Development Of Double Antibody Sandwich ELISA For Detection Of Swine Hepatitis E Virus Antigen And Epidemiological Investigation

Hepatitis E(HE)is a kind of zoonosis,caused by hepatitis E virus,which not only cause huge economic losses but also endanger human health.The study show that HE plays an important role in the epidemic and spread,indicating HEV could be transmitted between human and pigs.HEV is divided into four genotypes,the gene type 1 and type 2 infect human beings,the gene type 3 and type 4 infect swine and other domestic animals.In recent years,Hepatitis E has become global public health problem.There are many reports related to the laboratory diagnostic method for HEV detection such as RT-PCR,ELISA,immunoblotting,serum neutralization test,immunofluorescence microscopy,immune electron microscopy and immune colloidal gold.The most commonly diagnostic methods are RT-PCR and ELISA.RT-PCR has the advantages of simple operation,high sensitivity and strong specificity.However,RT-PCR is expensive and is prone to false positives and only for laboratory diagnosis and not suitable for detection of large-scale samples.Therefore,it is necessary to establish a rapid and simple method for HEV clinical diagnosis.In this study,we amplified partial sequences encoding HEV P1 protein and cloned it to the prokaryotic vector p GEX-4T-1 to construct the expression plasmid p GEX-4T-1-P1.The positive plasmid was transformed into the expression strain BL21.The recombinant protein was expressed under the IPTG induction and purified by urea.The BALB/c mice were immunized with the purified recombinant P1 protein emulsified with Freund’s complete adjuvant to generate either monoclonal or polyclonal antibodies.A double antibody sandwich ELISA method for detection of hepatitis E virus antigen was established,and the reaction conditions were optimized.Detection of the suspicious HEV samples collected from several pig farms in Jilin Province and Liaoning Province revealed a 9%-33% of pigs as HEV-positive.The results not only provide an effective method for rapid detection of HEV antigen but also provide a theoretical basis for the detection of hepatitis E infection.