Cloning, Expression And Activity Detection Of An Emulated Catfish Antimicrobial Peptide

Antimicrobial peptide produced by microorganisms, plants, mammals et al have severalproperties that have led them to be considered as potential new therapeutics including not only highantimicrobial activities ,wide activity spectrum and rapid killing action ,but also not easy to induceresistant pathogenic strains. According to reports ,the epithelial mucosal layer of the woundedcatfish Parasilurus asotus ,can secrete a broad spectrum of antimicrobial and no hemolyticactivities peptide –Parasin I. It has higher antimicrobial activities than others on differentmicroorganisms including Gram-negative,Gram-positive bacteria and fungi.Parasin I is product from the proteolytic cleavage of histone H2A, which can not gain genesequences from the histiocyte of catfish by cDNA. Emulational design and synthesized by peopleare taken in this study. Sequences XH1,XH2,XH3 coding for antimicrobial peptide with differentrestriction sites have been obtained by PCR amplification,and then cloned into the fusion vectorpGEX-5X-3 . The recombinant with three sequence segments was constructed and transformed intoE.coli BL21(DE3) after the expected sequences were checked. Induced by IPTG in 37℃, thecloned protein expressed in a high level in the form of inclusion body in cells and amounted toabout 30% of the total bacterial protein from desitometric scan analysis of SDS-PAGE.The fused protein was forced to be transformed and translated slowly so as to form solubleprotein in cells by low-temperature IPTG induction under the condition of: the temperature of 20℃,the induction time of 10 hrs. The emulated antimicrobial peptide has been obtained throughultrasonic disruption, saturation (NH4)2SO4 salting-out, CM-52 sepharose ion-exchangechromatography.The inhibition on many microbials such as Escherichia coli, Bacillus subtilis, Staphylococcusaureus, Saccharomyces cerevisiae have been tested by the purified and distilled peptide. The resultsshowed that it has strong inhibitation effect on Escherichia coli, Bacillus subtilis,Staphylococcusaureus, especially Bacillus subtilis shows most sensitivity to the peptide.