Secretion expression of antibiotic peptide CAD in
View Patent ↗The present invention relates to a method for expressing antimicrobial peptide CAD by means of a recombinant Bacillus subtilis expression system. The SUMO protease expression operon is first artificially synthesized. The protein expression operon genes of Saccharomyces cerevisiae small ubiquitin-related protein is then fused with the antibacterial peptide AD. The fusion protein is further cloned into the pNF11 plamid to be introduced into Bacillus subtilis , thereby ensuring the induced expression of recombined Bacillus subtilis in shake flasks. The method has the advantages of a simple expression system, large-scale production, low production cost, strong biological activity and no toxic or harmful substance production. Moreover, the method provides a medicine with low price and strong antibacterial capacity for clinic disease prevention and treatment. This invention can also be used as a feedstuff additive.
1. A method of producing a recombinant antimicrobial peptide Cecropin AD (CAD) comprising the following steps:
a. synthesizing the nucleic acid encoding a small ubiquitin-related modifier (SUMO) protease to produce a first synthetic DNA fragment;
b. synthesizing the nucleic acid encoding the fusion protein of a Saccharomyces cerevisiae small ubiquitin-related protein with the antimicrobial peptide cecropin AD (CAD) to produce a second synthetic DNA fragment wherein said fusion protein comprises the amino acid sequence of SEQ ID NO:1;
c. digesting the first synthetic DNA fragment from step a with the restriction enzymes EcoR I and BamHI, and digesting the second synthetic DNA fragment from step b with the restriction enzymes BamHI and SacI;
d. subcloning the products from step c into vector pBluesriptII SK(+) in order to produce a D1778-1 plasmid;
e. extracting the genome of Bacillus subtilis strain 168, and amplifying the highly active promoter p43 from the genome;
f. sub-cloning the amplified promoter p43 into a T-vector for sequencing;
g. introducing the promoter p43 from the T-vector into pGJ103 plasmid to produce pGJ284 plasmid;
h. digesting the pGJ284 plasmid and the D1778-1 plasmid with restriction enzymes EcoRI and SacI, respectively;
i. recovering the two restriction products from step h, and then ligating said two products with T4 DNA ligase in water-bath at 16° C. to obtain a recombinant pNF11 plasmid;
j. transforming Bacillus subtilis 1A747 strain with said pNF11 plasmid to produce a recombinant Bacillus subtilis 1A747 strain which comprises said pNF11; and
k. inducibly expressing said pNF11 which comprises the nucleic acid encoding the antimicrobial peptide CAD in the recombinant Bacillus subtilis 1A747 strain.
2. A Bacillus subtilis strain comprising the recombinant antimicrobial peptide cecropin AD (CAD) of claim 1 , which is deposited at China General Microbiological Culture Collection Center with number CGMCC No. 2373.