口蹄疫感染与免疫动物鉴别诊断方法的建立  

The establishment of differential diagnosis of infected and immuned animals of FMDV

作　　者：鲁会军[1] 金宁一[1] 韩松[1] 郑敏[1] 王凯[1] 贾雷立[1] 李昌[1] 

机构地区：[1]中国人民解放军军事医学科学院军事兽医研究所基因工程重点实验室,吉林长春130062

出　　处：《中国病原生物学杂志》2008年第2期81-85,共5页Journal of Pathogen Biology

基　　金：科技部攻关项目(No.2004BA519A40);科技部支撑项目(No.2007BAD55B05)

摘　　要：目的建立鉴别自然感染口蹄疫动物和口蹄疫灭活疫苗免疫动物的诊断方法。方法构建重组杆状病毒的转座质粒pFastBac-3AB,转化DH10 Bac感受态细菌,在体内进行重组,并经三重抗性与蓝白斑筛选,得到杆状病毒重组质粒Bacmid-3AB;将Bacmid-3AB转染Sf9细胞,获得重组杆状病毒。以重组杆状病毒表达的3AB蛋白为抗原建立鉴别口蹄疫感染与免疫动物的ELISA诊断方法,并对各种反应条件进行优化,包括抗原包被时间、包被浓度、封闭剂、酶标抗体工作浓度等。结果获得了重组杆状病毒,成功表达了口蹄疫病毒3AB蛋白,并用纯化的3AB蛋白建立了检测口蹄疫病毒抗体的间接ELISA方法。用该方法检测FMDV感染动物和免疫动物血清抗3AB蛋白抗体,猪和牛感染血清检测的阳性率分别为83.3%和91.7%。结论用纯化的口蹄疫病毒3AB蛋白建立了鉴别诊断口蹄疫感染与免疫动物的ELISA诊断方法。Objective To establish the differential diagnosis method for the infected animals and inactive-vaccine immuned animals of FMD. Methods To prepare antigen used for the detection of FMDV antibody of infected animals and immuned animals, 3AB gene were fused and cloned into baculoviruses transfer vector pFastbacI and recombinant plasmid pFastbac-3AB was constructed. After transforming it into competent Escherichia coli DH10bac cells, the recombinant Bacmid of E. coli-Baculovirus shuttle vector that can replicate in E. coli as a plasmid and can infect suscepitible sf9 insect cells was obtained. Co-transfecting the recombinant Bacmid with sf9 cells, and then the recombinant baculovirus carrying 3AB gene were obtained and the expression was confirmed by SDS-PAGE and western blotting. Then, the expressed 3AB protein was employed as the diagnosis antigen of the indirect ELISA to differential diagnosis the infected animals and immuned animals. The diagnosis method was established to detect the clinical samples after optimization of the reaction conditions. Results The 3AB protein of FMDV was expressed in recombinant baculovirus, and the purifed 3AB protein of FMDV was used as the diagnosis antigen to detect the nonstructural protein antibody of FMDV. The antibodies against 3AB protein were detected to differential diagnosis the infected and immuned pigs and cattles, and the results showed that the positive ratio of pig sera and cattle sera were 83.3% and 91.7% respectively. Conclusion The purifed 3AB protein of FMDV was used as the diagnosis antigen of the indirect ELISA to establish the differential diagnosis method for the infected animals and immuned animals of FMD.

关 键 词：重组杆状病毒 表达 蛋白 鉴别诊断 ELISA 

分 类 号：R373.9[医药卫生—病原生物学]