基于截短绵羊鹦鹉热衣原体r MOMP_(183-390)蛋白的间接ELISA检测方法建立与应用  

Establishment and application of an indirect ELISA detection method based on rMOMP_(183-390) of Chlamydia psittaci

作　　者：曲磊 朱日宁 鞠安琪 魏天 肖丹 王成宇 王思月 张芳毓[1] 王永志[1] QU Lei;ZHU Ri-ning;JU An-qi;WEI Tian;XIAO Dan;WANG Cheng-yu;WANG Si-yue;ZHANG Fang-yu;WANG Yong-zhi(Institute of Animal Science and Veterinary Medicine,Jilin Academy of Agricultural Sciences,Changchun 130118,China)

机构地区：[1]吉林省农业科学院畜牧兽医研究所,吉林长春130118

出　　处：《中国预防兽医学报》2023年第7期710-715,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：吉林省农业科技创新工程项目(CXGC2021TD008);吉林省科技厅重点研发项目(20210202040NC)。

摘　　要：为建立绵羊鹦鹉热衣原体(Cp)间接ELISA抗体检测方法,本实验将编码Cp主要外膜蛋白MOMP的omp A基因序列按大肠杆菌密码子偏好性优化后,合成质粒p ET-28b(+)-omp A,另外设计特异性引物,分别将omp A截短克隆至p ET-28b(+)载体中构建重组表达质粒p ET-28b(+)-omp A_(1-182)和p ET-28b(+)-omp A_(183-390),测序鉴定正确后分别转化至E.coli BL21(DE3)感受态细胞中,经IPTG诱导并纯化后,经western blot鉴定3个重组蛋白,结果显示,重组MOMP_(183-390)蛋白(r MOMP_(183-390))表达效果最好。以r MOMP_(183-390)作为包被抗原,采用方阵滴定法优化各反应条件,初步建立了检测Cp抗体的间接ELISA方法。采用建立的该方法检测羊痘病毒、布鲁氏菌、羊口疮病毒、小反刍兽疫病毒、口蹄疫病毒等羊临床常见病的阳性羊血清,结果显示,除Cp外,该方法与其他阳性羊血清均无交叉反应,特异性强;将Cp阳性血清2倍倍比稀释(1∶50~1∶6400)后采用该方法检测,结果显示,当Cp阳性血清稀释达1∶1600时,仍可检测到抗体,敏感性高;批内、批间重复性试验结果显示,变异系数均小于10%。采用本研究建立的ELISA方法,以及间接血凝(IHA)试验和western blot分别检测随机选取的120份临床羊血清,结果显示该方法与IHA、western blot的总符合率分别为85%和91.67%;进一步采用建立的间接ELISA方法检测381份吉林省5家羊场的绵羊血清样品,结果显示阳性率为22.83%(87/381)。本研究建立的绵羊Cp omp A重组截短蛋白的间接ELISA抗体检测方法敏感性高、特异性强、重复性好,为Cp临床样品检测及流行病学调查提供了可靠的技术手段。To establish an indirect ELISA method for detection of Chlamydia psittaci (Cp) in sheep,the omp A gene encoding the main outer membrane protein MOMP of Cp was optimized for expression in E.coli and synthesized into p ET-28b(+),and two truncated fragments of omp A were amplified and cloned into p ET-28b (+) to construct recombinant expression plasmids p ET-28b(+)-omp A_(1-182)and p ET-28b(+)-omp A_(183-390).The two plasmids were verified by sequencing and transformed into E.coli BL21(DE3),respectively.The three recombinant proteins were purified after induction by IPTG and identified by western blot.The results showed that the expression level of r MOMP_(183-390)protein was the highest.Using r MOMP_(183-390)protein as the coated antigen,an indirect ELISA method for detecting Cp antibody was preliminarily established after optimization the conditions by square titration.The established method was used to detect positive sheep serum for common clinical diseases in sheep,such as sheep pox virus,brucella,sheep mouth sore virus,small ruminant disease virus,and foot and mouth disease virus.The specificity test results showed that only Cp serum showed positivity,and there was no cross reaction with the other tested positive sheep sera,indicating that this method has high specificity.The sensitivity of this method was tested using 2×serially diluted (1∶50-1∶6400) Cp positive serum.The results showed that when the dilution ratio of Cp positive serum reached 1∶1600,the antibody could still be detected,indicating high sensitivity.The results of intra-batch and inter-batch repeatability tests showed that the coefficient of variation was less than 10%.The ELISA method,indirect hemagglutination (IHA) test and western blotting were used to detect 120 randomly selected clinical sheep serum samples.The results showed that the coincidence rates of this method with IHA and western blot test were 85%and 91.67%,respectively.The established ELISA was further used to detect 381 sheep serum samples from 5 sheep farms in Jilin Prov

关 键 词：鹦鹉热衣原体 omp A基因 间接ELISA 待检血清 抗体检测 

分 类 号：S852.67[农业科学—基础兽医学]