Expression, Purification And Applications Study Of Swine Hepatitis E Virus Genotype â…£ Recombinant ORF3 Proteins

SHEV-ORF3 has played an extremely important role in viral infection and cell signal transduction. The coding region of ORF3 from pMD18T-ORF3 was digested by EcoRâ… /Salâ… .Then the fragment was inserted into prokaryotic expression vector pET42a(+) with T4 ligase, which was named pET42a-ORF3. Positive clones were screened and identified by digestion and sequencing. The pET42a-ORF3 was transformed into E-coli BL21 and induced with IPTG After further optimization of IPTG induced conditions, the expressed protein was purified and identified by SDS-PAGE and Western Blot. The indirect ELISA for detection of HEV antibodies was established and the polyclonal antiserum was obtained by immunizing rabbits with the purified fusion protein as antigen.The results showed that the His/GST-ORF3 fusion protein were over-expressed in E-coli BL21 with molecular weight of about 45.5ku. The investigation for 190 swine serum samples from HaiKou and SanYa indicated that the coincidence rate between the fusion protein ELISA and available kit was up to 91.5% in detecting swine HEV antibody. The titer of the antiserum was above 1:12800.The His/GST-ORF3 fusion protein was successfully expressed and purified, high titer antiserum against SHEV-ORF3 was obtained. These data set a good basis for further study the function of SHEV-ORF3 and the indirect ELISA method is a sensitive and specific method in detecting HEV antibodies.