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

Eukaryotic Expression of Bovine Coronavirus S1 Protein and Establishment and Application of Indirect ELISA

为建立一种检测牛冠状病毒(BCoV)的抗体间接ELISA检测方法,本研究利用昆虫杆状病毒表达系统表达BCoV的S1蛋白,并作为包被抗原,建立BCoV的S1蛋白抗体间接ELISA检测方法,应用于临床样品检测。结果显示,S1蛋白大小约100 ku,可表达于昆虫High5细胞培养基上清,经Western blot鉴定S1蛋白抗原性良好;经反应条件优化,确定抗原包被浓度为0.125μg·mL^(-1),血清稀释度为1∶200;采用1%明胶,于37℃封闭3 h;血清样品于37℃孵育30 min;酶标二抗的稀释度1∶25000,37℃孵育45 min;37℃避光显色13 min;临界值为0.297;经检验,该方法特异性良好;批间及批内变异系数均小于10%。当临界值为OD_(450 nm)为0.297时,若以IFA为标准,ELISA的敏感性为96.88%(31/32),特异性为87.50%(7/8),与IFA具有很强的一致性(κ=0.844,P<0.01);若以中和试验为标准,ELISA的敏感性为100.00%(31/31),特异性为88.89%(8/9),与血清中和试验具有很强的一致性(κ=0.925,P<0.01)。采用该方法对303份牛血清进行检测,BCoV阳性率为74.91%。由此说明,本研究建立的ELISA方法特异性强,敏感性高,重复性好,可用于BCoV的血清学调查和临床诊断,并可以应用于替代中和试验检测BCoV免疫后抗体水平。

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 0.125μg·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∶25000,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 OD_(450 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 OD_(450 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.