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bmspi38 mutant and its application

A technology of mutants and site-directed mutagenesis, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of incompletely clear mechanism of activity, influence on genetic transformation and industrial application of inhibitors, and limited research

Active Publication Date: 2022-06-24
SHAANXI SCI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The activity and function of BmSPI38 have been relatively clear, but the mechanism of its activity is not completely clear, and the research on the potential amino acid sites that may affect the inhibitory specificity of TIL-like protease inhibitors is also relatively limited, which directly affects the genetics of the inhibitor. Retrofit and Industrial Applications

Method used

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  • bmspi38 mutant and its application
  • bmspi38 mutant and its application
  • bmspi38 mutant and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Construction and activity study of BmSPI38 mutant

[0028] 1. Mutation primer design

[0029] The amino acid sequence of BmSPI38 consists of positions 23 to 80 in SEQ ID NO.1, of which positions 1 to 22 are signal peptide sequences. Referring to the gene sequence of BmSPI38, as shown in SEQ ID NO.2, for BmSPI38 designed site-directed mutagenesis primers for 5' to 3' PCR amplification of this gene. The mutant template, desired mutation, DNA polymerase and primer sequences of BmSPI38 are shown in Table 1, respectively.

[0030] Table 1 P1 site mutation primers of BmSPI38

[0031]

[0032]

[0033] 2. PCR amplification

[0034] (1) When the DNA polymerase used in PCR amplification is FastPfu DNA Polymerase, the reaction system (25 μL) is shown in Table 2, and the amplification procedure is shown in Table 3. The amplification products were detected by 1% agarose gel electrophoresis.

[0035] Table 2 PCR reaction system

[0036]

[0037] Table 3 PCR amplificati...

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Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to BmSPI38 mutant and application thereof. BmSPI38 is composed of the 23rd to 80th positions in SEQ ID NO.1, and the BmSPI38 mutant is to mutate the glycine at the 54th position in the amino acid sequence of BmSPI38 as shown in SEQ ID NO.1 to arginine , lysine, serine, threonine, alanine, glutamine, aspartic acid, histidine, cysteine, proline, valine, asparagine, tyrosine, formazan from thionine, leucine, phenylalanine, isoleucine, tryptophan or glutamic acid. The BmSPI38 mutants of the present invention all have inhibitory activity on subtilisin and elastase, and when mutated into arginine or lysine, they also obtain trypsin inhibitory activity, and such mutants can be used to prepare trypsin inhibitors , the application prospect is good.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a BmSPI38 mutant and its application. Background technique [0002] Bombyx mori is a silk insect with great economic value. It has accumulated a lot of basic research and has become one of the best models of insect biochemistry, genetics and genomics. Our previous study systematically identified immune-related Bombyx mori protease inhibitors and found that many TIL (trypsin inhibitor-like cysteine-richdomain) protease inhibitors were up-regulated after microbial feeding infection, suggesting that TIL protease inhibitors may be involved in The immune process of silkworm. Further research showed that a silkworm TIL-like protease inhibitor BmSPI38 not only strongly inhibited the activities of subtilisin, proteinase K, Beauveria bassiana body wall-degrading proteases CDEP-1 and Aspergillus melis, but also blocked the activities of Coccid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C12N15/15C12N15/70C12N1/21C12R1/19
CPCC07K14/811C12N15/70
Inventor 李游山张杰
Owner SHAANXI SCI TECH UNIV