猪嵴病毒结构蛋白VP0与VP1原核表达及间接ELISA方法的建立

Porcine Kobuvirus Structural Proteins VP0 and VP1 Prokaryotic Expression and Establishment of Indirect ELISA Method

分别建立基于猪嵴病毒(PKV)结构蛋白VP0与VP1的间接ELISA检测方法。对PKV结构蛋白VP0与VP1的基因进行合成并连接至原核表达载体pET-32a后,转入感受态细胞BL21中,用IPTG诱导表达的重组蛋白经Ni柱纯化后,分别以重组蛋白pET-32a-VP0与pET-32a-VP1作为包被抗原,采用棋盘滴定法建立两种间接ELISA检测方法,并进行重复性、敏感性、特异性实验和临床检测。结果显示重组蛋白pET-32a-VP0与pET-32a-VP1抗原的最佳包被浓度分别为2 mg/mL和2.5 mg/mL,反应条件均为37℃、1 h;封闭液最佳条件为5%脱脂乳,37℃、2 h;血清最佳孵育条件为1∶200,37℃、1 h;二抗最佳孵育条件分别为1∶20000和1∶15000,37℃、1 h;最佳显色时间为10 min,两种间接ELISA检测方法临界值分别为0.306和0.277。试验结果表明本研究成功建立了能够有效检测PKV血清特异性抗体的间接ELISA方法,且方法具有重复性好、敏感性高、特异性强、稳定性好等特点,对今后PKV的临床诊断及抗体检测试剂盒的开发奠定了重要基础。

In this study,the indirect-ELISA method was established based on the structural proteins VP0 and VP1 of porcine Kobuvirus(PKV).The genes of structural proteins VP0 and VP1 of PKV were synthesized and ligated into the prokaryotic expression vector pET-32a,and then transferred into competent cells BL21.The expressions of two proteins were induced by IPTG and then were purified by Ni column.The two indirect ELISA assays were established while recombinant protein pET-32a-VP0 and pET-32a-VP as coating antigen through a checkerboard titration method for repeatability,sensitivity,specificity for experiments,and clinical testing.The results showed that the optimal coating conditions of recombinant protein pET-32a-VP0 and pET-32a-VP1 antigen were 2.0 mg/mL and 2.5 mg/mL at 37℃for 1 h,respectively.The optimal conditions for the blocking solution were 5%skimmed milk,37℃,2 h;the optimal incubation conditions for the serum were 1∶200,37℃,1 h;the optimal incubation conditions for the secondary antibody were 1∶20000 and 1∶15000,37℃,1 h;optimal colour development time was 10 min.The critical values of the two indirect-ELISA assays were 0.306 and 0.277 respectively.The results of experiment indicated that the indirect-ELISA methods were successfully established,which are accurate and efficient for detecting serum specific antibody of PKV.The methods are characterized as good reproducibility,sensitivity,specificity and stability.It is very helpful for future clinical diagnosis and the development of antibody detection kits of PKV.