埃博拉病毒核蛋白抗体间接ELISA检测方法的建立  

Development of an indirect ELISA technique based on recombinant protein for detection of antibodies against the nucleoprotein of the Ebola virus

作　　者：蔡建秋[1,2] 徐琳[2] 涂长春[1,2] 何彪[2] CAI Jian-qiu XU Lin TU Chang-chun HE Biao(College of Veterinary Medicine, Jilin University, Changchun 130062, China Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Institute, Academy of Military Medical Sciences)

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]军事医学科学院军事兽医研究所,吉林省人兽共患病防控重点实验室,吉林长春130122

出　　处：《中国病原生物学杂志》2017年第1期1-4,9,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金面上项目(No.31572529);中国军事医学科学院青年创新基金项目(No.2015CXJJ28)

摘　　要：目的建立快速而准确的检测蝙蝠埃博拉病毒(EBOV)核蛋白抗体ELISA方法。方法合成大肠埃希菌系统密码子优化的扎伊尔埃博拉病毒核蛋白羧基端序列,构建含His标签的原核表达载体pET-28(a)-Z-NP,并转化BL21(DE3)感受态细胞,用1.0mmol/L IPTG诱导表达目的蛋白,目的蛋白经亲和层析纯化后进行SDS-PAGE和Western blot鉴定,以纯化蛋白为包被抗原建立抗体间接ELISA方法。结果成功构建原核表达质粒pET-28(a)-Z-NP,可溶表达的目的蛋白纯化后经Western blot检测,能与His单抗和已知阳性血清特异性反应。建立的ELISA方法检测轮状病毒、来宾病毒、汉城病毒、玄松病毒感染蝙蝠血清均无交叉反应,已知阳性血清稀释至1∶1 600仍为阳性,试验的批内及批间变异系数均<10%。用此方法检测采自云南省西双版纳地区的15份棕果蝠血清,其中2份阳性,且与Western blot验证结果一致。结论建立的间接ELISA方法具有较高的敏感性和特异性,可用于蝙蝠扎伊尔埃博拉病毒核蛋白抗体的检测。Objective To establish a rapid and accurate method of detecting bat antibodies against the nucleoprotein（NP）of the Ebola virus. Methods Codon-optimized C-terminal sequences of the NP gene of the Zaire ebolavirus were chemically synthesized and inserted in pET-28（a）downstream of a His-tag.The recombinant was transformed into E.coli BL21,and its expression was induced with 1.0mmol/L of IPTG in a 37℃incubator.The expressed protein was purified using affinity chromatography and identified using SDS-PAGE and Western blot analysis.An indirect ELISA technique was established using apurified protein as a coating antigen. Results pET-28（a）-Z-NP was successfully constructed.Western blot analysis verified that the soluble recombinant protein was specifically recognized by anti-His monoclonal antibody and serum that was positive for the Zaire ebolavirus.Specific tests indicated that the new ELISA technique did not cross-react with bat sera that was positive for the group A rotavirus,the Laibin virus,the Seoul virus,of the Xuan Son virus,indicating a high level of specificity.This technique can detect the NP of the Ebola virus even when serum is diluted to 1：1 600.The intra-batch and inter-batch coefficients of variation were less than 10%.The indirect ELISA technique was used to screen 15 serum samples from Rousettus leschenaultii collected in Xishuangbanna,Yunnan.Two samples tested positive,and this finding agreed with the results of Western blot analyses. Conclusion The indirect ELISA technique developed here has excellent sensitivity and specificity and can be used to detect bat antibodies against the NP of the Zaire ebolavirus.

关 键 词：扎伊尔埃博拉 核蛋白 原核表达 蝙蝠 间接ELISA 

分 类 号：R373[医药卫生—病原生物学]