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非洲猪瘟间接ELISA诊断试剂盒的研究
引用本文:蒋正军,马世东,蔡丽娟,单文鲁,王树双,尹燕博,龚振华,李葳,宣华.非洲猪瘟间接ELISA诊断试剂盒的研究[J].中国预防兽医学报,2000(Z1).
作者姓名:蒋正军  马世东  蔡丽娟  单文鲁  王树双  尹燕博  龚振华  李葳  宣华
作者单位:农业部动物检疫所!266032(蒋正军,蔡丽娟,王树双,尹燕博,龚振华,李葳),新疆农业大学动物医学院!830000(马世东,单文鲁),解放军军需大学!长春 130000(宣华)
摘    要:用带有编码非洲猪瘟病毒衣壳蛋白P72 基因的重组杆状病毒(Bacp72)作载体,在sf9细胞中表达并得到重组P72蛋白,SDS-PAGE可得到分子量在 72kDa左右的电泳带。用标准阳性非洲猪瘟血清对 P72蛋白进行 ELISA检测,证明该蛋白具有生物学活性。用P72作为间接法的包被抗原,对ELISA反应条件进行了优化。确定最佳包被液为PBS(pH7.2)、最佳封闭液为1%PCT、最佳血清稀释液为4%PEG6000/PBS、最佳冲洗液为0.5M NaCl/0.5%Tween-20/PBS(pH7.2)。本实验反应体系采用50μl的微量法,可节约试剂及抗原。反应在2小时内即可完成,达到了快速诊断的目的。包被了抗原并用封闭液封闭后的酶标板密封后保存于-20℃的冰箱中,至少可以保存5个月。阻断试验和交叉试验表明ELISA法有良好的特异性。间接ELISA比Dot-ELISA法具有更高的灵敏性。血清学调查没有得到阳性结果,与我国实际情况相符。用Bacp72表达的非洲猪瘟病毒P72蛋白抗原作为间接ELISA的检测抗原来检测非洲猪瘟血清具有快速、简单、无感染的特点。本实验为非洲猪瘟ELISA检测试剂盒的最终组装提供了实验依据。

关 键 词:非洲猪瘟  P72蛋白  Bacp72  ELISA

Indirect ELISA Diagnostic Kit of African Swine Fever
JIANG Zhengjun,MA Shidong,CAI Lijuan,SHAN Wenlu,WANG Shushuang,YIN Yanbo,GONG Zhenhua,Li Wei,XUAN Hua.Indirect ELISA Diagnostic Kit of African Swine Fever[J].Chinese Journal of Preventive Veterinary Medicine,2000(Z1).
Authors:JIANG Zhengjun  MA Shidong  CAI Lijuan  SHAN Wenlu  WANG Shushuang  YIN Yanbo  GONG Zhenhua  Li Wei  XUAN Hua
Abstract:The highly antigenic major structural protein P72 of ASF virus was used as detecting antigen in indirect enzyme linked im- munosorbent assay (ELISA) to detect anti-ASFV serum. The protein P72 was expressed in sf insect cells by baculovirus (Bacp72). In SDSPAGE the molecular weight was about 72kDa. The recombinant proteins expressed in sf cells showed distinct antigenic properties when analysed by ELISA.The best ELISA react system were decided. The best coating buffer was 1% PGT. The best serum dillution buffer was 4% PEG 6000/ PBS, the best washing buffer was 0.5M NaCl/0.5% Tween - 70/ PBS. The ELISA method has higher specificity and sensitivity than Dot - ELISA and immunoelectroosmophoresis (IEOP). There were no positive serum in the 153 swine serums from different areas detected by ELISA. The indirect ELISA was quick.sensitive.specific and noninfectious.
Keywords:African swine  fever (ASF)  Protein P72  Bacp72  ELISA    
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