中国生物工程杂志

The ORF3 gene from a full-length swine HEV genomic clone was amplified by polymerase chain reaction(PCR) and inserted into vector pMD18-T, then subcloned to prokaryotic expression vector pET28a(+). The recombinant plasmid pET28a-ORF3 was transformed to E.coli BL21 (DE3) for expression under induction of IPTG. The expressed product was identified by SDS-PAGE, Western blot, then purified by Ni-NTA affinity chromatography and used as an antigen for the determination of HEV antibody in swine and human serum by ELISA. It indicated that the target gene was amplified by PCR. The results of restriction and DNA sequencing confirmed that the prokaryotic expression vector pET28a-ORF3 was constructed correctly. The expressed product, with a relative molecular weight 6 500~16 500, was consistent with the designed. Western blot showed specific reaction of the expressed fusion protein with mouse anti-His Tag serum. ELISA indicated that the expressed fusion protein can reacted with the HEV positive swine and human serum. The expressed product showed good reactionogenicity, which laid a foundation of developing a swine HEV diagnostic reagent.