| Porcine Epidemic Diarrhea Virus（PEDV）is an acute,severe and highly contact infectious disease.The mortality rate of piglets can reach 100%within one week of age,which has caused great harm to the development of pig industry.In this study,the isolated viruses were identified by RT-PCR and indirect immunofluorescence method,and a porcine epidemic diarrhea virus was confirmed to be isolated and the whole gene sequence was analyzed.Meanwhile,specific primers were designed for SID gene,and prokaryotic expression vector was constructed,and soluble recombinant protein was obtained after purification.The purified recombinant protein was used as the coated antigen to establish an indirect ELISA method for antibody detection of PEDV.1.Isolation,identification and complete gene sequence analysis of porcine epidemic diarrhea virus JS-2020-12 strainIn order to understand the variation of PEDV in Jiangsu province,this study conducted virus isolation and identification of diarrhea disease materials from a pig farm in Jiangsu Province:The virus was inoculated with Vero cells for blind transmission;A pair of specific primers were synthesized for PEDV S gene,and it was found that the specific bands could be amplified according to the expectation.A porcine epidemic diarrhea virus was identified by indirect immunofluorescence and named PEDV-JS-2020-12.Referring to the sequence published in Genbank,23 pairs of overlapping primers were designed and synthesized for full-gene amplification and splicing of PEDV-JS-2020-12.Genetic evolution and bioinformatics analysis of whole genes and structural proteins were performed.The genetic evolution results showed that PEDV-JS-2020-12 had the highest homology with HLJ,SC 1402 and SD-M（99.8%）.It was in the same branch with CV777 strain and was closely related.Bioinformatics analysis showed that S and M proteins were stable,S protein was hydrophobic,and S protein had good antigenicity.In this study,genetic evolution and bioinformatics analysis of the whole gene and structural gene of isolated strains were conducted to help understand the genetic variation of PEDV epidemic strains,enrich the PEDV epidemiological data,and provide reference for the prevention and control of PEDV in Jiangsu province.2.Cloning and expression of SID gene of porcine epidemic diarrhea virusIn order to obtain the recombinant protein for establishing indirect ELISA method,a pair of specific primers were designed and synthesized according to the PEDV S gene sequence published in GenBank,and the target gene fragment was amplified by RT-PCR,and cloned into the prokaryotic expression vector pCold-TF.The recombinant plasmid was identified by double digestion and sequencing.The recombinant plasmid identified by sequencing was transformed into BL21.The induced expression conditions were explored.At 16℃,22-24h,the recombinant protein was induced by IPTG with a final concentration of 0.1mM.Sds-page showed that most of the protein was soluble in supernatant.The recombinant protein was purified by nickel column after ultrasonic crushing.The results of Western-blot identification were correct,and it had good reactivity and could react specifically with porcine PEDV positive serum.The successful expression of recombinant protein provides raw material for indirect ELISA method of virus antibody detection.3.Development of indirect ELISA method for detection of antibody against porcine epidemic diarrhea virusIn order to better monitor the PEDV antibody level in pig farms,this study used the His-SID recombinant protein purified in Study 2 as the envelope,and established an indirect ELISA method for PEDV antibody detection.By optimizing the experimental conditions,the optimal antigen coating amount was determined to be 0.125 μg/mL,the optimal dilution of serum to be tested was 1:200,and the optimal incubation time of primary antibody was 60min.The optimal incubation time of secondary antibody was 45min,and the optimal chromogenic time of substrate was 15min.The established indirect ELISA method was used to detect 20 negative serum samples and determine the critical value:negative when OD450nm≤0.259,positive when OD450nm>0.306.The established indirect ELISA method was used for cross-reactivity test:the indirect ELISA method established in this study was negative for PEDV positive serum and porcine infectious gastroenteritis virus and porcine rotavirus positive serum,indicating that the method had good specificity.The coefficients of variation of the indirect ELISA method were all less than 10%,which proved that the method had good repeatability.The established indirect ELISA method was used to detect 53 pig serum samples.Compared with the commercial PEDV ELISA kit of a company,the overall coincidence rate was 96.2%.Therefore,the indirect ELISA method established in this study will add a simple and rapid detection method for the detection of PEDV antibodies in pigs. |
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