基于截短型Cap蛋白的猪圆环病毒3型间接ELISA检测方法的建立  

Establishment of an indirect ELISA for the detection of porcine circovirus type 3 based on truncated Cap protein

作　　者：郭钱菊 查云峰 陈永杰 钟文霞 高奎鹏 皮墨霖 宁章勇[1,3] GUO Qian-ju;ZHA Yun-feng;CHEN Yong-jie;ZHONG Wen-xia;GAO Kui-peng;PI Mo-lin;NING Zhang-yong(College of Veterinary Medieinet South China Agricultural University,Guangzhou 510642,China;Guangdong Center for Animal Disease Prevention and Control,Guangzhou 510230,China;Maoming Branch of Guangdong Laboratory of Lingncm Modem Agricultural Science and Technology,Maoming 525000,China)

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广东省动物疫病预防控制中心,广东广州510230 [3]岭南现代农业科学与技术广东省实验室茂名分中心,广东茂名525000

出　　处：《中国兽医科学》2021年第8期970-975,共6页Chinese Veterinary Science

基　　金：广东省现代农业产业技术体系生猪创新团队项目(2020KJ126)。

摘　　要：为建立猪圆环病毒3型(PCV3)的血清学诊断方法,本研究构建了去除核定位序列的截短型Cap蛋白重组质粒,并进行了原核表达,纯化的重组蛋白经SDS-PAGE及Western-blot确认后,将其作为包被抗原,建立了PCV3抗体检测的间接ELISA方法。SDS-PAGE结果显示,表达的Cap重组蛋白约为39ku,Western-blot结果表明,重组蛋白具有良好的反应原性。通过方阵滴定法确定了最佳抗原包被浓度为10g/mL,最佳血清稀释度为1∶20,当血清样本D450≥0.301时,可判为PCV3抗体阳性。该方法特异性强,仅与PCV3阳性血清发生特异性反应。该方法敏感性好,对1∶240稀释的血清仍可检出阳性;该方法重复性好,批内和批间试验的变异系数分别为1.24%~3.70%和3.25%~7.28%。用建立的间接ELISA方法对72份临床猪血清进行检测,检出阳性血清22份。结果表明,基于截短型Cap蛋白建立的间接ELISA方法具有良好的特异性、敏感性及重复性,检测方法稳定可靠,为临床PCV3血清学诊断提供了一种稳定可靠的技术手段,也为PCV3流行病学调查提供了有效技术支撑。To establish a serological diagnosis method for porcine circovirus type 3(PCV3),a recombinant plasmid of truncated Cap gene without nuclear localization signal sequence was constructed and expressed in prokaryotic system.After SDS-PAGE and Western-blot analysis confirrmed,the purified recombinant protein was used as the coating antigen to establish an indirect ELISA method for detection of PCV3 antibody.SDS-PAGE showed that the molecular weight of the recombinant protein is 39 ku which has good reactogenicity analysized by western blot.The optimal antigen coating concentration was 10 g/m L confirmed by square titration and the optimal serum dilution was 1∶20.When the serum sample D450 is equal to or greater than 0.301,PCV3 antibody of the sample was identified as positive.The method ishighly specific and only reacts specifically with PCV3 positive serum.The method has good sensitivity,and the 1∶240 diluted serum can still be detected as positive.The coefficient of variation in intra-batch and inter-batch tests were 1.24%—3.70%and 3.25%—7.28%,respectively,showing that the detection method was stable.72 clinical porcine sera were detected using the established indirect ELISA method and 22 of them were positive.The results showed that the established indirect ELISA method based on truncated Cap protein had good specificity,sensitivity,repeatability and reliable which provides a stable method for clinical serological diagnosis and effective technical support for epidemiological investigation and research of PCV3.

关 键 词：猪圆环病毒3型 CAP蛋白 原核表达 间接ELISA 

分 类 号：S852.659.2[农业科学—基础兽医学]