Purification Of GP5 Recombinant Protein And Establishment And Application Of An Indirect ELISA For Detecting The Antibody To PRRSV

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by PRRS virus(PRRSV)in swine.The main clinical manifestations are respiratory diseases of weaned piglets and reproductive disorders of sows in late pregnancy.It causes huge economic losses to the pig industry all over the world every year.The viral GP5 protein is the most important immune protection protein of PRRSV,which induces the body to produce virus neutralizing antibodies.At present,the main measure for the prevention and control of PRRSV is vaccine immunization.The anti-PRRSV N protein ELISA antibody detection kit mainly used in the market cannot effectively determine the immune protection level of pigs after vaccination.And it also cannot be used to detect the immune antibody against GP5 subunit vaccine.In order to construct an ELISA antibody detection method for this protein,our laboratory has modified the GP5 gene to construct an E.coli recombinant expression plasmid(p ET-28a-GP5/3m),which can effectively express the GP5 recombinant protein and has high antigenicity.In order to further construct the ELISA antibody detection kit,this study used p ET-28a-GP5/3m prokaryotic expression and prepared GP5 recombinant protein,and then optimized the ELISA reaction conditions.By using ROC curve,the critical value of negative and positive in ELISA method was determined.This method has high specificity,sensitivity and reproducibility,and can be used for PRRSV antibody detection.The specific contents are as follows:In this study,p ET-28a-GP5/3m recombinant expression plasmid was used to obtain PPRSV GP5 recombinant protein through the expression of target gene and purification of inclusion body.Using the recombinant protein as coating antigen,an indirect ELISA assay was developed by optimizing reaction conditions.Firstly,the coating concentration of GP5 protein at 1μg/m L was best and the optimum dilution of serum was 1:100 by square matrix titration test.The effect of antigen coating at 37℃ for 1h and then overnight at 4℃ was the best.When 5% skim milk is used as a sealant and sealed at 37℃ for 2h,the effect is the best.The best effect of enzyme-labeled SPA was 15000 times diluted and then at 37℃ for 45 min.Lastly,the effect of substrate at 37 ℃ for 8 min was the best.371 PRRSV negative and positive serum samples determined by IFA were used for antibody detection by the GP5 ELISA method.ROC curve results show that AUC of the ELISA detection assay was 0.842,indicating a higher diagnostic value.The cut-off value of ELISA was as follows: S/P≥0.4 was considered to be positive and S/P < 0.4 was considered to be negative.Sensitivity experiments show that this method can detect positive serum diluted 160 times,and its diagnostic sensitivity is 87.1%.It has no cross reaction with the antibodies against porcine circovirus type 2,classical swine fever virus,African swine fever virus,foot-and-mouth disease virus and pseudorabies virus.The intra-batch and inter-batch repeatability coefficient of variation was 0.98%～7.54%,less than 10%.So the stability of method is good.Compared with the commercial PRRSV ELISA kit,this method has a coincidence rate of 88.4%.This method was used to detect against PRRSV in 962 immunized clinical pig serum samples from East China.And the results were statistically analyzed in different years,seasons and provinces.The results indicated that in the past three years,the year with more serious infection was 2019,with an annual positive rate of 53.00%.And the positive detection rate of clinical pig serum antibodies was 49.38%.The epidemic season of PRRSV infection was in summer and autumn.And the infection in Anhui,Fujian and Jiangsu provinces were more serious in East China.This method can not only detect the anti-GP5 antibody,but also indirectly reflect the protection level of pigs against PRRSV infection,which being helpful to control this disease in the future.