猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用  

Establishment and application of an indirect ELISA based on protein by expression and cloning of CbpB gene of Erysipelothrix rhusiopathiae

作　　者：刘君雯 王迪 朱艳艳 邢刚 占松鹤[3] 刘晓露 魏建忠[1] 孙裴[1] 刘雪兰[1] 李郁[1] LIU Junwen;WANG Di;ZHU Yanyan;XING Gang;ZHAN Songhe;LIU Xiaolu;WEI Jianzhong;SUN Pei;LIU Xuelan;LI Yu(College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;Maanshan Shiji Animal Health Management Co., Ltd., Maanshan 238251, China;Anhui Animal Disease Prevention and Control Center, Hefei 230091, China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]马鞍山史记动物健康管理有限公司,安徽马鞍山238251 [3]安徽省动物疫病预防与控制中心,安徽合肥230091

出　　处：《浙江农业学报》2021年第5期816-824,共9页Acta Agriculturae Zhejiangensis

基　　金：国家星火计划重点项目(2014GA710002);安徽省重点研究与开发计划(面上攻关)项目(201904a06020013);安徽省长三角联合科技攻关项目(1101c0603065);安徽省生猪产业体系基金(皖农科〔2016〕84号)。

摘　　要：利用表达纯化的猪丹毒丝菌(ER)胆碱结合蛋白B(CbpB)建立一种检测ER抗体的间接ELISA方法。克隆扩增ER的CbpB基因并与原核表达载体pGEx-6P-1连接,经PCR、双酶切及测序鉴定,将阳性重组质粒转入E.coil BL21,IPTG诱导表达,SDS-PAGE和Western-blot鉴定产物。以纯化重组CbpB蛋白作为包被抗原,通过一系列反应条件优化,建立检测ER抗体的间接ELISA方法(CbpB-ELISA),并用于临床猪血清样品检测。建立该方法的最适条件:包被抗原浓度为1.0μg·mL^(-1),4℃过夜;5%脱脂奶粉于37℃封闭2 h;血清稀释度为1:100,37℃孵育60 min;酶标二抗稀释度为1∶1000,37℃作用45 min;显色时间为37℃作用10 min。该方法可特异性地检测ER抗体,与其他猪病原阳性血清均无交叉反应性;阳性血清稀释至1:12800时仍可检出;批内及批间变异系数均小于10.00%;与全菌体-ELISA、SpaA-ELISA、商品化ELISA试剂盒及Western-blot比较,该方法总符合率分别为91.67%、93.33%、86.67%和90.00%,其中阳性符合率分别为92.45%、94.55%、88.89%、90.91%,阴性符合率分别为85.71%、80.00%、66.67%、80.00%。对进行和未进行ER免疫的猪血清样品检测,免疫抗体阳性率为86.67%,感染抗体阳性率为30.50%。该方法具有良好的特异性、敏感性和重复性以及临床应用的可靠性,为ER的免疫监测和流行病学调查提供了技术手段。Using the expression of purified choline binding protein B(CbpB)of Erysipelothrix rhusiopathiae(ER)to establish an indirect ELISA method for detecting ER antibodies,the CbpB gene of ER was cloned and linked to pGEx-6P-1 prokaryotic expression vector.The positive recombinant plasmid was identified by PCR,double enzyme digestion and sequencing.The positive recombinant plasmid was transferred into E.coil BL21,induced by IPTG,and the product was identified by SDS-PAGE and Western-blot.Using purified recombinant CbpB protein as coating antigen,an indirect ELISA method(CbpB-ELISA)for detection of ER antibody was established through a series of optimization of reaction conditions,and was applied to the detection of clinical pig serum samples.The optimal conditions for this method were as follows:the concentration of encapsulated antigen was 1.0μg·mL^(-1) at 4℃overnight;5%skim milk powder was sealed at 37℃for 2 hours.The serum dilution was 1∶100 which was incubated at 37℃for 60 min.The dilution of the secondary antibody was 1∶1000,and it was treated at 37℃for 45 min.The color development time was 10 min at 37℃.This method could specifically detect ER antibody,and had no cross reactivity with other pig pathogen positive serum.The method was sensitive to detect antibody even when the positive serum was diluted to 1∶12800.Both the intra-and inter-coefficients of variation were less than 10.00%.Compared with the methods of whole cell-ELISA,SpaA-ELISA,commercial ELISA kit and Western-blot,the total coincidence rate of these methods were 91.67%,93.33%,86.67%and 90.00%,respectively.The positive coincidence rates were 92.45%,94.55%,88.89%and 90.91%,respectively.The negative coincidence rates were 85.71%,80.00%,66.67%and 80.00%,respectively.The detection of pig serum samples with and without ER immunization showed that the immune antibody positive rate was 86.67%,and the infection antibody positive rate was 30.50%.The indirect ELISA detection method of ER antibody based on recombinant CbpB protein had good speci

关 键 词：猪丹毒丝菌 胆碱结合蛋白B 间接ELISA 抗体检测 

分 类 号：S858.28[农业科学—临床兽医学]