bmspi39 mutants and their applications
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.
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[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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