| Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious intestinal infectious disease in swine. It is one of the worldwide porcine diseases at present. Since October 2010, the epidemic intensity and area of PED have continuously enlarged. Especially, the mortality of suckling piglets can be up to 100%. In many immunized pig herds, the morbidity and mortality still maintained high levels, and PED is often found to be co-infected with other porcine diseases, which increased the difficulty to prevent and control PED and caused significant economic losses in worldwide swine industry. The diagnosis and antibody detection of PEDV play an important role in prevention and control of PED. Although, various methods can be used for diagnosis and antibody monitoring of PEDV, such as ELISA coated by whole virus of PEDV, IFA etc. There are some disadvantage in whole virus coated ELISA, including that lots of antigen are difficult to prepare and there are potential risk for spreading of PEDV particles. IFA is not suitable for clinical testing since it requires expensive equipment. Indirect ELISA methods based on coating antigen of expressed protein are becoming the new direction for PEDV antibody detection.To furthermore characterize the Jiangsu prevalent strains, diarrheic samples were intermittently collected from one pig farm in Jiangsu province and subjected to molecular epidemiological investigation of PEDV, an indirect ELISA antibody detection method was developed to evaluate infection or immune antibody by using purified recombinant S protein of PEDV. This study will provide theoretical guidance and detection means for choice of PED vaccine and antibody monitoring.1. Molecular epidemio logical investigation of PEDV from a pig farm in Jiangsu ProvinceIn order to study the current prevalence of PEDV in a pig farm in Jiangsu province, 106 suspected porcine epidemic diarrhea samples which were collected between Mar,2015 and Mar,2016 were detected for PEDV using RT-PCR method. The positive rate was 30.19% (32/106), and PEDV could be detected from the pigs at all ages. Specific primers were designed for the amplification of M gene, N gene and ORF3 gene from two positive samples, and PCR products were subjected to sequence and analyze. Results showed that the PEDV reference strains were divided into two groups, and PEDV strains from this pig farm were belonged to group Ⅰ. The PEDV strains were field strains based on the phylogenetic analysis of the ORF3 genes, which were homologous to prevalent strains in our country.2. Prokaryotic expression of PEDV partial S geneSince PEDV S protein plays a pivotal role in immune protection, the gene was selected for expression in prokaryotic expression system. Part of S gene, which encodes a protein for induction of neutralization antibody, was amplified for a band of 711 bp, and cloned into pET32a to form recombinant prokaryotic plasmid (pET-Sp). The recombinant plasmid was transferred to the competent cell BL21. Under the condition of 28 ℃,200 rpm, and 0.2 mM IPTG induction, soluble recombinant Sp protein can be obtained. Western-Blot result showed that recombinant protein could be recognized by PEDV positive serum, suggesting that the recombinant protein has good antigenicity.3. Establishment of an indirect ELISA method for detection of PEDV antibodyFor survey of the PEDV antibody levels in pig herds, an indirect ELISA method against PEDV antibody was established with purified Sp protein as the coating antigen. The optimal antigen concentration and serum samples dilution rate were 0.5 μg/ml and 1:200, respectively. The coated time was overnight at 4℃. The sealing buffer was 10% FBS and the incubation time was 2 h at 37℃. The serum samples were incubated for 1 h at 37℃. The dilution of the conjugate was set as 1:8000 and the reaction time was 1 h at 37℃. The reaction was stopped after 10 min when adding TMB. The serum was defined as positive when the OD450 value of sample was equal or more than 0.273, as negative when the OD450 value was less than 0.273. When PRRSV, CSFV, PRV, PCV, and FMDV antibody from commercial ELISA kits were detected by this indirect ELISA method, all results were negative, indicating that the method has good specificity. Serum test results showed that it had good inter- and intra-batch reproducibility. 47 sera were tested by established indirect ELISA, the positive rate was 72.34% (34/47). Compared with the result detected by an imported indirect ELISA kit, the coincidence rate was 95.74%. These data indicated that the established indirect ELISA method could be used for PEDV epidemiological surveys and diagnosis in the future. |
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