Construction Of ELISA Detection Of Swine Anti-HEV Antibody And Survey Of Its Molecular Epidemiology In Shanghai

Hepatitis E(HE) was an amphixenosis caused by Hepatitis E Virus(HEV).HE occured on breakout epidemic stage in Asia and Africa with poor sanitation condition,whereas on sporadic stage in developed countries,China is on of the HE-epidemic country of the world.Various animals are susceptible to HE.Swine is one of the most important intermediate hosts and play an important role in interspecies transmission.The samples of this study were derived from the epidemic strains of Shanghai area. The ORF2 target gene fragment was amplified by RT-PCR and cloned into pMD-18T 5imple vector to construct cloning vector pMD-ORF2.The segment size of the cloning vector was 666bp which code 222 amino acid.The result showed most highly homolgous with genotype 4 by comparing the sequencs of amino acids with all different genotypes,the better hydrophilicity and antigenicity were also proven.The cloning vector pMD-ORF2 was performed double digestion.The result segment was cloned into pET-30a(+) with corret open reading flame.The reconstruvtive plasmid was transformed into the host bacterial BL₂₁ and then was induced by under 37℃for 1.0h The result of SDS-PAGE demostrated the expressed fusion proteins was 33kD approximately,which consisted with the expected results.The single target protein band was obtained by Ni-purification.The result of immunoblot technique demostrate that the reconstructive protein could be identified by HEV- swine serum of humen or porcine. Supernant of the reconstructive protein washed by urea was performed SDS-PAGE and Western blot;the expressed proteins showed good antigenicity.The China-derived porcine HEV recombinant proteins-ORF2(394-617aa) was applied as coated antigens;HEV-positive serum of porcine was applied as first antibody;, PSA marked by HRP(HRP-SPA) was applied as enzyme-labeled antibody;indirect ELISA was performed to assay the IgG of swine serums.Results of square titration showed the best coated concentration of protein was 2μg per well;the best dilution rate of swine serum was 1:100;the potent concentration Of HRP-SPA was 1:10000.The method of indirect ELSA was then established.87 swine serums were sampled randomly and assayed HEV by the established ELISA kits and total HEV assay kits(Beijing Wantai antigen sandwiched ELISA kit) respectively.70 of 87(80.4%)) samples showed IgG positive by established ELISA kit, 64 of 87(73.5%) samples showed IgG and IgM positive by Wantai ELISA kit.Coincident rate of the two methods was 91.4%(64/70).553 swine serum samples from Shanghai area were assayed by established ELISA kit to detect IgG antibody of HEV;results showed a 64.56%(357/553) positive rate.61 swine feces samples were collected form Shanghai area and measured by RT-PCR;10 of 61 samples showed positive.Results of sequencing showed that all clones belong to genotype 4,which suggested the prevalent genotype of HEV in Shanghai area belong to genotype 4 in major.