家养野猪源EMCV VP1基因的原核表达及其间接ELISA方法的建立和应用

Development and application of indirect ELISA for detection of antibodies against encephalomyocarditis virus based on prokaryotic expression of VP1 protein

目的以原核表达的脑心肌炎病毒(EMCV)VP1蛋白作为检测抗原,建立间接ELISA方法用来检测EMCV抗体。方法应用原核表达载体pET-28a构建家养野猪源EMCV VP1基因的重组表达质粒,并在感受态细胞BL21中表达,将该重组蛋白纯化后作为包被抗原,建立间接ELISA标准化检测程序,并应用于临床检测。结果经双酶切、PCR和测序鉴定,重组质粒pET-28a-VP1构建成功,转化后诱导表达,经SDS-PAGE电泳分析得到高效表达的VP1蛋白,该重组蛋白可被EMCV阳性血清特异性识别,具有良好的反应原性;通过优化反应条件,确定抗原浓度为1.25ug/mL、待检血清以1∶80倍稀释为最佳抗原包被浓度和待检血清最佳稀释度,5%脱脂乳作为封闭液,待检血清的最佳作用时间为37℃作用60min,酶标抗体稀释度的最佳工作浓度和最佳作用时间分别为1∶8 000和37℃30min,底物最佳显色时间为20min,特异性强,敏感性较高,重复性良好;应用该间接ELISA方法分别检测河南省126个猪场送检的1 618份血清和527份PCV-2抗体阳性血清,发现阳性场检出率为65.87%,血清总阳性率为45.30%,不同规模和不同阶段猪群均存在EMCV感染,中小型猪场的阳性率显著高于规模化猪场,EMCV与猪圆环病毒2型的混合感染率高达75.14%。结论应用原核表达的重组VP1蛋白作为包被抗原建立的间接ELISA方法可用于检测EMCV抗体水平,临床检测结果填补了河南省EMCV血清流行病学监测的空白。

We developed an indirect-ELISA method to detect the antibodies against encephomyocarditis virus（EMCV）using the prokaryotic expression of viral protein 1as antigen.VP1 gene of EMCV was amplified and cloned into the prokaryotic expression vector pet-28 ato obtain the recombinant expressed plasmid,and then were transformed into competent cells BL21 for expression.The recombinant protein purified was used as antigen to develop the indirect-ELISA standard detection procedure for clinical application.Results showed that the recombinant expression plasmid pet-28a-VP1 was identified by double digestion,PCR and sequence analysis.They were transformed into competent cells for expression,and VP1 protein was highly expressed by SDS-PAGE analysis.The recombinant protein was recognized specifically by positive serum of EMCV,the optimal antigen concentration was 1.25ug/mL,the optimal reaction condition of serum samples was 60 min at 37℃,the optimal dilution concentration and time of antibodies labeled with enzyme was 1∶8 000 and 30min at 37℃respectively,and the optimal color time of substrate was 20 min.The indirect-ELISA method was with high specificity and sensitivity,and good repeatability.The survey results showed that the detection rate of positive farm was 65.87%,and total serum positive rate was 45.3%in Henan Province.EMCV infection existed in different size and stages pig farms,compared with large-scale ones,small and medium-sized had higher positive rate,moreover,mixed infection rates with PCV-2as high as 75.14%.In conclusion,the developed indirect ELISA method based on recombinant VP1 protein as coating antigen is useful for the detection of the EMCV antibody in clinical samples,which filled the surveillance gap of serum epidemiological of EMCV in Henan Province.