A construction method of a recombinant bacteria expressing a mutant protein of a nicotinamide phosphoribosyltransferase Nampt, wherein the mutant protein of the nicotinamide phosphoribosyltransferase Nampt has only one point mutation on an amino acid sequence of the nicotinamide phosphoribosyltransferase Nampt, the amino acid sequence of the nicotinamide phosphoribosyltransferase Nampt is the sequence as set forth in SEQ ID NO:1, wherein the only one point mutation is any one of N67K, N164L, R166W, A208G, A245T, S248A, V365L and S382M,
- the construction method comprising the following steps:
- (1) performing a codon optimization on a gene encoding the amino acid sequence of SEQ ID NO:1, to obtain an optimized gene;
- (2) performing a PCR amplification using a site-directed mutation primer and a KOD-Plus-Neo enzyme respectively by taking the optimized gene as a template, to obtain amplification products;
- (3) digesting the amplification products with a DpnI enzyme respectively, and then connecting to the pPSUMO vector respectively, to obtain a recombinant expression vector;
- and
- (4) transforming the recombinant expression vector into E. coli competent cells respectively, and picking positive clones, to obtain the recombinant bacteria,
- wherein the site-directed mutation primer in step (2) comprises:
- a forward site-directed mutation primer N67K-F and a reverse site-directed mutation primer N67K-R directed at a point mutation of N67K, the nucleotide sequence of the forward site-directed primer N67K-F as set forth in SEQ ID NO:
- 3, and the nucleotide sequence of the reverse site-directed primer N67K-R as set forth in SEQ ID NO:4;
- a forward site-directed mutation primer N164L-F and a reverse site-directed mutation primer N164L-R directed at a point mutation of N164L, the nucleotide sequence of the forward site-directed mutation primer N164L-F as set forth in SEQ ID NO:5, and the nucleotide sequence of the reverse site-directed primer N164L-R as set forth in SEQ ID NO:6;
- a forward site-directed mutation primer R166W-F and a reverse site-directed mutation primer R166W-R directed at a point mutation of R166W, the nucleotide sequence of the forward site-directed mutation primer R166W-F as set forth in SEQ ID NO:7, and the nucleotide sequence of the reverse site-directed primer R166W-R as set forth in SEQ ID NO:8;
- a forward site-directed mutation primer A208G-F and a reverse site-directed mutation primer A208G-R directed at a point mutation of A208G, the nucleotide sequence of the forward site-directed mutation primer A208G-F as set forth in SEQ ID NO:9, and the nucleotide sequence of the reverse site-directed primer A208G-R as set forth in SEQ ID NO:10;
- a forward site-directed mutation primer A245T-F and a reverse site-directed mutation primer A245T-R directed at a point mutation of A245T, the nucleotide sequence of the forward site-directed mutation primer A245T-F as set forth in SEQ ID NO:11, and the nucleotide sequence of the reverse site-directed primer A245T-R as set forth in SEQ ID NO:12;
- a forward site-directed mutation primer S248A-F and a reverse site-directed mutation primer S248A-R directed at a point mutation of S248A, the nucleotide sequence of the forward site-directed mutation primer S248A-F as set forth in SEQ ID NO:13, and the nucleotide sequence of the reverse site-directed primer S248A-R as set forth in SEQ ID NO:14;
- a forward site-directed mutation primer V365L-F and V365L-R directed at a point mutation of V365L, the nucleotide sequence of the forward site-directed mutation primer V365L-F as set forth in SEQ ID NO:15, and the nucleotide sequence of the reverse site-directed primer V365L-R as set forth in SEQ ID NO:16;
- or
- a forward site-directed mutation primer S382M-F and a reverse site-directed mutation primer S382M-R directed at a point mutation of S382M, the nucleotide sequence of the forward site-directed mutation primer S382M-F as set forth in SEQ ID NO:17, and the nucleotide sequence of the reverse site-directed primer S382M-R as set forth in SEQ ID NO:18.