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

Porcine epidemic diarrhea virus(PEDV), dehydration and high mortality in seronegative neonatal piglets. For the last three decades, PEDV infection has resulted in significant economic losses in the European and Asian pig industries, but in 2013–2014 the disease was also reported in the US, Canada and Mexico. The PED epidemic in the US, from April 2013 to the present, has led to the loss of more than 10% of the US pig population.In order to study the antigenicity of prokaryotic expression of the fragment N gene of porcine epidemic diarrhea virus(PEDV), and lay a foundation of an indirect ELISA method of PEDV. The segment was amplified with RT-PCR. Then we constructed the recombinant with the vector pET-30a(+) named pET-30a-PEDV-N-450, which was induced to express in E.coli BL21(DE3) by 0.5 mmol/L IPTG at 37 ℃. Furthermore, the expressed product was analyzed by SDS-PAGE and Western blotting. Sequencing results proved that recombinant plasmids were correctly constructed. The result showed that the PEDV polyclonal antibody could specifically bind to PEDV the fragment N protein, which indicated that the recombinant fusion protein had excellent immunogenicity. A prokaryotic expression vector for the fragment N protein was successfully constructed. This work lays a foundation for the development of diagnosis of PEDV.In order to develop an indirect ELISA to detect antibody to PEDV. The experiment using the recombinant and purified N protein as antigen expressed in E.coli BL21( DE3), the indirect ELISA was named rnPED-ELISA. The recombinant N protein antigen showed no cross-reaction with the positive sera of other 7 kinds of swine diseases, CV% of intro-batch duplicativity test was less 13%; Sensitivity and specificity of rnPED-ELISA relative to SN was respectively 93.33% and 90.00%; rnPED-ELISA compared with TSZ PEDV antibody diagnosis Kit, 91.67% concordance was obtained. 200 serum samples were detected by this method, the total masculine ratio was 83.5%. Therefore, this rnPED-ELISA based on recombinant N protein antigen has good sensitivity and specificity, can afforded a simple and rapid means for assessment of vaccination in the field and investigation of PED epidemiology.