Development Of An Indirect ELISA Kit To Detect SHEV

Hepatitis E is an acute,self-limited,icteric viral disease that occurs in many countries. Hepatitis E is a sort of acute viral hepatitis which prevalences by the intestinal.In developing country it is widespread.Human healthy will be severe harmed.The newest research shows that the infection of human HE roots in swine.To prevent the transmission of HEV from swine to human and the prevalence of HE among the crowd,it is essential to press the study on diagnosis method and reagent of HE.RT-PCR was used to amplified swine hepatitis E virus ORF2 fragment.Its size is 681bp,which was sequened by cloning into the pMD18-T vector.This fragment was inserted into pET-32a express vector.The recombinant expression plasmid was constructed and transferred into BL21 and induced by IPTG,the SDS-PAGE result showed that the 45kD recombinant protein was expressed.The Western blot analysis showed that the recombinant protein has a good antigenicity with positive SHEV serum the same time.An indirect ELISA for detection of swine HEV antibodies using effectively fusion expressed recombinant antigen was developed.The optimal coating concentration of antigen was 6μg/mL.The dilution of sera was 1:100.The reaction time of sera was 60min.The reaction time of conjugate was 60min.The standard of judgment was that S/P≥0.34 was positive and S/P≤0.29 was negative.The recombinant antigen had no cross reactions with sera of TGEV,HCV,PEDV,PRV,PCV,PRRSV,and it showed this assay was highly specific.The result suggested the assay was sensitive.In the intro-bateh duplieativity test,the variation coeficient is less than 10%and in the inter-batch duplicativity test,the vartiation coecient is less than 15%.The result showed the assay had good replicativity.The HEV IgG detection of person and animal serum in Qinghai Province showed that the infection rate of HEV were remarkably different among animals(x~2 =38.78,P<0.01).The highest rate(95.96%) appears in swine,which is followed by watchdog whose infection rate is 30%.There is no HEV detected in horse and chicken and the infection rate of the same animal is no remarkable difference(x~2 =5.78,P>0.05).Detection of animal and crowd HEV IgG results showed that indirect ELISA method and double antigen sandwich ELISA method of Wan Tai compony are uniform when it was used to detect the serum of person,horse,chicken,cattle,pig hepatitis E,and there is no remarkble difference when it was used to detect the serum of watchdog and goat.