CN1237173C - Hemolytic peptide precursor gene of Asian-African wasp aptoxin, polypeptide coded by it and its preparing process - Google Patents

Abstract

The invention discloses a coding sequence of the precursor gene of the Asian-African wasp venom hemolytic peptide, its coded polypeptide and a preparation method. The protein encoded by the cDNA sequence is a homologue of the precursor of Apis mellifera melitin hemolytic peptide, which has at least 70% homology with the nucleotide sequence from nucleotide 1 to 213 in SEQ ID No.5. The sequence encodes a polypeptide having the sequence shown in SEQ ID No.6. In addition, the carrier and host cell containing the 130-213 nucleotide sequence of the hemolytic peptide precursor of the Asian and African wasp venom and the end of the hemolytic peptide precursor gene, the antibody related to the hemolytic peptide of the Asian and African wasp venom, and the protein The preparation method of the polypeptide of the active sub-African hornet bee venom hemolytic peptide and related antibodies. The invention lays a foundation for further studying the molecular action mechanism of the Asian-African wasp venom hemolytic peptide, and developing biomedicine, diagnostic reagents, biochemical reagents and biological insecticides related to the hemolytic peptide.

Description

Asian-African hornet venom hemolytic peptide precursor gene and its encoded polypeptide and preparation method

                      

The invention relates to the field of genetic engineering, in particular to a gene of the Asia-African wasp venom hemolytic peptide (Prepromelittin) and its coded polypeptide and a preparation method.

Background technique

Italian bee (Apis mellifera) venom hemolytic peptide (Melittin) is the basic component of Italian bee venom, accounting for about 40% to 50% of bee venom (Vol.3.New York: Academic, 1971, 535), according to the source And biochemical properties, it is an active polypeptide, wherein 90% of the hemolytic peptide exists in a free state, while the other 10% exists in the form of Melittin N1-formylation (Biochem.Biophys.Res.Commun., 1967, 27 : 275-280). It has very important biological activity, can cause erythrocyte lysis (Z.Physiol.Chem., 1970,351,884), liposome releases label ion (marker ions) (J.Biol.Chem., 1969,244, 3575) and histamine released by mast cells (J.Pharmacol, 1965, 25, 29) and so on. There are two precursors in the synthesis of Melittin. One is Promelittin, which is found in bee venom or frog eggs infused with queen bee venom extract. When synthesized in frog eggs, Promelittin is a stable end product, and in the poison gland, it will slowly transform into Melittin; the second is called Prepromelittin, which has a larger molecular weight than Promelittin and can only be transcribed in a cell-free system. Melittin mRNA Found on (Proc. Natl. Acad. Sct. USA, 1978, 75, 701-704).

In 1983, Vlasak et al. reverse-transcribed the total RNA of the queen bee venom gland of the Italian honeybee into cDNA, then cloned the synthesized double-stranded cDNA into the pBR322 vector, constructed a cDNA library, and screened out positive clones with inserts greater than 200 bp with probes. Then they were subcloned into the PUC8 vector, and then 2 positive clones were screened out through enzyme digestion identification, and the PUM13/4 containing the largest insert was sequenced, and the results showed that the insert sequence was the nucleotide sequence of Prepromelittin, The mature peptide encoded by the cDNA, the melittin hemolysin precursor protein, has 49 amino acid residues (Eur. J. Biochem., 1983, 135: 123-126). Prepromelittin protein is essentially a natural fusion protein of bee venom hemolytic peptide, which is synthesized in bee venom gland cells and then hydrolyzed into bee venom melittin by dipeptide protease.

Contents of invention

One of the objectives of the present invention is to provide a polynucleotide of Apis venom Prepromelittin, which encodes a homologue of Apis mellifera prepromelittin protein.

The second object of the present invention is to provide a final product encoded by the Asia-African hornet venom Prepromelittin polynucleotide - the Asia-African hornet venom Melittin protein.

The object of the present invention also provides the preparation of the Asia-African wasp venom Prepromelittin nucleotide probe, the 130-213 nucleotide sequence containing the Asia-African wasp venom Prepromelittin nucleotide and the end of the Prepromelittin gene (i.e. the core of this part of Melittin) nucleotide sequence), a host cell containing the recombinant vector, and a method for polypeptide antibody.

In the present invention, an isolated DNA molecule is provided, which includes a nucleotide sequence encoding a polypeptide having the sub-African wasp venom Prepromelittin protein, and the nucleotide sequence is the same as that of SEQ ID No.5 from the nucleoside The nucleotide sequence of acid 1-213 has at least 70% homology, or the nucleotide sequence can be under moderately stringent conditions with the core of nucleotide 1-213 in SEQ ID No.5 Nucleotide hybridization, preferably, the polypeptide has the sequence shown in SEQ ID No.6. More preferably, the sequence has the nucleotide sequence from nucleotide 1-213 in SEQ ID No.5.

The present invention also provides a final expression product of isolated sub-African hornet venom Prepromelittin--Melittin protein active polypeptide, which includes: a polypeptide with an amino acid sequence of SEQ ID No.6, or an active fragment thereof, or an active derivative thereof things. Preferably, the polypeptide is a polypeptide having the sequence of SEQ ID No.6. The present invention also provides a vector containing the above-mentioned isolated DNA; a host cell transformed with the vector.

The present invention also provides a method for preparing an active polypeptide having the final expression product of the sub-African wasp venom Prepromelittin-Melittin protein, the method comprising:

(a) the nucleotide sequence encoding the polypeptide having the sub-African hornet venom Prepromelittin protein and the 130-213 nucleotide sequence encoding the end of the Prepromelittin gene (i.e. the nucleotide sequence of this part of Melittin) are operably linked to Expression control sequences to form expression vectors for Asia-African wasp venom Prepromelittin and Melittin, the nucleotide sequence has at least 70% homology with the nucleotide sequence at nucleotide 1-213 of SEQ ID No.5 ;

(b) transferring the expression vector in the step (a) into the host cell to form recombinant cells of the sub-African wasp venom Prepromelittin and Melittin;

(c) cultivating the recombinant cells in step (b) under conditions suitable for expressing the sub-African wasp venom Prepromelittin and Melittin;

(d) isolating the polypeptide having the activity of the sub-African wasp venom Melittin protein.

The present invention has discovered a gene encoding the venom Prepromelittin of the Asian-African hornet, the Prepromelittin encoded by the gene is a natural fusion protein of the venom Melittin, and the Melittin is a biologically active peptide, which can be applied to the heart and blood organs in medicine. It can be used as an antigen for the preparation of bee venom immune preparations and diagnostic reagents for bee venom allergy in the treatment of diseases, tumors, etc. It can be used as a natural antimicrobial peptide for the prevention and control of plant diseases and insect pests, and can also be used as a model peptide It is used for research on the mechanism of membrane action and the mechanism of calmodulin action. The Prepromelittin gene of Asia-African wasp venom can be used to study the molecular mechanism related to Melittin in Asia-African wasp venom, and provide a new basis for the development and utilization of Asia-African wasp venom resources.

Detailed ways

In a specific embodiment of the present invention, the isolated polynucleotide of the present invention has a full length of 213 nucleotides, its detailed sequence is shown in SEQ ID No.5, and the open reading frame is located at nucleotides 1-213. In the present invention, "isolated", "purified" or "substantially pure" DNA means that the DNA or fragment has been separated from the sequences flanking it in its natural state; it also means that the DNA or fragment has been Separated from the components that naturally accompany nucleic acids and that have been separated from the proteins that accompany them in cells.

In the present invention, the term "sub-African wasp venom Prepromelittin protein (or polypeptide) coding sequence" refers to the nucleotide sequence encoding a polypeptide having sub-African wasp venom Prepromelittin protein, such as the 1-213 core in SEQ ID No.5 Nucleotide sequence and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 1-213 nucleotides of the coding frame of the SEQ ID No.5 sequence. Due to the degeneracy of codons, the homology with the 1-213 nucleotide sequence in SEQ ID No.5 is as low as about 70%. The degenerate sequence can also encode the sequence described in SEQ ID No.6. The term also includes a nucleotide sequence capable of hybridizing to the nucleotide sequence from nucleotides 1-213 in SEQ ID No.5 under moderately stringent conditions, preferably under highly stringent conditions. The term also includes at least 70%, preferably at least 80%, and more preferably at least 90% of the nucleotide sequence of the nucleotide sequence from nucleotide 1-213 in SEQ ID No.5 .

The term also includes variants of the SEQ ID No. 6 sequence that encodes a protein that has the same function as the sub-African wasp venom Prepromelittin. These variations include (but are not limited to): deletions, insertions and/or substitutions of several nucleotides, and additions of several nucleotides at the 5' and/or 3' ends.

In the present invention, "substantially pure" protein or polypeptide means that it accounts for at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight or wet weight). Purity can be measured by any suitable method, such as measuring the purity of the polypeptide by column chromatography, PAGE or HPLC. A substantially pure polypeptide is substantially free of components that accompany it in its native state.

In the present invention, the term "sub-African wasp venom Prepromelittin protein polypeptide" refers to a polypeptide having the sequence of SEQ ID No. 6 of sub-African wasp venom Prepromelittin protein. The term also includes variants of the SEQ ID No. 6 sequence having the same function as the sub-African wasp venom Prepromelittin protein. These variant forms include (but are not limited to): deletion, insertion and/or substitution of several amino acids, and addition of one or several amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes fragments and derivatives of the sub-African wasp venom Prepromelittin protein.

The variant forms of the polypeptide include: homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with sub-African hornet venom Prepromelittin DNA under high or low stringency conditions, And the polypeptide or protein obtained by using the antiserum against the final expression product of the Asian-African wasp venom Prepromelittin-Melittin polypeptide. The present invention also provides other polypeptides, such as a fusion protein comprising the sub-African wasp venom Prepromelittin polypeptide or a fragment thereof. In addition to the nearly full-length polypeptide, the present invention also includes soluble fragments of the sub-African wasp venom Prepromelittin polypeptide. Typically, the fragment has at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, and most preferably 70 contiguous amino acids of the sub-African wasp venom Prepromelittin polypeptide coding sequence.

The invention also provides analogs of the Asia-African wasp venom Prepromelittin protein or polypeptide, and the difference between these analogs and the natural Asia-Africa wasp venom Prepromelittin polypeptide may be a difference in amino acid sequence, or a difference in a modified form that does not affect the sequence , or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs having residues other than natural L amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, beta, gamma amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include; chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

The present invention also includes the antisense sequence of the polypeptide coding sequence of the sub-African wasp venom Prepromelittin and its fragment, and the antisense sequence can be used to inhibit the expression of the sub-African wasp venom Prepromelittin in cells.

The present invention also includes a probe molecule, which generally has 8-100, preferably 1-50, continuous nucleotides of the coding sequence of the sub-African wasp venom Prepromelittin polypeptide. The probe can be used to detect whether there is a nucleotide molecule encoding sub-African hornet venom Prepromelittin in a sample.

The present invention also includes a method for detecting the nucleotide sequence of the sub-African hornet venom Prepromelittin, which comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined, preferably, the sample is PCR-amplified The product, wherein the PCR amplification primers correspond to the coding sequence of the sub-African wasp venom Prepromelittin polypeptide, and can be located on both sides or in the middle of the coding sequence, and the length of the primers is generally 15-50 nucleotides.

In the present invention, various vectors known in the art, such as commercially available vectors, can be used.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, such as Tn cells, CHO cells, and COS cells.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, which are specific to the sub-African wasp venom Prepromelittin or the polypeptide encoded by its fragment. Here, "specificity" means that the antibody can bind to the gene product or fragment of the wasp venom Prepromelittin. Preferably, it refers to those antibodies that can bind to the gene product or fragment of the sub-African wasp venom Prepromelittin but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules that can bind to and inhibit the final expression product of the sub-African wasp venom Prepromelittin gene - Melittin protein, and those that do not affect the final expression product of the sub-African wasp venom Prepromelittin gene - Melittin protein function antibodies. The present invention also includes those antibodies that can bind to Melittin, the final expression product of the sub-African wasp venom Prepromelittin gene in modified or unmodified form.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from Wasp wasp.

The antibody of the present invention can be prepared by various techniques known to those skilled in the art, for example, the purified sub-African wasp venom Prepromelittin gene product or its antigenic fragment can be administered to animals to induce polyclonal antibodies generation. Similarly, cells expressing the sub-African wasp venom Prepromelittin or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J. Immunol , 6:511, 1976; Kohler et al., Eur.J. Immunol . , 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibody of the present invention includes the antibody capable of blocking the function of Melittin, the final expression product of the Asia-African wasp venom Prepromelittin gene, and the antibody that does not affect the function of the Asia-African wasp venom Melittin. All kinds of antibodies of the present invention can be obtained by conventional immunization techniques by using fragments or functional regions of the sub-African wasp venom Prepromelittin gene product. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to the unmodified form of the final product of the sub-African hornet venom Prepromelittin gene can be produced by immunizing animals with the gene product produced in prokaryotic cells (such as E. coli); antibodies that bind to the post-translationally modified form (such as glycosyl or phosphorylated protein or polypeptide), which can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (eg, yeast or insect cell).

In one embodiment of the present invention, the cDNA nucleotide sequence of the Asia-African wasp venom Prepromelittin is obtained by using the cDNA reverse-transcribed from the total RNA of the Asia-African wasp venom as a template, and using a pair of oligonucleotides as primers -A: 5'-GCTCGAGATGAAATTCTTAGTCAACGTT-3' is the forward primer, oligonucleotide B: 5'-GAAGCTTCTAACCCTGTTGCCTCTT-3' is the reverse primer, and PCR is performed. After sequencing the amplified product, the cDNA sequence of SEQ ID No.5 was obtained. The Asian-African wasp venom Melittin is the final protein expressed by the Prepromelittin gene in the Asian-African wasp venom, and the Asian-African wasp venom Prepromelittin provides a basis for studying the molecular action mechanism related to Melittin in the Asian-African wasp venom.

Below in conjunction with specific example, further set forth the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Example 1, the cloning and determination of the cDNA of the Asia-African wasp venom Prepromelittin

1. Primer amplification

Using the cDNA reverse-transcribed from the total RNA of the sub-African wasp venom as a template, a pair of oligonucleotides were used as primers-A: 5'-GCTCGAGATGAAATTCTTAGTCAACGTT-3'(SEQ ID No.1) was used as a forward primer, oligonucleotide Acid B: 5'-GAAGCTTCTAACCCTGTTGCCTCTT-3' (SEQID No.2) was used as a reverse primer for PCR. The PCR condition of A/B is 94°C for 3 minutes, followed by 35 cycles of 94°C for 40 seconds, 54°C for 40 seconds and 72°C for 1 minute, and finally 72°C for 10 minutes, and the target of about 220bp was detected by electrophoresis fragment.

2. Sequencing of PCR products

The above-mentioned PCR amplification product A/B was connected with PGEM® ^- T carrier (Promega), transformed into Escherichia coli JM109, and the plasmid was extracted by alkaline method, and the extracted plasmid was carried out with the BigDye terminator v2.0 (Applied BiosystemIncorporation) sequencing kit. Sequencing, and finally splicing the sequence with computer software to obtain a cDNA sequence, a total of 213bp, the detailed sequence is shown in SEQ ID No.5, wherein the open reading frame is located at nucleotides 1-213.

According to the obtained cDNA sequence, the amino acid sequence of the sub-African wasp venom Prepromelittin was deduced, with a total of 70 amino acid residues, and its amino acid sequence is shown in SEQ ID No.6.

Embodiment 2, homologous comparison

With the cDNA sequence of the Asia-Africa wasp venom Prepromelittin of the present invention and the encoded protein thereof, in the Non-redundant GenBank+EMBL+DDBJ+PDB database and the Non-redundant GenBankCDS translations+PDB+SwissProt+Spupdate+PIR database, use the Blast program Perform nucleic acid and protein homology searches. It was found that it has significant homology with Italian bee mee venom Prepromelittin. Analysis by GENTYX software shows that the homology of their proteins is 95% (see attached table 1). Therefore, it can be speculated that the sub-African wasp venom Prepromelittin protein of the present invention is a homologue of Apis mellifera prepromelittin and has similar functions.

In 1983, Vlask and others applied biotechnology methods to reverse transcribe the total RNA of the royal venom of Apis mellifera into cDNA. By constructing a cDNA library and then using probes for library screening, they obtained a new gene encoding the venom of Apis mellifera Prepromelittin. The signal peptide and mature peptide of Prepromelittin encoded by cDNA are 70 amino acid residues, of which the first 21 amino acid residues are signal peptide, and the remaining 49 are mature peptides, including the amino acid sequence (44-70) of terminal Melittin. The amino acid sequence encoded by the Asia-African wasp venom Prepromelittin gene contains the above-mentioned corresponding signal peptide and mature peptide, including the amino acid sequence (44-70) of the terminal Melittin. The final expression product of Prepromelittin in Asian-African wasps is Melittin. Melittin is currently a very useful active polypeptide in medicine, agriculture and biology. It can affect the signal transduction system of cells through multiple channels, and can induce ceramide synthesis and cell apoptosis. It has anti-inflammatory, antibacterial, antiviral and other properties. At the same time, Melittin has also become a model peptide, which is widely used in the research on the mechanism of membrane action and the mechanism of calmodulin action. The inventor's Asian-African wasp Prepromelittin gene provides a new basis for studying the molecular action mechanism related to Melittin in Asian-African wasp venom and for developing and utilizing Asian-African wasp venom resources.

Embodiment 3, the fusion expression of the 130～213 nucleotide sequence (being the nucleotide sequence of this part of Melittin) of the sub-African wasp venom Prepromelittin gene end in Escherichia coli

In this example, the PCR amplification product in Example 1 was used as the insert.

The 5' oligonucleotide primer sequences used in the PCR reaction are:

5'-CGTGGATCCGGAATTGGAGCAGTTCTG-3' (SEQ ID No.3), this primer contains the restriction endonuclease restriction endonuclease site of BamH I, after this restriction site is 21 parts of the Melittin sequence encoded by the sub-African hornet venom Nucleotide;

The 3' end primer sequence is:

5'-CGGGAATTCCTAACCCTGTTGCCTCTT-3' (SEQ ID No.4), this primer contains the restriction endonuclease restriction site of EcoR I, the translation terminator and the partial nucleotide sequence of the coding sub-African wasp venom Melittin.

The restriction endonuclease cutting site on the primer corresponds to the restriction endonuclease cutting site on the bacterial expression vector pGEX-4T-3, and this plasmid vector encodes antibiotic resistance (Amp ^r ), a bacterial Origin of replication (Ori), an IPTG regulated promoter/operator (P/O), a ribosome binding site (RBS), a glutathione tag (GST), and restriction enzyme cloning sites .

The pGEX-4T-3 vector and the insert were digested with BamHI and EcoRI, and the insert was subsequently ligated into the pGEX-4T-3 vector keeping the open reading frame at the beginning of the bacterial RBS. The ligation mix was subsequently used to transform an E. coli strain commercially known as BL21, which contains multiple copies of the recombinant plasmid expressing the lac I repressor and carrying ampicillin resistance (Amp ^r ), for transformation selection on LB dishes containing Amp ^r The progeny was extracted from the plasmid and sequenced to verify that the cDNA fragment encoding Melittin in the venom of the Asian-African hornet was correctly inserted into the vector. Positive transformant clones containing the desired construct were cultured overnight (O/N) in LB liquid medium supplemented with Amp ^r (100 μg/ml). The overnight (O/N) culture was diluted with a dilution ratio of 1:10, and then inoculated into a large volume of LB liquid medium, and the cultured cells were grown to 600 optical density (OD ₆₀₀ ) of 0.4-0.6, and IPTG ("isopropyl -β-D-thiogalactoside") to a final concentration of 1.0 mM. By inactivating the lacI repressor, IPTG-induced initiation of P/O leads to increased gene expression levels. After continuing to culture the cells for 3-5 hours, centrifuge the pelleted cells and suspend them with 1/20 volume of PBS; then sonicate to lyse the cells; then add Triton X-100 to a final concentration of 1% at room temperature (25°C), gently Gently mix for 30 minutes; then centrifuge at 10,000g for 5 minutes to collect the supernatant and purify the expressed fusion protein with GST Purification Modules. Finally, the protein was kept in a stock solution with a final concentration of 10% (w/v) glycerol.

Electrophoresis was carried out with 15% SDS-PAGE gel, and the molecular weight of the expressed protein was identified as about 29.0 KDa.

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and it was found to be consistent with the sequence of SEQ ID No.6.

Embodiment 4, the expression of sub-African wasp venom Prepromelittin in eukaryotic cells (Tn cell strain, i.e. Trichoplusia spp cell strain)

In this example, the PCR amplification product in Example 1 was used as the insert.

The 5' oligonucleotide primer sequences used in the PCR reaction are:

5'-GCTCGAGATGAAATTCTTAGTCAACGTT-3'(SEQ ID No.1), this primer contains the restriction endonuclease restriction endonuclease site of Xho I, after this restriction site is the sub-African hornet venom Prepromelittin code that signal peptide begins 21 nucleotides of the sequence;

The 3' end primer sequence is:

5'-GAAGCTTCTAACCCTGTTGCCTCTT-3'(SEQ ID No.2), the primer contains the restriction endonuclease restriction site of Hind III, translation terminator and part of the coding sequence of Prepromelittin from the sub-African wasp venom.

The restriction endonuclease cutting sites on the primers correspond to the restriction endonuclease cutting sites on the Tn cell expression vector pBacFastHTb, which encodes antibiotic resistance (Amp ^r and Gm ^r ), a phage replication origin ( Ori), a viral origin of replication (SV40), a T7 promoter, a viral promoter (P-CMV), an SV40 promoter, an SV40 tailing signal and the corresponding polyA sequence, a BGH tailing signal and the corresponding polyA order.

Digest the pBacFastHTb vector and insert with Xho I and Hind III, then use the ligation mixture to transform the E.coli TG I strain, screen transformants on LB plates containing Amp ^r and Gm ^r , and add Amp ^r and Gm ^r to the LB Cultivate (O/N) clones containing the required constructs in liquid medium, extract plasmids, and sequence to verify that the cDNA fragment of the sub-African hornet venom Prepromelittin has been correctly inserted into the vector. Then the E.coli DH10Bac strain was transformed with the recombinant plasmid, and the transformants were screened on LB culture dishes containing Kan ^r , Gm ^r , and tetracycline, and cultured in LB liquid medium supplemented with Kan ^r , Gm ^r , and tetracycline (O/N ) the clone containing the desired construct, the plasmid was extracted, purified by Sepharose 2B column, and the recovered plasmid was stored at -70°C.

Plasmid transfection into Tn cells was carried out by lipofection with Lipofectin (GiBco Life). After 48 hours of transfection, the cells and cell supernatant were collected, and the cell supernatant containing recombinant virus particles was used for the next round of infection. After 2-3 weeks of continuous subculture in TC-100, the cells were collected, and the cells were broken by ultrasonic lysis, and the 20mM Tris-CI (PH8.0) solution containing NaCl 0.5mM, imidazola 5mM, and PMSF 1mM was used as the balance solution and washed. Remove the liquid, and pass the protein solution through the pre-equilibrated Ni ²⁺ Sepharose 6B column; then use a solution of 20mM Tris-CI (pH 8.0) containing imdazola 50mM, NaCl 0.5mM, and PMSF 1mM to wash away the impurity protein, specifically The fusion protein of 6×His adsorbed, and then eluted with a solution of 20mM Tris-CI (PH8.0) containing imdazola 500mM, NaCl 0.5mM, PMSF 1mM. Then use PBS (PH7.4) as the dialysate Dialyze the expressed protein solution. Finally freeze-dried and stored.

Use 16%T containing 6mol/L urea (or 24% glycerol), 6%C and 8%T, 2L-T-SDS-PAGE of 3%C to carry out electrophoresis, and identify the molecular weight of the expressed protein to be about 3.0KDa .

In addition, the N-terminal and C-terminal 10 amino acid lengths of the expressed protein were sequenced by conventional methods, and the sequence was found to be consistent with the protein of SEQ ID No.6.

Example 5, preparation of antibodies

The recombinant proteins obtained in Examples 3 and 4 were used to immunize animals to produce antibodies, and the specific method was as follows. The recombinant molecules are separated by affinity chromatography for further use. It can also be separated by SDS-PAGE gel electrophoresis, and the electrophoresis bands are excised from the gel and emulsified with an equal volume of complete Freund's adjuvant. With 200-300μg/head emulsified protein, inject subcutaneously to 12-week-old rabbits. After 21 days, the same antigen emulsified with incomplete Freund's adjuvant was injected subcutaneously at multiple points and leg muscles at a dose of 100-200 μg/head to boost immunization. After 21 days, a buttock intramuscular injection was given to boost the immunization. The specific reactivity of the antiserum was assessed by its ability to precipitate in vivo the translation product of the sub-African wasp venom Prepromelittin gene.

Description of SEQ ID No.1∽6,

Information on SEQ ID NO.1

(i) Sequential features

CA) length: 28 bases

CB) type: nucleic acid

(C) chain: single chain

(D) topology; linear

(ii) Molecular type: oligonucleotide

(vi) Sequence description: SEQ ID NO.1

GCTCGAGATGAAATTCTTAGTCAACGTT

Information on SEQ ID NO.2

(i) Sequential features

(A) Length: 25 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(vi) sequence description; SEQ ID NO.2

GAAGCTTCTAACCCTGTTGCCTCTT

Information on SEQ ID NO.3:

(i) Sequential features

(A) Length: 27 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(vi) sequence description; SEQ ID NO.3

CGTGGATCCGGAATTGGAGCAGTTCTG

Information on SEQ ID NO.4:

(i) Sequential features

(A) Length: 27 bases

(B) type: nucleic acid

(C) chain: single chain

(D) Topology: Linear

(ii) Molecular type: oligonucleotide

(vi) sequence description; SEQ ID NO.4

CGGGAATTCCTAACCCTGTTGCCTCTT

Information on SEQ ID NO.5:

(i) Sequential features

(A) Length: 213bp

(B) type: nucleic acid

(C) chain: single chain

(D) topology; linear

(ii) Molecular type: cDNA

(vii)(vi) Sequence description: SEQ ID NO.5

1 ATGAAATTCT TAGTCAACGT TGCCCTTGTT TTTATGGTTG TATACATTTC TTTCATCTAT

60 GCGGCCCCTG AACCAGAACC GGCACCGGAG GCAGAGGCAG AGGCAGACGC GGAGGCAGAT

121 CCGGAAGCAG GGATTGGAGC AGTTCTGAAG GTATTAGCCA CAGGATTGCC TGCCCTTATA

181 AGTTGGATTA AACGTAAGAG GCAACAGGGT TAG

Information on SEQ ID NO.6:

(i) Sequential features

(A) Length: 70 amino acids

(B) Type: amino acid

(C) topology; linear

(ii) Molecular 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 Ala Thr Gly Leu Pro Ala Leu Ile

61 Ser Trp Ile Lys Arg Lys Arg Gln Gln Gly

Table 1 The comparison of Prepromelittin protein of Asia-African wasp venom Prepromelittin and Apis mellifera

The two sequences for homology comparison are:

PhPrepromelittin PhPrepromelittin

Total number of residues: 70

Italian honeybee bee venom AmPrepromelittin

Total number of residues: 70

The symbol for consensus residues is "*"

AmPrepromelittin 1: MKFLVNVALVFMVVYISYIYAAPEPEPAPEPEAEADAEADPEAGIGAVLKVLTTGLPALI

PhPrepromelittin 1: MKFLVNVALVFMVVYISFIYAAPEPEPAPEAEAEADAEADPEAGIGAVLKVLATGLPALI

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AmPrepromelittin 61: SWIKRKRQQG

PhPrepromelittin 61: SWIKRKRQQG

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Homology: 95%

Claims (5)

1. An isolated sub-African wasp venom hemolytic peptide precursor protein coding sequence is characterized in that its sequence is as shown in SEQ ID No.5 nucleotide sequence. 2. An isolated sub-African hornet venom hemolytic peptide precursor protein, characterized in that it has the amino acid sequence of SEQ ID No.6. 3. A vector comprising the DNA according to claim 1. 4. A host cell, characterized in that it is a host cell transformed with the vector according to claim 3. 5. a method for preparing the isolated sub-African hornet venom hemolytic peptide precursor protein as claimed in claim 2, characterized in that the method comprises: (a) The coding sequence of claim 1 is operably connected to the expression control sequence to form an expression vector for the precursor protein of wasp venom hemolytic peptide; (b) transferring the vector expressed in the step (a) into a host cell to form a recombinant cell of the wasp venom hemolytic peptide precursor protein; (c) cultivating the recombinant cells in step (b) under conditions suitable for expressing the precursor protein of wasp venom hemolytic peptide; (d) Isolate the precursor protein with sub-African wasp venom hemolytic peptide.