羊流产嗜性衣原体重组ompA蛋白的原核表达及间接ELISA抗体检测方法的建立  

Prokaryotic expression of ompA protein of Chlamydophila abortus and establishment of indirect ELISA for the detection of antibodies against the C.abortus

作　　者：付明哲[1] 吴浩阳 许信刚[1] 张琪[1] FU Ming-zhe;WU Hao-yang;XU Xin-gang;ZHANG Qi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China;College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《中国兽医学报》2019年第11期2184-2189,共6页Chinese Journal of Veterinary Science

基　　金：陕西省重点研发计划资助项目(2018ZDXM-NY-039);陕西省农业科技创新转化资助项目(NYKJ-2018-YL02);杨凌示范区农业科技示范推广资助项目(2017-TS-23);西北农林科技大学试验示范站(基地)科技成果推广资助项目(TGZX2017-43)

摘　　要：原核表达羊流产嗜性衣原体(Chlamydophila abortus,C.abortus)ompA蛋白,包被ompA蛋白抗原,筛选反应条件,成功建立了血清抗体ELISA检测方法。PCR扩增ompA蛋白基因并进行原核表达,经纯化、复性和Western blot鉴定后作为包被抗原,通过反应条件筛选、灵敏性、特异性、重复性检验和临床样品检测,创建了羊流产嗜性衣原体血清抗体间接ELISA检测方法。结果表明,建立pET-28α-ompA阳性质粒并表达约为40000的ompA蛋白,Western blot显示纯化复性的ompA蛋白具备抗原性与特异性。反应条件筛选结果为ompA蛋白包被360ng;血清及二抗分别稀释1∶50,1∶5000并37℃作用1h;TMB显色10min。在优化条件下,D450≥0.327为阳性,反之为阴性;特异性、敏感性和重复性结果显示,对布氏杆菌、羊痘、羊口疮、小反刍兽疫和产气荚膜梭菌阳性血清的检测结果均为阴性且50份流产性衣原体阳性血清检出率为98%,批内和批间D450值变异系数分别为1.56%~2.56%和2.35%~3.25%。应用建立的方法对临床175份血清检测,阳性率为52%,商品化衣原体间接血凝检测阳性率为49.1%,阳性符合率为96.5%。本试验建立的间接ELISA检测方法可用于羊流产性衣原体血清抗体水平检测。The ompA protein of Chlamydophila abortus(C.abortus)was prokaryotic expressed in E.coli and ELISA method was sucessfully established by optimizing reaction conditions of the recombinant ompA protein which regarded as coating antigen.The ompA protein gene was amplified by PCR and expressed in E.coli.After the recombinant ompA protein was purified and identified by Western blot and as an antigen,the indirect ELISA was established to test the level of Chlamydiatrachomatis antibody in serum by optimizing reaction condition,specificity,sensitivity and reproducibility.The clinical samples were tested.The results showed that recombinant ompA protein was successfully expressed in the size of 40000.Western blot results showed that ompA protein has specificity and reactivity.The optimized reaction condition results of indirect ELISA showed that coating 360ng ompA protein;serum and HRP labeled mouse anti-goat IgG dilution were 1∶50,1∶5000;TMB reacting time was 10min.Under the optimal conditions,D450≥0.327 was determined to positive results,otherwise negative;specific results showed that the positive serum of brucellosis,goat poxvirus,sheep mouth virus,pestileria virus and Clostridium perfringens displayed negative result and 50positive sera were tested and the sensitivity was 98%.The reproducibility test showed that the coefficients of variation of intra-and inter-batch D450values were between 1.56%to 2.56%and 2.35%to 3.25%.Results of 175clinical serum samples showed,the positive rate of established method in this study was 52%,whereas the positive rate of commercial Chlamydiaindirect hemagglutination test was 49.1%,and positive coincidence rate was 96.5%.In conclusion,indirect ELISA detection method established in this study could be applied to the detection of anti-Chlamydia pneumoniae antibody.

关 键 词：羊流产性衣原体 ompA蛋白 原核表达 间接ELISA 

分 类 号：S852.67[农业科学—基础兽医学]