CN1233830C - China bee and hornet hemolysis peptide precursor gene and coded polypeptide and preparing method - Google Patents
China bee and hornet hemolysis peptide precursor gene and coded polypeptide and preparing method Download PDFInfo
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- CN1233830C CN1233830C CNB021107483A CN02110748A CN1233830C CN 1233830 C CN1233830 C CN 1233830C CN B021107483 A CNB021107483 A CN B021107483A CN 02110748 A CN02110748 A CN 02110748A CN 1233830 C CN1233830 C CN 1233830C
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Abstract
The present invention discloses an Apis cerana and hornet venom melittin precursor gene coding sequence, coded polypeptide thereof and a preparing method thereof. The cDNA sequence coded protein is a homolog of an Italian hornet venom melittin precursor, and has the homology of at least 70% with the nucleotide sequence of the nucleotide from 1 to 213 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 host cells containing the Apis cerana and hornet venom melittin precursor and the 130 to 213 nucleotide sequence of the gene terminals of the melittin precursor, antibodies related to the Apis cerana and hornet venom melittin, Apis cerana and hornet venom melittin polypeptide having protein activity, and related antibodies. The present invention provides a basis for further researching the molecular action mechanism of Apis cerana and hornet venom melittin and developing biological medicines, diagnostic reagents, biochemical reagents and biological pesticides related to the melittin.
Description
Technical field
The present invention relates to the genetically engineered field, relate in particular to apis cerana, Vespa magnifiac (Sonan). melittin precursor (Prepromelittin) gene and encoded polypeptides and preparation method.
Background technology
Apis mellifera (Apis mellifera) melittin (Melittin) is the ultimate constituent of apis mellifera bee venom; account for 40%～50% (Vol.3.New York:Academic of bee venom; 1971; 535), by sources and biochemical property, it is a kind of active polypeptide; wherein 90% haemolysis peptide exists with unbound state; other 10% then with Melittin N1-formylation form exist (Biochem.Biophys.Res.Commun., 1967,27:275-280).It has very important biologic activity, can cause erythrolysis (Z.Physiol.Chem., 1970,351,884), liposome release mark ion (marker ions) (J.Biol.Chem., 1969,244,3575) and mastocyte discharge histamine (J.Pharmacol, 1965,25,29) or the like.Melittin has two kinds of precursors to exist when synthetic.A kind of is Promelittin, finds at the frog's egg of bee venom or injection queen bee poison gland extract.When synthesizing in frog's egg, Promelittin is stable end product, and in poison gland, it will change Melittin lentamente into; Second kind is called Prepromelittin, and molecular weight ratio Promelittin is big, find on the MelittinmRNA that can only in cell-free system, transcribe (Proc.Natl.Acad.Sct.USA, 1978,75,701-704).
Nineteen eighty-three, Vlasak etc. utilize the total RNA reverse transcription of apis mellifera queen bee poison gland to become cDNA, then the double-stranded cDNA of synthetic is cloned in the pBR322 carrier, the construction cDNA library, filter out to have with probe and insert segmental positive colony greater than 200bp, and then with their subclones in the PUC8 carrier, cut evaluation through enzyme again and therefrom filter out 2 positive colonies, check order to wherein containing the maximum segmental PUM13/4 of insertion, the result shows that this insertion sequence is the nucleotide sequence of Prepromelittin, the mature peptide that this cDNA is coded--melittin precursor protein be 49 amino-acid residues (Eur.J.Biochem., 1983,135:123-126).Prepromelittin albumen comes down to the natural fusion rotein of melittin, and it is synthetic after the dipeptides protease hydrolysis becomes bee venom Melittin in the bee venom cell.
Summary of the invention
One of the object of the invention provides the polynucleotide of a kind of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin, the proteic homologue of this polynucleotide encoding apis mellifera bee venom Prepromelittin.
Two of the object of the invention provides a kind of final product-apis cerana by apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin polynucleotide encoding, Vespa magnifiac (Sonan). bee venom Melittin albumen.
The object of the invention also provides this recombinant vectors for preparing apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin nucleotide probe, contains the 130-213 nucleotide sequence (being this a part of nucleotide sequence of Melittin) of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin Nucleotide and Prepromelittin gene end, 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 that coding has the nucleotide sequence of apis cerana, the proteic polypeptide of Vespa magnifiac (Sonan). bee venom Prepromelittin, show at least 70% homology from the nucleotides sequence of Nucleotide 1-213 position among described nucleotide sequence and the SEQ ID No.5, perhaps described nucleotide sequence can be hybridized from the Nucleotide of Nucleotide 1-213 position under the moderate stringent condition 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 Nucleotide 1-213 position.
Final expression product--the Melittin protein active polypeptide of a kind of isolating apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin also is provided in the present invention, it comprises: have polypeptide or its active fragments of SEQ ID No.6 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID No.6 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 final expression product--the method for the proteic active polypeptide of Melittin of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin, and this method comprises:
(a) coding had the nucleotide sequence of apis cerana, the proteic polypeptide of Vespa magnifiac (Sonan). bee venom Prepromelittin and 130～213 nucleotide sequences that coding has Prepromelittin gene end (being this a part of nucleotide sequence of Melittin) operationally are connected in expression regulation sequence, form the expression vector of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin and Melittin, the nucleotides sequence of described nucleotide sequence and SEQ ID No.5 Nucleotide 1-213 position is shown at least 70% homology;
(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin and Melittin;
(c) be fit to express under the condition of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin and Melittin the reconstitution cell in the culturing step (b);
(d) isolate have apis cerana, the polypeptide of Vespa magnifiac (Sonan). bee venom Melittin protein-active.
The present invention has found a kind of coding apis cerana, the gene of Vespa magnifiac (Sonan). bee venom Prepromelittin, the Prepromelittin of this coded by said gene is the natural fusion rotein of bee venom Melittin, bee venom Melittin then is a kind of biologically active peptides, can be applicable to medically painstaking effort official disease, treatment of diseases such as tumour, can be used as antigen and be used for the bee venom immunological reagent, the preparation of bee venom anaphylodiagnosis reagent, can be used as a kind of natural antimicrobial peptide and be used for phytopathy, the control of insect pest also can be used as a kind of model peptide and is used for membrane interaction mechanism, the research of aspects such as the calmodulin mechanism of action.Apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene can be the molecular mechanisms of action relevant with Melittin in research apis cerana, the Vespa magnifiac (Sonan). bee venom, for development and use apis cerana, Vespa magnifiac (Sonan). bee venom resource provide new foundation.
Embodiment
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 213 Nucleotide, and its detailed sequence is seen SEQ ID No.5, and open reading frame is positioned at 1-213 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, Vespa magnifiac (Sonan). bee venom Prepromelittin albumen (or polypeptide) encoding sequence " refer to encode have apis cerana, the nucleotide sequence of the proteic polypeptide of Vespa magnifiac (Sonan). bee venom Prepromelittin, as 1-213 position nucleotide sequence and degenerate sequence thereof among the SEQ ID No.5.This degenerate sequence is meant the encoder block 1-213 position Nucleotide 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 1-213 position nucleotide sequence homology be low to moderate about 70%.The 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 Nucleotide 1-213 position.This term also comprise with SEQ ID No.5 in from the homology of nucleotide sequence at least 70% of Nucleotide 1-213 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.
This term also comprises encoding to have variant form with the proteic SEQ ID No.6 sequence of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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, Vespa magnifiac (Sonan). bee venom Prepromelittin protein polypeptide " refer to have apis cerana, the proteic SEQ ID of Vespa magnifiac (Sonan). bee venom Prepromelittin No.6 polypeptide of sequence.This term also comprises the variant form that has with the SEQ ID No.6 sequence of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin albumen identical function.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 apis cerana, the proteic fragment of Vespa magnifiac (Sonan). bee venom Prepromelittin and 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, Vespa magnifiac (Sonan). bee venom Prepromelittin DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of final expression product-Melittin polypeptide of anti-apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin to obtain.The present invention also provides other polypeptide, as comprises apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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, Vespa magnifiac (Sonan). bee venom Prepromelittin polypeptide.Usually, this fragment have apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin polypeptid coding sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids,, be 70 continuous amino acids best preferably at least about 50 continuous amino acids.
Invention also provides the analogue of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin albumen or polypeptide, the difference of these analogues and natural apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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, Vespa magnifiac (Sonan). bee venom Prepromelittin polypeptid coding sequence and segmental antisense sequences thereof, and this antisense sequences can be used for suppressing the expression of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin in the cell.
The present invention also comprises a kind of probe molecule, and this molecule has apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin polypeptid coding sequence 8-100, preferably 1-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample coding apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin nucleic acid molecule.
The present invention also comprises the method that detects apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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, Vespa magnifiac (Sonan). bee venom Prepromelittin 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 comprises that also apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin or the polypeptide of its fragment coding are had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene product or fragment.Preferably, refer to that those can combine with apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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 final expression product--the proteic molecule of Melittin of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene, comprise that also those do not influence final expression product--the antibody of Melittin protein function of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene.The present invention comprises that also those can be with modification or without the apis cerana of modified forms, final expression product--the Melittin bonded antibody of Vespa magnifiac (Sonan). bee venom Prepromelittin gene.
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, Vespa magnifiac (Sonan). bee venom.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art, for example, the apis cerana of purifying, Vespa magnifiac (Sonan). bee venom Prepromelittin gene product or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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.Jmmunol, 6:511,1976; People such as Kohler,
Eur.J.Immunol, 6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the final expression product that can block apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene--the antibody of Melittin function and the antibody that does not influence apis cerana, Vespa magnifiac (Sonan). bee venom Melittin function.Each antibody-like of the present invention can utilize the fragment or the functional zone of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin 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 final product unmodified form bonded antibody of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene; 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, Vespa magnifiac (Sonan). bee venom Prepromelittin is so to obtain, cDNA with apis cerana, the total RNA reverse transcription of Vespa magnifiac (Sonan). bee venom poison gland is a template, with a pair of oligonucleotide is that primer-A:5 '-GCTCGAGATGAAATTCTTAGTCAACGTT-3 ' is that forward is to primer, oligonucleotide B:5 '-GAAGCTTCTAACCCTGTTGCCTCTT-3 ' is a reverse primer, carries out PCR.Obtain the cDNA sequence of SEQ IDNo.5 after amplified production checked order.Apis cerana, Vespa magnifiac (Sonan). bee venom Melittin are the final albumen of Prepromelittin genetic expression in apis cerana, the Vespa magnifiac (Sonan). poison gland, and apis cerana of the present invention, Vespa magnifiac (Sonan). bee venom Prepromelittin provide the foundation for the molecular mechanisms of action relevant with Melittin in research apis cerana, the 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 apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin
1. primer amplification
CDNA with apis cerana, the total RNA reverse transcription of Vespa magnifiac (Sonan). poison gland is a template, with a pair of oligonucleotide is that primer-A:5 '-GCTCGAGATGAAATTCTTAGTCAACGTT-3 ' (SEQ ID No.1) is that forward is to primer, oligonucleotide B:5 '-GAAGCTTCTAACCCTGTTGCCTCTT-3 ' (SEQ IDNo.2) is a reverse primer, carries out PCR.The PCR condition of A/B be 94 ℃ 3 minutes, thereupon with 94 ℃ of 40 second, 54 ℃ of 40 second and 72 ℃ carried out 35 circulations in 1 minute, last 72 ℃ were extended 10 minutes, electrophoresis detection obtains the purpose fragment of about 220bp.
2.PCR the order-checking of product
With above-mentioned pcr amplification product A/B and PGEM
-T carrier (Promega) connects, transformed into escherichia coli JM109, extract plasmid with alkaline process, with BigDye terminator v2.0 (Applied Biosystem Incorporation) sequencing kit extractive plasmid is checked order, with computer software splicing order, obtain the cDNA sequence at last, altogether 213bp, detailed sequence is seen SEQ ID No.5, and wherein open reading frame is positioned at 1-213 position Nucleotide.
Derive the aminoacid sequence of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin according to the cDNA sequence that obtains, totally 70 amino-acid residues, its aminoacid sequence sees SEQ ID No.6 for details.
Embodiment 2, and homology relatively
CDNA sequence and proteins encoded thereof with apis cerana of the present invention, Vespa magnifiac (Sonan). bee venom Prepromelittin, 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 Prepromelittin have significant homology.With the GENTYX software analysis as can be seen, their proteic homology is 95% (seeing attached list 1).Therefore, can infer that apis cerana of the present invention, Vespa magnifiac (Sonan). bee venom Prepromelittin albumen are the homologue of apis mellifera bee venom Prepromelittin, and have similar function.
Nineteen eighty-three, people's Applied Biotechnology methods such as Vlask, utilize the total RNA reverse transcription of apis mellifera queen bee poison gland to become cDNA, by making up the eDNA library, do the library screening with probe then, obtained the new gene of coding apis mellifera bee venom Prepromelittin, the signal peptide of the Prepromelittin that this eDNA is coded and mature peptide are 70 amino-acid residues, 21 amino-acid residues that wherein begin are signal peptide, remaining 49 is mature peptide, comprises the aminoacid sequence (44～70) of terminal M elittin.The aminoacid sequence of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin coded by said gene contains above-described corresponding signal peptide and mature peptide among the present invention, comprises the aminoacid sequence (44～70) of terminal Melittin.Prepromelittin last expression product in apis cerana, Vespa magnifiac (Sonan). bee venom is Melittin.And Melittin is present medical science, the very useful active polypeptide of agricultural and biology aspect, it can influence the signal transducting system of cell by multipath, and can induce the synthetic and natural death of cerebral cells of ceramide, have anti-inflammatory, various biological effect such as antibiotic, antiviral; Simultaneously, Melittin has also become a kind of model peptide, is widely used in the research of aspects such as membrane interaction mechanism, the calmodulin mechanism of action.The inventor's apis cerana, Vespa magnifiac (Sonan). Prepromelittin gene are the molecular mechanisms of action relevant with Melittin in research apis cerana, the Vespa magnifiac (Sonan). bee venom, for development and use apis cerana, Vespa magnifiac (Sonan). bee venom resource provide new foundation.
Embodiment 3, the amalgamation and expression of 130～213 nucleotide sequences of apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene end (being this a part of nucleotide sequence of Melittin) in intestinal bacteria
In this embodiment, with pcr amplification product among the embodiment 1 as inserting fragment.
PCR. 5 ' the Oligonucleolide primers sequence of using in the reaction is:
5 '-CGTGGATCCGGAATTGGAGCAGTTCTG-3 ' (SEQ ID No.3), this primer contains the restriction enzyme site of BamHI restriction enzyme, is 21 Nucleotide of apis cerana, Vespa magnifiac (Sonan). bee venom coding Melittin sequence after this restriction enzyme site;
3 ' end primer sequence is:
5 '-CGGGAATTCCTAACCCTGTTGCCTCTT-3 ' (SEQ ID No.4), this primer contains the restriction enzyme site of EcoR I restriction enzyme, the partial nucleotide sequence of translation termination and coding apis cerana, Vespa magnifiac (Sonan). bee venom Melittin.
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 ribosome bind site (RBS), a gsh marker (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 bacterium RBS.With connecting the E.coli bacterial strain that mixture transforms commodity BL21 by name, BL21 contains the recombinant plasmid of multiple copied subsequently, and it is expressed lac I repressor and carries ammonia benzyl resistance (Amp
r), containing Amp
rThe LB culture dish on screen transformant, the extracting plasmid, in sequence verification apis cerana, the Vespa magnifiac (Sonan). bee venom coding Melittin the cDNA fragment 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 0.4-0.6, add IPTG (" isopropyl-β-D-thiogalactoside ") to final concentration be 1.0mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continue culturing cell after 3-5 hour, centrifugation cell, and with the PBS of 1/20 volume it is suspended; Supersound process lysing cell subsequently; Add Triton X-100 down to final concentration 1% in room temperature (25 ℃) again, mixed gently 30 minutes; Subsequently 10, centrifugal 5 minutes of 000g collects supernatant, the fusion rotein of having expressed with GSTPurification Modules purifying again.At last, protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.
SDS-PAGE glue with 15% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 29.0KDa.
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.6 with ordinary method.
Embodiment 4, apis cerana, the expression of Vespa magnifiac (Sonan). bee venom Prepromelittin in eukaryotic cell (Tn cell strain, i.e. cabbage looper cell strain)
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 '-GCTCGAGATGAAATTCTTAGTCAACGTT-3 ' (SEQ ID No.1), this primer contains the restriction enzyme site of Xho I restriction enzyme, is the apis cerana that begins of signal peptide, 21 Nucleotide of Vespa magnifiac (Sonan). bee venom Prepromelittin encoding sequence after this restriction enzyme site;
3 ' end primer sequence is:
5 '-GAAGCTTCTAACCCTGTTGCCTCTT-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 apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin.
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 Xho I and Hind III digestion pBacFastHTb 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, Vespa magnifiac (Sonan). bee venom Prepromelittin has correctly been inserted carrier.Use recombinant plasmid transformed E.coli DHl0Bac 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, be balance liquid and elutriant with 20mM Tris-CI (PH8.0) solution that contains NaCl 0.5mM, imidazola 5mM, PMSF lmM, the Ni of protein liquid through pre-equilibration
2+Sepharose 6B post is crossed post; Use the solution of the 20mM Tris-Cl (PH8.0) that contains imdazola 50mM, 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.
With the 16%T that contains 6mol/L urea element (or 24% glycerine), 6%C and 8%T, the 2L-T-SDS-PAGE of 3%C carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 3.0KDa.
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.6 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, 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.The ability that sero-fast specific reaction activity precipitates apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin gene translation product in vivo with it is assessed.
The explanation of SEQ ID No.1 ∽ 6,
The information of SEQ ID NO.1
(i) sequence signature
CA) length: 28 bases
CB) type: nucleic acid
(C) chain: strand
(D) topology; Linear
(ii) molecule-type: oligonucleotide
(vi) sequence description: SEQ ID NO.1
GCTCGAGATGAAATTCTTAGTCAACGTT
The information of SEQ ID NO.2
(i) sequence signature
(A) length: 25 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: oligonucleotide
(vi) sequence description; SEQ ID NO.2
GAAGCTTCTAACCCTGTTGCCTCTT
The information of SEQ ID NO.3:
(i) sequence signature
(A) length: 27 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: oligonucleotide
(vi) sequence description; SEQ ID NO.3
CGTGGATCCGGAATTGGAGCAGTTCTG
The information of SEQ ID NO.4:
(i) sequence signature
(A) length: 27 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: oligonucleotide
(vi) sequence description; SEQ ID NO.4
CGGGAATTCCTAACCCTGTTGCCTCTT
The information of SEQ ID NO.5:
(i) sequence signature
(A) length: 213bp
(B) type: nucleic acid
(C) chain: strand
(D) topology; Linear
(ii) molecule-type: cDNA
(vii) (vi) sequence description: SEQ ID NO.5
1 ATGAAATTCT?TAGTCAACGT?TGCCCTTGTT?TTTATGGTTG?TATACATTTC?TTTCATCTAT
61 GCGGCCCCTG?AACCAGAACC?GGCACCGGAG?GCAGAGGCAG?AGGCAGACGC?GGAGGCAGAT
121?CCGGAAGCAG?GGATTGGAGC?AGTTCTCAAA?GTATTAACCA?CAGGATTGCC?TGCCCTTATA
181?AGTTGGATTA?AACGTAAGAG?GCAACAGGGT?TAG
The information of SEQ ID NO.6:
(i) sequence signature
(A) length: 70 amino acid
(B) type: amino acid
(C) topological framework; Linear
(ii) molecule-type: polypeptide
(vi) sequence description: SEQ ID NO.6
1 Met?Lys?Phe?Leu?Val?Asn?Val?Ala?Leu?Val?Phe?Met?Val?Val?Tyr?Ile?Ser?Phe?Ile?Tyr
21?Ala?Ala?Pro?Glu?Pro?Glu?Pro?Ala?Pro?Glu?Ala?Glu?Ala?Glu?Ala?Asp?Ala?Glu?Ala?Asp
41?Pro?Glu?Ala?Gly?Ile?Gly?Ala?Val?Leu?Lys?Val?Leu?Thr?Thr?Gly?Leu?Pro?Ala?Leu?Ile
61?Ser?Trp?Ile?Lys?Arg?Lys?Arg?Gln?Gln?Gly
Table I apis cerana, Vespa magnifiac (Sonan). bee venom Prepromelittin and apis mellifera bee venom Prepromelittin are proteic
Relatively
Two sequences of carrying out the homology comparison are:
Apis cerana, Vespa magnifiac (Sonan). bee venom AcVmPrepromelittin
Residue sum: 70
Apis mellifera bee venom AmPrepromelittin
Residue sum: 70
The symbol of representing consistent residue is " * "
AcVmPrepromelittin1：MKFLVNVALVFMVVYISFIYAAPEPEPAPEAEAEADAEADPEAGIGAVLKVLTTGLPALI
AmPrepromelittin 1：MKFLVNVALVFMVVYISYIYAAPEPEPAPEPEAEADAEADPEAGIGAVLKVLTTGLPALI
*****************?************?*****************************
AcVmPrepromelittin 61：SWIKRKRQQG 70
AmPrepromelittin 61：SWIKRKRQQG 70
**********
Homology: 97%
Claims (5)
1. an isolated apis cerana, Vespa magnifiac (Sonan). melittin precursor protein encoding sequence is characterized in that its sequence is shown in SEQ ID No.5.
2. an isolating apis cerana, Vespa magnifiac (Sonan). melittin precursor protein is characterized in that it has SEQ ID No.6 aminoacid sequence.
3. a carrier is characterized in that, it contains the described DNA of claim 1.
4. a host cell is characterized in that, it is with the described carrier transformed host cells of claim 3.
5. the preparation method of an isolating apis cerana as claimed in claim 2, Vespa magnifiac (Sonan). melittin precursor protein is characterized in that this method comprises:
(a) encoding sequence with claim 1 operationally is connected in expression regulation sequence, forms the expression vector of apis cerana, Vespa magnifiac (Sonan). melittin precursor protein;
(b) change the carrier of expressing in the step (a) over to host cell, form the reconstitution cell of apis cerana, Vespa magnifiac (Sonan). melittin precursor protein;
(c) be fit to express under the condition of apis cerana, Vespa magnifiac (Sonan). melittin precursor protein the reconstitution cell in the culturing step (b);
(d) isolate have apis cerana, Vespa magnifiac (Sonan). melittin precursor protein.
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