| Porcine epidemic diarrhea(PED)is a highly contagious disease which caused by porcine epidemic diarrhea virus(PEDV),vomiting and severe diarrhea dehydration as the main clinical features.Different age and different varieties of pigs are susceptible,within 7 days piglet mortality is highest,therefore,the mortality rate up to 100%.In recent years,the popular area of porcine epidemic losses to the pig industry.Therefore,timely diagnosis and prevention of PED’s of great significance.S protein as the structural protein on the surface of PEDV,it’s encoded by the S gene.S1 have multiple B cell neutralize epitopes,which can induce strong neutralizing antibodies,and thus a set of primers specific to S gene of PEDV(CHYJ130331)strain that was isolated from the lab was designed.Utilizing designed primers,a truncated S1 gene which have neutralized epitopes.The fragment of 1062 bp amplified by PCR was purified and recovered.Afterwards,the truncated S1 gene was cloned into the PMD-18-T vector and sequencing analysis,and then sub cloned the truncated S1 into pGEX-4T-1 vector to construct a recombinant expression plasmid referred as pGEX-4T-S.The plasmid was transformed into E.coli BL21(DE3),after then S1 truncated protein expression induced with IPTG.Optimizing the protein induced expression conditions,the truncated protein expression optimum conditions which were as follows: the IPTG concentration was 0.6mmol/L,and under 37℃ induction 4 hours,the expression S1 truncated protein was purified by GST purification Kit,and highly pure S1 truncated protein was gained.Western-blotting analyses showed that the recombinant S1 truncated protein could specifically react with PEDV positive sera and induce high titers of antibody reactogenicity.Breast colostrum contains a lot of sIgA,therefore,In this study,an AKATA preparative liquid chromatography protein analyzer was used to isolate and purify from the milk to obtain sIgA.After the purified sIgA was identified,two SPF New Zealand rabbits were immunized to prepare rabbit anti-pig sIgA positive serum,and the HRP marker was carried out for the positive serum,and the enzyme resistance of rabbit anti-pig sIgA was obtained.The purified S1 truncated protein was used as coating antigen,and optimizing the reaction conditions,establishing the indirect ELISA conditions for the detection of lactic sIgA.The optimum ELISA conditions as follows: the concentration of coating antigen was 12.5μg/mL,and coated 1h at 37℃,after put it 4℃ overnight;blocking time was 90 min at 37℃ with 1% BSA;the colostrum test was diluted 40-fold with 100mg/mL pepsin and sodium acetate solution,and it was incubated at 37℃ for 60min;1:1000 diluted HRP-antibody conjugate was incubated at 37℃ for 45min;and TMB substrate solution was incubated at 37℃ for 10 min in dark room.The cut off value of the positive results was 0.371.A seria of test results showed that this method was used to detect the PEDV antibody in lactic,and it had good specificity and repeatability.This method was used to detect 120 samples of lactic from Guangdong province,and the positive rate for PEDV was 38.33(46/120),compared with PEDV anti-IgA test kit made by Antigen company,the coincidence rate was 80.0%. |
