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BmSPI39 mutant and application thereof

A mutant and site-directed mutation technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems affecting the genetic transformation and industrial application of inhibitors, the mechanism of activity is not completely clear, and the research is limited.

Active Publication Date: 2022-07-29
SHAANXI SCI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The activity and function of BmSPI39 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 limited, which directly affects the genetics of the inhibitor. Transformation and Industrial Applications

Method used

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  • BmSPI39 mutant and application thereof
  • BmSPI39 mutant and application thereof
  • BmSPI39 mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Construction and activity study of BmSPI39 mutant

[0029] 1. Mutation primer design

[0030] The amino acid sequence of BmSPI39 consists of positions 25 to 98 in SEQ ID NO.1, of which positions 1 to 24 are signal peptide sequences. Referring to the gene sequence of BmSPI39, as shown in SEQ ID NO.2, for BmSPI39 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 BmSPI39 are shown in Table 1, respectively.

[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 amplification program

[0038]

[0039] (2) When the DNA poly...

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Abstract

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to a BmSPI39 mutant and application thereof. The BmSPI39 is composed of the 25th site to the 98th site in SEQ ID NO.1. The BmSPI39 mutant is obtained by synthesizing a BmSPI39 amino acid sequence into a BmSPI39 amino acid sequence, a BmSPI39 amino acid sequence and a BmSPI39 amino acid sequence. The amino acid mutant is obtained by mutating the 56th alanine as shown in SEQ ID NO.1 into arginine, lysine, serine, threonine, glutamine, tyrosine, methionine, leucine, aspartic acid, glutamic acid, histidine, cysteine, valine, asparagine, isoleucine, phenylalanine, tryptophan, proline or glycine. The amino acid mutant is obtained by mutating the 56th alanine as shown in SEQ ID NO.1 into arginine, lysine, serine, threonine, glutamine, tyrosine, methionine, leucine, aspartate, glutamic acid, histidine, cysteine, valine, asparagine, isoleucine, phenylalanine, tryptophan, proline or glycine. The BmSPI39 mutant disclosed by the invention has inhibitory activity on subtilisin and elastase, trypsin inhibitory activity is also obtained when the BmSPI39 mutant is mutated into arginine or lysine, and the mutant can be used for preparing a trypsin inhibitor and has a good application prospect.

Description

technical field [0001] The present invention is a divisional application of the application number 202111080025.3, and the filing date is September 15, 2021. [0002] The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a BmSPI39 mutant and its application. Background technique [0003] 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-rich domain) 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 BmSPI39 not only strongly inhibited the activities of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K14/435C12N15/70
CPCC07K14/81C07K14/43586C12N15/70Y02A50/30
Inventor 李游山张杰
Owner SHAANXI SCI TECH UNIV
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