Design, Expression And Activity Study Of Hybrid Antimicrobial Peptides CC31 And CC34

According to the principle of antibacterial peptides molecular design, this study designed two hybrid antimicrobial peptides CC31å'ŒCC34 through hybrided antibacterial peptides Cec Md and Rana chensinensis Chensirin with the methods and technology of bioinformatics and molecular biology. Their genes were synthesized by splicing overlap extension PCR and cloning vectors of their genes were constructed. Their recombinant expression vectors were constructed using expression vectors pET32a and pGEX-6-p-1, and recombinant expression vectors were transformed Escherichia coli BL21 (DE3) forming genetic engineering bacteria. Fermentation conditions of genetic engineering bacteria pGEX-6P-1/CC31/BL21 (DE3) and pGEX-6P-1/CC34/BL21 (DE3) were optimized. Their expressional products CC31-GST and CC34-GST were purified through affinity chromatography and cut by formic acid. Then antibacterial activity, hemolytic activity, thermal and differet pH buffer solutions stability and immune activity of two hybrid antimicrobial peptides CC31å'ŒCC34 were initial researched.The results showed that:(1) Two hybrid peptides CC31 and CC34 were designed with the potential forming of antimicrobial peptides.(2) The gene of two hybrid antimicrobial peptides CC31å'ŒCC34 were synthesized and their cloning vectors pMD 18-T/ CC31and pMD 18-T /CC34 were constructed.(3) The hybrid antimicrobial peptides genetic engineering bacteria pET32a/CC31/BL21 (DE3), pET32a/CC34/BL21 (DE3), pGEX-6p-1/CC31/BL21 (DE3) and pGEX-6p-1/CC34/BL21 (DE3) were constructed, then the fusion proteins were expressed by induced with IPTG. The fusion proteins of pGEX-6p-1/CC31/BL21 (DE3) and pGEX-6p-1/CC34/BL21 (DE3) were acquired after affinity chromatography and their molecular weight were 29.6 kDa and 29.7 kDa, respectively. The quantity of the products of two hybrid antimicrobial peptides genetic engineering bacteria expressed respectively were 76 mg and 37 mg per litter broth. The molecular weight of hybrid antimicrobial peptides CC31 and CC34 were 3.6 kDa and 3.7 kDa after cut their fusion proteins by formic.(4) The best fermentation conditions of genetic engineering bacteria of pGEX-6p-1/CC31/BL21 (DE3) and pGEX-6p-1/CC34/BL21 (DE3) were 1) time of persistently fermention: 6 h; concentration of IPTG were 0.4 mmol/L; temperature: 37℃; culture: TB.(5) Two hybrid antimicrobial peptides CC31å'ŒCC34 posses characteristics of thermal (p>0.05) and different pH buffer solutions stability (p>0.05), compared to Cec Md, faint hemolytic activity (p<0.01), resistance against different pathogenic bacteria and increasing (p<0.01) lymphocyte transformation rate in vitro of rats and sheep.