Recombination And Fusion Expression Of Porcine Defensin Genes (PBD-Ⅰ,PBD-Ⅱ) In E.Coli

Objective: New cationic antimicrobial polypeptides named as defensins are recently discovered. They are widely distributed in animals and plants.They have broad antimicrobial spectrum and highly efficient antimicrobial activity, because of its special antimicrobial mechanism, they have drug resistance to pathogen. So, developing the research and exploiting of defensins has significant economical and social benefits. Based on cDNA sequence of porcine defensin P -defensin-1 gene reported in this study, We designed two pairs of primers with restriction enzyme sites, P -defensin-1 gene was amplified by RT-PCR. The gene was inserted into expression vector and recombinant vector transformed into E.coli ,which recombinant vector effectively produce fusion protein.Finally ,we hope antimicrobial peptide would be obtained from the fusion protein and explore the influence of the sequence of signal peptide on expression of antimicrobial peptide.MethodrThe P-defensin-1 gene which was amplified by RT-PCR ligated with vector PinPoint æ©·a-3 which was proceedingly digested with restriction enzymes. Then recombinant vector named ppd-1 and ppd-2 were transformed into JM109 strain. The positive clones were screened out with the method of extraction plasmid and restriction enzyme analysis. The expected results were inspected with DNA sequencing. The positive clones were induced with IPTG, we hope to discover the fusion protein by SDS-PAGE electrophoresis.Results:The RT-PCR product was about 130bp by DNA agarose gel electrophoresis and accorded with the anticipated results. After the PBD I and PBD II gene ligated with the expression vector PinPoint æ©·a-3, the recombinated plasmid ppd-1 and ppd-2 was transformed into JM109 strains, 4positive clones were screened by restriction enzyme analysis, DNA sequencing showed that two out of 4 positive clones inserted sequence of the constructed plasmid, which was the same as that of PBD- I and PBD- II gene respectively, and its reading frame was correct, thus its could be used to express fusion protein. The positive recombinants of PBD- I gene expressed 17kDa fusion protein after its were induced by IPTG, expression of PBD-1 gene didn't increase distinctly along with time, but the protein wasn't expressed in the positive recombinant of PBD-2 gene.Conclusion:By restriction enzyme secting, ligating, transforming, restriction enzyme analysis, and final DNA sequencing, the PBD- I and PBD-II gene were proved to be recombinated with the expression vector and the recombinated vector ppd-1 and ppd2 were transformed successfully. We successfully expressed the PBD-1 gene, which lays down the foundation in research of antimicrobial activities , antimicrobial mechanism of the Defensin.