO:15, SEQ ID NO:42, perhaps listed nucleotide sequence among the SEQ ID NO:47.

150. the method for claim 146, wherein allos polymerized nucleoside acid encoding reporter protein.

151. the method for claim 150, wherein reporter protein comprises green fluorescent protein or luciferase.

152. the method for claim 151, wherein the allos polynucleotide comprises SEQ ID NO:1, the nucleotide sequence of coding SEQ ID NO:2, SEQ ID NO:45, the nucleotide sequence of the SEQ IDNO:46 that perhaps encodes.

153. the method for claim 150, the wherein first polymerized nucleoside acid encoding, first polypeptide and at least a second polypeptide.

154. the method for claim 153, wherein first polypeptide and second polypeptide comprise the subunit of albumen composition.

155. the method for claim 154, wherein albumen composition is a heterodimer.

156. the method for claim 150 further comprises importing at least a second recombinant nucleic acid molecules in plastid.

157. the method for claim 156, wherein second recombinant nucleic acid molecules comprise effective connection coding at least the one RBS first nucleotide sequence and the coding at least a second polypeptide the second allos polynucleotide, wherein a RBS can instruct the translation of described polypeptide in chloroplast(id).

158. the method for claim 157, wherein first recombinant nucleic acid molecules and second recombinant nucleic acid molecules coexpression in chloroplast(id).

159. the method for claim 140, wherein first recombinant nucleic acid molecules is comprised in the carrier.

160. the method for claim 159, wherein carrier is the chloroplast(id) carrier, and it comprises the nucleotide sequence of one section chloroplast gene group thymus nucleic acid (DNA), this section sequence can with chloroplast genomic dna generation homologous recombination.

161. the method for claim 160, wherein carrier further comprises the prokaryotic organism replication origin.

162. the method for claim 140 further comprises and separate described polypeptide from plastid.

163. pass through the polypeptide separated that the method for claim 162 obtains.

164. the polypeptide separated of claim 163, it is a kind of antibody or reporter protein.

165. the synthetic polynucleotide comprises at least the first nucleotide sequence of coding at least the first polypeptide, the codon that wherein has at least a codon to be tended to show chloroplast(id) in first nucleotide sequence uses.

166. the polynucleotide of claim 165, wherein each codon of first nucleotide sequence is all tended to show the codon use of chloroplast(id).

167. the polynucleotide of claim 165, wherein polynucleotide further comprises at least the second nucleotide sequence of second polypeptide of encoding.

168. the polynucleotide of claim 167, wherein at least one codon of second nucleotide sequence is tended to show the codon use of chloroplast(id).

169. the polynucleotide of claim 167, wherein first nucleotide sequence effectively is connected with second nucleotide sequence.

170. the polynucleotide of claim 169, its coding comprises the fusion rotein of first polypeptide and second polypeptide.

171. the polynucleotide of claim 169, wherein first nucleotide sequence effectively is connected with second nucleotide sequence by the trinucleotide sequence.

172. the polynucleotide of claim 171, wherein a kind of connection peptides of trinucleotide sequence encoding.

173. the polynucleotide of claim 172, its coding comprise the fusion rotein of first polypeptide that is connected with second polypeptide by connection peptides.

174. the polynucleotide of claim 165, wherein first polypeptide comprises immune globulin variable region, constant region for immunoglobulin, the perhaps combination of the two.

175. the polynucleotide of claim 167, its single-chain antibody of encoding, described antibody comprises the variable region of heavy chain and the variable region of light chain of effective connection.

176. the polynucleotide of claim 175, wherein single-chain antibody has the IDNO:16 at SEQ, SEQ ID NO:43, perhaps listed aminoacid sequence among the SEQ ID NO:48.177. the polynucleotide of claim 175, it has the NO:15 at SEQ ID, SEQ IDNO:42, perhaps listed nucleotide sequence among the SEQ ID NO:47.

178. the polynucleotide of claim 165, its coding report polypeptide.

179. the polynucleotide of claim 178 reports that wherein polypeptide is a luciferase.

180. the polynucleotide of claim 179, wherein luciferase has the listed aminoacid sequence at SEQ IDNO:46.

181. the polynucleotide of claim 180, it has listed nucleotide sequence in SEQ ID NO:45.

182. polypeptide, it is included in SEQ ID NO:16, SEQ ID NO:43, SEQ ID NO:46, perhaps listed aminoacid sequence among the SEQ ID NO:48.

183. a method of producing heterologous polypeptide in plastid, described method are included in the synthetic polynucleotide that imports claim 165 at least in plastid under the condition that permission the first polypeptide expresses to plastid.

184. the method for claim 183, wherein the synthetic polynucleotide effectively is connected with the nucleotide sequence of at least a ribosome binding sequence of coding (RBS).

185. the method for claim 184, wherein RBS can instruct the translation of described polypeptide in plastid.

186. the method for claim 184, wherein said polynucleotide further comprise at least the second nucleotide sequence of second polypeptide of encoding.

187. the method for claim 186, wherein first nucleotide sequence effectively is connected with second nucleotide sequence.

188. the method for claim 187, wherein heterologous polypeptide comprises fusion rotein, and described fusion polypeptide comprises first polypeptide and second polypeptide.

189. the method for claim 187, wherein first nucleotide sequence effectively is connected with second nucleotide sequence by the trinucleotide sequence.

190. the method for claim 189, wherein a kind of connection peptides of trinucleotide sequence encoding.

191. the method for claim 190, wherein heterologous polypeptide comprises and contains first polypeptide that connects by connection peptides and the fusion rotein of second polypeptide.

192. the method for claim 183, wherein heterologous polypeptide comprises immune globulin variable region, constant region for immunoglobulin, the perhaps combination of the two.

193. the method for claim 183, wherein heterologous polypeptide comprises and contains the variable region of heavy chain that effectively links together and the single-chain antibody of variable region of light chain.

194. the method for claim 193, wherein single-chain antibody has the NO:16 at SEQ ID, SEQID NO:43, perhaps listed aminoacid sequence among the SEQ ID NO:48.

195. the method for claim 193, wherein single-chain antibody is by SEQ ID NO:15, SEQ IDNO:42, perhaps listed nucleotide sequence coded of SEQ ID NO:47.

196. the method for claim 183, wherein heterologous polypeptide comprises the report polypeptide.

197. the method for claim 196 reports that wherein polypeptide is a luciferase.

198. the method for claim 197, wherein luciferase has listed aminoacid sequence in SEQ ID NO:46.

199. the method for claim 198 reports that wherein polypeptide is by listed nucleotide sequence coded in SEQ ID NO:45.

200. the method for claim 183, wherein plastid is a chloroplast(id).

201. the method for claim 200, wherein chloroplast(id) is in a kind of algae.

202. the method for claim 201, wherein algae is a kind of little algae.

203. pass through the heterologous polypeptide that the method for claim 183 is produced.

204. a method that detects vegetable cell is included under the condition that allows the report polypeptide to express in chloroplast(id) and imports the polynucleotide of claim 178 in the vegetable cell chloroplast(id), and detects described report polypeptide expression.

205. the method for claim 204 reports that wherein polypeptide is a luciferase.

206. the method for claim 205, wherein luciferase has listed aminoacid sequence in SEQ ID NO:46.

207. the method for claim 204, wherein said polynucleotide has nucleotide sequence listed in SEQ IDNO:45.

Images