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A mutant and site-directed mutagenesis technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of incompletely clear active mechanism, affecting the genetic modification and industrial application of inhibitors, and limited research.
Active Publication Date: 2022-06-28
SHAANXI SCI TECH UNIV
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[0003] 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. Retrofit and Industrial Applications
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Embodiment 1
[0027] Construction and activity study of BmSPI39 mutant
[0028] 1. Mutation primer design
[0029] 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.
[0030]
[0031] 2. PCR amplification
[0032] (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.
[0033] Table 2 PCR reaction system
[0034]
[0035] Table 3 PCR amplification program
[0036]
[0037] (2) When the DNA polymerase used ...
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Abstract
The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to BmSPI39 mutant and application thereof. BmSPI39 is composed of the 25th to 98th positions in SEQ ID NO.1. The BmSPI39 mutant is the amino acid sequence of BmSPI39, as shown in SEQ ID NO.1, the alanine at the 56th position is mutated to arginine, Lysine, Serine, Threonine, Glutamine, Tyrosine, Methionine, Leucine, Aspartic Acid, Glutamic Acid, Histidine, Cysteine, Valine, Astragalus Paragine, Isoleucine, Phenylalanine, Tryptophan, Proline or Glycine. The BmSPI39 mutants of the present invention all have inhibitory activity on subtilisin and elastase, and when mutated to 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 BmSPI39 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-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 subtilisin, proteinase K, Beauveria bassiana body wall-degrading proteases CDEP-1 and Aspergillus melis, but also blocked the activities of Cocci...
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