The Initial Establishment Of Indirect ELISA For Antibody To TGEV And PRCV

In this study, Recombinant TGEV SBC (deleted in PRCV) protein and N protein (consist in TGEV and PRCV) was expressed for developing an method for antibody to TGEV and PRCV differential diagnosis. The main contents were as follows:1.Expression of recombinant SBC proteins and N proteinsThe expression plasmids pET32a-SBC and pET32a-N was expressed. SDS-PAGE analysis showed that the molecular weight.of the SBC and protein was approximately 38KD and 67KD.Furthermore, The results of western blotting analysis demonstrated that both of SBC and N protein possesed antigenic activity and could be recognized by antiserum against TGEV.2.TGEV and PRCV differential diagnosis ELISA was developedThe indirect ELISA were developped by the recombinant SBC and N antigen respectively. The optimal result of ELISA as follow:coating concentration were 1.4μg/ml(1/160) and 1.1μg/ml(1/320);The optimal dilution of serum examined was 1/320 and 1/640;5% calf serumin was determined the best blocking reagent of the heat-stable antigen,37℃,90min was the best reaction condition; 2% milk and 5% calf serumin was determined to be the best blocking reagent of antigen,37℃90min was the best reaction condition.The optimal dillution of HRP were 1:4800 and 1:9600,37℃30min for ELISA; The cut-off OD450 value of SBC and N to judge result were 0.1417 and 0.1958 respectively. The recombinant SBC and N protein showed no reactivity with antisera to PEDV, CSFV, PRV, PRRSV, PPV, PrV, ECPS and PBS.The recombinant N protein showed reactivity with antisera to PRCV, but the recombinant SBC protein do not reactivity. With different batches of 96 well plate in the same time and with same batch of 96 well plate in different time, six swine sera were examined by indirect SBC base ELISA and N base ELISA to determine the repeat efficiency. The result demonstrated that the coefficient of variability of SBC base ELISA were 10% and 10%, N base ELISA were 10% and 11%,Above result showed that the indirect ELISA method had advantages of good repetition, high sensibility, strong specificity, economic practicality and safety. So that, the indirect ELISA method developed in this study might be widely used as a serological diagnostic method to TGEV and PRCV differential diagnosis.3.Detection of clinical serumThe TGEV and PRCV differential diagnosis ELISA menthod was used to detected TGEV and PRCV antibody of 548 serum which is from 28 swine farms in SiChuan province, at the same time SNT was the contrast test. Coincidence of indirect ELISA and VN were 84.0% and 86.0% respectively. The Clinic pig serums were detected indirect ELISA, The results show that positive of TGEV samples were 253 and the positive of PRCV samples were 7.