基于截短MOMP蛋白的绵羊流产衣原体抗体间接ELISA方法的建立  

Establishment of an indirect ELISA method for antibodies against Chlamydia abortus in sheep based on truncated MOMP protein

作　　者：刘欣钊 张芳毓[2] 魏天 王成宇 王思月 刘悦 曲可 朱日宁 鞠安琪 马惠海[2] 张建峰[1] 王永志[2] LIU Xinzhao;ZHANG Fangyu;WEI Tian;WANG Chengyu;WANG Siyue;LIU Yue;QU Ke;ZHU Rining;JYU Anqi;MA Huihai;ZHANG Jianfeng;WANG Yongzhi(College of Life Science,Jilin Agricultural University,Changchun 130118,China;Institute of Animal Science and Veterinary Medicine,Jilin Academy of Agricultural Sciences,Changchun 130118,China)

机构地区：[1]吉林农业大学生命科学学院,长春130118 [2]吉林省农业科学院畜牧兽医研究所,长春130118

出　　处：《黑龙江畜牧兽医》2023年第1期78-84,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：吉林省科技厅重点研发项目(20210202040NC);吉林省畜牧业管理局乡村振兴专项。

摘　　要：为了建立检测绵羊流产衣原体MOMP(main outer membrane protein)蛋白抗体的间接ELISA方法,试验将编码MOMP蛋白的基因序列经密码子优化后进行基因合成,并克隆和原核表达截短蛋白,以该蛋白为抗原建立绵羊流产衣原体抗体的间接ELISA方法,优化该方法的反应条件,同时验证其特异性、敏感性和重复性,最后用该方法对临床待检羊血清进行检测,并与间接血凝试验试剂盒的检测结果进行比较。结果表明:截短蛋白MOMP201~390的表达、纯化效果最好。经探索,优化后的间接ELISA方法最优反应条件:抗原包被浓度为1 mg/L,37℃包被2 h,待检血清最佳稀释度为1∶100,血清和酶标二抗的最佳作用时间分别为45 min、60 min。建立的间接ELISA方法的敏感性较高,特异性和重复性较好,使用该方法从209份临床待检羊血清样本中检出47份阳性血清,与间接血凝试验试剂盒分别检测临床待检羊血清样本86份,总符合率为80.23%。说明基于截短蛋白MOMP201~390建立的绵羊流产衣原体抗体间接ELISA方法可用于绵羊流产衣原体临床血清抗体的检测及流行病学调查。In order to establish an indirect ELISA method for the detection of antibodies to the MOMP(main outer membrane protein) protein of Chlamydia abortus in sheep, in the experiment, the gene sequence encoding the MOMP protein was codon-optimized for gene synthesis, and the truncated protein was cloned and prokaryoticly expressed, which was used as an antigen to establish an indirect ELISA method for antibodies against Chlamydia abortus in sheep. The reaction conditions of the method were optimized, and its specificity, sensitivity and reproducibility were also verified. Finally, the method was used to test the clinical sheep sera to be detected, as well as to compare the results with those of the indirect hemagglutination assay(IHA) kit. The results showed that the truncated protein MOMP201-390were best expressed and purified. After exploration, the optimal reaction conditions of the optimized indirect ELISA method were: the antigen coating concentration being 1 mg/L, the coating at 37 ℃ for 2 h, the optimal dilution of the serum to be tested being 1∶100, and the optimal action time of the serum and the enzyme-labeled secondary antibody being 45 min and 60 min, respectively. The established indirect ELISA method had high sensitivity, good specificity and good reproducibility. The established indirect ELISA method was able to detect 47 positive sera from 209 clinical sheep serum samples;86 clinical sheep serum samples were tested separately with the indirect hemagglutination assay kit, with an overall compliance rate of 80.23%. The results suggested that the indirect ELISA method based on the truncated protein MOMP201-390could be used for the detection of clinical serum antibodies against Chlamydia abortus in sheep and epidemiological investigation.

关 键 词：流产衣原体 原核表达 WESTERN-BLOT 间接ELISA 抗体检测 

分 类 号：S852.67[农业科学—基础兽医学] Q78[农业科学—兽医学]