Expression Of The Major Antigenic Domains On Protein VP₂ Of Porcine Parvovirus In E.coli And Purification Of Recombinant Products

Based on VP2 gene sequence data, a fragment 848bp in VP2 gene was amplified by Polymerase Chain Reaction(PCR) from the genome of Porcine Parvovirus(PPV) and cloned into plasraid pGEM T-easy. The result of PCR showed that the designed fragment was amplified with expected molecular weight and named as VP2I , The fragment contained a major antigenic domain on VP2 protein calculated by software DNAstar. Two enzyme sites BamHl^ Xho I were introduced at 5' terminal and 3' terminal of VP2 I.The VP2 I was subcloned into BamHI, Xho I enzyme sites of a prokaryotic expression vector pET32-a(+). The recombinant plasmid was named as pET VP21 .Expression vector of major antigenic domain on VP2 protein (VP2I) was transformed into E.coli strain BL-21, The host was name BLVPJ .After inducing by IPTG, the expression production experiment showed that fused protein TrxA- VP2I was expressed in BL VP2I with expected molecular weight 45KD. The result of Western-blot showed that TrxA- VP2I had the antigen tic characteristic of PPV. Incubation of TrxA- VP2I for expression was studied. The optimized conditions for induction of TrxA- VP2I was :37 C,0.2mM IPTG, incubating 4-5hrs after induction. The TrxA- VP2I protein amounted to about 30% of the total protein. The purification process of the recombinant TrxA- VP2I fusion protein was studied. Similar to majority of eukaryotic proteins, TrxA- VPJ was expressed as inclusion bodies in E.coli. A series of purification steps, including cell breakage, inclusion body washing, inclusion body solubilization, protein renaturation and gel filtration chromatographies of sephacryl S-200HR and metal chelated chromatography. The result showed that 6M urea was capable to dissolve inclusion body, and the relative recovery of gel filtration chromatographies was about 25%. The production of purification was lyophilized as antigen for future study.