Development Of Antibody Test Kit For Porcine Epidemic Diarrhea Virus (PEDV)

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). It is characterized by diarrhea, vomiting and dehydration, and is responsible for high mortality rate in different ages of piglets. Since 2011, the new strain of PEDV has been widely epidemic in our country, caused a large number of death to piglets, leading to great economic losses to the pig rearing industry in China.S protein is a major structural protein that induces neutralizing antibodies and provides immune protection. In this study, a pair of gene specific primers was designed based on the GeneBank data of S gene of CV777 strain. An expression method was used for amplifying the truncated S1 gene by PCR, the amplicon was cloned into pET-28a expression vector for obtaining the recombinant expression plasmid pET28a-PEDV-S1-600.The recombinant construct pET28a-PEDV-S 1-600 was then transformed into the host strain Rosetta for expressing recombinant protein via IPTG induction. The most optimized reaction condition for protein induction was obtained experimentally. The condition of the most effective protein expression was 0.2 mmol/L IPTG in 25 ℃for 4 hours. The form of the expressed protein was analyzed by SDS-PAGE, and the target protein was expressed in the form of inclusion body with the relative molecular weight about 26 KDa. The recombinant protein was analyzed by western blotting for the specific reaction with PEDV positive serum, which showed good immunogenicity.The recombinant protein was used as an antigen after purifying via Ni column and was used for optimizing the most effective condition of indirect ELISA. The optimum working conditions of ELISA was:0.5μg/mL antigen concentration for coating in 37℃ for 2 h and incubate overnight in 4 ℃; 3% skim milk was used as the blocking buffer for incubation in 37℃ for 30 min; 1st antibody serum was 1:2000 diluted and incubated in 37℃ for 30 min; enzyme labeled 2nd antibody was 1:10000 diluted and reacted in 37℃ for 15 min; the TMB substrate solution was used for staining in 37℃ for 30 min in dark. The critical value of the negative and positive serum was 0.175. The results show that this optimized ELISA method has the characteristics of high specificity, high sensitivity and good reproducibility. The established method was then used to detect 300 serum samples from Yueyang, Hunan province. The positive rate of detection was 77%(231/300), which showed a good accuracy of this method.In the present study, the protein expressed from truncated Rosetta-pET28a-PEDV-S1-600 gene was used as an antigen and showed good antigenicity. An indirect ELISA method was established and used for the development of a porcine epidemic diarrhea virus (PEDV) antibody test kit. This research provided technical support to the PED epidemiological investigation and clinical evaluation of vaccine immune effect, which bears high social significance and will lead to bright market prospects.