鸭圆环病毒无核定位信号衣壳蛋白的原核表达及其在间接ELISA中的应用

Prokaryotic expression of the duck circovirus capsid protein of no-nuclear localization signal and its application in indirect ELISA

鸭圆环病毒(DuCV)是一种具有长期免疫抑制特性的病原体,其Cap蛋白是DuCV的主要结构蛋白和主要免疫保护抗原,构成病毒的核衣壳,对DuCV的血清学诊断具有重要意义。为了开发出适合DuCV的检测方法,本研究在重组Rosetta大肠杆菌细胞内对无核定位信号的DuCV Cap蛋白进行了表达,建立了基于无核定位信号Cap蛋白检测DuCV血清抗体的间接ELISA方法(Cap-ELISA)。结果:Cap-ELISA包被抗原的最佳浓度为280 ng/孔,血清稀释比为1∶1600;检测样本的阴阳临界值为0.339,灵敏度达到1∶12800,并对DuCV抗体检测具有特异性;与常规PCR方法的符合率为92.7%,用Cap-ELISA对山东部分地区的樱桃谷鸭血清进行了间接ELISA检测,总阳性率为88.9%,说明该方法适合在DuCV血清学诊断中应用。

Duck circovirus(DuCV)is a pathogen with long-term immunosuppressive characteristics.The cap protein of DuCV is the main structural protein and the main immune protective antigen,and constitutes the nucleocapsid of the virus.The protein is important for serological diagnosis of DuCV.In order to establish an effective virus detection method which is suitable for DuCV,the Cap protein of DuCV without nuclear localization signal was expressed in the recombinant Rosetta E.coli cells,and an indirect ELISA method(Cap-ELISA)based on the recombinant protein to detect DuCV in serum antibody was established in this study.The results of the established indirect ELISA detection showed that the optimal concentration of the coated antigen and the serum dilution ratio were 280 ng/well and 1∶1600,respectively.The negative and positive cut-off value of the Cap-ELISA for detecting the samples was 0.339,the sensitivity was 1∶12800,and it was specific for detecting the DuCV antibody;which laid a foundation for effectively monitoring the DuCV antibody.At the same time,the compliance rate of the Cap-ELISA method with conventional PCR methods was 92.7%.The indirect ELISA was used to detect the serum of Cherry Valley ducks in some areas of Shandong Province with a total positive rate of 88.9%,which indicated that this indirect ELISA was helpful for serological diagnosis of DuCV.