兔病毒性出血症病毒抗体间接ELISA检测方法的建立.pdfVIP

畜牧与兽医 2013 年第45 卷第1 期 .29. 兔病毒性出血症病毒抗体间接 ELISA 检测方法的建立 邱立，郝华芳，王兴龙，陈胜利，王佳 (西北农林科技大学动物医学院，陕西杨凌 712100) 摘要:建立一种检测兔病毒性出血症病毒 (RHDV) 抗体的间接 ELISA 方法c 对 RHDV 陕西分离株 VP60 基因进行原核表达， Westem blot 分析表 达产物的免疫反应性;以纯化的蛋白为包被抗原建立 ELISA 方法，并对反应条件进行优化。结果表明， VF吧。蛋白在大肠杆菌中成功表达，产物 约为42.34 ku 的融合蛋白，具有良好的反应原性;优化的 ELISA 最佳工作条件为:重组抗原包被浓度2.9μ邑/mL ， 37 c 2 h 后 4 C过夜， 1% BSA 37 c封闭 2 h ，待检血清37 c孵育 1 h ，酶标抗体 1 : 10 0ω 稀释， 37 c作用 1 h , 37 c显色5 min ，临界值为0.340: 建立的 ELISA 方法特 异性强、重复性好、敏感性高;临床检测 180 份样品，与血凝抑制试验的符合率为74.1% ，与商品化试剂盒检测结果符合率为94.8% 。该方法可 用于临床样品的大批量检测。 关键询:兔病毒性出血症;重组V问0 蛋白: ELISA 中图分类号: S855.3 文献标识码:A 文章编号: 0529-5130(2013)01-0029-05 Establishment of indirect ELISA for detection of antibody against rabbit hemorrhagic disease virus 。IU Li , HAO Hua-fang , WANG Xing-long , CHEN Sheng-li , WANG Jia (College ofVeterinary Medicine , Northwest Agriculture and Forestry University , Yangling712100 , China) Abstract: The aim of this study is to develop an indirect ELISA for detecting the antibody against rabbit hemorrhagic disease virus ( RHDV). The VP60 gene of RHDV Shanxi strain was expressed. The immunoreactivity of recombinant protein was analyzed by Westem blot. Then the purified recombinant protein was used for establishment of an indirect ELISA for detection of anti-RHDV antibody. The results showed that the recombinant protein with the molecular weight of 42. 34 ku was successfully expressed in Escherichia coli. The optimal reac- tion condition were determined as followings: coating antigen with a concentration of 2. 9μgImL at 4 c ovemight after 37 c 2 h , 1 % BSA as blocking agent at 37 c 2