天蚕素A截短肽在大肠埃希菌中的高效表达及活性分析

将构建好的含抗菌肽天蚕素A(cecropin A,CA)截短肽CA19(36、210)1～5倍串连体的pET-31b(+)载体进行诱导表达,其表达量比单体表达明显提高.将融合蛋白用亲和层析法将其纯化,其纯度达到90%以上.纯化融合蛋白免疫BALB/c小鼠,制备了其特异的多克隆抗体.纯化串连融合蛋白用溴化氰切割成单体检测其活性,发现均具有抑菌活性.本研究为抗菌肽的基因工程制备提供了新途径.

High-level expression of antimicrobial peptide cecropin A-derivatives in E.coli and analysis of their antibiacterial activities

The pET-31b (+) vectors including tandem repetitive sequences of cecropin A (CA)-derivative CA19 (36,210)1-5 were expressed in the E. coli strain BL21 (DE3). High-level expression products were obtained after inducing by IPTG. The expression products were purified by His·Bind chromatography. SDS-PAGE and Bandscan analysis showed that the target proteins were highly purified and the purity was higher than 90%. Using the recombinant proteins of CA'derivatives to immune BALB/c mice, the CA derivatives-specific antibodies were obtained which can detect the CA expression in heterogeny expression systems. Purification products were cleaved into monomer by CNBr and the antibacterial activities of monomer were investigated by inhibition experiment with Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia, Pseudominas aeruginosa and Staphylococcus aureus. Our results provide a new approach for the preparation of antibiotic peptides.