牛冠状病毒S1蛋白的真核表达及间接ELISA方法的建立与应用OA北大核心CSTPCD

In order to establish a specific indirect ELISA method that can replace the detection of neutralizing antibodies against bovine coronavirus(BCoV),the recombinant S1 protein of BCoV was prepared by expression of insect baculovirus expression system.The purified recombinant S1 protein was used as coating antigen to establish an indirect ELISA method for detection of BCoV S1 protein antibody.The results showed that the recombinant S1 protein was about 100 ku and could be expressed in the supernatant of insect High5 cell culture medium,and the antigenicity of S1 protein was identified by Western blot.After the reaction conditions were optimized,the antigen coating concentration was 0.125 μg·mL-1,and the dilution of serum was 1∶200.The type of sealant is 1％gelatin and the sealing time is 3 h at 37 ℃.The action time of serum is incubating 30 min at 37 ℃.The dilution of enzyme-labeled second antibody was 1∶25 000,and the time was incubated at 37 ℃ for 45 min.The time for substrate coloration was 13 min at 37 ℃.The critical value was 0.297.This method had a good specificity,the maximum coefficients of variation of inter-assay and intra-assay repeatability tests were less than 10％.Taking IFA as the standard,the results showed that when the critical value OD450 nm was 0.297,the sensitivity of IFA was 96.88％(31/32)and the specificity was 87.50％(7/8),which was highly consistent with the serum neutralization test(κ=0.844,P＜0.01).Taking the neutralization test as the standard,the results showed that when the critical value OD450 nm was 0.297,the sensitivity of ELISA was 100.00％(31/31)and the specificity was 88.89％(8/9),which was highly consistent with the serum neutralization test(κ=0.925,P＜0.01).Three hundred and three samples of bovine serum were detected by this method,and the positive rate was 74.91％.Therefore,the established ELISA method has strong specificity,high sensitivity and good repeatability,which can be used as an alternative to neutralization test to detect BCoV antibody levels after immunization.