CN1194089C - Large wasp sedative peptide precursor gene and its coded polypeptide and preparation method - Google Patents
Large wasp sedative peptide precursor gene and its coded polypeptide and preparation method Download PDFInfo
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- CN1194089C CN1194089C CNB021107491A CN02110749A CN1194089C CN 1194089 C CN1194089 C CN 1194089C CN B021107491 A CNB021107491 A CN B021107491A CN 02110749 A CN02110749 A CN 02110749A CN 1194089 C CN1194089 C CN 1194089C
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Abstract
The present invention discloses a hornet venom sedative peptide precursor (Preprosecapin) gene sequence, coded polypeptide thereof and a preparing method of a processed product sedative peptide (Secapin) of genes after translation. The cDNA sequence coded protein is a homolog of Apis mellifera venom Preprosecapin, and has at least 70% of homology with the nucleotide sequence from nucleotide 1 to 234 sites in SEQ ID No. 5. The sequence codes have polypeptide with a sequence showed in SEQ ID No. 6. Additionally, the present invention also provides a method for preparing carriers and hose cells containing the genes of hornet venom sedative Preprosecapin, antibodies related to the hornet venom sedative Preprosecapin, hornet venom Secapin polypeptide having protein activity and related antibodies. The present invention provides a basis for further researching polypeptide active molecular mechanisms related to Secapin and developing biological medicines, diagnostic reagents and biological reagents related to the secapin.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new bee venom gene nucleotide series.More particularly, the present invention relates to the cDNA sequence of Vespa magnifiac (Sonan). bee venom sedative peptide precursor (Preprosecapin) gene, the polypeptide of this cDNA sequence encoding is the proteic homologue of apis mellifera Preprosecapin, and this polypeptide is the homologue of the calm peptide of apis mellifera (Secapin) through the translation resulting polypeptide of post-treatment (mature peptide).The invention still further relates to this nucleotide sequence coded polypeptide, the application of these polynucleotide and polypeptide, and the preparation method of described polynucleotide and described polypeptide.
Background technology
The calm peptide of apis mellifera (Apis mellifera) bee venom (Bee venomous Secapin) is one of four polypeptide of apis mellifera bee venom, accounts for 1% of bee venom dry weight, by 25 amino acid form (
Eur.J. Biochem.1978,83:405-410;
Abstr.15 Eur.Pepetide.Symp.1978, p84).By sources and biochemical property, it belongs to basic polypeptide, and its biological activity of Secapin is similar to the haemolysis peptide, have effects such as anti-inflammatory, antibiotic, step-down, calmness (
Eur.J.Biochem.1978,83:405-410).
1984, pst I and Cla I site that Vlask and Kreil insert pBR322 with Apis mellifera queen bee poison gland cDNA are used
32The cDNA probe of P mark filters out positive colony (PUM9/24, PBMC1), it is inferior more grand after enzyme is cut to PUC8, obtained bee venom Preprosecapin gene, sequencing result to this gene carries out the aminoacid sequence derivation, obtain 77 amino-acid residues, wherein last 25 amino-acid residues and known Secapin consensus amino acid sequence, preceding 32 amino-acid residues are signal peptide, middle 20 amino acid are polarity peptide precursor, the Preprosecapin gene is through the translation post-treatment, promptly in the malicious capsule of honeybee through the cracking of a kind of lytic enzyme in the honeybee body just obtained active polypeptide Secapin (
Eur.J.Biochem.1984,145:279～242.).Secapin and other several bee venom polypeptide have important use to be worth on medical science and biophysics.
Summary of the invention
One of the object of the invention provides a kind of polynucleotide of Vespa magnifiac (Sonan). bee venom Preprosecapin gene, the proteic homologue of this polynucleotide encoding apis mellifera bee venom Preprosecapin, the active polypeptide that this precursor protein obtains after processing is a homologue of apis mellifera Secapin polypeptide.
Two of the object of the invention provides a kind of new proteic homologue of apis mellifera Preprosecapin, and this polypeptide is a Vespa magnifiac (Sonan). Preprosecapin albumen.
The object of the invention also provides this preparation Vespa magnifiac (Sonan). bee venom Preprosecapin nucleotide probe, contain the recombinant vectors of Vespa magnifiac (Sonan). bee venom Preprosecapin Nucleotide, contain 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 proteic nucleotide sequence of Vespa magnifiac (Sonan). bee venom Preprosecapin, show at least 70% homology from the nucleotides sequence of 1～234 in Nucleotide among described nucleotide sequence and the SEQ ID No.5, perhaps described nucleotide sequence can be hybridized from the Nucleotide of 1～234 in Nucleotide under the rigorous condition of moderate with among the SEQ ID No.5, preferably, this polypeptide has the sequence shown in the SEQ ID No.6.More preferably, this sequence has among the SEQ ID No.5 nucleotide sequence from 1～234 in Nucleotide.
Also provide a kind of isolating Vespa magnifiac (Sonan). bee venom Secapin protein active polypeptide in the present invention, it comprises: have polypeptide or its active fragments of 53～77 aminoacid sequences among the SEQ ID No.6, or its reactive derivative.Preferably, this polypeptide is the polypeptide with 53～77 aminoacid sequences among the SEQ ID No.6.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 Vespa magnifiac (Sonan). bee venom Secapin active polypeptide, and this method comprises:
(a) nucleotide sequence that coding is had a Vespa magnifiac (Sonan). bee venom Preprosecapin gene operationally is connected in expression regulation sequence, form Vespa magnifiac (Sonan). bee venom Preprosecapin expression vector, the nucleotides sequence that described nucleotide sequence and SEQID No.5 Nucleotide are 1～234 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 Vespa magnifiac (Sonan). bee venom Preprosecapin;
(c) be fit to express under the condition of Vespa magnifiac (Sonan). bee venom Preproecapin protein polypeptide the reconstitution cell in the culturing step (b);
(d) isolate polypeptide with Vespa magnifiac (Sonan). bee venom Secapin protein-active.
The present invention has found a kind of coding Vespa magnifiac (Sonan). bee venom Preprosecapin protein gene, the Preprosecapin polypeptide of this coded by said gene (Vespa magnifiac (Sonan). bee venom sedative peptide precursor) has just obtained active polypeptide Secapin through the cracking of a kind of lytic enzyme in the wasp body, this active polypeptide is a kind of biologically active substance, can be applicable to the assisting therapy of medically sacroiliitis, nervous system disease, drug abuse etc., can be used as antigen and be used for the preparation of bee venom immunological reagent, can be used as the toolenzyme of biological study.Vespa magnifiac (Sonan). Preprosecapin gene can be the molecule mechanism of research Vespa magnifiac (Sonan). bee venom immunity, for development and use Vespa magnifiac (Sonan). bee venom resource provides new foundation.
Embodiment
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 234 Nucleotide, and its detailed sequence is seen SEQ ID No.5, and open reading frame is positioned at 1～234 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 " Vespa magnifiac (Sonan). bee venom Preprosecapin albumen (or polypeptide) encoding sequence " refers to that coding has the nucleotide sequence of Vespa magnifiac (Sonan). bee venom Preprosecapin albumen (or polypeptide), as 1～234 nucleotide sequence among the SEQ ID No.5 and degenerate sequence thereof.This degenerate sequence is meant 1～234 Nucleotide of encoder block that is arranged in SEQ ID No.5 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.5 in 1～234 nucleotide sequence homology be low to moderate about 70% degenerate sequence described sequence of SEQ ID No.6 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.5 from the nucleotide sequence of the nucleotide sequence hybridization of 1～234 in Nucleotide.This term also comprise with SEQ ID No.5 in from the homology of nucleotide sequence at least 70% of 1～234 in Nucleotide, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have variant form with proteic, the SEQ ID No.6 sequence of Vespa magnifiac (Sonan). bee venom Preprosecapin 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 " Vespa magnifiac (Sonan). bee venom Secapin protein polypeptide " refers to have the polypeptide of the SEQ ID No.6 sequence 53-77 of Vespa magnifiac (Sonan). bee venom Preprosecapin protein-active.This term also comprises having and variant form Vespa magnifiac (Sonan). bee venom Preprosecapin albumen identical function, SEQ ID No.6 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 Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of the wasp aptoxin Preprosecapin of Chinese People's Anti-Japanese Military and Political College polypeptide to obtain.The present invention also provides other polypeptide, as comprises Vespa magnifiac (Sonan). bee venom Preprosecapin polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of Vespa magnifiac (Sonan). bee venom Preprosecapin polypeptide.
Invention also provides the analogue of Vespa magnifiac (Sonan). bee venom Preprosecapin albumen or polypeptide, the difference of these analogues and natural Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin polypeptid coding sequence and segmental antisense sequences thereof, and this antisense sequences can be used for suppressing the expression of Vespa magnifiac (Sonan). bee venom Preprosecapin in the cell.
The present invention also comprises a kind of probe molecule, and this molecule has 8～100 of Vespa magnifiac (Sonan). bee venom Preprosecapin polypeptid coding sequence, preferably 1～50 continuous nucleotide usually.This probe can be used for whether existing in the test sample coding Vespa magnifiac (Sonan). bee venom Preprosecapin nucleic acid molecule.
The present invention also comprises the method that detects Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin gene product or fragment.Preferably, refer to that those can combine with Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin, comprise that also those do not influence the antibody of Vespa magnifiac (Sonan). bee venom Preprosecapin protein function.The present invention also comprise those can with modify or without the Vespa magnifiac (Sonan). bee venom Preprosecapin 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 vespine antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art, for example, the Vespa magnifiac (Sonan). bee venom Preprosecapin gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Vespa magnifiac (Sonan). bee venom Preprosecapin 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 prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol, 6:511,1976; People such as Kohler,
Eur.J.Immunol, 6:292,1976; People such as Hammerling, I
N Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block Vespa magnifiac (Sonan). bee venom Preprosecapin function and the antibody that does not influence Vespa magnifiac (Sonan). bee venom Preprosecapin function.Each antibody-like of the present invention can utilize the fragment or the functional zone of Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Vespa magnifiac (Sonan). bee venom Preprosecapin gene is so to obtain, cDNA with Vespa magnifiac (Sonan). poison gland and the total RNA reverse transcription of malicious capsule is a template, with a pair of oligonucleotide is that primer-A:5 '-GCTCGAGATGAAGAACTATTCAAAAAATGCA-3 ' is that forward is to primer, oligonucleotide B:5 '-GAAGCTTTTAAGGCACTATGACTCTACTACA-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID No.5 after amplified production checked order.
Vespa magnifiac (Sonan). bee venom Preprosecapin is a Vespa magnifiac (Sonan). poison gland expressed proteins, and Vespa magnifiac (Sonan). bee venom Preprosecapin of the present invention provides the foundation for the relevant molecule mechanism of research Vespa magnifiac (Sonan). 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 Vespa magnifiac (Sonan). bee venom Preprosecapin
1. primer amplification
CDNA with Vespa magnifiac (Sonan). poison gland, the total RNA reverse transcription of malicious capsule is a template, with a pair of oligonucleotide is that primer-A:5 '-GCTCGAGATGAAGAACTATTCAAAAAATGCA-3 ' (SEQ IDNo.1) is a forward primer, oligonucleotide B:5 '-GAAGCTTTTAAGGCACTATGACTCTACTACA-3 ' (SEQ ID No.2) is a reverse primer, carries out PCR.The PCR condition of A/B be 94 ℃ 1 minute, thereupon with 94 ℃ of 40 second, carry out 35 circulations in 54 ℃ of 40 second and 72 ℃ of 60 second, last 72 ℃ were extended 10 minutes, electrophoresis detection obtains the purpose fragment of about 250bp.
2.PCR the order-checking of product
With above-mentioned pcr amplification product A/B and pGEM
-T Easy carrier (Promega) connects, and transformed into escherichia coli JM109 extracts plasmid with alkaline process, with BigDye terminator v2.0.Sequencing kit (Epicentre Teclnologies) checks order to extractive plasmid, obtains the cDNA sequence, and total length is 234bp altogether, and detailed sequence is seen SEQ ID No.5, and wherein open reading frame is positioned at 1～234 Nucleotide.
Derive the proteic aminoacid sequence of Vespa magnifiac (Sonan). bee venom Preprosecapin according to the full length cDNA sequence that obtains, totally 77 amino-acid residues (comprising 25 amino acid whose mature peptides, 32 amino acid whose signal peptides and 20 amino acid whose precursor peptides), its aminoacid sequence sees SEQ ID No.6 for details.
Embodiment 2, and homology relatively
Full length cDNA sequence and proteins encoded thereof with Vespa magnifiac (Sonan). bee venom Preprosecapin 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 Preprosecapin have significant homology.With the Blast software analysis as can be seen, their proteic homologys are 93% (seeing attached list 1).Therefore, can infer that Vespa magnifiac (Sonan). bee venom Preprosecapin albumen of the present invention is the homologue of apis mellifera bee venom Preprosecapin, and have similar function.
1984, pst I and Cla I site that Vlask and Kreil insert pBR322 with Apis mellifera queen bee poison gland cDNA are used
32The cDNA probe of P mark filters out positive colony, and (PUM9/24, PBMCl), the inferior more grand PUC8 that arrives carried out aminoacid sequence to sequencing result and derives after enzyme was cut, and had obtained the proteic new gene of coding apis mellifera bee venom Preprosecapin.Coded Preprosecapin mature peptide Secapin, signal peptide and the pre-pro-peptide of this cDNA is 77 amino-acid residues.Secapin and other several mellitin mixtures at the biologically active substance that medically is applied to assisting therapy such as cardiovascular, segmental bronchus, tumour, also are the antibiotic radioprotective biologically active substances of a class at present.Secapin and other several mellitins still are the biologically active substance that a class improves human immunity function in addition.The inventor's Vespa magnifiac (Sonan). Preprosecapin gene is the molecule mechanism of research Vespa magnifiac (Sonan). bee venom allergy, immunity, for development and use Vespa magnifiac (Sonan). bee venom resource provides new foundation.
Embodiment 3, the expression of Vespa magnifiac (Sonan). bee venom Preprosecapin mature peptide Secapin 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 '-GCTCGAGATGAAGAACTATTCAAAAAATGCA-3 ' (SEQ ID No.1), this primer contains the restriction enzyme site of Xhol I restriction enzyme, is 24 Nucleotide of the Vespa magnifiac (Sonan). bee venom Preprosecapin encoding sequence that begins of initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5 '-GAAGCTTTTAAGGCACTATGACTCTACTACA-3 ' (SEQ ID No.2), this primer contains the restriction enzyme site of Hind III restriction enzyme, the part encoding sequence of translation termination and Vespa magnifiac (Sonan). bee venom Preprosecapin.
Restriction enzyme digestion sites is corresponding to the restriction enzyme digestion sites on the Tn fibrocyte expression vector pBacFastHTb 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 XholI and Hind III 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 Vespa magnifiac (Sonan). bee venom Preprosecapin has correctly been inserted carrier.Use recombinant plasmid transformed E.coli DH10Bac bacterial strain subsequently, containing Amp
r, Gm
r, tsiklomitsin the LB culture dish on screen transformant, adding Amp
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.The 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 2.8KDa.
In addition, check order to expressing proteic amino acid, find consistent with 53～77 (mature peptides) of SEQ ID No.6 sequence with ordinary method.
Embodiment 4, the expression of the proteic mature peptide Secapin of Vespa magnifiac (Sonan). bee venom Preprosecapin in intestinal bacteria
In this embodiment, hybridize with the Nucleotide of 1～234 in the Nucleotide among the SEQ ID No.5
5 ' the Oligonucleolide primers sequence of using in the PCR reaction is:
5 '-GGATCCTTACATTATTGATGTTCCT-3 ' (SEQ ID No.5), this primer contains the restriction enzyme site of BamH I restriction enzyme, 18 Nucleotide of the mature peptide sequence of Vespa magnifiac (Sonan). bee venom Preprosecapin coding;
3 ' end primer sequence is:
This primer of 5 '-CGGGAATTAAGGCACTATGACTCT-3 ' (SEQ ID No.6) contains the restriction enzyme site of EcoR I restriction enzyme, the part encoding sequence of translation termination and Vespa magnifiac (Sonan). bee venom Preprosecapin.
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 pGEX-4T-3, this plasmid vector coding antibiotics resistance (Amp
r), a bacterium replication orgin (Ori), an adjustable promotor/operon of IPTG (P/O), a zymoplasm recognition site, warm protein marker of a glutathione S transferring enzyme (GST) and restriction enzyme cloning site.
With BamH I and EcoR I digestion pGEX-4T-3 carrier and insertion fragment, will insert fragment subsequently and be connected to the pGEX-4T-3 carrier and keep open reading frame initial at the zymoplasm recognition site.With connecting the E.coli bacterial strain that mixture transforms commodity JM105 by name, JM105 contains the recombinant plasmid of multiple copied subsequently, and it is expressed the lacIq repressor and carries resistance (Amp
r), containing Amp
rThe LB culture dish on screen transformant, the extracting plasmid, the cDNA fragment of sequence verification Vespa magnifiac (Sonan). bee venom Secapin, this sequence are 160～234 of SEQ ID No.5 sequence, and have correctly inserted carrier.
Adding Amp
rIncubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of (100 μ g/ml).(O/N) culture that spends the night dilutes with 1: 10 thinning ratio, is inoculated into then in the large volume LB liquid nutrient medium, and culturing cell grows to 600 optical density(OD) (OD
600) when being 0.6-1.0, add IPTG (" isopropylthio-β-D galactoside ") to final concentration be 1.0mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-5 hour, the centrifugal collecting cell precipitation is resuspended among the PBS of 1/20 volume ratio, by the ultrasonic treatment cell, adds TritonX-100 to final concentration 1%30min soluble protein.Low-temperature centrifugation crude extract (10000g) 5min, the supernatant thing is with glutathione S transferring enzyme (GST) purification unit purifying dissolved glutathione-Vespa magnifiac (Sonan). bee venom Secapin fusion rotein from solution, and Vespa magnifiac (Sonan). bee venom Secapin is cut down from warm albumen.Vespa magnifiac (Sonan). bee venom Secapin albumen carries out electrophoresis with 15% SDS-PAGE glue, identifies that this proteic molecular weight size of expression is about 2.8KDa.
In addition, check order to expressing proteic amino acid, find consistent with 53～77 (mature peptides) of SEQ ID No.6 sequence with ordinary method.
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, and electrophoretic band is downcut from gel. also with isopyknic complete Freund ' s adjuvant emulsion.With the albumen of g/ emulsification of 200-300 μ, to the rabbit subcutaneous injection in 12 ages in week.After 21 days,, carry 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.Sero-fast specific reaction activity precipitates Vespa magnifiac (Sonan). bee venom Preprosecapin gene translation in vivo with it to be assessed through the ability of processing after product.
The explanation of SEQ ID No.1 ∽ 6,
The information of SEQ ID NO.1
(i) sequence signature
(A) length: 31 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology; Linear
(ii) molecule-type: oligonucleotide
(xi) sequence description: SEQ ID NO.1
GCTCGAGATGAAGAACTATTCAAAAAATGCA
The information of SEQ ID NO.2
(i) sequence signature
(A) length: 31 bases
(B) type: nucleic acid, (C) chain: strand
(D) topology: linearity
(ii) molecule-type: oligonucleotide
(xi) sequence description; SEQ ID NO.2
GAAGCTTTTAAGGCACTATGACTCTACTACA
The information of SEQ ID NO.3
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology; Linear
(ii) molecule-type: oligonucleotide
(xi) sequence description: SEQ ID NO.3
5’-GGATCCTTACATTATTGATGTTCCT-3’
The information of SEQ ID NO.4
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology; Linear
(ii) molecule-type: oligonucleotide
(xi) sequence description: SEQ ID NO.4
5’-CGGGAATTAAGGCACTATGACTCT-3’
The information of SEQ ID NO.5:
(i) sequence signature
(A) length: 234 bp
(B) type: nucleic acid
(C) chain: strand
(D) topology; Linear
(ii) molecule-type: cDNA
(xii) (xi) sequence description: SEQ ID NO.5
1 ATGAAGAACT?ATTCAAAAAA?TGCAACATAT?TT?AATTACTG?TT?CTGTTATT?CAGTTTTGTT
61 GCTATGCTTT?TAATTATTCC?ATCAAAATGT?GAAGCCGTTA?GCAATGATAT?GCAACCATTG
121?GAAGCACGAT?CTGCTGATTT?AGTCCCGGAA?CCAAGATACA?TTATTGATGT?TCCTCCTAGA
181?TGTCCTCCAG?GTTCTAAATT?CATTAAGAAC?AGATGTAGAG?TCATAGTGCC?TTAA
The information of SEQ ID NO.6:
(i) sequence signature
(A) length: 77 amino acid
(B) type: amino acid
(C) topological framework; Linear
(ii) molecule-type: polypeptide
(xi) sequence description: SEQ ID NO.6
1 Met?Lys?Asn?Tyr?Ser?Lys?Asn?Ala?Thr?Tyr?Leu?Ile?Thr?Val?Leu?Leu?Phe?Ser?Phe?Val
21 Ala?Met?Leu?Leu?Ile?Ile?Pro?Ser?lys?Cys?Glu?Ala?Val?Ser?Asn?Asp?Met?Gln?Pro?Leu
41 Glu?Ala?Arg?Ser?Ala?Asp?Leu?Val?Pro?Glu?Pro?Arg?Tyr?Ile?Ile?Asp?Val?Pro?Pro?Arg
61 Cys?Pro?Pro?Gly?Ser?Lys?Phe?Ile?Lys?Asn?Arg?Cys?Arg?Val?Ile?Val?Pro
Table I Vespa magnifiac (Sonan). bee venom Preprosecapin and the proteic comparison of apis mellifera bee venom PreprosecapinTwo sequences of carrying out the homology comparison are:
Preprosecapin
Residue sum: 77
Preprosecapin residue sum: 77
The symbol of representing consistent residue is " * "
Apis mellifera 1:MKNYSKNATHLITVLLFSFVVILLIIPSKCEAVSNDRQSLEARSADLVPEPRYI IDVPPR 60
Vespa magnifiac (Sonan). 1:MKNYSKNATYLITVLLFSFVAMLLIIPSKCEAVSNDMQPLEARSADLVPEPRYI IDVPPR 60
*********?**********?************** *?*********************
Apis mellifera 61:CPPGSKFIKNRCRVIVP 77
Vespa magnifiac (Sonan). 61:CPPGSKFIKNRCRVIVP 77
*****************
Homology: 93%
Claims (7)
1. isolated dna molecular, it is characterized in that: sequence is a SEQ ID No.5 nucleotide sequence, and coding Vespa magnifiac (Sonan). bee venom sedative peptide precursor albumen.
2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID No.6.
3. an isolating Vespa magnifiac (Sonan). bee venom sedative peptide precursor albumen is characterized in that it has the polypeptide of SEQ IDNo.6 aminoacid 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 Vespa magnifiac (Sonan). bee venom sedative peptide precursor gene its lytic activity polypeptide is characterized in that this method comprises:
(a) coding is had the proteic nucleotide sequence of Vespa magnifiac (Sonan). bee venom sedative peptide precursor and operationally be connected in expression regulation sequence, form Vespa magnifiac (Sonan). bee venom sedative peptide precursor protein polypeptide expression vector, described nucleotide sequence has the nucleotide sequence of SEQ ID No.5;
(b) change the carrier of expressing in the step (a) over to host cell, form the proteic reconstitution cell of Vespa magnifiac (Sonan). bee venom sedative peptide precursor;
(c) be fit to express under the condition of Vespa magnifiac (Sonan). bee venom sedative peptide precursor gene product polypeptide the reconstitution cell in the culturing step (b);
(d) isolate and have the calm peptide of Vespa magnifiac (Sonan). bee venom sedative peptide precursor protein gene product active polypeptide.
7. an antibody is characterized in that, it be can with the described Vespa magnifiac (Sonan). bee venom of claim 3 sedative peptide precursor protein-specific bonded antibody.
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| KR101638863B1 (en) * | 2015-05-21 | 2016-07-13 | 동아대학교 산학협력단 | Secapin peptide from Apis cerana |
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