Cloning Of VP4 Gene And Prokaryotic Expression Of Major Antigen Site Gene From Porcine Rotavirus JL94

Porcine Rotavirus(PRV) belongs to the family reoviridae. It is one of the major pathogens of diarrhea in piglets and causes of significant losses in graziery worldwide. According to the reported complete VP4 nucleotide sequences of group A PRV in GenBank,a pair of primers was designed to amplify VP4 gene of JL94. The segments of complete gene 4 of JL94 were obtained from RT-PCR using dsRNA extracted from the virus as template. The product of PCR named VP4 is approximate 2.3kb in length. The VP4 gene was cloned into pMD18-T vector and sequenced. The results of sequencing showed that JL94 isolate complete gene 4 was 2362bp and had a complete open reading frame which encoded 776 amino acides. The sequence was analyzed homologously in comparison with the VP4 nucleotide sequences of two reference porcine rotavirus BEN-307 and Gottfried. The deduced animo acid sequences were 96.06% and 71.88% correspondingly. The result affirmed that JL94 isolate is more similar to BEN-307 than Gottfired in gene type. According to date specific major antigen site of VP4 is in 5' extremity 750bp in legs, another pair of primers was designed to amplify VP4 gene of major antigen site(1bp-756bp). The sequence was analyzed homologously in comparison with the VP4 nucleotide sequences of two reference porcine rotavirus CRW-8 and Gottfried. The deduced animo acid sequences were 96.43% and 67% correspondingly. The most divergence of amino acid sequence is located in a region delimited by aa81-aa207. The major antigen site of VP4 gene was into expression plasmid pGEX-6P-1. The recombinant plasmid VP4-pGEX-6P-1 was transformed into E.coliBL21(DE3)plysS and induced with IPTG. A fusion protein about 50kDa as we expected was found. The amount of the goal protein was evaluated by densitometric scanning. It indicated that the 50kDa product of the VP4 gene was 26.6% of total bacterial protein of BL21. The expressed products were purified by Glutathion-Sepnarose chelation affinity chromatography and tested by western blot. The results showed that GST-VP4 fusion proteins have a positive reaction with anti-JL94 serum.This study is first time in amplify complete JL94/VP4 gene and get expression of major antigen site of JL94-VP4, it provides the basis evidence and material basis for PRV identification, molecular epdiemiology investigation research of diagnostic reagent and genetic engineering vaccine.Canditate: Song YanSpeciality: Preventive Veterinary ScienceSupervisor: Prof.Li Yijing...