Construction, Expression And Purification Of Prokaryotic Vectors Of Hev ORF3and Its Candidate Interactive Proteins

Hepatitis E is an important Zoonosis, which is epidemic mainly in developing countries. China is a vulnerable region with both outbreaks and sporadic cases. Hepatitis E virus (HEV) is a small non-enveloped virus with a size of27-34nm, and has a positive-sense, single-stranded,7.2kb, RNA genome, which contains three open reading frames (ORF). ORF3encodes a protein of114aminoacids, involved in virion morphogenesis and release, distinction among virus genotypes, which makes sence in development of HEV vaccine and diagnosis. In this reasearch, recombinant ORF3pokaryotic expression vectors were expressed and ORF3protein is purified successfully with sobility and purity improved. Recombinant RPS11pokaryotic expression vectors were also expressed and the protein is purified.HEV ORF3gene was amplified by PCR, the amplified product was double digested and then inserted pET28a, pGEX-4T-l, His-pGEX4T1and pET43vectors with the same restriction sites, constructing prokaryotic expression vectors. The recombinant plasmids were transformed into E.coli DH5a. The vectors with correct sequence were transferred into E.coli BL21(DE3), and was induced with IPTG. The expression of the protein was analyzed by SDS-PAGE and western-blot, the protein was purified by affinity chromatography. The expression proportion and purity of the fusion protein were analyzed in BandScan5.0. That provided foundation for the study of function and structure of ORF3, the research of interaction with receptor, and the development of vaccine.RPS11gene was amplified by PCR, the amplified product was double digested and then inserted pGEX-4T-1and pET43a vectors with the same restriction sites, constructing prokaryotic expression vectors. The recombinant plasmids were transformed into E.coli DH5a. The vectors with correct sequence were transferred into E.coli BL21(DE3), and was induced with IPTG. The expression of the protein was analyzed by SDS-PAGE and western-blot, and the protein was purified by affinity chromatography. The purifed protein was analyzed in BandScan5.0. The purified protein was used in the study of interaction between RPS11and ORF3through GST Pull-down.