苏丹型埃博拉病毒糖蛋白原核表达及抗体间接ELISA检测方法的建立  

Prokaryotic expression of Sudan ebolavirus GP and the development of recombinant protein-based indirect ELISA for antibody detection

作　　者：吴芳芳[1] 曹增国 焦翠翠 王琪[3] 迟航[1] 侯朋飞 黄培[3] 金宏丽 赵永坤[1] 李忠义[1] 王化磊[1] 杨松涛[1] 夏咸柱[1] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [2]吉林大学动物医学学院 [3]吉林农业大学动物科学技术学院

出　　处：《中国病原生物学杂志》2018年第1期1-4,10,共5页Journal of Pathogen Biology

基　　金：国家科技重大专项(重大新药创制)(No.2015ZX09102025).

摘　　要：目的原核表达苏丹型埃博拉病毒(Sudan virus,SUDV)糖蛋白(GP),并作为包被抗原建立抗体间接ELISA检测方法。方法 PCR扩增SUDV GP基因片段,克隆至pET30a(+)表达载体,IPTG诱导表达SUDV GP,经HisNi柱纯化,SDS-PADE和Western blot鉴定正确的SUDV GP作为包被抗原建立SUDV抗体间接ELISA方法,并对实验条件进行优化。结果SUDV GP在大肠杆菌中以包涵体的形式表达,建立的抗体间接ELISA检测方法最佳SUDV GP包被量为2μg/孔,待检血清最佳稀释度为1∶320,最佳作用时间1.5h,HRP标记羊抗鼠IgG最佳稀释度1∶10 000,TMB最佳显色时间为7min。该方法的特异性强,样品检测结果与已知血清的符合率为100%,检测SUDV阳性血清的敏感度为1∶40 960,SUDV GP批间及批内的实验结果变异系数(CV)均小于10%。结论成功表达SUDV GP并建立SUDV抗体间接ELISA检测方法,为疫苗免疫效果的评价、疫病的监测及SUDV抗体检测试剂盒的研发奠定了基础。Objectives To express SUDV GP in a prokaryotic system and to develop recombinant protein-based indirect ELISA for antibody detection. Methods A fragment of SUDV GP was amplified with PCR and then cloned into the prokaryotic expression vector pET30a（＋）. Expression of GP was induced with IPTG. The IPTG concentration and time required for induction were optimized. Expressed products were purified with His-Ni affinity chromatography, fol- lowed by SDS-PAGE and Western blotting. An indirect ELISA technique was devised using the purified protein as a coat- ing antigen. Results SUDV GP was efficiently expressed in the form of inclusion bodies. The conditions for the ELISA technique were as follows： the antigen preparation was 2/xg/pool, dilution of sera was 1 ： 320, the duration o{ ser- um inoculation was 1.5 h, HRP-labeled goat anti-mouse IgG was 1：10 000, and the duration of TMB incubation was 7 min at room temperature. ELISA specifically detected recombinant GP, and the rate of concordance between test results and positive serum samples was 100 ~. The reproducibility of ELISA was 1：40,960, and the intra- and inter-assay CVs for test results were both less than 10%. Conclusion A SUDV GP fragment was efficiently expressed and an indirect ELISA technique was successfully developed for detection of SUDV antibodies. This technique could be used to evaluate vaccines, monitor SUDV, and develop SUDV antibody detection kits.

关 键 词：苏丹型埃博拉病毒 原核表达 糖蛋白 ELISA 

分 类 号：R373.9[医药卫生—病原生物学]