重组汉坦病毒核衣壳蛋白抗原用于胶体金标记免疫层析法检测肾综合征出血热抗体

目的制备高表达量、高活性的HV NP重组抗原,以此为基础建立一个可同时检测HFRs患者血清中IgM和lgG抗体的快速胶体金标记诊断试剂盒。方法从TA-A537中扩增出截短的HV S基因片段P6-119,定向克隆至原核表达载体pET-30a中进行原核表达,采用亲和层析法纯化重组蛋白。以胶体金标记重组抗原,应用金标快速免疫层析法同时检测HFRS患者血清中IgM和IgG抗体。结果金标快速免疫层析法可同时检测HFRS患者血清中IgM和IgG抗体,与IFA比较,IgG抗体检测的敏感性和特异性分别为94.9%、100%。与ELISA比较,IgM抗体检测的敏感性和特异性均为100%。结论截短的重组汉坦病毒NP蛋白表达量高且具有良好的抗原性。以此为基础建立的金标快速免疫层析法显示出很高的敏感性和特异性,且具有简便、快速等优点,非常适用于各种层次尤其是缺乏实验条件和专业人员的基层医疗单位对HFRS疑似患者作出早期诊断。

Preparation of recombinant nucleocapsid protein of Hantavirus as the antigen for colloidal gold immunochromatographic assay to detect antibodies in hemorrhagic fever with renal syndrome(HFRS)

Objective To prepare recombinant nucleocapsid protein (rNP) of Hantavirus A537 and to use it as the antigen in colloidal gold immunochromatographic assay for the detection of IgM and IgG antibodies in hemorrhagic fever with renal syndrome (HFRS). Methods From the recombinant plasmid TA-A537, truncated genes p6-119 of HV S gene was amplified and subcloned into the expression vectors pET-30a, and constructed recombinant expression vectors of pET-6-119. The antigen of truncated rNP was expressed in E. coli BL21(DE3) and purified by Ni-NTA affinity chromatography. The immunochromatographic assay was established with purified recombinant antigen conjugated with colloidal gold. Results The colloidal gold immunochromatographic assay can detect IgM and IgG antibodies simultaneously in serum sample. Compared with IFA, the assay showed 94.9% in sensitivity and 100% in specificity. Compared with ELISA, the sensitivity and the specificity of the assay for the detection of IgM antibodies are all 100% . Conclusion The antigen of truncated rNP has higher expression and better antigenicity. The immunochromatographic assay used it as antigen showed higher sensitivity and specificity and had the advantages of rapid, simple and convenient, so it has been applied in fast diagnosis, especially in the countryside or hospitals without essential experimental equipments.