CN108840918B

CN108840918B - Cloning method of gene encoding PLA2 inhibitor

Info

Publication number: CN108840918B
Application number: CN201810613259.1A
Authority: CN
Inventors: 杨仙玉; 应加富; 赵含笑; 宋源普
Original assignee: Jiyang College of Zhejiang A&F University
Current assignee: Jiyang College of Zhejiang A&F University
Priority date: 2018-06-14
Filing date: 2018-06-14
Publication date: 2021-07-23
Anticipated expiration: 2038-06-14
Also published as: CN108840918A

Abstract

The present application relates to a toad phospholipase A2 inhibitor PLI and its encoding gene and cloning method, belonging to the technical field of assay or detection methods comprising enzymes or microorganisms. The gene sequence of the toad phospholipase A2 inhibitor PLI is SEQ ID No.1, and the translated amino acid sequence is SEQ ID No.2; and/or, the gene sequence is SEQ ID No.3, and the translated amino acid sequence is SEQ ID No.3 No.4; and/or, the gene sequence is SEQ ID No.5, and the translated amino acid sequence is SEQ ID No.6. The application of the present application to the preparation of inhibitors has the advantages of no side effects, convenient acquisition, and the like.

Description

Cloning method of PLA2 inhibitor encoding gene

Technical Field

The application relates to a cloning method of a PLA2 inhibitor encoding gene, belonging to the technical field of determination or detection methods containing enzymes or microorganisms.

Background

Phospholipase A2(PLA2) is a group of numerous esterases, is present in almost all eukaryotic cells, and plays an important role in maintaining homeostasis in the body. Also plays an important role in the breakdown digestion of phospholipids, the deacylation/acylation pathway and the equilibration of the cellular phospholipid pool. More than 30 subtypes of PLA2 and related enzymes have been found to separate PLA2 into six families based on structure, catalytic mechanism, site of action, evolutionary relationships, and the like: cytosolic PLA2(cPLA2), calcium-independent PLA2(iPLA2), secretory PLA2(sPLA2), lysosome PLA2(LyPLA2), platelet activating factor acetylhydrolase (PAF-AH), and fat-specific PLA2 (adpa 2). PLA2 preferentially cleaves acyl bonds at the sn-2 position of glycerophospholipids, releasing fatty acids and lysophospholipids (LPLs, see formula (1)).

Fatty acids are primarily Arachidonic Acid (AA), which produces the proinflammatory mediators Leukotrienes (LTs) and Prostaglandins (PGs), respectively, through activation of Lipoxygenases (LOXs) and cyclooxygenases (COX-1 and COX-2), which are called arachidonic acids, playing an important role in the mediation of inflammation and a variety of cancers. In addition AA and LPL may be used as precursors of arachidonic acid biosynthesis cascade. Various studies have confirmed that PLA2 is involved in the pathological processes of various diseases such as arthritis, gastroenteritis, asthma, atherosclerosis and cancer, and have shown that blocking the reaction of PLA2 to enzymatically hydrolyze glycerophospholipids to produce arachidonic acid and lysophospholipids has a good effect in preventing and treating inflammation-related diseases and cancers.

In view of the good effect of blocking the activity of PLA2 in preventing and treating inflammation-related diseases and cancers, the development and innovation of anti-inflammatory drugs become the research hotspots of researchers at home and abroad in treating inflammation. The earliest anti-inflammatory drugs were steroidal anti-inflammatory drugs (SAIDs), which had a significant effect on anti-inflammation, but had large side effects. Recent studies have shown that the combination of sPLA2-IIA with cell membrane surface adhesion molecule integrin avb3 induces tumor cell migration, while peptidomimetic molecules consisting of phenyl ring-thiazole-pyrazole-AWD I can antagonize the interaction between the proteins to inhibit tumor cell migration, so that the development of PLA2 inhibitors is in the field of researchers. Arachidonic acid AA has been adopted at home and abroad as a blocking agent of PLA2, lipoxygenase and cyclooxygenase epoxide blocking agents, Platelet Activating Factor (PAF) and Leukotriene (LT) receptor antagonists are adopted to resist inflammation, but the medicines cannot solve the influence of PLA2 activated on membrane structure and on membrane receptor function, and particularly, AA and lysophospholipid have adverse effects on the stability and fusion of most inflammatory reaction cells and visceral parenchymal cell membranes. At present, the development of PLA2 inhibitors (PLI) is an important direction for anti-inflammatory therapy, and the development and development of several inhibitors is imperative.

Earlier PLI studied belonged to sPLA2 inhibitors, mainly natural extracts. Such as manoalide, which is an anti-inflammatory drug such as sPLA2 that has emerged in the late century by extraction from marine sponges, and has been shown to have an anti-inflammatory effect. The mechanism of action of this inhibitor is that the two modified aldehyde groups bind covalently irreversibly to the lysine residue of sPLA2, thereby inhibiting enzymatic activity. The inhibitors extracted from the sponge are mostly similar to manoalide, have similar action mechanism and function, and produce inhibition effect through covalent bonding with lysine residue of sPLA 2. Tetrandrine (alkaloid) is extracted from radix Stephaniae Japonicae root, has wide anti-inflammatory and antiallergic effects, and can reduce production of PLA2, cyclooxygenase and PGE 2. In addition, the lysophospholipase of snake venom can cause various toxicological activities such as neurotoxicity, hemorrhage, muscle toxicity, hemolysis and anticoagulation, but PLA2 in the viper can not cause the reaction to be generated, because the viper generates the toxin and generates antitoxin factors during the long-term evolution process of the viper, and the factors are PLI. Interestingly, this anti-virus factor is not limited to vipers, and Phisalix et al found anti-snake venom proteins in some animals, such as possums, hedgehogs and ferrets as early as 1922, and later discovered that these proteins are either anti-snake venom metalloprotein factors or anti-snake venom PLA2 factors. Indeed, researchers have found that these PLIs are resistant to many toxins, including muscle tissue damage, neurotoxicity, inflammation, and that these antitoxic proteins can be extracted from avirulent snakes, mammals, and more recently, from drug-resistant plants.

The present application was made based on this.

Disclosure of Invention

Aiming at the defects in the preparation and acquisition of the existing inhibitor, the first purpose of the cloning method of the PLA2 inhibitor coding gene is to provide a toad phospholipase A2 inhibitor PLI, the gene sequence of which is SEQ ID No.1, and the translated amino acid sequence of which is SEQ ID No. 2.

The second purpose of the application is to provide a toad phospholipase A2 inhibitor PLI, the gene sequence of which is SEQ ID No.3, and the translated amino acid sequence of which is SEQ ID No. 4.

The third purpose of the application is to provide a toad phospholipase A2 inhibitor PLI, the gene sequence of which is SEQ ID No.5, and the translated amino acid sequence of which is SEQ ID No. 6.

The fourth aspect of the application aims at providing a cloning method of a toad phospholipase A2 inhibitor PLI, which adopts PCR amplification, (1) a PCR template is a first strand cDNA of the skin of a Chinese toad; primers for PCR: the sequence of the upstream primer is as follows: 5'-GGATTTGGTCCACTGCAAAC-3', downstream primer sequence: 5'-CTGACAACCGGAGACACAG-3', the concentration is 2 pmol/. mu.L; (2) the PCR reaction system was 30. mu.L, where 10 × Buffer: 3 mu L of the solution; 10mmol/L dNTPS: 1.5 mu L; 1.5 mu L of each of the upstream primer and the downstream primer; template DNA: 0.2 mu L; 5U/. mu.L Taq: 0.3 mu L; sterilizing 21.7 μ L of pure water; (3) PCR program for Taq DNA polymerase PCR amplification of Bufo bufo PLI Gene fragments: denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 30 s/1 min, 40 cycles.

Wherein, the cDNA selected in the step (1) can be from Japanese toads besides Chinese toads; in addition to the skin, the expression of PLI was also confirmed in the brain, heart, liver, lung, pancreas, stomach, intestine, kidney, testis, oviduct and spleen of chinese toads and japanese toads.

It is an object of the fifth aspect of the present application to provide a toad phosphatase a2 inhibitor, the active ingredient of which comprises any one of a PLI gene sequence, amino acids translated from said PLI gene sequence, protein polypeptides of amino acids translated from said gene sequence having the above characteristics.

The project group has been engaged in gene cloning research of toad skin-related functional proteins for many years to analyze polypeptide active ingredients contained in toad skin. In the process, two cDNAs obtained by cloning from a skin cDNA library of an amphibious toad (Bufo), namely a Chinese big toad, have certain homology with PLI of other species, and organisms with the highest homology with the coding proteins of SEQ ID No.1 and SEQ ID No.2 are Tibet plateau frogs. Until now, no report on successful cloning of toad PLI has been available, which indicates that we successfully clone a cDNA encoding PLI in Chinese toad for the first time in view of low homology with other species, and that the gene encoding PLI is a novel type of PLI in view of low homology with other species.

Toads are animals of the family bufonidae in the amphibia and without tails, are widely distributed in China, are common experimental animals in medical and pharmaceutical research, have high medicinal value, are famous and precious Chinese traditional medicinal materials in China since ancient times, such as Shennong Ben Cao Jing, Ben Cao gang mu and modern monograph Chinese materia Medica, have detailed records on toads, and are cool in nature and pungent in taste, and can diminish inflammation, relieve pain, treat swelling and sore vulva, detoxify and dissipate carbuncles. The Chinese medicinal composition is clinically used for treating diphtheria, chronic bronchitis, anthrax and other diseases, and has good clinical effect. At present, toads are clinically used for tumor treatment in modern medicine, including malignant tumors, and have good curative effects on patients with various tumors such as gastric cancer, lung cancer, bladder cancer, leukemia and the like. Therefore, the PLI is probably an important component contained in the toad skin and the secretion thereof and related to the curative effects of diminishing inflammation, resisting tumors and the like, and has good potential for developing new anti-inflammatory and anti-tumor medicines. The protein coded by the gene can be used for developing a PLA2 inhibitor without side effects, is used for treating various inflammation-related diseases, and is a candidate of a potential inflammation treatment drug.

Drawings

FIG. 1 is a comparison graph of the similarity between SEQ ID No.2 and Tibet plateau frogs;

FIG. 2 is a comparison graph of the similarity between SEQ ID No.4 and Tibet plateau frogs;

FIG. 3 is a comparison graph of the similarity between SEQ ID No.6 and Tibet plateau frogs;

FIG. 4 is an electrophoretogram of expression of different organs.

Detailed Description

Example 1: chinese giant toad

The project group has been engaged in gene cloning research of toad skin-related functional proteins for many years to analyze polypeptide active ingredients contained in toad skin. In the process, two cDNAs are cloned from a skin cDNA library of an amphibious toad (Bufo), namely a Chinese big toad, and the two gene sequences are respectively SEQ ID No.1 and SEQ ID No.3, the translated amino acid sequence of the SEQ ID No.1 is SEQ ID No.2, and the translated amino acid sequence of the SEQ ID No.3 is SEQ ID No. 4.

Meanwhile, the PCR amplification product is cloned in the project, and the PLI cloning is determined by TA cloning and DNA sequencing of the PCR amplification product. The specific experimental conditions (parameters) were as follows:

(1) the PCR template is the first strand cDNA of the skin of the Chinese toad; primers for PCR: the sequence of the upstream primer is as follows: 5'-GGATTTGGTCCACTGCAAAC-3', downstream primer sequence: 5'-CTGACAACCGGAGACACAG-3', at a concentration of 2 pmol/. mu.L each.

(2) The PCR reaction system was 30. mu.L, where 10 × Buffer: 3 mu L of the solution; 10mmol/L dNTPS: 1.5 mu L; 1.5 mu L of each of the upstream primer and the downstream primer; template DNA: 0.2 mu L; 5U/. mu.L Taq: 0.3 mu L; sterilized purified water 21.7. mu.L.

(3) PCR program for Taq DNA polymerase PCR amplification of Bufo bufo PLI Gene fragments: denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 30 s/1 min, 40 cycles.

The gene sequence, the translated nucleotide of the gene sequence and the protein polypeptide formed by the nucleotide can be applied to the preparation of inhibitors.

The protein coded by the gene sequence has certain homology with PLI of other species, the organism with the highest homology with the coded protein of SEQ ID No.1 and SEQ ID No.2 is Tibet plateau frog, the identity is 53 percent, and the homology is 69 percent (see figure 1 and figure 2, and tables 1 and 2). The organisms with the highest homology with the encoded proteins of SEQ ID No.3 and SEQ ID No.4 are Rana tibetana with identity of 55% and homology of 71% (see FIG. 1, FIG. 2 and tables 1 and 2). Until now, no report on successful cloning of toad PLI has been available, which indicates that we successfully clone a cDNA encoding PLI in Chinese toad for the first time in view of low homology with other species, and that the gene encoding PLI is a novel type of PLI in view of low homology with other species.

TABLE 1 homology of SEQ ID No.2 with Tibet plateau frog

TABLE 2 homology of SEQ ID No.4 with Tibet plateau frog

TABLE 3 homology of SEQ ID No.2 to Cyprinus carpiod

TABLE 4 homology of SEQ ID No.4 to Cyprinus citrinopileatus

Example 2: japanese toad

In the process of carrying out the gene cloning research of the toad skin related functional protein, the gene sequence of a cDNA cloned from a Japanese skin cDNA library is SEQ ID No.5, and the translated amino acid sequence of the SEQ ID No.5 is SEQ ID No. 6.

Meanwhile, the cloning method of the Bufo siccus in the project is shown in the cloning method of the Bufo siccus in example 1, and the homology and similarity of the Bufo siccus are tested, and particularly shown in the figure 3, the tables 5 and the tables 6.

TABLE 5 homology of SEQ ID No.6 with Tibet plateau frog

TABLE 6 homology of SEQ ID No.6 to Cyprinus citrinopileatus

Furthermore, we have also performed experiments on organs other than the skin, and the results show that: the expression of PLI in the brain, heart, liver, lung, pancreas, stomach, intestine, kidney, testis, oviduct and spleen of chinese bufo gargarizans and japanese bufo gargarizans was also confirmed, see fig. 4, where A, B, C, D, E, F, G, H, I, J, K, L refers to skin, brain, heart, liver, lung, pancreas, stomach, intestine, kidney, testis, oviduct and spleen, respectively.

The above description is provided for the purpose of describing the preferred embodiments of the present invention in more detail, and it should not be construed that the embodiments of the present invention are limited to the description above, and it will be apparent to those skilled in the art that the present invention can be implemented in many different forms without departing from the spirit and scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang agriculture and forestry university

<120> Bufo siccus phospholipase A2 inhibitor PLI, and coding gene and cloning method thereof

<130> 1

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 996

<212> DNA

<213> toad (Bufo)

<400> 1

GGATTTGGTCCACTGCAAACCATCCCTGTGCAACCATGAGGACTGTACTGAACGTCTGCCTCCTACTCTCCATCACTATTGCAACAGGGTCATGTCTGGTTTGTCAGACTTGTAAGAACTACACTGGAGACATCTGCACAGAGCCGACCACTAAGACGTGCGACCCGTCGGTCACCAGATGCCTCACCACCTTGGCAGTTACGATGAATGGTAAAAACTTTTATCCGATGGTCGAAAAATCCTGTGCAACCAGTCCACAGTTGTGTGAGGCAACGTTCAACATGTCTACAGGAGTCGAATGGTATAGTGTCTCGAAGTGCTGCCAAGGAGACCTGTCCAACAAAGGATCGATCAAGTTGCCTCCTATAAACAGCACCGAGAACGGGGTGCAGTGTCCGGCCTGCTATGCTAAAGGAAAAACCTGCACACCCACCGGGACCATCAAATGTCGCGGACCGCAGACTAAATGCTTCAACTTCTCCGGCGGAATCTACAACGGCACTCAGTTTGAAGATTGGGCCTTCCAGGGATGCACCACAGAGAATATCTGTGAGTATCCAGCTCTAACCTACCCTGAGAATTACTTACAAGAAGACTACAAACTGACCTGCTCCAACTCCAAACCCTGAAGAAACCAGCAGCGGAGGAGCCCAGAATATTTGTTAAAGGGGTTGTCTGACATTTTCATACTGATAGGCCTGGCCTATCCTCAGGACGGATCATCAATATCAGGTGGGGGGGGCGACACCCAGCACCCCCGCCGATCAGCTGTATGAAGGAGACCTCCTTCTACCATATCCAGCACAGCGCCACAGAAGGTGTAGCAGAGCCGCAGACTCTTCAAACAGCTGATTGGAAGTGGTGTCAGGAGTCAGATCCCCACCACCACCACCACCTATTATCTGAGGATAGTTCCTCAAAATCATAGTCTTGGACAACCCCTTTAAACCTTTCACTGTCAGTTGTTACAGTCAAATCTGTGTCTCCGGTTGTCAG

<210> 2

<211> 198

<212> PRT

<213> toad (Bufo)

<400> 2

MRTVLNVCLLLSITIATGSCLVCQTCKNYTGDICTEPTTKTCDPSVTRCLTTLAVTMNGKNFYPMVEKSCATSPQLCEATFNMSTGVEWYSVSKCCQGDLSNKGSIKLPPINSTENGVQCPACYAKGKTCTPTGTIKCRGPQTKCFNFSGGIYNGTQFEDWAFQGCTTENICEYPALTYPENYLQEDYKLTCSNSKP.

<210> 3

<211> 1072

<212> DNA

<213> toad (Bufo)

<400> 3

GGATTTGGTCCACTGCAAACCATCCCTGTGCAACCATGAGGACTGTACTGAACGTCTGCCTCCTACTCTCCATCACTATTGCAACAGGGTCATGTCTGGTTTGTCAGACTTGTAAGAACTACACTGGAGACGCCTGCGCAGAGCCGACCACTAAGACGTGCGACCCGTCGGTCACCAGATGCCTCACCACCTTGACAGTTATGCAGATTGGTGAAAAACTTTATCCGATGGTCGAAAAATCCTGTGCAACCAGTCCACTGTTGTGTGAGCTAGCGTACAACATGTCTTCAGGAATCGAACTGTATAGTGTCTCGAAGTGCTGCCAAGGAGACCTGTGCAACAAAGGATCGATCAAGTTGCCTCCTATAAACAGCACCGAGAACGGGGTGCAGTGTCCGGCCTGCTATGCTAAAGCAAAAACCTGCACACCCACCGGGACCATCAAATGTCGCGGATCGCAGACTAAATGCTTCAACTTCTCCGGCGGAATCTACAACGGCACTCAGTTTGAAGATTGGGCCTACCAGGGATGCACCACAGAGAATGTCTGCCAGCATCCAGCTCCAACCTACCCTGAGACTTACTTACAAGAAGGCTACAAACTGAGCTGCTCCGACTCCAAACCCTGAAGAAACCAGCAGCGGAGGAGCACAGAATATTTGTTAAAGGGGTTGTCTGACATTTTCATACTGATGGCCTATCCTCAGGACGGATCATCAATATCGGGGGGGGGGGAAGGTCAGTCTCTGGATATGATAGACTGACCTTCTACCATAGCCAGCACAGCGCCACAGATGGTGTAGTGGCTATACTTGGTATTGCAGTCTAGGCTGATACACAATGTTGCGCCCGGCCATGTGACTTCGCTTGCCCTAGAAAGAGCCGTGGTACTCACCAGAGCGCCGCAGACTCTTCAAACAGCTGACTGGAAGTGGTGTCAGGAGTCAGATCCCCACCACCACCACCACCACCGATTATCTGAGGATAGTTCCTCAAAATCAAGGTCTTGGACAACCCCTTTAAACCTTTCACTGTCAGTTGTTACAGTCAAATCTGTGTCTCCGGTTGTCAG

<210> 4

<211> 198

<212> PRT

<213> toad (Bufo)

<400> 4

MRTVLNVCLLLSITIATGSCLVCQTCKNYTGDACAEPTTKTCDPSVTRCLTTLTVMQIGEKLYPMVEKSCATSPLLCELAYNMSSGIELYSVSKCCQGDLCNKGSIKLPPINSTENGVQCPACYAKAKTCTPTGTIKCRGSQTKCFNFSGGIYNGTQFEDWAYQGCTTENVCQHPAPTYPETYLQEGYKLSCSDSKP.

<210> 5

<211> 1087

<212> DNA

<213> toad (Bufo)

<400> 5

GGATTTGGTCCACTGCAAACCATCCCTGTGCCACCATGAGGACTGTACTGAACGTCTGCCTCCTACTCTCCATCACTATTGCAACAGGGTCATGTCTGGTTTGTCAGACTTGTAAGAACTACACTGGAGACGCCTGCGCAGAGCGGACCACTAAGACGTGCGACCCGTCGGTCAACAGCTGCATCACCACCTTGACAGTTATGCAGATTGGTGAAAAACGTTATCCGATGGTCGAAAAATCCTGTGCAACCAGTCCACTGTTGTGTGAGCTAGCGTACAACATGTCTTCAGGAATCGAACTGTATAGTGTCTCGAAGTGCTGCCAAGGAGACCGGTGCAACAAAGGATCGATCAAGTTGCCCCCGATAAACAGCACCGAGAACGGGGTGCAGTGTCCGGCCTGCTATGCTAAAGCAAAAACCTGCACACCCACCGGGACCATCAAATGTCGTGGATCGCAGACTAAATGCTTCAACTTCTCCGGCGGAATCTACAACGGCACTCAGTTTGAAGATTGGGCCTACCAGGGATGCACCACAGAGAATGTCTGCCAGCATCCAGCTCCAACCTACCCTGAGACTTACTTACAAGAAGGCTACAAACTGACCTGCTTCAACTCCAAACCCTGAAGAAACCAGCAGCGGAGGAGCCCAGAATATTTGTTAAAGGGGTTGTCTGACATTTTCATACTGACGACCTATCCTCAGGACGGATCATCAATATCAGGTGGGGGGGCGACACCCAGCACCCCTGCCGATCAGCTGTATGAAGGAGACCTCCTTCTACCATATCCAGCACAGCGCCACAGATTGTGTAGTGGTTATACTTGGTATTGCAGTCTAGGCTGATACACAATGTTGCGCCCAGCCATGTGACTTCACTTGCCCTAGAAAGAGCCGTGGTACTCACCAGAGCCGCAGACTCTTCACACAGCTGACTGGATGTGGTGTCAGGAGTCAGATCCCCACCACCACCACCACCACCACAGATTATCTGAGGATAGTTCCTCAAAATCAAGGTCTTGGACAACCCCTTTAAACCTTTCACTGTCAGTTGTTACAGTCAAATCTGTGTCTCCGGTTGTCAG

<210> 6

<211> 198

<212> PRT

<213> toad (Bufo)

<400> 6

MRTVLNVCLLLSITIATGSCLVCQTCKNYTGDACAERTTKTCDPSVNSCITTLTVMQIGEKRYPMVEKSCATSPLLCELAYNMSSGIELYSVSKCCQGDRCNKGSIKLPPINSTENGVQCPACYAKAKTCTPTGTIKCRGSQTKCFNFSGGIYNGTQFEDWAYQGCTTENVCQHPAPTYPETYLQEGYKLTCFNSKP.

Claims

A method for cloning a gene encoding a PLA2 inhibitor, comprising the steps of:

cloning SEQ ID No.1 and SEQ ID No.3 obtained from a bufo gargarizans bunge skin cDNA library, translating the SEQ ID No.1 to obtain an amino acid sequence SEQ ID No.2, and translating the SEQ ID No.3 to obtain an amino acid sequence SEQ ID No. 4;

step two, adopting PCR amplification:

(1) the PCR template is first strand cDNA of Bufo bufo gargarizans Cantor; primers for PCR: the sequence of the upstream primer is as follows: 5'-GGATTTGGTCCACTGCAAAC-3', downstream primer sequence: 5'-CTGACAACCGGAGACACAG-3', the concentration is 2 pmol/. mu.L;

(2) the PCR reaction system was 30. mu.L, where 10 × Buffer: 3 mu L of the solution; 10mmol/L dNTPS: 1.5 mu L; 1.5 mu L of each of the upstream primer and the downstream primer; template DNA: 0.2 mu L; 5U/. mu.L Taq: 0.3 mu L; sterilizing 21.7 μ L of pure water;

(3) PCR program for Taq DNA polymerase PCR amplification of the Chinese toad PLI gene fragment: denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 30 s/1 min, 40 cycles.
2. The method for cloning a gene encoding a PLA2 inhibitor according to claim 1, wherein the method comprises: the cDNA in the first step is from any part of skin, brain, heart, liver, lung, pancreas, stomach, intestine, kidney, testis, oviduct and spleen of the Chinese giant toad.
3. The method for cloning a gene encoding a PLA2 inhibitor according to claim 1 or 2, wherein the method comprises: the gene sequence listed in the cloning process, the amino acid translated by the gene sequence and the protein polypeptide formed by the amino acid translated by the gene sequence can be applied to the preparation of the inhibitor.
A method for cloning a gene encoding a PLA2 inhibitor, comprising the steps of:

cloning an obtained SEQ ID No.5 from a Japanese toad skin cDNA library, and translating the SEQ ID No.5 to obtain an amino acid sequence SEQ ID No. 6;

step two, adopting PCR amplification:

(1) the PCR template is Japanese toad first chain cDNA; primers for PCR: the sequence of the upstream primer is as follows: 5'-GGATTTGGTCCACTGCAAAC-3', downstream primer sequence: 5'-CTGACAACCGGAGACACAG-3', the concentration is 2 pmol/. mu.L;

(2) the PCR reaction system was 30. mu.L, where 10 × Buffer: 3 mu L of the solution; 10mmol/L dNTPS: 1.5 mu L; 1.5 mu L of each of the upstream primer and the downstream primer; template DNA: 0.2 mu L; 5U/. mu.L Taq: 0.3 mu L; sterilizing 21.7 μ L of pure water;

(3) PCR procedure for Taq DNA polymerase PCR amplification of the Japanese Bufo bufo PLI Gene fragment: denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 30 s/1 min, 40 cycles.
5. The method for cloning a gene encoding a PLA2 inhibitor according to claim 4, wherein the method comprises: the cDNA in the first step is from any part of skin, brain, heart, liver, lung, pancreas, stomach, intestine, kidney, testis, oviduct and spleen of the Japanese toad.
6. The method for cloning a gene encoding a PLA2 inhibitor according to claim 4 or 5, wherein the method comprises: the gene sequence listed in the cloning process, the amino acid translated by the gene sequence and the protein polypeptide formed by the amino acid translated by the gene sequence can be applied to the preparation of the inhibitor.