Gene Reformation And Prokaryotic Expression Of Sheep Antimicrobial Peptide SMAP-29

Bone marrow cells of sheep antimicrobial peptide SMAP-29 (Sheep myeloid antibacterial peptides with 29 amino acids), is from Italy and the United States by Mahoney and Bagella two different research groups found at the same time in 1995. It has a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, viruses, parasites, spirochetes, chlamydia are killing effect. Its unique mechanism of action, having the characteristics of rapid kill bacteria, but it also has a known family members Cathelicidin strongest antibacterial activity. It is understood that the relevant domestic antimicrobial peptide SMAP-29 study reported relatively small, and therefore the use of these advantages SMAP-29 to the synthesis of new antibacterial drugs is imperative, however, SMAP-29 level structure includes 29 amino acids, the molecular weight of 3 256 Da According to Chen Chen et al statistics,0.1 g SMAP-29 synthesis of pure need 12 000-17 000 yuan, so that will certainly limit the peptide drug research and development, therefore, part of the active interception of natural antimicrobial peptides gene fragment used the synthesis of proteins bound to reduce production costs.Objective:This study aimed to reform the gene of sheep antimicrobial peptide SMAP-29 in its active gene fragments,and highly express fusion protein in E.coli BL21.Methods:According to the published amino acid sequence of SMAP-29(1-18),a gene encoding SMAP-29 (sheep myeloid antibacterial pepirdes-29) peptide was syn-thesized and was based codon usage of E.coli,using the protein diagnostic tools, which can predict physicochemical and transmembrane properties of proteins.The synthesized gene was inserted into E.coli expression vector pET-30a,transformed into E.coli strain DH5a, Used kanamycin resistance screening positive clones,identified a little constructed recombinant plasmids by restriction enzyme digestion,PCR, and DNA sequencing. Transformed the constructed recombinant plasmids to E.coli BL21 (DE3) plyS for expression under induction of IPTG, and the expressed product was analyzed by SDS-PAGE,purified by nickel ion affinity chromatography, and cut by enterokinase.Results:The synthesized gene was expressed in E.coli BL21 (DE3) successfully. Using Ni-NTA column can get higher purity of fusion proteins.Conclusion:After reformation, the gene of antimicrobial peptide SMAP-29 was hightly expressed in E.coli BL21 (DE3).The target proteins had higher purity provide a theory and practice foundation for further normalized production of SMAP-29.