Prokaryotic Expression On The Fragment S Gene Of Porcine Epidemic Diarrhea Virus, The Development Of Indirect ELISA

Porcine Epidemic Diarrhea Virus(PEDV) is the pathogen of Porcine Epidemic Diarrhea(PED),which is a highly contagious enteric disease in piglets.PED is infectious of different ages and different breeds of porcines,hazard suckle piglets severely,causing vomiting,severe yellowish diarrheas,weight loss, dehydration.Within 1 week after birth,suckle pig died after 2～4 days diarrhea,the lethality up to 90%.High mortality and resulting in severe economical losses to the swine farms.At present,main diagnostic methods to PED are virus neutralization(VN),immunofluorescence(IF),immune electron microscopy(IEM),ELISA and RT-PCR.With the development of molecular biology,the diagnostic methods of indirect ELISA is becoming more important than before for their sensitivity and specialization.The spike(S) glycoprotein is the main structure protein of PEDV and the predominant inducer of neutralization antibody.The sequence analysis suggests that the S gene of PEDV is surprisingly conventional, hence the recombinant of S protein may be useful as an effective diagnositic antigen for detecting PEDV infection.In this work,a pair primer P1 and P2 are devised according to conservative region of S gene to amplify the fragment of S gene for expression.Different vector systems were applied to explore express effect of S gene.The fragment S gene of PEDV was subcloned into SalⅠand NotⅠmultiple cloning sites of the expression vector into pGEX-6p-1 and pET-32a.The recombinant expression plasmids were transformed into the receptive cells of E.coli BL21 and induced by IPTG with a final concentration 1 mmol/L.The expression protein had a high production and could react with polyclonal antibody against PEDV by SDS-PAGE and Western-blot,sharing a good reactionogencity.An indirect enzyme-linked immunosorbent assay(ELISA) based on the recombinant protein was developed.Using the purified recombinant protein as coating antigen,the optimal coating conditions were determined.100 serum samples were detected from JiLin Province by this method,the total masculine ratio was 88%.The result showed that the developed indirect ELISA had the advantages such as high sensibility,strong specificity and the method can be used to detect the porcine serum antibodies to PEDV..It will be found for developing a PEDV ELISA diagnostic kit,and provide scientific method for diagnosing PEDV quickly in wide scope.