Research On Expression And Biological Activity Of Antimicrobial Peptide Ranalexin From Rana Catesbeiana

Antimicrobial peptides are a kind of bioactive peptides that exist in organism and biosphere widely.They are important components of the innate defenses of all species life. Antimicrobial peptides show the multiple biological functions including broad spectrum of antibacterial activities and potent antimicrobial activities against viruses,fungi and protozoa. Antimicrobial peptides also exhibit anti-tumor activities,as well as exhibiting unique mechanism.Compared with mammals , the acquired immune system in amphibians is very fragile . The innate system is mainly composed of antimicrobial peptides in amphibian skins that act as very important defensive function against the invasiveness of microorganisms,which have high effective and broad-spectrum antibacterial activities and hardly inducing drug-resistance. So it is important significance to study the antimicrobial peptides in amphibian skins and their application in clinic. Nevertheless , preparation of AMPs at a large scale is a significant challenge in order to develop commercial products. Therefore, this research produced the antimicrobial peptide by the method of gene engineering preparation. Antibiotic peptide ranalexin gene was obtained from the skin of the bullfrog, the recombinant strain DE3/pGEX-3X -Ranalexin and yeast engineeing strains of GS115/ pPIC9K-Ranalexin were constructed respectively and induced to express antimicrobial peptide of the bullfrog skin with antibacterial activities.This study laid the foundations for the application research on antimicrobial peptide from the skin of the bullfrog and large scale production.In this study, a pair of specific primers were designed and synthesized according to the ranalexin gene sequence reported in GenBank and antibiotic peptide ranalexin gene was amplified from the skin of the bullfrog ( Rana catesbeiana )by RT-PCR. Amplified DNA fragments were ligated into pMD18-T vector, and transformed into E.coli strain DH5αby the calcium chloride method. The sequence was confirmed by DNA sequencing. The result of sequence analysis displayed that this gene contained 201 nucleotides which encode 66 amino acides. Compared with the ranalexin gene sequence reported in Genbank, the homology of the complete DNA sequence was 99.57%. The molecular weight was 7.6 kDa.The mature peptide of ranalexin contained 20 amino acides, and the sequence was consistent with ranalexin publicated in GenBank.The predicted second structure was mainlyα-spiral and the protein has highly amphipathic property.Redesign the primers by adding the restriction site , then the ranalexin gene was cloned into the expression vector pGEX-3X to construct the recombinant expression plasmids. Positive plasmid was identified and transformed into the competent cell BL21(DE3)which was induced with IPTG. The expressed products was identified with Tris-SDS-PAGE and the molecular weight of the expressed products was 34 kDa.The optimal conditions were determined that the induction time was 5~6 hours, the temperature was 37℃, the pH was 7.4 and the IPTG concentration was 1mmol/L. Under this conditions, the expressed recombinant protein existed in the form of inclusion bodies. After refolding in vitro and purifying, the expressed recombinant protein exhibited the antibacterial activity to Bacillus subtilis and Staphylococcus aureus in vitro.The recombinant protein GST-Ranalexin existed in the form of inclusion bodies so that it is hard to refold in vitro,then the biological activities of ranalexin were affected. Therefore, yeast expression plasmid pPIC9K-Ranalexin were constructed, using the expression plasmid pPIC9K as vector. Positive plasmid was identified, then the linearized recombinant plasmid was electrotransformed into the competent yeast strain GS115 of Pichia pastoris. The positive clones were screened by the menthods of plate G418 pressure and His auxotroph,then identified by PCR. Tris-Tricine SDS-PAGE analysis of the protein bands indicated that the interest proteins were soluble proteins in the culture supernatants, and the molecular weight of the expressed products was 7.7 kDa. The result of susceptibility tests in vitro showed that the expressed products exhibited the antibacterial activity to Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Candida albicans.