Prokaryotic Expression Of Antibacterial-immunomodulating Fusion Peptide And Primary Research On Its Antibacterial Function

Objective To express Lactoferricin B and immuno-modulating peptide (PGPIPN) fusion peptide (antimicrobial-immunomodulating fusion peptide, AIFP) in bacteria, preparatorily research on its antimicrobial activity in vitro.Methods We design a soft truss arm GGGGS to join Lactoferricin B and immuno- modulating peptide (PGPIPN) for constructing antimicrobial-immunomodulating fusion peptide (AIFP) with computer and biology soft Insight II after analysis of the structure of the tow pepetides. AIFP gene was obtained through gene splicing by overlap extension polymerase chain reaction (SOE-PCR). The expression plasmid was constructed by inserting AIFP cDNA into pGEX-KG. The positive recombinants were identified by restriction endonuclease digestion and expressed in E. coli BL21 induced by isopropyl-beta D-thiogalactopyranoside (IPTG). The desired fusion proteins were identified by SDS- PAGE and Western blot. The expressed products were purified by affinity chromatography using Glutathione Sepharose 4B. AIFP was obtained after digestion by Factor Xa protease, and identified by Tricine-SDS-PAGE. The antimicrobial activities were tested by paper agar diffusion method.Results AIFP cDNA was cloned into pGEX-KG vector and expressed in E coli BL21 successfully. SDS-PAGE and Western blot showed the molecular weight of the fusion protein recognized by the specific monoclonal anti-GST antibody was coincident with prediction. The fusion proteins were purificated prolifically. After digestion by Factor Xa, and identified by Tricine-SDS-PAGE, AIFP was obtained. The antimicrobial activities were proved by paper agar diffusion method.Conclusion AIFP is obtained by prokaryotic expression and it show antimicrobial activity in vitro.