Objective: S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology. The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum. Methods: Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z10S-TN was constructed by using the routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP. WB assay was established to detect the serum samples from 95 confirmed HFRS patients. Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method. Results: rBAC-Z10S-TN was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV IgG could efficiently recognize rNP and hybridize with the recombinant protein. 97.67% of the serum samples from the HFRS patients were positive confirmed by WB. Conclusions: We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10. WB assay which was established in this study could be used as a new serological test for HFRS diagnosis, thanks to the simplicity, safety, sensitivity and specificity of this method.
目的： 应用基因工程技术，在昆虫细胞中表达汉坦病毒（HV）S基因，表达产物作为诊断抗原，用于检测血清中抗HV特异性抗体IgG。 方法： PCR扩增HV-Z10株N蛋白（NP）编码基因，基因工程方法构建NP编码基因昆虫表达系统rBAC-Z10S-TN。间接荧光法了解重组NP（rNP）表达及与特异性免疫反应情况，SDS-PAGE观察重组蛋白表达情况。建立免疫印迹法检测肾综合征出血热（HFRS）患者血清样品，并与常规间接免疫荧光法进行比较。 结果： rBAC-Z10S-TN高效表达rNP，SDS-PAGE显示rNP的蛋白表达带，此抗原仅与HFRS患者血清起反应。经双盲试验，两法检测血清143份，HFRS阳性符合率为97.67%。 结论： 成功构建了HV-Z10株NP编码基因高效真核表达系统。所建立的免疫印迹法可作为HFRS简便、安全、敏感、特异的血清学诊断新方法。.
Keywords: Baculovirus expression system; Hantavirus; Nucleocapsid protein; Recombinant expression; Western-blot.