狐源兔脑炎微孢子虫极管蛋白2(PTP2)的原核表达及间接ELISA方法的建立

Prokaryotic expression of fox-derived Encephalitozoon cuniculi polar tubulin protein 2(PTP2)and establishment of an indirect ELISA method

为了建立一种特异性好、敏感性高、重复性好的可检测蓝狐兔脑炎微孢子虫病的间接ELISA方法,试验首先从疑似感染兔脑炎微孢子虫(Encephalitozoon.cuniculi)的蓝狐脑组织DNA中扩增PTP2基因,并将其与原核表达载体pET-32a(+)连接,构建重组质粒pET32a-PTP2,转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,将菌体超声后对表达的重组蛋白进行可溶性分析,并纯化目的蛋白;然后以重组蛋白作为包被抗原,通过优化反应条件和临界值的确定建立蓝狐兔脑炎微孢子虫的间接ELISA方法,并进行特异性、敏感性和重复性试验;最后采用建立的间接ELISA方法对28份经常规PCR方法验证已经感染兔脑炎微孢子虫病的蓝狐血清样品进行检测,并计算符合率。结果表明:表达的重组蛋白的分子质量为45.8 ku,且表达产物以可溶性蛋白和包涵体两种形式存在,经纯化杂带明显减少,纯化后的重组蛋白质量浓度为1.55 mg/mL。间接ELISA方法的最佳包被抗原质量浓度为8μg/ml,最佳一抗稀释度为1∶100,最佳封闭液为5%脱脂乳,最佳封闭条件为37℃、1 h,最佳一抗孵育时间为120 min,最佳二抗稀释度为1∶5 000,最佳二抗孵育时间为60 min,最佳显色时间为20 min。阴、阳性血清OD_(450)临界值分别为0.268和0.302。建立的间接ELISA方法检测蓝狐犬瘟热病毒、蓝狐伪狂犬病毒、蓝狐细小病毒阳性血清均为阴性,可检测到稀释度为1∶3 200的蓝狐兔脑炎微孢子虫阳性血清,批内重复与批间重复变异系数均小于10%。用建立的间接ELISA方法检测28份蓝狐血清样品的阳性样品数为23份,与常规PCR方法的符合率为82.14%。说明试验成功建立了一种可检测蓝狐兔脑炎微孢子虫病的间接ELISA方法,该方法特异性好、敏感性高、重复性好,适合在基层推广。

In order to establish an indirect ELISA method with good specificity,high sensitivity and good reproducibility for the detection of blue fox Encephalitozoon cuniculi disease,in the test,firstly,the PTP2 gene was amplified from brain tissue DNA of the blue fox suspected to be infected with Encephalitozoon cuniculi,and the recombinant plasmid pET32a-PTP2 was constructed by ligating it with the prokaryotic expression vector pET-32a(+),which was transformed into Escherichia coli BL21(DE3)competent cells for induction expression.The expressed recombinant protein was analyzed for solubility analysis after ultrasonication,and the target protein was purified.Then,using the recombinant protein as the coating antigen,an indirect ELISA method for blue fox Encephalitozoon cuniculi was established by optimizing the reaction conditions and determining the negative and positive serum cut-off values,and specificity,sensitivity and repeatability tests were carried out.Finally,the established indirect ELISA method was used to detect 28 serum samples of blue foxes infected with rabbit encephalitis microsporidiosis confirmed by conventional PCR method,and the compliance rate between this method and PCR method was calculated.The results showed that the expressed recombinant protein had a molecular mass of 45.8 ku and the expression product existed in both soluble protein and inclusion body forms;the heterobands were significantly reduced after purification,and the purified recombinant protein amount concentration was 1.55 mg/mL.The optimal mass concentration of the encapsulated antigen was 8μg/ml;the optimal dilution of primary antibody was 1∶100;the optimal sealing solution was 5%skimmed milk;the optimal sealing conditions were 37℃for 1 h;the optimal primary antibody incubation time was 120 min;the optimal dilution of enzyme-labeled secondary antibody was 1∶5000;the optimal enzyme-labeled secondary antibody incubation time was 60 min;and the optimal color development time was 20 min.The critical values of OD_(450) for negative and positive sera were 0.268 and 0.302,respectively.The established indirect ELISA method was negative for detection and blue fox canine distemper virus,blue fox pseudorabies virus,and blue fox parvovirus standard-positive sera;blue fox Encephalitozoon cuniculi-positive standard sera at a dilution of 1∶3200 could be detected;and the coefficients of variation for intra-and inter-assay duplicates were less than 10%.The established indirect ELISA method was used to detect 23 positive samples from 28 blue fox serum samples,and the coincidence rate with the conventional PCR method was 82.14%.The results indicated that an indirect ELISA method had been successfully established for the detection of blue fox Encephalitozoon cuniculi,which had good specificity,high sensitivity and reproducibility,and was suitable for popularization at the grass-roots level.