CN1366049A - Chinese bee venom AcHA gene, polypeptide coded by it and its preparing process - Google Patents
Chinese bee venom AcHA gene, polypeptide coded by it and its preparing process Download PDFInfo
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- CN1366049A CN1366049A CN01135659A CN01135659A CN1366049A CN 1366049 A CN1366049 A CN 1366049A CN 01135659 A CN01135659 A CN 01135659A CN 01135659 A CN01135659 A CN 01135659A CN 1366049 A CN1366049 A CN 1366049A
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- bee venom
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- apis cerana
- polypeptide
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Abstract
A Chinese bee venom AcHA gene sequence, the polypeptide coded by it and its preparing process are disclosed. The protein coded by the said cDNA sequence is a homologue of Italian bee venom AmHA, and it at least has 70% humology with nucleotide in SEQ ID No.3 from nucleotide I-1164 site. The said sequence code has the polypeptide with sequence expressed by SEQ ID No.4. The carrier or host cell containing AcHA gene, the polypeptide associated with AcHA, the polypeptide of AcHA with protein activity, and the process for preparing relative antibody are also disclosed.
Description
Technical field
The present invention relates to the genetically engineered field, relate in particular to apis cerana bee venom AcHA gene and encoded polypeptides and preparation method.
Background technology
Apis mellifera (Apis mellifera) bee venom Unidasa (Bee venomous hyaluronidase, BvHA) be one of two main anaphylactogens of apis mellifera bee venom, (Science 1972 to account for the 2%-3% of bee venom dry weight, 177:314-322) by sources and biochemical property, it belongs to glycoprotein, be β-1 between N-acetyl-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) in a kind of catalysis hyaluronic acid long-chain, 4 glycosidic link hydrolysis, produce a kind of β-N-acetyl-D-amino hexoside enzyme (β-N-acetyl-D-hexosaminidase), or claim hyaluronic acid hexosaminidase (Hyaluronoglucosaminidase) of 4 sugared product GlcA-GlcNAc-GlcA-GlcNAc.BvHya has very strong biological activity, make bee venom between local organization, permeate and spread (Structure.2000,8:1025-1035).
1993, people's Applied Biotechnology methods such as Gmachl, utilize the total RNA reverse transcription of apis mellifera bee venom gland to become cDNA, by phage construction cDNA library, and according to the synthetic degenerate core nucleotide sequence of this honeybee bee venom Unidasa N end amino sequence as primer, with the Apis mellifera genomic dna is that template is carried out the PCR reaction, amplifies the upstream sequence of one section 47bp.Do the library screening with this sequence as probe then, obtained 3 positive colonies, wherein two clone's insertion fragments are 1.45kb, and it is 1.25kb that a clone inserts fragment.Insert segmental sequencing result according to preceding two clones, the coded HA mature peptide of this cDNA is 349 amino-acid residues.The back clone insert in the fragment, except that initiating terminal 198bp and 199-416 position are the intron, all the other be among preceding two clones encoding sequence (Insect Biochem.1988,18:511-514).Their cDNA is named as AmH.
Summary of the invention
One of the object of the invention provides the polynucleotide of a kind of apis cerana bee venom AcHA, the proteic homologue of this polynucleotide encoding apis mellifera bee venom AmHA.
Two of the object of the invention provides a kind of apis cerana bee venom AcHA albumen by apis cerana bee venom AcHA polynucleotide encoding.
The object of the invention also provides this recombinant vectors for preparing apis cerana bee venom AcHA nucleotide probe, contains apis cerana bee venom AcHA Nucleotide, has contained the host cell of recombinant vectors, and the method for polypeptide antibody.
In the present invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the polypeptide of apis cerana bee venom AcHA protein-active, show at least 70% homology from the nucleotides sequence of Nucleotide 1-1164 position among described nucleotide sequence and the SEQ ID No.3, perhaps described nucleotide sequence can be hybridized from the Nucleotide of Nucleotide 1-1164 position under the tight condition of moderate with among the SEQ ID No.3, preferably, this polypeptide has the sequence shown in the SEQ ID No.4.More preferably, this sequence has among the SEQ ID No.3 nucleotide sequence from Nucleotide 1-1164 position.
Also provide a kind of isolating apis cerana bee venom AcHA protein polypeptide in the present invention, it comprises: have polypeptide or its active fragments of SEQ ID No.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID No.4 polypeptide of sequence.
The present invention also provides a kind of carrier, and it contains above-mentioned isolated DNA; A kind of described carrier transformed host cells.
The present invention also provides a kind of preparation to have the method for the polypeptide of apis cerana bee venom AcHA protein-active, and this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of apis cerana bee venom AcHA protein-active operationally is connected in expression regulation sequence, form apis cerana bee venom AcHA expression vector, the nucleotides sequence of described nucleotide sequence and SEQ ID No.3 Nucleotide 1-1164 position is shown at least 70% homology;
(b) change the expression vector in the step (a) over to host cell, form the proteic reconstitution cell of apis cerana bee venom AcHA;
(c) be fit to express under the condition of apis cerana bee venom AcHA protein polypeptide the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with apis cerana bee venom AcHA protein-active.
The present invention has found the gene of a kind of apis cerana bee venom AcHA that encodes, the AcHA of this coded by said gene (apis cerana bee venom Unidasa) is a kind of biologically active substance, can be applicable to medically treatment of diseases such as painstaking effort official disease, tumour, the transdermal factor that can be used as medicine is used for pharmaceutical preparation, can be used as antigen and be used for the preparation of bee venom immunological reagent, bee venom anaphylodiagnosis reagent, can be used as the toolenzyme of biological study.Apis cerana AcHA gene can be research apis cerana bee venom molecule mechanism hypersensitive, for development and use apis cerana bee venom resource provides new foundation.
Embodiment
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1164 Nucleotide, and its detailed sequence is seen SEQ ID No.3, and open reading frame is positioned at 1-1164 position Nucleotide.
In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant that this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state; Refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, term " apis cerana bee venom AcHA albumen (or polypeptide) encoding sequence " refer to the encode nucleotide sequence of polypeptide with apis cerana bee venom AcHA protein-active is as 1-1164 position nucleotide sequence and degenerate sequence thereof among the SEQ ID No.3.This degenerate sequence is meant the encoder block 1-1164 position Nucleotide that is arranged in SEQ ID No.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID No.3 in 1-1164 position nucleotide sequence homology be low to moderate about 70% degenerate sequence described sequence of SEQ ID No.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID No.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 1-1164 position.This term also comprise with SEQ ID No.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 1-1164 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have variant form with proteic, the SEQID No.4 sequence of apis cerana bee venom AcHA identical function.These variant forms comprise (but being not limited to): the disappearance of several Nucleotide, insertion and or replace, and add several Nucleotide at 5 ' and/or 3 ' end.
In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with post layer folding, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.
In the present invention, term " apis cerana bee venom AcHA protein polypeptide " refers to have the SEQ ID No.4 polypeptide of sequence of apis cerana bee venom AcHA protein-active.This term also comprises having and variant form apis cerana bee venom AcHA albumen identical function, SEQ ID No.4 sequence.These variant forms comprise (but being not limited to): several amino acid whose disappearances, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of apis cerana bee venom AcHA and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, the induced mutation body, under high or low rigorous degree condition can with the coded albumen of the DNA of apis cerana bee venom AcHA DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-apis cerana bee venom AcHA polypeptide to obtain.The present invention also provides other polypeptide, as comprises apis cerana bee venom AcHA polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of apis cerana bee venom AcHA polypeptide.Usually, this fragment have apis cerana bee venom AcHA polypeptid coding sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of apis cerana bee venom AcHA albumen or polypeptide, the difference of these analogues and natural apis cerana bee venom AcHA polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L (as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, γ amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises; The chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylation or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
The present invention also comprises apis cerana bee venom AcHA polypeptid coding sequence and segmental antisense sequences thereof, and this antisense sequences can be used for suppressing the expression of apis cerana bee venom AcHA in the cell.
The present invention also comprises a kind of probe molecule, and this molecule has 8-100 of apis cerana bee venom AcHA polypeptid coding sequence, preferably 1-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample coding apis cerana bee venom AcHA nucleic acid molecule.
The present invention also comprises the method that detects apis cerana bee venom AcHA nucleotide sequence, it comprises with above-mentioned probe and sample and hybridizing, whether detection probes combination has taken place then, preferably, this sample is the product behind the pcr amplification, wherein the pcr amplification primer is corresponding to the encoding sequence of apis cerana bee venom AcHA polypeptide, and can be positioned at the both sides or the centre of this encoding sequence, and primer length is generally 15-50 Nucleotide.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell, and preferably, this host cell is an eukaryotic cell, as Tn cell, Chinese hamster ovary celI, COS cell etc.
On the other hand, the present invention also comprises Chinese bee variety poison AcHA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into apis cerana bee venom AcHA gene product or fragment.Preferably, refer to that those can combine with apis cerana bee venom AcHA gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of apis cerana bee venom AcHA, comprise that also those do not influence the antibody of apis cerana bee venom AcHA protein function.The present invention also comprise those can with modify or without the apis cerana bee venom AcHA gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from the antibody moiety of apis cerana.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art, and for example, the apis cerana bee venom AcHA gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing apis cerana bee venom AcHA or its has antigenic segmental cell and can be used to immune animal and prepare antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nnature 256; 495,1975; People such as Kohler, Eur.J.Immunol, 6:511,1976; People such as Kohler, Eur.J.Immunol, 6:2921976; People such as Hammerling, In Monoclonai Antibodies andT Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block apis cerana bee venom AcHA function and the antibody that does not influence apis cerana bee venom AcHA function.Each antibody-like of the present invention can utilize the fragment or the functional zone of apis cerana bee venom AcHA gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of preparation in the prokaryotic cell prokaryocyte (for example E.coli) with the unmodified form bonded antibody of apis cerana bee venom AcHA gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
In one embodiment of the invention, the cDNA nucleotide sequence of apis cerana bee venom AcHA is so to obtain, cDNA with the total RNA reverse transcription of apis cerana poison gland is a template, or be template with apis cerana poison gland λ gtllcDNA library, with a pair of oligonucleotide is that primer-A:5 '-AGAATTCATGTCTCGGCCTCTCGTGATC-3 ' is that forward is to primer, oligonucleotide B:5 '-GAAGCTTACACTTGGTCCACGCTCA-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID No.3 after amplified production checked order.
Apis cerana bee venom AcHA is an apis cerana poison gland expressed proteins, and apis cerana bee venom AcHA of the present invention provides the foundation for the relevant molecule mechanism of research apis cerana bee venom.
Below in conjunction with specific examples, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the invention.
Embodiment 1, clone and the mensuration of the cDNA of apis cerana bee venom AcHA
1. primer amplification
CDNA with the total RNA reverse transcription of apis cerana poison gland is a template, or be template with apis cerana poison gland λ gtllcDNA library, with a pair of oligonucleotide is that primer-A:5 '-AGAATTCATGTCTCGGCCTCTCGTGATC-3 ' (SEQ ID No.1) is that forward is to primer, oligonucleotide B:5 '-GAAGCTTACACTTGGTCCACGCTCA-3 ' (SEQ IDNo.2) is a reverse primer, carries out PCR.The PCR condition of A/B be 94 ℃ 1 minute, thereupon with 94 ℃ of 45 second, carry out 30 circulations in 58 ℃ of 45 second and 72 ℃ of 90 second, last 72 ℃ were extended 10 minutes, electrophoresis detection obtains the purpose fragment of about 1200bp.
2.PCR the order-checking of product
With above-mentioned pcr amplification product A/B and PGEM
-T carrier (Promega) connects, and transformed into escherichia coli TG1 extracts plasmid with alkaline process, uses SequiTherm EXCEL
TMDna sequencing kit (EpicentreTeclnologies) checks order to extractive plasmid, with computer software splicing order, obtain full length cDNA sequence at last, altogether 1164bp, detailed sequence is seen SEQ ID No.3, and wherein open reading frame is positioned at 1-1164 position Nucleotide.
Derive the aminoacid sequence of apis cerana bee venom AcHA according to the full length cDNA sequence that obtains, totally 387 amino-acid residues, its aminoacid sequence sees SEQ ID No.4 for details.
Embodiment 2, and homology relatively
Full length cDNA sequence and proteins encoded thereof with apis cerana bee venom AcHA of the present invention, in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBankCDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with the Blast program.Found that it and apis mellifera bee venom AmHA have significant homology.With the GENTYX software analysis as can be seen, their proteic homology is 90% (seeing attached list 1).Therefore, can infer that apis cerana bee venom AcHA albumen of the present invention is the homologue of apis mellifera bee venom AmHA, and have similar function.
1993, people's Applied Biotechnology methods such as Gmachl utilized the total RNA reverse transcription of apis mellifera poison gland to become cDNA, by the construction cDNA library.Do the library screening with probe then, obtained the new gene of coding apis mellifera bee venom AmHA.AmHA mature peptide that this cDNA is coded and signal peptide are 382 amino-acid residues, wherein contain 4 Cys, 3 N-glycosylation site Asn-Leu-Thr.Apis cerana bee venom AcHA coded by said gene aminoacid sequence contains corresponding 4 Cys among the present invention, 3 N-glycosylation site Asn-Leu-Thr.Apis mellifera Apis mellifera bee venom AmHA, apis cerana bee venom AcHA and people's seminal fluid HA (PH20) have 30% homology, and according to homology and biological activity characteristics, apis mellifera Linnaeus bee venom AmHA and Mammals testis HA belong to Glycosylase 56 families together.HA is the biologically active substance that medically has been applied to treatments such as painstaking effort official disease, tumour at present, also is a kind of toolenzyme of biological study.The inventor's apis cerana AcHA gene is research apis cerana bee venom molecule mechanism hypersensitive, for development and use apis cerana bee venom resource provides new foundation.
Embodiment 3, the expression of apis cerana bee venom AcHA in intestinal bacteria
In this embodiment, with pcr amplification product among the embodiment 1 as inserting fragment.
5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:
5 '-AGAATTCATGTCTCGGCCTCTCGTGATC-3 ' (SEQ ID No.1), this primer contains the restriction enzyme site of EcoR I restriction enzyme, is 18 Nucleotide of the apis cerana bee venom AcHA encoding sequence that begins of initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
This primer of 5 '-GAAGCTTACACTTGGTCCACGCTCA-3 ' (SEQ ID No.2) contains the restriction enzyme site of HindIII restriction enzyme, the part encoding sequence of translation termination and apis cerana bee venom AcHA.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme site of the restriction enzyme on the bacterial expression vector pET-28a (+), this plasmid vector coding antibiotics resistance (Kan
r), a bacterium replication orgin (Ori), an adjustable promotor/operon of IPTG (P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.
With EcoR I and Hind III58 digestion pET-28a (+) carrier and insertion fragment, will insert fragment subsequently and be connected to pET-28a (+) carrier and keep open reading frame initial at bacterium RBS.With connecting the E.coli bacterial strain that mixture transforms commodity TG I by name, TG I contains the recombinant plasmid of multiple copied subsequently, and it is expressed lac I repressor and carries kalamycin resistance (Kan
r), containing Kan
rThe LB culture dish on screen transformant, the extracting plasmid, the cDNA fragment of sequence verification apis cerana bee venom AcHA has correctly been inserted carrier.
Adding Kan
rIncubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume LB liquid nutrient medium, culturing cell grows to 600 optical density(OD) (OD
600) when being 0.4-0.6, add IPTG (" isopropylthio--D galactoside ") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M, after the clarification, by containing under the condition that 6His marker albumen combines closely making, with nickel-NTA post layer folding purifying dissolved apis cerana bee venom AcHA from solution.With 6M Guanidinium hydrochloride (PH7.9) wash-out apis cerana bee venom AcHA from post, available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use dialysis step distiller's yeast to remove Guanidinium hydrochloride.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.
SDS-PAGE glue with 10% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 46.3KDa.
In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID No.4 with ordinary method.
Embodiment 4, the expression of apis cerana bee venom AcHA in eukaryotic cell (Tn cell strain, the i.e. wild moth cell strain of powder pattern)
In this embodiment, with pcr amplification product among the embodiment 1 as inserting fragment.
5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:
5 '-AGAATTCATGTCTCGGCCTCTCGTGATC-3 ' (SEQ ID No.1), this primer contains the restriction enzyme site of EcoR I restriction enzyme, is 18 Nucleotide of the apis cerana bee venom AcHA encoding sequence that begins of initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5 '-GAAGCTTACACTTGGTCCACGCTCA-3 ' (SEQ ID No.2), this primer contains the restriction enzyme site of HindIII restriction enzyme, the part encoding sequence of translation termination and apis cerana bee venom AcHA.
Restriction enzyme digestion sites is corresponding to the restriction enzyme digestion sites on the Tn fibrocyte expression vector pBacFastHTa on the primer, this plasmid vector coding antibiotics resistance (Amp
rAnd Gm
r), a phage replication starting point (Ori), a virus replication starting point (SV40), T7 promotor, a viral promotors (P-CMV), a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and corresponding polyA order.
With EcoR I and HindIII digestion pBacFastHTa carrier and insertion fragment, with linking mixture Transformed E .coli TG I bacterial strain, containing Amp subsequently
rAnd Gm
rThe LB culture dish on screen transformant, add Amp
rAnd Gm
rThe LB liquid nutrient medium in cultivate the clone that (O/N) contains required construction, the extracting plasmid, the cDNA fragment of sequence verification apis cerana bee venom AcHA has correctly been inserted carrier.Use recombinant plasmid transformed E.coli DH10Bac bacterial strain subsequently, containing Kan
r, Gm
r, tsiklomitsin the LB culture dish on screen transformant, adding Kan
r, Gm
r, tsiklomitsin the LB liquid nutrient medium in cultivate the clone that (O/N) contains required construction, the extracting plasmid through Sepharose 2B column purification, reclaims plasmid-70 ℃ preservation.
Plasmid transfection Tn cell is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, collecting cell and cell conditioned medium, the cell conditioned medium that contains the recombinant virus particle is used for next round to be infected.Cultivate through the TC-100 in 2-3 week continuous passage, collecting cell with ultrasonic degradation method smudge cells, is balance liquid and elutriant with 20mM Tris-CI (PH8.0) solution that contains NaCl0.5mM, imidazola 5mM, PMSF 1mM, the Ni of protein liquid through pre-equilibration
2+Sepharose 6B post is crossed post; Use the solution of the 20mM Tris-CI (PH8.0) that contains imdazola50mM, NaCl 0.5mM, PMSF 1mM again, the flush away foreign protein, the fusion rotein of 6 * His of narrow spectrum absorption, the solution with the 20mM Tris-CI (PH8.0) that contains imdazola 500mM, NaCl 0.5mM, PMSF 1mM carries out wash-out again. and be that dialyzate is dialysed to expressing protein solution with PBS (PH7.4) then.Last freeze-drying is preserved.
SDS-PAGE glue with 10% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 45.3KDa.
In addition, with ordinary method the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order the albumen of discovery and the white ID No.4 of SEQ, sequence unanimity.
Embodiment 5, preparation antibody
The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with affinity chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.With the albumen of g/ emulsification of 200-300 μ, to subcutaneous injection after 21 days,, the rabbit in 12 ages in week is carried out subcutaneous multiple spot and leg muscle injection with booster immunization with the dosage of 100-200 μ g/ head with the same antigen of non-complete Freund ' s adjuvant emulsion.Carry out one time the intragluteal injection booster immunization after 21 days again.The ability that sero-fast specific reaction activity precipitates apis cerana bee venom AcHA gene translation product in vivo with it is assessed.
The explanation of SEQ ID No.1 ∽ 4, information (i) the sequence signature CA of SEQ ID NO.1) length:type 28 base CB): nucleic acid (C) chain:strand (D) topology; Linearity is molecule-type (ii): (vi) sequence description:information (i) sequence signature (A) length of SEQ ID NO.1AGAATTCATGTCTCGGCCTCTCGTGATCSEQ ID NO.2:25 bases (B) type:nucleic acid, (C) chain:strand (D) topology:linearity is molecule-type (ii): oligonucleotide (vi) sequence description for oligonucleotide; The information of SEQ ID NO.2GAAGCTTACACTTGGTCCACGCTCASEQ ID NO.3:(i) sequence signature (A) length:1164bp (B) type:nucleic acid (C) chain:strand (D) topology; ( ii ) ：cDNA ( vii ) ( vi ) ：SEQ ID NO.3 1 ATGTCTCGGC CTCTCGTGAT CGCGGAAGAG ATGATGGTTG GAGTGTTGCT AATGCTAGCC 61 CCCGTACTTC GGATAAACGC GTTATTACTC GGCTTCGTAC CGAGCACCCC CAACGACAAC 121 AACAAAACCA AACGGGAGTT CAACGTTTAC TGGAACGTGC CCACCTTTAT GTGCCATAAA 181 TACGGGCTAC GCTTCGAAGA AGTATCGGAG AAATATGGTA TTCTACAGAA CTGGATGGAT 241 AAGTTTTGGG GCGAAGAGAT CGCGATCCTT TACGACCCTG GAATGTTCCC GGCGTTGCTG 301 AAAGACTCGA ACGGGAACGT AGTGGCGAGG AACGGCGGTG TCCCGCAACT GGGCAATCTC 361 ACCAAGCATC TCCAAGTATT TCGGGACCAC TTGATCAATC AGATCCCGGA CAAATCGTTC 421 CCCGGCGTGG GGGTGATCGA TTTCGAAAGT TGGAGGCCGA TATTCAGACA GAATTGGGCC 481 TCCCTCCAAC CCTACAAGAA ACTCTCCATA GACGTGGTTC GCCGTGAGCA TCCGTCCTGG 541 GGCGATCAGA GGGTGGAGCA GGAGGCGAAA CGAAGGTTCG AGAAATACGG GAAACTTTTT 601 ATGGAGGAGA CGTTGAAAGC TGCGAAACGG ATGAGGCCGG CCGCCAATTG GGGACACTAC 661 GCTTACCCTT ATTGCTACAA TCTGACGCCG AATCAACCGA GCTCCCAATG CGAAGCAACC 721 ACCATGCAGG AAAACGATAA AATGTCATGG CTGTTCGAGT CGGAAGACGT CCTCCTTCCG 781 TCCGTTTATT TGAGATGGAA TCTGACGAGC GGCGAACGAG TGGGCCTGGT CGGTGGCCGC 841 GTGAAGGAGG CGTTGAGAAT AGCGAGGCAA ATGACGACGA GCCGAAAGAA GGTTCTACCC 901 TATTACTGGT ACAAATATCA GGATCGAAGG GACACGGATT TGAGCAGGGC TGACCTCGAG961 GCAACTTTAC GAAAAATCAC GGACCTCGGC GCCGATGGGT TTATCATTTG GGGAAGTTCC1021 AACGATATAA ACACGAAAGC GAAATGCGTA CAATTCAGGG AATACCTGAA CAACGATTTG1081 GGCCCTGTCG TTAAACGGAT CGCGTTGAAC AGCAACGCCC CTGCCGCGAA AAGTCGACTC1141 GACGTGAGCG TGGACCAAGT GTAASEQ ID NO.4： ( i ) ( A ) ：387 ( B ) ： ( C ) ； ( ii ) ： ( vi ) ：SEQ ID NO.41 Met Ser Arg Pro Leu Val Ile Ala Glu Glu Met Met Val Gly Val Leu Leu Met Leu Ala21 Pro Val Leu Arg Ile Asn Ala Leu Leu Leu Gly Phe Val Pro Ser Thr Pro Asn Asp Asn41 Asn Lys Thr Lys Arg Glu Phe Asn Val Tyr Trp Asn Val Pro Thr Phe Met Cys His Lys61 Tyr Gly Leu Arg Phe Glu Glu Val Ser Glu Lys Tyr Gly Ile Leu Gln Asn Trp Met Asp81 Lys Phe Trp Gly Glu Glu Ile Ala Ile Leu Tyr Asp Pro Gly Met Phe Pro Ala Leu Leu101 Lys Asp Ser Asn Gly Asn Val Val Ala Arg Asn Gly Gly Val Pro Gln Leu Gly Asn Leu121 Thr Lys His Leu Gln Val Phe Arg Asp His Leu Ile Asn Gln Ile Pro Asp Lys Ser Phe141 Pro Gly Val Gly Val Ile Asp Phe Glu Ser Trp Arg Pro Ile Phe Arg Gln Asn Trp Ala161 Ser Leu Gln Pro Tyr Lys Lys Leu Ser Ile Asp Val Val Arg Arg Glu His Pro Ser Trp181 Gly Asp Gln Arg Val Glu Gln Glu Ala Lys Arg Arg Phe Glu Lys Tyr Gly Lys Leu Phe201 Met Glu Glu Thr Leu Lys Ala Ala Lys Arg Met Arg Pro Ala Ala Asn Trp Gly His Tyr221 Ala Tyr Pro Tyr Cys Tyr Asn Leu Thr Pro Asn Gln Pro Ser Ser Gln Cys Glu Ala Thr241 Thr Met Gln Glu Asn Asp Lys Met Ser Trp Leu Phe Glu Ser Glu Asp Val Leu Leu Pro261 Ser Val Tyr Leu Arg Trp Asn Leu Thr Ser Gly Glu Arg Val Gly Leu Val Gly Gly Arg281 Val Lys Glu Ala Leu Arg Ile Ala Arg Gln Met Thr Thr Ser Arg Lys Lys Val Leu Pro301 Tyr Tyr Trp Tyr Lys Tyr Gln Asp Arg Arg Asp Thr Asp Leu Ser Arg Ala Asp Leu Glu321 Ala Thr Leu Arg Lys Ile Thr Asp Leu Gly Ala Asp Gly Phe Ile Ile Trp Gly Ser Ser341 Asn Asp Ile Asn Thr Lys Ala Lys Cys Val Gln Phe Arg Glu Tyr Leu Asn Asn Asp Leu361 Gly Pro Val Val Lys Arg Ile Ala Leu Asn Ser Asn Ala Pro Ala Ala Lys Ser Arg Leu381 Asp Val Ser Val Asp Gln ValI HAHA：AcHA：387AmHA：382“*”AcHA 1：MSRPLVIAEEMMVGVLLMLAPVLRINALLLGFVPSTPNDNNKTKREFNVYWNVPTFMCHK 60AmHA 1：MSRPLVITEGMMIGVLLMLAP---INALLLGFVQSTPDNNKTV-REFNVYWNVPTFMCHK 56
*******?*?**?******** *********?*** * ****************AcHA 61：YGLRFEEVSEKYGILQNWMDKFWGEEIAILYDPGMFPALLKDSNGNVVARNGGVPQLGNL?120AmHA 57：YGLRFEEVSEKYGILQNWMDKFRGEEIAILYDPGMFPALLKDPNGNVVARNGGVPQLGNL?116
**********************?*******************?*****************AcHA 121：TKHLQVFRDHLINQIPDKSFPGVGVIDFESWRPIFRQNWASLQPYKKLSIDVVRREHPSW?180AmHa 117：TKHLQVFRDHLINQIPDKSFPGVGVIDFESWRPIFRQNWASLQPYKKLSVEVVRREHPFW?176
************************************************* *******?*AcHA 181：GDQRVEQEAKRRFEKYGKLFMEETLKAAKRMRPAANWGHYAYPYCYNLTPNQPSSQCEAT?240AmHA 177：DDQRVEQEAKRRFEKYGQLFMEETLKAAKRMRPAANWGYYAYPYCYNLTPNQPSAQCEAT?236
****************?********************?***************?*****AcHA 241：TMQENDKMSWLFESEDVLLPSVYLRWNLTSGERVGLVGGRVKEALRIARQMTTSRKKVLP?300AmHA 237：TMQENDKMSWLFESEDVLLPSVYLRWNLTSGERVGLVGGRVKEALRIARQMTTSRKKVLP?296
************************************************************AcHA 301：YYWYKYQDRRDTDLSRADLEATLRKITDLGADGFIIWGSSNDINTKAKCVQFREYLNNDL?360AmHA 297：YYWYKYQDRRDTDLSRADLEATLRKITDLGADGFIIWGSSDDINTKAKCLQFREYLNNEL?356
****************************************?********?********?*AcHA 361：GPVVKRIALNSNAPAAKSRLDVSVDQV 387AmHA 357：GPAVKRIALNNNANDRLT-VDVSVDQV 382
* * * * * * * * * * * * * * * * * homology: 90%
Claims (10)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of apis cerana bee venom AcHA active polypeptide.
Show at least 70% homology from the nucleotides sequence of Nucleotide 1-1164 position among described nucleotide sequence and the SEQ ID No.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID No.3 in from the nucleotide sequence hybridization of Nucleotide 1-1164.
2. according to the dna molecular of claim 1 SEQ ID No.3, it is characterized in that described encoded polypeptides has the sequence shown in the SEQ No.4, have among the SEQ ID No.3 nucleotide sequence from Nucleotide 1-1164 position.
3. an isolating apis cerana bee venom AcHA protein polypeptide is characterized in that it has polypeptide or its active fragments of SEQ IDNo.4 aminoacid sequence, or its reactive derivative, and this polypeptide has SEQID No.4 sequence.
4. a carrier is characterized in that, it contains the described DNA of claim 1.
5. a host cell is characterized in that, it is with the described carrier transformed host cells of claim 4.
6. preparation method with apis cerana bee venom AcHA protein active polypeptide is characterized in that this method comprises:
(a) nucleotide sequence that coding is had a polypeptide of apis cerana bee venom AcHA protein-active operationally is connected in expression regulation sequence, form apis cerana bee venom AcHA protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 1-1164 position among described nucleotide sequence and the SEQ ID No.3;
(b) change the carrier of expressing in the step (a) over to host cell, form the proteic reconstitution cell of apis cerana bee venom AcHA;
(c) be fit to express under the condition of apis cerana bee venom AcHA protein polypeptide the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with apis cerana bee venom AcHA protein-active.
7. an antibody is characterized in that, it be can with the described apis cerana bee venom of claim 3 AcHA protein polypeptide specificity bonded antibody.
8. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1.
9. probe molecule according to claim 8; It is characterized in that being meant can combine with apis cerana bee venom AcHA gene product or segment but nonrecognition and in conjunction with the antibody of other uncorrelated antigen molecule.
10. probe molecule according to claim 8 is characterized in that, it has 15-20 successive Nucleotide in the apis cerana bee venom AcHA polypeptid coding sequence.
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|---|---|---|---|
| CN01135659A CN1366049A (en) | 2001-10-12 | 2001-10-12 | Chinese bee venom AcHA gene, polypeptide coded by it and its preparing process |
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|---|---|---|---|
| CN01135659A CN1366049A (en) | 2001-10-12 | 2001-10-12 | Chinese bee venom AcHA gene, polypeptide coded by it and its preparing process |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112794895A (en) * | 2021-01-06 | 2021-05-14 | 中国医学科学院医药生物技术研究所 | Application of exogenous ATG10S protein in preparation of antiviral drugs |
| CN114350691A (en) * | 2021-03-05 | 2022-04-15 | 华熙生物科技股份有限公司 | Gene for efficiently expressing hyaluronidase and expression method thereof |
| CN116179578A (en) * | 2022-09-30 | 2023-05-30 | 华熙生物科技股份有限公司 | Gene for high-level expression of aglycosylated hyaluronidase and application thereof |
-
2001
- 2001-10-12 CN CN01135659A patent/CN1366049A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112794895A (en) * | 2021-01-06 | 2021-05-14 | 中国医学科学院医药生物技术研究所 | Application of exogenous ATG10S protein in preparation of antiviral drugs |
| CN114350691A (en) * | 2021-03-05 | 2022-04-15 | 华熙生物科技股份有限公司 | Gene for efficiently expressing hyaluronidase and expression method thereof |
| CN114350691B (en) * | 2021-03-05 | 2023-12-08 | 华熙生物科技股份有限公司 | Gene for efficiently expressing hyaluronic acid hydrolase and expression method thereof |
| CN116179578A (en) * | 2022-09-30 | 2023-05-30 | 华熙生物科技股份有限公司 | Gene for high-level expression of aglycosylated hyaluronidase and application thereof |
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