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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 ELISA method for detection of Ebola virus (EBOV) in bats. Methods The carboxyl terminal sequence of Ebola virus Zaire Ebola virus codon optimized by Escherichia coli was synthesized and a His-tagged prokaryotic expression vector pET-28 (a) -Z-NP was constructed and transformed into BL21 (DE3) The target protein was induced with 1.0 mmol / L IPTG. The target protein was purified by affinity chromatography and identified by SDS-PAGE and Western blot. The indirect ELISA was established by using purified protein as coating antigen. Results The prokaryotic expression plasmid pET-28 (a) -Z-NP was constructed successfully. After purification, the expressed protein was detected by Western blot and specifically reacted with His monoclonal antibody and known positive sera. The established ELISA method for detection of rotavirus, guest virus, Seoul virus, Xuan Song virus infected bat serum did not cross reactions, known positive serum dilution to 1: 600 is still positive, the experimental intra-assay and inter-assay coefficient of variation <10%. Fifteen copies of the sera collected from Xishuangbanna Prefecture of Yunnan Province were detected by this method. Two of them were positive, and were consistent with the results of Western blot. Conclusion The established indirect ELISA method has high sensitivity and specificity and can be used for the detection of antibody to nucleoprotein of Zai Ebola virus in bats.