汉坦病毒核蛋白的重组表达及其免疫印迹法在肾综合征出血热血清抗体检测中的应用

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody

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。

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.