Expression And Activity Identification Of Porcineβ-defensin-1Gene In E.Coli

Defensins was an important antimicrobial peptides generated by the organism during the defense reaction. It possessed broad-spectrum antimicrobial, including many microorganisms like bacteria, fungi, enveloped viruses. Antibacterial peptides didn’t induce the resistance of bacteria because of its distinctive antibacterial mechanism, which suggest that it can be a potential substitute of traditional antibiotics.Porcine β defensin-1(PBD-1) was an antibacterial peptide that played an important role in defense system of porcine. PBD-1had a high expression in porcine respiratory and digestive tracts, liver and kidney etc. Researches also proved that PBD-1can be used as food preservatives and feed additives. With the further study on PBD-1, it would play more important role in animal breeding, illness prevention and quality of livestock products. The purpose of this study was to clone porcine tongue epithelium defensin by a traditional techniques and to realize high expression of pBD-1depend on E.coli expression system, and then look forward to obtain high yield of active pBD-1product, which lay a foundation for the mass production of PBD-1and application in animal husbandry.In this study, the fresh porcine tongue epithelium mucosa was obtained from healthy porcine. Primers were designed based on porcine β-defensin1cDNA sequence reported in GenBank and multiple cloning sites in pET-30a vector, containing BamHI and Xhol on the upstream and downstream, respectively. The gene of pBD-1was amplified using RT-PCR, and sequenced after clone into pMD18-T vector. The mature peptide gene of pBD-1,126bp, was obtained from recombinant plasmid of pMD18-T-pBD-1by digested with BamHI and Xhol restriction enzymes, and then inserted into the BamHI and Xhol sites of pET-30a vector to construct recombinant plasmid of pET-30a-pBD-1. The expression system of fusion pBD-1was constructed successfully after recombinant plasmid pET30a-pBD-1transformed into "E.coli Rossetts (DE3) strains and sequenced correctly. The positive clone was fermented with flask-shaking and induced with IPTG, and then identified using Tricine SDS-PAGE after sonicated. The fusion proteins were purified with Ni-NTA resin, and digested with recombinant enterokinase to remove His-tag. The antimicrobial activities of His-pBD-1and pBD-1proteins were performed after refolding according to Agarose Diffusion Assay. The results indicated that fusion protein of His-pBD-1was expression with soluble components in the cell lysates, and about50mg His-pBD-1proteins were obtained per liter medium; both of His-pBD-1and pBD-1proteins were showed as a high antibacterial activity against streptococcus, so active pBD-1was not effected by the presence of his-tag, and the minimum inhibitory concentrations (MICs) of active pBD-1was40±5.2μg/ml. This study lay a good foundation for future cytotoxicity assay, immunogenicity and development of antimicrobial agents.The streptococcus infection model was performed with Bama pigs. Expression level of pBD-1in porcine tongue epithelium was performed using qPCR method at different times after infection. The results indicated that the level of pBD-1was initially changed with significant up-regulation (P<0.001) at3h compared to after infection, and reached the highest at6h, however, the pBD-1reduced to normal level at12h. The expression level of pBD-1against Streptococcus is up-regulation in the porcine tongue.