绵羊家畜衣原体血清抗体间接ELISA检测方法的建立  

Establishment of Indirect ELISA for Detection of Serum Antibody Against Chlamydophilia pecorum in Sheep

作　　者：曲可 刘欣钊 马惠海[1] 曲磊 王成宇 马俊 吕礼良[1] 朱日宁 张芳毓[1] 王永志[1] QU Ke;LIU Xin-zhao;MA Hui-hai;QU Lei;WANG Cheng-yu;MA Jun;LYU Li-liang;ZHU Ri-ning;ZHANG Fang-yu;WANG Yong-zhi(Institute of Animal Husbandry and Veterinary Medicine,Jilin Academy of Agricultural Sciences,Changchun 130118,China;College of Veterinary Medicine,Jilin Agricultural University,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 [2]吉林农业大学动物医学院,吉林长春130118

出　　处：《中国兽医杂志》2023年第7期62-69,共8页Chinese Journal of 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资金项目。

摘　　要：为了建立检测绵羊家畜衣原体血清抗体的间接ELISA方法,本试验构建pET-28b-ompA重组表达质粒,经诱导表达和纯化后,对重组主要外膜蛋白(MOMP)进行Western blot鉴定;以MOMP为包被抗原,通过反应条件筛选建立绵羊家畜衣原体血清抗体间接ELISA检测方法,分析其敏感性、特异性、重复性和符合性,并进行临床样品检测。结果显示,MOMP以包涵体形式表达,经Western blot鉴定重组蛋白具有良好的反应原性。优化反应条件为:抗原包被浓度为1 mg/L,37℃静置孵育1 h;血清抗体稀释倍数为1∶100,37℃反应1 h;酶标二抗稀释倍数为1∶5000,37℃孵育1 h;底物显色条件为37℃避光5 min。利用52份绵羊血清确定ELISA方法的阳性判定临界值为P/N=2.156。该方法的敏感性、特异性和重复性较高。用该方法与间接血凝试验对86份血清进行检测,两者符合率为73.2%。用该方法对临床采集的205份绵羊血清样品进行检测,阳性率为34.1%。结果表明,本试验建立的绵羊家畜衣原体血清抗体间接ELISA检测方法具有较高的特异性和敏感性,可应用于临床检测。To establish an indirect ELISA method for detecting serum antibodies against Chlamydophilia pecorum(C.pecorum)in sheep,the pET-28b-ompA recombinant expression plasmid was constructed in this study.After expression and purification,the recombinant major outer membrane protein(MOMP)was identified by Western blot.Using MOMP as the coating antigen,the indirect ELISA method for detecting serum antibodies against C.pecorum in sheep was established by optimizing reaction conditions.The sensitivity,specificity,repeatability,and conformity of the method were analyzed,and clinical samples were tested.The results showed that MOMP was expressed in inclusion bodies and exhibited good antigenicity as confirmed by Western blot.The optimized reaction conditions were as follows:antigen coating concentration of 1mg/L,incubation at 37℃for 1 h;serum antibody dilution ratio of 1∶100,reaction at 37℃for 1 h;enzymic conjugate secondary antibody dilution ratio of 1∶5000,incubation at 37℃for 1 h;substrate color development at 37℃in the dark for 5 min.The critical value for the ELISA method was determined as P/N=2.156 using 52 sheep serum samples.The method exhibited high sensitivity,specificity,and repeatability.86 serum samples were detected by established ELISA and indirect hemagglutination assay(IHA),and the coincidence rate between the two methods was 73.2%.The positive rate of 205 collected clinic sheep serum samples was 34.1%detected by the ELISA.The results indicate that the established indirect ELISA method have high specificity and sensitivity for the detection of serum antibodies against C.pecorum in sheep,and can be applied in clinical detection.

关 键 词：绵羊家畜衣原体 主要外膜蛋白(MOMP) 原核表达 间接ELISA 

分 类 号：S852.67[农业科学—基础兽医学]