| 重组牛分枝杆菌CFP-10蛋白间接ELISA检测方法的建立 |
| |
| 引用本文： | 谢志勤,;谢芝勋,;刘加波,;庞耀珊,;邓显文,;谢丽基,;范晴,;罗思思,;牟群,;莫文胜.重组牛分枝杆菌CFP-10蛋白间接ELISA检测方法的建立[J].广西农业科学,2014(7):1291-1295. |
| |
| 作者姓名： | 谢志勤 ;谢芝勋 ;刘加波 ;庞耀珊 ;邓显文 ;谢丽基 ;范晴 ;罗思思 ;牟群 ;莫文胜 |
| |
| 作者单位： | [1]广西兽医研究所/广西畜禽疫苗新技术重点实验室,南宁530001; [2]桂林市动物疫病预防诊断中心,广西桂林541001; [3]永福县动物疫病预防诊断中心,广西桂林541800 |
| |
| 基金项目： | 新世纪百千万人才工程国家级人选专项基金项目（945200603）;公益性行业（农业）科研专项项目（201103008）;广西特聘专家专项项目（2011B020）;广西重大科研专项项目（桂科重1222003-2） |
| |
| 摘 要： | 目的]建立检测牛分枝杆菌的间接ELISA方法,快速区分致病性与非致病性牛分枝杆菌,为净化牛结核病提供技术支撑.方法]以体外扩增表达并纯化的重组牛分枝杆菌CFP-10蛋白为包被抗原,建立检测牛分枝杆菌的间接ELISA,并应用方阵法优化间接ELISA的检测条件.结果]重组CFP-10蛋白间接ELISA的最佳检测条件为：以8μg/mL重组CFP-10蛋白包为被抗原,37℃下包被1h,再以5％脱脂奶封闭1h,血清（一抗）经1∶200倍稀释后作用1h,然后以1：500倍稀释的辣根过氧化物酶标记羊抗兔IgG（二抗）作用1h.该间接ELISA的阴阳临界值为0.281,仅与牛分枝杆菌呈阳性反应,与牛布鲁氏杆菌、牛口蹄疫病毒无交叉反应;其敏感性能检测1：320倍稀释的血清,批内与批间的变异系数均小于5.0％.结论]建立的重组牛分枝杆菌CFP-10蛋白间接ELISA具有特异、敏感等优点,临床上可用于致病性牛分枝杆菌的检测.
|
| 关 键 词： | 牛分枝杆菌 CFP-10蛋白 间接ELISA 阴阳临界值 |
Development of an indirect ELISA using the recombinant culture filter protein 10 from Mycobacterium bovis |
| |
| Institution: | XIE Zhi-qin, XIE Zhi-xun , LIU Jia-bo, PANG Yao-shan, DENG Xian-wen, XIE Li-ji, FAN Qing, LUO Si-si, MOU Qun, MO Wen-sheng (1Guangxi Veterinary Research Institute, Guangxi Key Laboratory of Animal Vaccine and New Technology, Nanning 530001 China; 2Gnilin Animal Disease Diagnostic Center, Guilin, Guangxi 541001, China; 3yongfu Animal Disease Diagnostic Center,Yongfu, Guangxi 541800, China) |
| |
| Abstract: | Objective] An indirect enzyme-linked immunosorbent assay （ELISA） with the recombinant culture filter protein 10 （CFP-10） from Mycobacterium bovis（M.bovis） was developed to detect M.bovis as a rapid and sensitive method.Method]The CFP-10 gene from M.bovis was amplified and transferred to competent cell.The expressed CFP-10 gene protein from recombinant was purified and used as the coating antigen in this ELISA.The best conditions for this ELISA were tested with CFP-10 recombinant protein as an antigen.Result]The best coating concentration of the CFP-10 protein was 8 μg/mL at 37 ℃ for 1 h and it was enclosed with 5％ skimmed milk for 1 h.The best antibody （serum） was diluted to 200 and the goat-rabbit antibody IgG labeled with enzyme HRP was diluted to 500 before incubated at 37℃ for 1 h.The critical value was 0.281.It did not cross with the serum from brucellosis,foot and mouth disease,and it was only positive to M.bovis.The sensitive test of 1∶320 dilution of serum showed the variable coefficient within and among the batch was less than 5.0％.Conclusion] Due to its specificity and sensitivity,this indirect ELISA assay established here will be a good method for detecting M.bovis in future. |
| |
| Keywords: | Mycobacterium bovis （M bovis） recombinant CFP-10 protein indirect ELISA critical value |
| 本文献已被 维普 等数据库收录！ |
|
