埃博拉病毒核蛋白抗体间接ELISA检测方法的建立

发布时间：2017-12-30 20:20

本文关键词：埃博拉病毒核蛋白抗体间接ELISA检测方法的建立　出处：《中国病原生物学杂志》2017年01期 　论文类型：期刊论文

【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。
[Abstract]:Objective to establish a rapid and accurate method for the detection of bat Ebola virus (EBOV). Methods the nucleoprotein ELISA method was used to synthesize the carboxyl terminal sequence of Zairian Ebola virus nucleoprotein from Escherichia coli. His tagged prokaryotic expression vector pET-28AL-Z-NPs was constructed and transformed into BL21DE3) -receptive cells. The target protein was induced by 1.0 mmol / L IPTG. The target protein was purified by affinity chromatography and identified by SDS-PAGE and Western blot. Indirect ELISA method was established with purified protein as coating antigen. Results the prokaryotic expression plasmid pET-28(a)-Z-NP was successfully constructed. After purification of the soluble expressed target protein, Western blot was used to detect the specific reaction with His monoclonal antibody and known positive serum. The established ELISA method was used to detect rotavirus and guest virus. There was no cross reaction in the sera of bats infected with Seoul virus and Hyun Sung virus. The known positive serum diluted to 1: 1 600 was still positive. This method was used to detect 15 samples of Rhinolophus rubrum serums collected from Xishuangbanna area of Yunnan Province, of which 2 were positive. Conclusion the indirect ELISA assay has high sensitivity and specificity, and can be used for the detection of nuclear protein antibodies against Ebola virus in bats and Zaire.
【作者单位】：吉林大学动物医学学院;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;
【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)
【分类号】：R373
【正文快照】： 埃博拉病毒(Ebolavirus)是世界上公认的致命的病毒之一,由于其引起的疾病-埃博拉出血热(EbolaHemorrhagic Fever)对人具有高度的传染性和致死性,所以世界卫生组织(World Health Organization,WHO)将其列为潜在的生物战剂之一[1]。埃博拉病毒属于单股负链RNA病毒目(Mononegavir

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