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Novel gene of virus original protease inhibitor as well as expression and application thereof

A technology of protease inhibitors and amino acids, which is applied in the fields of genetic engineering and protein engineering, can solve the problems of few researches on protease inhibitors derived from microorganisms, and achieve strong poisonous killing effect and the effect of inhibiting protease activity

Inactive Publication Date: 2009-11-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on protease inhibitors at home and abroad is mainly concentrated in animals and plants, while the research on microbial protease inhibitors is very little.

Method used

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  • Novel gene of virus original protease inhibitor as well as expression and application thereof
  • Novel gene of virus original protease inhibitor as well as expression and application thereof

Examples

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Effect test

example 1

[0024] Genomic DNA was extracted from overnight cultured bacterial liquid of Pathogenic Bacillus burgdorferi burgdorferi "BJFS" collected and screened in our laboratory, and PCR amplification was performed using the extracted genomic DNA as a template. Recover the PCR product and connect it with pMD18-Tvector to transform the competent cell DH5α, inoculate the transformed bacterial liquid on LB solid medium containing ampicillin, culture overnight at 37°C, pick positive colonies and inoculate them on LB liquid culture containing ampicillin solution, cultured at 200rpm / min for about 9h, extracted the plasmid, carried out double digestion and identification with restriction endonucleases BamHI and EcoRI, and sent the identified clones for sequencing. From the mass sequence sequencing results, protease inhibitor genes were obtained. The DNA sequence SEQ ID NO.1 of the gene (XbPI-1) of the present invention is the first report in the pathogenic bacteria of Nematophila burgdorferi,...

example 2

[0026] The DNA sequence SEQ ID NO.1 of Xenorhabdus bovienii protease inhibitor gene XbPI-1 obtained above, the encoded protein contains 119 amino acids, SEQ ID NO.2. After comparison in the database, the protein encoded by the protease inhibitor gene XbPI-1 has an amino acid sequence similarity of 77% and 60% to the protein encoded by the protease inhibitor gene Xenorhabdus nematophila and Photorhabdus sp.Az29 in the database. , Pectobacterium chrysanthemi amino acid sequence similarity is less than 60%.

example 3

[0028] According to the full-length sequence of the XbPI-1 gene obtained in Example 1, the pQE30UA expression vector was used to recover the PCR product obtained in Example 1 by electrophoresis, and the recovered gene fragment was ligated with the pQE30UA expression vector at 16°C for 2 hours to transform the large intestine Bacillus M15 competent cells were spread on LB solid medium (containing Amp and Kana antibiotics), and the obtained resistant colonies were picked and cultured in liquid LB medium for preliminary identification by PCR, extraction of plasmids for sequencing, and confirmation The reading frame of the constructed expression vector was correct.

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Abstract

The invention relates to a novel gene of virus original protease inhibitor as well as the expression and the application thereof. The invention discloses a novel virus original protease inhibitor DNA sequence and an encoding protein sequence thereof, relates to a virus insect resistance key gene and enzyme and belongs to the field of gene engineering and protein engineering. The gene DNA sequence and the encoding amino acid sequence thereof are the first report of xenorhabdus nematophila. The protein expressed by the gene has remarkable inhibitory effect to prolease activity. A biological activity test shows that the protein encoded by the gene can reduce the reproduction rate of green peach aphids, improves the death of the green peach aphides and has good insecticide resistance function. The invention provides a new novel for the research of biological pesticides and transgenic zoophobous and has favorable application prospect in the field of pest control. The gene and the encoding protein thereof can also be applied in the field of medicament and biochemistry.

Description

technical field [0001] The invention discloses a protease inhibitor gene DNA sequence and its coded protein sequence, belonging to the fields of genetic engineering and protein engineering. The invention relates to the key genes and enzymes of microbial insect resistance, and also relates to the research, production and application of protease inhibitors in the fields of medicine, biochemistry and the like. Background technique [0002] The application of protease inhibitor genes in the fields of agriculture and medicine has attracted more and more attention. Protease inhibitor genes can not only be directly used to produce biopesticides, but also can be used in transgenic plants to improve plant insect resistance; protease inhibitors are safe for mammals, have no environmental pollution and residue problems in agricultural products, and can provide A safe and effective new way to control insect pests; protease inhibitor genes can also be used in medicine such as the treatm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C07K14/195A61K39/02A61P1/18A61P35/00A61P9/10A01N63/00A01P7/04A01H1/00
Inventor 曾凡荣薛仁风
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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