| Porcine epidemic diarrhea is an acute,highly contact intestinal infectious disease caused by a-coronavirus belonging to the subfamily of coronavirus.The virus can cause disease in all stages of pigs,but the most serious harm is that it often causes dehydration,vomiting and watery diarrhea in piglets within 10 days of age.The disease was first reported in the UK in 1971,followed by outbreaks in several countries.Due to the use of inactivated or attenuated vaccine cv777,the control of PEDV is better.However,this phenomenon changed in the winter of 2010.The epidemic diarrhea caused by the variation strain of PEDV broke out and prevailed in most of the pig raising provinces in China,resulting in great loss of pig raising industry in China.It was found that the gene variation of PEDV was the main cause of immune failure and high mortality of piglets.However,due to the lack of effective vaccine against the current strain,the disease is still the most serious disease endangering the pig industry.Therefore,the prevention and control of PED is of great significance,and detection is the main means of prevention and control,but at present,there is a lack of clinical detection methods for the currently popular mutation of PEDV.In this study,a pair of specific primers were designed to amplify the N gene of PEDV according to the sequence of PEDVN(AF353511)gene registered in GenBank.The target fragment was cloned into pQE-30 expression vector for prokaryotic expression and purification.The purified recombinant N protein(rN)was used as coating antigen.The ELISA reaction conditions were optimized by matrix method and PEDV was established.The indirect ELISA method was used to test the sensitivity,specificity and repeatability.500 clinical serum samples from different pig farms were tested and compared with imported commercial kits.The results showed that the length of 1221 BP N gene was amplified successfully,and the rN expressed was about 50.4 ku.Western blot results showed that the reaction was good.The optimum concentration of rN was 2 g/mL,the best dilution of serum was 1:100,the time of action was 1 h,the optimum dilution of Sheep anti pig HRP-IgG was 1:5000,the time of action was 30 min,and the best time of TMB was 10 min.This method can specifically detect PEDV antibody,but there is no cross-reaction with positive serum of PRV,PRRSV,CSFV,PCV,TGEV and other pathogens.The highest dilution multiple of the same serum detected by this method is basically consistent with the neutralization test and has good sensitivity.The coefficient of variation of repeatability test within and between batches is less than 10%,which is good.Repeatability and stability.The detection rate of 500 clinical pig serum was 99.6%compared with imported commercial kit.This study provides a technical means for the serological detection of PEDV,and lays a foundation for the next screening of monoclonal antibodies(McAbs).In this study,mice were immunized with routine techniques.The serum of mice was diluted by indirect ELISA.The spleens of mice with detection titer greater than 1:80000 were fully ground and then fused with pre-cultured SP/20 cells under the action of PEG1500.The fused cells were then laid on 96-well plate.The cell condition was observed day by day.The supernatant of each pore was screened and identified by the indirect ELISA method established.At the same time,the monoclonal antibody subtypes were identified.After five subclones of the positive pore,Two hybridoma cell lines with IgG subtype and stable secretion of mAb against PEDV N protein were screened.Hybridoma cells were cultured and injected into the abdominal cavity of mice,Ascites were collected and purified.SDS-PAGE,stability and specificity were detected.The results showed that there was only one strain(6c6)in the two strains after ascites purification,the purity was over 95%,and it could react with PEDV antigen specifically,but it did not cross react with PRV,PRRSV,CSFV,PCV,TGEV,Escherichia coli and other antigens,the specificity was good;after several successive generations,its titer was still 1:102400,and its stability was good.The results showed that only one of the three strains(6C6)could react specifically with PEDV antigen,but not with PRV,PRRSV,CSFV,PCV,TGEV and E.coli.After several successive passages,the titer of the three strains was still up to 1.102400,good stability.It laid a foundation for the further establishment of an antibody blocking ELISA based on PEDV N protein and the rapid detection of test strip by colloidal gold applied in clinic.In this study,the inactivated vaccine prepared by the virus isolated and identified in our laboratory was used for the study of immune efficacy test.10 piglets of 5-day-old were divided into 2 groups,5 in each group.The experimental group was given virus solution orally and the control group was given DMEM of the same dose orally.The incidence of piglets was observed within 3 days after the attack.The results showed that all the patients in the experimental group met the requirements of diarrhea,and none in the control group.The pregnant sows were immunized with the inactivated vaccine three times at a specific time point before delivery.The control group was injected with the same dose of DMEM at the same time.The experimental group and the control group were selected to study the immune protection of the piglets.The results showed that the protection rate of sows in the experimental group was 90%,and that of piglets in the control group was 100%,and the mortality rate was 30%.The experimental group was better than the control group in the aspects of mental state,weight gain rate,etiology detection and autopsy symptoms.This shows that the inactivated vaccine prepared by our laboratory can provide effective protection for piglets,and it can be used for the research and development of the variant vaccine of swine epidemic diarrhea. |
