CN100540667C - Utilize rice-embryo milk cell to produce recombination human serum albumin as bio-reactor - Google Patents
Utilize rice-embryo milk cell to produce recombination human serum albumin as bio-reactor Download PDFInfo
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Abstract
The invention discloses the method for utilizing rice-embryo milk cell to produce recombination human serum albumin, belong to gene engineering technology field as bio-reactor.It relates to and utilizes the cereal seed rice-embryo milk cell to produce medical recombination human serum albumin as bio-reactor.The proteoplast of the rice-embryo milk cell of one of main food that utilization is human is as the storage place of recombinant protein, adopt rice endosperm specific expression promoter and signal peptide, the mediation recombination human serum albumin enters the endomembrane system of rice-embryo milk cell, and be stored in the proteoplast of paddy endosperm, thereby recombination human serum albumin can be accumulated in rice paddy seed in a large number, finally reach higher level.Its expression level reaches more than 0.3% of rice paddy seed weight at least.Have higher output and lower production cost than other plant expression system.
Description
Technical field
The invention belongs to gene engineering technology field, it relates to the method for utilizing the cereal seed rice-embryo milk cell to produce medical recombination human serum albumin as bio-reactor.Promptly with paddy rice as the host, with albuminous cell as bio-reactor, by the DNA recombinant technology, the carrier of the specific expressed human serum albumin of rice-embryo milk cell is imported rice cell, the recombination human serum albumin of specific expressed and a large amount of methods that accumulate human serum albumin and the plant origin produced by this method in the albuminous cell of transgenic paddy rice.
Background technology
The production of main pharmaceutical prod is as bio-reactor with bacterium in the fifties in last century.But because bacterium belongs to prokaryotic organism and does not possess Eukaryotic protein synthesis and system of processing.And some proteinic biological activitys rely on proteinic modification to reach, so its application is restricted.Yeast begins to be applied to pharmaceutical prod production as s-generation bio-reactor in the seventies in last century, owing to yield poorly, its body modification/system of processing imperfection has also limited its widespread use.Third generation bio-reactor be utilize highly plant, zooblast is as bio-reactor.At present the eukaryote reactor can be divided into animal and plant bio-reactor two big classes, and animal bioreactor has two kinds of cell cultures and transgenic animal, and some main medical antibody use animal Chinese hamster ovary celIs are that (or mouse) cell cultures is produced at present.The Study on Transgenic Animal aspect mainly be the mammary gland cell express recombinant protein matter of transgenosis cow and in egg white express recombinant protein matter.Yet, animal cell culture and transgenic animal add that the cost of animal cell culture is high owing to can not overcome the pollution problem of animal pathogen, and scale operation needs huge investment, someone estimates that the whole world has only the ability of 1000 kilograms manufacture order clonal antibody at present; It is calculated that the throughput that every expansion is 1000 kilograms needs 4,000,000,000 dollars of investments, and will spend the time in 10 years to be constructed and put into operation, the throughput of the recombinant protein that these data declarations are present is widely less than the market requirement.Aspect medical applications, do not have competitive edge, make to be restricted yet in pharmaceutically application.Present application is nonoptional selection.Thereby be badly in need of efficient, a safe expression system and satisfy great demand.
Human serum albumin is made up of 585 amino acid, it is a non-glucosidesization, the goodish monomer molecule of solubility, and its molecular weight is 65kD, is the major protein composition in the human blood, it is proteic 60% to account for total serum, and concentration is up to every milliliter 40 milligrams in human blood.After it synthesizes, the normal osmotic pressure that keeps blood had important effect in liver; It also in blood as the carrier of multiple functional molecular, the intravital different physiological roles of mediator.External, it is widely used in treatment, pharmaceutical carrier, animal cell culture, blood plasma surrogate of various diseases etc. as the stablizer of multiple medicine.Have huge demand in the international market, annual demand is up to more than 500 tons.Yet almost everyone sero-abluminous source is to extract from human plasma at present.Because the restriction in blood plasma source, especially from the threat of disease such as acquired immune deficiency syndrome (AIDS), the hepatitis etc. of blood propagation, make people produce huge worry as medicinal use, thereby reorganization human serum protein's production and application produced great interest using the serum albumin that extracts in the blood plasma.At present, existing in the world people attempts to adopt the prokaryotic organism intestinal bacteria, and eukaryote such as yeast, animal cell culture, transgenosis milk cow produce recombination human serum albumin.But these methods are lower or production cost is too high because of expression level, can not be used for producing.As German GTC biological medicine company at the expression level of the human serum albumin of transgenic cattle about every liter 40 gram.Chicken gene genetic (AviGenic) company limited of the U.S. utilizes the transgenosis egg to come recombinant Human Serum Albumin Expression, and the expression level in Ovum Gallus domesticus album is very low, is every milliliter 1.65 microgram.The Delta of Britain (Delta) biotech company utilizes yeast to produce recombination human serum albumin, its output has reached 150 milligrams for every liter, though on market, begun to use, but produce human serum albumin as bio-reactor with zooblast, owing to can not avoid the pollution of animal pathogen, on using, be restricted.And become organ to come recombinant Human Serum Albumin Expression to have bigger advantage with vegetable cell, at present have only two testing laboratories to carry out the research of this respect in the world, one is that Hispanic national Agricultural biotechnologies institute utilizes the potato tuber recombinant Human Serum Albumin Expression, it is expressed in the per kilogram potato tuber is 10 milligrams level, its expression level does not still reach commercial level, thereby can not be used for producing; Another family is that the Florida State University of the U.S. utilizes the chloroplast expression technology at the chloroplast expression recombination human serum albumin, though its expression level per kilogram reaches 250 milligrams, a deadly defect of this system is that recombination human serum albumin is expressed the inclusion body that formation is similar to escherichia expression system in chloroplast(id), recombination human serum albumin is insoluble form, thereby make the loss of bioactivity of human serum albumin, and to solve its solubility problem, will improve its production cost widely.
Summary of the invention
The inventor is that host's the expression amount of bio-reactor is low at existing protokaryon and eukaryote, poorly soluble, active and the uneasy congruent shortcoming of lifeless matter, the proteoplast of the rice-embryo milk cell of one of main food that utilization is human is as the storage place of recombinant protein, adopt rice endosperm specific expression promoter and signal peptide, the mediation recombination human serum albumin enters the endomembrane system of rice-embryo milk cell, and be stored in the proteoplast of paddy endosperm, thereby recombination human serum albumin can be accumulated in a large number in rice paddy seed, finally reach higher level, the expression amount that the present invention not only can overcome other expression systems is low, poorly soluble, problems such as lifeless matter activity, the pollution problem that can also stop animal pathogen fully.
The promotor of utilizing paddy rice storage prolamin gene Gt13a and signal peptide mediation recombination human serum albumin have been an object of the present invention is to provide in the albuminous cell specific expression and be stored in the intravital method of albumen of rice-embryo milk cell, make recombination human serum albumin not attacked by intracytoplasmic proteolytic enzyme, thereby in paddy endosperm, accumulate in a large number, finally obtain the expression technology of higher output.
Second purpose of the present invention is the genetic codon of human serum albumin gene to be converted to the genetic codon of paddy rice preference, thereby improved the translation skill of recombinant protein, finally improves expression and the accumulation of human serum albumin at rice-embryo milk cell.
The 3rd purpose of the present invention provided the new system of a recombinant Human Serum Albumin Expression, and this system utilizes paddy rice or cereal albuminous cell to produce recombination human serum albumin.This system is not only than safer, the virus-free pollution of transgenosis milk cow and chicken, and mass-producing easily; Have higher output and lower production cost than other plant expression system.
The 4th purpose of the present invention provided a perfect paddy gene conversion system, this system utilizes the promotor of L-Cysteine HCL Anhydrous B (the Cysteine Proteinase β) gene of paddy rice, the selected marker is at the rice callus tissue expression for the special mediation of this promotor, selects effect to improve widely thereby make.The effect of other adverse environments of avoiding simultaneously using the promotor of non-paddy rice and producing.
Below method of the present invention is described in detail:
The acquisition of paddy rice specificity promoter and signal peptide: in order to obtain stronger rice endosperm specific expression promoter and signal peptide, research according to protein science, find a member Gt13a gene among the prolamin gene man bunch of paddy rice storage protein in the protein expression amount of rice-embryo milk cell for the highest, this gene member that derives has stronger promoter activity.In order to obtain the signal peptide sequence of Gt13a promotor and it, synthesized a pair of nucleotide primer according to the Gt13a (gene pool registration number AP003256) of gene database and be used for pcr amplification ( sequence 1,2).For the ease of gene clone, hold the restriction enzyme site that has added a HindIII at 5 ' of forward primer, added the restriction enzyme site of a NaeI at 5 ' end of reverse primer.Extracting genomic dna from any rice varieties blade, is masterplate with this DNA, according to the PCR program of standard, utilizes above-mentioned primer amplification to go out the dna fragmentation of one 1284 base.Through dna sequence analysis, this dna fragmentation has tangible promoter structure.Relatively have evident difference with the Gt1 promotor, at AT content apparently higher than existing Gt1 promotor and long nucleotide sequence.
The structure of rice-embryo milk cell specific expression vector: obtain the sequence of Gt13a promotor and signal peptide through PCR after, the PCR product is after HindIII and NaeI digestion, this fragment is connected with pBI221 fragment (U.S. clone technology Clontech company limited) through HindIII and NaeI digestion, import intestinal bacteria strain DH10B then, formed the underlying carrier of the rice endosperm specific expression that contains Gt13a promotor, Gt13a signal peptide and Nos terminator, and with this vector plasmid called after pOsPMP1 (seeing sequence 3).
The gene of the human serum albumin gene that rice genetic is codon optimized synthesizes the structure with expression vector: obtain higher expression in order to make recombination human serum albumin at paddy endosperm, the genetic codon that must use paddy rice to optimize, thereby, obtained the aminoacid sequence of sophisticated human serum albumin gene (gene pool registration number CAA01491) in U.S. biotechnology information center (NCBI) gene pool, by DNA analysis software mark carrier MacVector (the Acker Er Lesi Accelrys company product of Britain) aminoacid sequence of human serum albumin gene is converted to and to contain the nucleotide sequence of optimizing the rice genetic codon, adopt the PCR extension method then, synthetic human serum albumin gene (seeing sequence 4).The nucleotide sequence of the human serum albumin gene after codon optimized has changed 25.5% than original human serum albumin gene, and genetic code has changed 71.1%, but its amino acid whose sequence constant (seeing Table 1).
The comparison of table 1, the human serum albumin gene after codon optimized
| The genetic code sum | 585 |
| Change the genetic code subnumber | 416 |
| Change genetic codon per-cent (%) | 71.1 |
| The deoxynucleotide sum | 1755 |
| Change the deoxynucleoside acid number | 447 |
| Change deoxynucleotide per-cent (%) | 25.5 |
| Aminoacid sequence changes (%) | 0 |
When the synthetic human serum albumin gene, add restriction enzyme MylI and XhoI site respectively at gene synthetic primer two ends, be cloned into pUC19 carrier (the clone technology Clontech company limited of the U.S.) then, produced the carrier pOsHSA that has the recombination human serum albumin gene; POsHSA is through restriction enzyme MylI and XhoI digestion, produced a concordant end and produced a sticky end at 5 ' end at 3 ' end, use NaeI and XhoI digested vector plasmid pOsPMP1 simultaneously, also produced a concordant end and produced a sticky end at 3 ' end at 5 ' end, separate through agarose gel electrophoresis, reclaim the human serum albumin gene fragment, the segment of this recombination human serum albumin gene is connected to vector plasmid pOsPMP1 through NaeI and XhoI digestion, transformed into escherichia coli strain DH10B then produces the vector plasmid pOsPMP2 (Fig. 2 and sequence 5) of rice endosperm specific recombinant Human Serum Albumin Expression gene.
The structure of selected marker's carrier: improve the selection effect in the rice callus tissue for the selected marker is efficiently expressed, (cysteineproteinase β, promotor mediation selected marker (the mould plain gene-hygromix phosphotransferase of moisture resistance) CP) expresses in callus to adopt rice cysteine proteinase gene β.At first, synthesize one couple of PCR primers (seeing sequence 6,7), added HindIII and SmaI site at the two ends of PCR primer; With the oryza sativa genomic dna is masterplate, adopt Standard PC R reaction, amplifying a length is the segment that 1103 bases contain promotor, the PCR product is after HindIII and SmaI digestion, this PCR fragment is connected with cloning vector pBI221 (the clone technology Clontech company limited of the U.S.), transformed into escherichia coli strain DH10B has produced middle interstitial granules pOsPMP4 then.The CP promoter sequence has tangible promotor feature and element (sequence 10) through sequential analysis.The selected marker adopts hygromycin phosphotransferase gene (Hygromycin β Phosphotransferas, Hpt), this gene is from plasmid pCAMBIA1301 (Australian taxi driver brother Lun Biya CAMBIA company) amplification, (see sequence 8 at forward primer, 9) 5 ' end has added that a flat end limit restriction endonuclease is site SmaI, added the restriction endonuclease sites XhoI of a sticky end at 5 ' end of reverse primer, with the pCAMBIA1301 plasmid is masterplate, adopt Standard PC R reaction, amplify the hygromycin phosphotransferase gene segment, the PCR product digests with SmaI and XhoI, pOsPMP4 DNA digests with NaeI and XhoI, then the hygromycin phosphotransferase gene segment is connected with the dna fragmentation of XhoI digestion through NaeI with the pOsPMP4 plasmid with CP promotor, transformed into escherichia coli fungus strain DH10B, finally produced the specific expressed selected marker expression vector of rice tissue, called after pOsPMP5.
Paddy gene genetic transformation: after rice paddy seed shells, sterilization is 20 minutes in 20% clorox, with aqua sterilisa rinsing 3 times, induces on callus inducing medium then 20-25 days, the inductive callus is used for genetic transformation after transferring to and growing 9-10 days on the pre-treatment substratum.Human serum albumin expression vector plasmid pOsPMP2 and DNA with 0.5 microgram with selected marker plasmid pOsPMP5, after the 0.1M spermidine of the 1M calcium chloride of the bronze of 50 microlitres, 250 microlitres and 50 microlitres mixes, reacted 30 minutes, it is inferior to give a baby a bath on the third day after its birth with alcohol, DNA is wrapped on the bronze, then according to the particle gun method of du pont company, with two plasmid cotransformations to by in spend the callus that produces for No. 11.In the screening that contains on the selection substratum of hygromycin B through 45 days, callus with hygromycin B resistance on the regeneration culture medium under illumination, inducing through about 20 days, callus is divided into green plant, then immature plant is forwarded on the root media, through 15-20 days induce, form complete plant, obtain transfer-gen plant, these transfer-gen plants detect through PCR, after proving that it contains human serum albumin gene, forward the experimental plot to and grow into sophisticated seed, this is the T1 seed.
The screening of high expression level transfer-gen plant: transgenic paddy rice after heading is bloomed, through one month time, forms sophisticated transgenic paddy rice seed through about 4 months g and D.Wherein there is the transfer-gen plant of 50-60% normally solid.After T1 seed results, utilize the protein detection technology---enzyme linked immunological (ELISA) medicine box (the northern rope laboratory Bethyl Laboratory of the U.S.) detection technique, at first, every strain transgenosis is got 10 seeds, adds 10 milliliters extraction damping fluid (50mMTris, pH8.0,50mM NaCl, 10mM EDTA) homogenate, per minute 14000 changes on whizzer, centrifugal 10 minutes, supernatant liquor carried out enzyme linked immunosorbent detection with the quantitative medicine box of human serum albumin ELISA.The expression level of the recombination human serum albumin of every seed is between the 9.82-282.22 microgram after testing.The expression amount of the recombination human serum albumin of a wherein the highest transgenosis individual plant pPMP2-68 is on average up to 174.18 micrograms.Amount to into every gram rice paddy seed and contain the 8.29mg recombination human serum albumin, promptly account for 0.83% seed dry weight.The high transgenosis individuality of screening expressing human serum albumin from the T1 seed through the T1 continuation selection in generation, forms the transgenic strain of stably express recombination human serum albumin.Produce the usefulness of the recombination human serum albumin of plant origin for big area.
The present invention utilizes paddy endosperm to produce recombination human serum albumin soluble, biologically active as bio-reactor, make its expression level reach more than 0.3% of rice paddy seed weight at least, being that the human serum albumin output of per kilogram seed reaches more than 3 grams, is 300 times of maximum amount of 12 times of maximum amount of the chloroplast expression system and potato tuber expression system.
What the present invention described utilizes bread crop-rice-embryo milk cell as bio-reactor, 1), it has the system of expressing efficiently the method for producing recombination human serum albumin with rice paddy seed compared with prior art has the excellent characteristics of the following aspects:, vegetable cell has the protein synthesis system identical with the human and animal, has the potentiality of expressing exogenous protein efficiently.2), it has the similar animal mankind's protein processing modification system; 3), its product safety is reliable.Because the proteinaceous product that plant seed such as paddy rice, wheat and barley etc. all are human main food, produced by it is definitely without any the pollution of pathogeny bacterium, therefore safe and reliable; 4), production cost is extremely low, its production cost is respectively 1/20 and 1/200 of bacterium and an animal system; 5), store easily and process.Because recombinant protein is stored in the seed, do not need the processing in time of picture other plant system needs, do not need special storage facilities and low temperature requirement yet; 6), production in enormous quantities is very easy.This technology in case need, enlarges cultivated area and gets final product after forming stable transformed variety, does not need to increase any workshop facility.On the contrary, bacterium or yeast need be acquired a large amount of drill shacks and fermentation equipment; And the animal reproduction of animal system adds for very difficult with breeding.
The code name of expression vector plasmid and function thereof and purposes contrast is as follows:
The underlying carrier of pOsPMP1----rice endosperm specific expression
POsHSA-----has the carrier of recombination human serum albumin gene
The vector plasmid of pOsPMP2------rice endosperm specific recombinant Human Serum Albumin Expression gene
Interstitial granules among the pOsPMP4-----
The selected marker expression vector that the pOsPMP5-----rice tissue is specific expressed
Description of drawings
The pcr amplification product of Fig. 1, Gt13a promotor and signal peptide.The arrow indication is the nucleotide fragment of 1284 bases of pcr amplification.Swimming lane 1 is a molecule marker, and swimming lane 2 is the PCR product, and the arrow indication is the promoter sequence of the Gt13a of 1284 bases.
The restriction map spectrum of the human serum albumin expression vector of the rice endosperm specific expression of Fig. 2, structure.
The selected marker's of Fig. 3, rice callus tissue specific expression restriction map spectrum.
The polyacrylamide gel and the Western blotting collection of illustrative plates of the recombination human serum albumin of expressing in Fig. 4, the demonstration paddy endosperm.The recombination human serum albumin that extracts from transgene paddy rice endosperm, 10 transgenic paddy rice seeds extract the recombination human serum albumin of buffer extraction with 10 ml proteins.15 microlitre point samples are at 10% polyacrylamide gel.Detect through coomassie brilliant blue staining and Western blotting then.Figure A is the polyacrylamide gel collection of illustrative plates of coomassie brilliant blue staining; Figure B is the Western blotting collection of illustrative plates.The arrow indication is that human serum albumin is apparent in the transgenosis endosperm, but lacks corresponding protein belt in the control sample Taibei 309.
Embodiment
Below the specific embodiment of the present invention is described in further detail, but present embodiment does not limit protection scope of the present invention.
[embodiment 1] clone's Gt13a promotor and signal peptide
Promotor and signal peptide for the Gt13a gene of clone's prolamine in the rice genome sequence, utilize the primer of sequence 1, the polymerize chain reaction of employing standard (PCR) reaction, the dna fragmentation (Fig. 1) of 1284 bases that from the genomic dna of the Taibei 309 kinds, increased, the fragment of amplification is through restriction enzyme NaeI and XhoI digestion, be cloned among the vector plasmid pBI221, produced the specific expressed expression vector pOsPMP1 of rice-embryo milk cell, through dna sequence analysis, this dna fragmentation has the sequence (sequence 3) of tangible promotor feature and signal peptide.
[embodiment 2] synthetic human serum albumin gene that contains the rice genetic codon.
The aminoacid sequence of human serum albumin gene sequence (the gene pool registration number is CAA01491), use DNA analysis software mark carrier MacVector human serum albumin gene to be converted to the nucleotide sequence of rice genetic codon, the deoxynucleoside acid sequence of its amended human serum albumin gene has changed 25.5%, genetic code has changed 71.1%, but its aminoacid sequence is identical.Then,, adopt the PCR extension method according to codon optimized deoxynucleoside acid sequence, synthetic the recombination human serum albumin gene.In the gene building-up process, added MylI and XhoI restriction enzyme site at the gene two ends, be cloned into the pUC19 plasmid vector, produced and contained the codon optimized human serum albumin gene of paddy rice (pOsHSA).
[embodiment 3] make up the carrier of the specific expressed human serum albumin of paddy rice.
The pOsHSA plasmid DNA digests with restriction enzyme MylI and XhoI, simultaneously with NaeI and XhoI digestion pOsPMP1 plasmid DNA, human serum albumin gene is connected with vector plasmid pOsPMP1, transformed into escherichia coli strain DH10B then, producing the expression vector plasmid is pOsPMP2.The restriction map spectrum of its plasmid is seen Fig. 2.
[embodiment 4] clone rice cysteine proteolytic enzyme B (cysteine proteinase B) promotor.
Adopt the promotor of the method for PCR from oryza sativa genomic dna clone rice cysteine proteolytic enzyme β gene, nucleotide sequence according to gene pool has designed two PCR primers, the primer nucleotide sequence is seen sequence 6,7, utilize Standard PC R reaction, from the artificial bacterial chromosome of paddy rice library, screen a positive colony 42M2.The BAC clone is after XhoI digestion, the segment Southern hybridization of a 5kb confirms to contain the full DNA sequence of L-Cysteine HCL Anhydrous β gene, with this BAC cloned DNA is template, adopt Standard PC R reaction with primer (seeing sequence 6,7), obtained the dna fragmentation of one 1113 base, this fragment cloning in the pBI221 carrier, has been produced an intermediate carrier (pOsPMP4).
[embodiment 5] make up selected marker's carrier.
The DNA of pOsPMP4 digests with NaeI and XhoI, use primer (seeing sequence 8,9), with pCAMBIA1301 DNA is masterplate, use standard pcr, the PCR fragment that amplifies is through SmaI and XhoI digestion, be cloned into then on the pOsPMP4 carrier of NaeI and XhoI digestion, produced the specific expressed selected marker carrier pOsPMP5 (Fig. 3) of paddy rice.
[embodiment 6] are with the gene transformation of particle gun mediation
Spend No. 11 seed in the rice varieties, slough husk after, through 20% clorox sterilization 20 minutes, through rinsed with sterile water 3 times, each 10 minutes, be placed on then on the callus inducing medium through 20-30 days induce, produce callus.The DNA that the plasmid pOsPMP2 and 0.5 microgram of 0.5 microgram had selected marker plasmid pOsPMP5, after the 0.1M spermidine of the 1M calcium chloride of the bronze of 50 microlitres, 250 microlitres and 50 microlitres mixes, at room temperature reacted 30 minutes, it is inferior to give a baby a bath on the third day after its birth with alcohol, then according to the particle gun method of E.I.Du Pont Company, to be enclosed with two plasmid DNA bronzes, cotransformation is in the cell of the callus of therefrom spending No. 11 generations; On the selection substratum of the hygromycin B that contains 50 mcg/ml through 45 days screening, the callus of continued growth is the positive callus of moisture resistance mycin B, the callus of hygromycin B resistance is transferred on the regeneration culture medium, inducing through about 20 days under illumination, callus is divided into green plantlet, then immature plant is forwarded on the root media, through 15-20 days induce, form complete plant, forward greenhouse or experimental plot at last to and grow into sophisticated seed.
The high transfer-gen plant of [embodiment 7] screening expressing human serum albumin.
Transfer-gen plant in the greenhouse or the land for growing field crops through growth with grow, the knot seed of blooming then, form transgenosis generation seed, after T1 seed results, every strain transfer-gen plant is got 10 seeds, is extracting (50mM Tris in the buffered soln, pH 8.0,50mM NaCl, 1mM EDTA) extracting protein, through the expression amount of enzyme linked immunosorbent detection (ELISA) technology for detection human serum albumin.Its expression level at the recombination human serum albumin of transgenic paddy rice strain is summed up (seeing Table 3).
The result of the recombinant Human Serum Albumin Expression of table 3, different transfer-gen plants
| Transfer-gen plant number | Average expression level (microgram/grain ± standard error) | Variation range (microgram/grain) | Variation coefficient % (3-4 repetition) |
| pPMP2-1 | 9.82±4.5 | 3.86-15.48 | 7.79 |
| pPMP2-4 | 110.65±39.1 | 55.13-173.84 | 4.24 |
| pPMP2-6 | 9.38±6 | 2.62-18.56 | 13.14 |
| pPMP2-18 | 165.92±62.7 | 69.03-282.22 | 3.58 |
| pPMP2-20 | 89.91±22.04 | 70.3-116.92 | 4.91 |
| pPMP2-21 | 56.37±10.84 | 42.15-69.12 | 2.18 |
| pPMP2-23 | 1.36±0.19 | 10.6-1.57 | 21.95 |
| pPMP2-26 | 67.29±19.53 | 37.72-105.11 | 4.4 |
| pPMP2-27 | 53.23±19.16 | 28.98-82.02 | 3.39 |
| pPMP2-29 | 25.53±20.86 | 10.01-76.84 | 6.02 |
| pPMP2-31 | 29±11.2 | 19.52-42.87 | 5.62 |
| pPMP2-43 | 56.06±24.6 | 22.52-83.69 | 5.7 |
| pPMP2-55 | 75.17±26.6 | 44.9-126.42 | 4.7 |
| pPMP2-58 | 13.54±5.6 | 9.87-18.88 | 16.26 |
| pPMP2-62 | 71.36±25 | 39.25-123.47 | 3.74 |
| pPMP2-64 | 16.92±2.2 | 15.37-18.47 | 4.43 |
| pPMP2-68 | 174.18±71.7 | 72.22-286.20 | 6.11 |
| pPMP2-70 | 79.61±28.7 | 34.15-147.72 | 4.6 |
The highest transgenosis individuality of screening recombinant Human Serum Albumin Expression from the T1 seed.In order to check the transgenic line that efficiently expresses human serum albumin, the T1 of different transgenic strains for the crude extract of seed through SDS-PAGE polyacrylate hydrogel electrophoresis, coomassie brilliant blue staining and Western blotting detect, human serum albumin is high-visible in polyacrylate hydrogel, has identical molecular weight (Fig. 4) with positive control, in Western blotting, show the banding pattern identical, illustrate that the specific protein band in the transgenosis endosperm is a human serum albumin with coomassie brilliant blue staining.
The sequence of relevant Nucleotide
＜110〉Yang Daichang
＜120〉utilize rice-embryo milk cell to produce recombination human serum albumin as bio-reactor
<140>2005100190844
<141>2005-07-13
<160>10
<170>PatentIn version 3.3
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gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 3240
ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 3300
gtcatctatg ttactagatc gggaattcac tggccgtcgt tttacaacgt cgtgactggg 3360
aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 3420
gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 3480
aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 3540
ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 3600
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 3660
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 3720
cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 3780
tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 3840
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 3900
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 3960
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 4020
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 4080
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 4140
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 4200
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 4260
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 4320
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 4380
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 4440
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 4500
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 4560
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 4620
ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 4680
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 4740
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 4800
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 4860
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4920
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4980
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 5040
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 5100
tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 5160
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 5220
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 5280
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 5340
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 5400
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 5460
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 5520
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 5580
tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 5640
gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 5700
agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 5760
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 5820
gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 5880
ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 5940
atgaccatga ttacgcc 5957
<210>6
<211>25
<212>DNA
＜213〉synthetic
<400>6
cgccaagctt gcatgcctgc agcca 25
<210>7
<211>25
<212>DNA
＜213〉synthetic
<400>7
ccatgccggc gacgtggaga cgagc 25
<210>8
<211>33
<212>DNA
＜213〉synthetic
<400>8
aaagtactat gaaaaagcct gaactcaccg cga 33
<210>9
<211>35
<212>DNA
＜213〉synthetic
<400>9
ggagaaactc gagcttgtcg atcgacagat ccggt 35
<210>10
<211>1135
<212>DNA
＜213〉paddy rice
<400>10
aagcttgcat gcctgcagcc aggcttcatc ctaaccatta caggcaagat gttgtatgaa 60
gaagggcgaa catgcagatt gttaaactga cacgtgatgg acaagaatga ccgattggtg 120
accggtctga caatggtcat gtcgtcagca gacagccatc tcccacgtcg cgcctgcttc 180
cggtgaaagt ggaggtaggt atgggccgtc ccgtcagaag gtgattcgga tggcagcgat 240
acaaatctcc gtccattaat gaagagaagt caagttgaaa gaaagggagg gagagatggt 300
gcatgtggga tccccttggg atataaaagg aggaccttgc ccacttagaa aggagaggag 360
aaagcaatcc cagaagaatc gggggctgac tggcactttg tagcttcttc atacgcgaat 420
ccaccaaaac acaggagtag ggtattacgc ttctcagcgg cccgaacctg tatacatcgc 480
ccgtgtcttg tgtgtttccg ctcttgcgaa ccttccacag attgggagct tagaacctca 540
cccagggccc ccggccgaac tggcaaaggg gggcctgcgc ggtctcccgg tgaggagccc 600
cacgctccgt cagttctaaa ttacccgatg agaaagggag gggggggggg gaaatctgcc 660
ttgtttattt acgatccaac ggatttggtc gacaccgatg aggtgtctta ccagttacca 720
cgagctagat tatagtacta attacttgag gattcggttc ctaatttttt acccgatcga 780
cttcgccatg gaaaattttt tattcggggg agaatatcca ccctgtttcg ctcctaatta 840
agataggaat tgttacgatt agcaacctaa ttcagatcag aattgttagt tagcggcgtt 900
ggatccctca cctcatccca tcccaattcc caaacccaaa ctcctcttcc agtcgccgac 960
ccaaacacgc atccgccgcc tataaatccc acccgcatcg agcctatcaa gcccaaaaaa 1020
ccacaaacca aacgaagaag gaaaaaaaaa ggaggaaaag aaaagaggag gaaagcgaag 1080
aggttggaga gagacgctcg tctccacgtc gccggccccg gcggggtggt cagtc 1135
Claims (7)
1, is used for mediating the Gt13a promotor of the paddy rice storage protein that recombination human serum albumin expresses at the transgene paddy rice endosperm cell and the Nucleotide of signal peptide, shown in sequence 3.
2, the Nucleotide of rice endosperm specific expression vector Gt13a-sp-HSA-Nos that comprises the called after pOsPMP1 of described Gt13a promotor of claim 1 and signal peptide is shown in sequence 5.
3, a kind of method of utilizing rice-embryo milk cell to produce recombination human serum albumin as bio-reactor, its step is as follows:
(1) acquisition of paddy rice specificity promoter and signal peptide: obtain to contain the Gt13a promotor of rice endosperm specific expression and the length shown in sequence 3 of signal peptide is the dna fragmentation of 1284 bases with PCR reaction;
(2) structure of rice-embryo milk cell specific expression vector: with above-described length is that the dna fragmentation of 1284 bases is cloned into the pBI221 carrier, interstitial granules in the formation, import intestinal bacteria strain DH10B then, made up the specific expressed underlying carrier new system of rice-embryo milk cell, called after pOsPMP1;
(3) the synthetic structure with expression vector of the codon optimized human serum albumin gene of rice genetic: the aminoacid sequence that obtains human serum albumin gene in the gene pool, the aminoacid sequence of human serum albumin gene is converted to the nucleotide sequence of the rice genetic codon that contains optimization by DNA analysis software, adopt the PCR extension method then, synthetic recombination human serum albumin gene, be cloned into the pUC19 carrier, form pOsHSA; Then the recombination human serum albumin gene is connected to paddy endosperm expression vector pOsPMP1, produces the vector plasmid pOsPMP2 of rice endosperm specific recombinant Human Serum Albumin Expression gene;
(4) structure of the specific expressed selected marker's carrier of rice tissue: make up the specific expressed selected marker expression vector of rice tissue, called after pOsPMP5 with the CP promotor; At first be template with the oryza sativa genomic dna, with the segment of sequence 6, the 7 CP promotor that to amplify a length be 1103 bases, above-mentioned PCR fragment is connected with cloning vector pBI221, has produced middle interstitial granules pOsPMP4; Adopt Standard PC R reaction, amplify selected marker's hygromycin phosphotransferase gene segment, then the hygromycin phosphotransferase gene segment is connected with the middle interstitial granules with CP promotor, finally produced the specific expressed selected marker expression vector of rice tissue, called after pOsPMP5;
(5) carry out the paddy gene genetic transformation: with the vector plasmid pOsPMP2 of the expressing human Serum Albumin Gene of some amount with have the DNA of the expression plasmid pOsPMP5 of selected marker, method cotransformation with particle gun arrives the callus that is produced by rice varieties, by selecting-cultivate-screening-induce, form complete plant.
4, method according to claim 3, the preferred method of the acquisition of paddy rice specificity promoter and signal peptide is: adopt the primer shown in sequence 1,2, the polymerase chain reaction,PCR of employing standard is PCR, and the Gt13a promotor that contains the rice endosperm specific expression from increasing to the genomic dna of the Taibei 309 kinds and the length shown in sequence 3 of signal peptide are the dna fragmentation of 1284 bases.
5, method according to claim 3, the structure preferred method of rice-embryo milk cell specific expression vector is: after obtaining to contain the sequence of Gt13a promotor and signal peptide, this fragment is connected with pBI221 fragment through HindIII and NaeI digestion, import intestinal bacteria strain DH10B then, form the underlying carrier pOsPMP1 of the rice endosperm specific expression that contains Gt13a promotor, Gt13a signal peptide and Nos terminator.
6, method according to claim 3, the method that obtains the vector plasmid pOsPMP2 of rice endosperm specific recombinant Human Serum Albumin Expression gene is: the carrier pOsHSA that will have the recombination human serum albumin gene is through digestion with restriction enzyme, produce a concordant end and produce a sticky end at 5 ' end at 3 ' end, use NaeI and XhoI digested vector plasmid pOsPMP1 simultaneously, also produce a concordant end and produce a sticky end at 3 ' end at 5 ' end, separate through agarose gel electrophoresis, reclaim the above-mentioned fragment that has the recombination human serum albumin gene, the segment of this recombination human serum albumin gene is connected to vector plasmid pOsPMP1 through NaeI and XhoI digestion, transformed into escherichia coli strain DH10B then produces the vector plasmid pOsPMP2 of rice endosperm specific recombinant Human Serum Albumin Expression gene.
7, method according to claim 3, preferred method with the specific expressed selected marker expression vector of the rice tissue of CP promotor structure called after pOsPMP5 is: the synthetic one couple of PCR primers has added HindIII and SmaI site at the two ends of PCR primer shown in sequence 6,7; Genomic dna with the rice varieties Taibei 309 is a masterplate, adopts the PCR reaction of standard, and amplifying a length is the segment that 1103 bases contain promotor; The PCR product is after HindIII and SmaI digestion, and this PCR fragment is connected with cloning vector pBI221, and transformed into escherichia coli strain DH10B has produced middle interstitial granules pOsPMP4 then; The hygromycin phosphotransferase gene segment is connected with the dna fragmentation of XhoI digestion through NaeI with the pOsPMP4 plasmid with CP promotor, transformed into escherichia coli fungus strain DH10B, finally produced the expression vector of the specific expressed selected marker of rice tissue, called after pOsPMP5.
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