绵羊梅迪-维斯纳病毒CA-TM融合蛋白的原核表达及间接ELISA检测方法的建立  

Prokaryotic expression of CA-TM fusion protein of sheep Maedivisna virus and establishment of an indirect ELISA method

作　　者：陈思旭 张培 史晓娜[1,2,3] 刘然 刘淑英 CHEN Si-xu;ZHANG Pei;SHI Xiao-na;LIU Ran;LIU Shu-ying(Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Discase,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease,Ministry of Agriculture,Hohhot 010018,China;Inner Mongolia Key Laboratory of Basic Veterinary Science,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业农村部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018 [3]内蒙古自治区基础兽医学重点实验室,内蒙古呼和浩特010018

出　　处：《中国预防兽医学报》2022年第12期1290-1297,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金面上项目(32072819);内蒙古科技重大专项计划项目(2021ZD0010);内蒙古应用研究项目(2019GG240);内蒙古草原英才创新团队项目(20151031)。

摘　　要：为建立一种快速、准确的梅迪-维斯纳病毒(MVV)抗体检测方法,本研究利用DNAStar生物学软件对MVV衣壳蛋白(CA)和MVV跨膜蛋白(TM)的抗原表位分析,利用重叠延伸PCR(SOE PCR)获得CA-TM融合基因,构建表达质粒p Easy-E1-CA-TM,将其转化大肠杆菌BL21后诱导蛋白表达并纯化,经SDS-PAGE检测结果显示,得到重组CA-TM蛋白(rCA-TM),r CA-TM以包涵体形式表达。以获得的rCA-TM作为包被抗原,采用方阵法优化各反应条件,建立了MVV间接ELISA检测方法。利用该方法检测羊临床常见病原阳性血清,结果显示,除MVV阳性血清以外,口蹄疫病毒、小反刍兽疫病毒、绵羊肺炎支原体等绵羊临床常见病原的阳性血清均为阴性,特异性较强,且该方法能识别感染MVV病畜体内产生的CA蛋白和TM蛋白抗体。将MVV阳性血清2倍倍比稀释(1:500~1:32 000)后,分别利用建立的MVV间接ELISA方法及商品化MVV抗体ELISA试剂盒检测,结果显示商品化MVV抗体ELISA试剂盒最低可检测出1:40 00倍稀释的阳性血清,而本研究建立的间接ELISA方法最低可检测出1:16 000倍稀释的阳性血清,敏感性较高。重复性试验结果显示,该方法批内、批间试验的变异系数分别为3.6%~6.9%、2.3%~8.5%,均小于10%,重复性较好。利用本实验建立的MVV间接ELISA方法和商品化MVV抗体ELISA试剂盒对69份绵羊临床样品检测,结果显示,本实验建立的ELISA方法检测出31份阳性样品,38份阴性样品;商品化MVV抗体ELISA检测试剂盒检出37份阳性样品,32份阴性样品。二者的阳性符合率为83.7%,阴性符合率为100%,总符合率为91.3%。本研究首次基于MVV rCA-TM建立了MVV间接ELISA检测方法,且该方法能够分别识别CA蛋白抗体和TM蛋白抗体,不仅减少了因为血清转换而导致血清抗体检测不准确的影响,还扩大了检测范围,为内蒙地区绵羊梅迪-维斯纳病的诊断及净化提供了检测手段。This study aims to establish a rapid and accurate method for detecting Maedi-visna virus(MVV) antibodies. Here,the antigenic epitopes of MVV capsid protein(CA) and MVV transmembrane protein(TM) were analyzed by DNAStar bioinformatics software, and the fusion CA-TM gene was obtained by gene splicing overlap extension PCR(SOE PCR). The expression plasmid pEasy-E1-CA-TM was constructed and transformed into E. coli BL21. After induction, SDS-PAGE results showed the recombinant CA-TM protein(rCA-TM) expressed as an inclusion body. The purified rCA-TM was used as the coating antigen, and an indirect ELISA method for detecting MVV was established by optimizing the reaction conditions. The specificity test showed that this ELISA method only detected MVV and was negative for other pathogens, including foot-and-mouth disease virus, peste des petits ruminants virus, and Mycoplasma pneumonia in sheep. The method could detect CA and TM protein antibodies produced in animals infected with MVV. The sensitivity test showed that the commercial ELISA kit for detecting antibodies to MVV could detect at least 4000 times diluted positive serum. In comparison, this ELISA method could detect at least16000 times diluted positive serum with good sensitivity. The repeatability results showed that the intra-batch and inter-batch coefficients of variation are 3.6%-6.9% and 2.3%-8.5%, respectively, less than 10%. The repeatability is good. Sixty-nine sheep clinical samples were used to evaluate the established indirect ELISA method and the commercial ELISA kit for detecting MVV antibodies. The results showed that the ELISA method established in this experiment detected 31 positive samples and 38 negative samples. In contrast, the commercial ELISA kit detected 37 positive and 32 negative samples. The positive coincidence rate of the two kits is 83.7%, the negative coincidence rate is 100%, and the overall coincidence rate is 91.3%. In summary, this study established an indirect ELISA method for detecting MVV antibodies, which can recognize CA

关 键 词：梅迪-维斯纳病毒 间接ELISA方法 原核表达 融合蛋白 

分 类 号：S852.65[农业科学—基础兽医学]