Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody
10.3760/cma.j.issn.0254-6450.2017.04.023
- VernacularTitle: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
- Author:
Pingping YAO
1
;
Fang XU
;
Yisheng SUN
;
Zhangnv YANG
;
Yun ZHANG
;
Ming YUE
;
Hanping ZHU
Author Information
1. 浙江省疾病预防控制中心浙江省肾综合征出血热疫苗研究重点实验室
- Keywords:
Hantavirus;
Nucleocapsid protein;
Recombinant expression;
Baculovirus expression system;
Western-blot
- From:
Chinese Journal of Epidemiology
2017;38(4):528-530
- CountryChina
- Language:Chinese
-
Abstract:
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-Z 10S-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.
