Cloning, Expression And Application Of The ORF2 C-Terminal Encoding Fragment Of Swine HEV

RNA as template of RT-PCR was extracted from fecal supernatant containing the swCH25 strain of Hepatitis E virus, and then the C-terminal 916bp of viral capsid protein (ORF2) gene was amplified. The PCR products were inserted into pMD18-T vector routinely, and the positive recombinants were identified by endonuclease digestion, PCR and DNA sequencing. Compared to that of other genetypes, the predicted 305 amino acids of swCH25 strain showed identity of 92.5-92.8%, 90.8%, 95.1-95.7% and 98.0%-98.7% to that of Genetype I , II, III and IV respectively. The results of DNAStar Protean analysis indicated that ORF2 C-terminal fragment of swCH25 strain shared similar antigenicity with other strains of HEV.Digested with EcoRI and SalI, a fragment 926bp of the PCR product was cloned into the expression plasmid vector pET-32a(+). The recombinant was transformed into the BL21 and induced to express by 1.0mM IPTG at 37℃. The expression product was identified by SDS-PAGE and found to be 55.9kD as expected one and confirmed by Western blotting with swine or human HE positive serum. A single band of p305ORF2 was collected in elution after purification by affinity chromatography on a nikel-agarose resin. The results revealed that expression products of the partial fragment of ORF2 gene in vitro had the critical antigenitic epitopes and immunological cross reaction with human HEV. The recombinant protein was found to form homodimers or higher oligomers in vitro, which were demonstrated by SDS-PAGE and Western blot.An indirect ELISA in which the purified recombinant protein p305ORF2 was used as coating antigen, was developed to detect anti-HEV IgG in serum samples. Results of array test suggested a coating concentration of 0.62 μg/ml of p305ORF2, a 1:10 dilution (in PBST) of swine sera, a 1:10,000 dilution (in PBST) of horseradish peroxide conjugated staphylococcus A protein (HRP-SPA). The established ELISA was named as p305ORF2 ELISA.To evaluate sensitivity and specificity of the ELISA method, 91 serum samples...