SARS荧光免疫诊断技术研究

An Immunofluorescence Assay for the Detection of SARS Associated Coronavirus Antibody Based on the Recombinant Viral Antigen

目的 应用基因工程技术 ,在昆虫细胞中表达SARS核蛋白基因 ,产生无感染性的核蛋白抗原 ,利用此蛋白制备抗原片 ,用于检测血清中SARS特异性抗体。方法 从非典型性肺炎病人血清中提取病毒RNA ,通过RT PCR方法扩增出SARS核蛋白基因片段 ,将核蛋白基因插入杆状病毒 ,构建重组昆虫杆状病毒 ,转染昆虫细胞 ,收获细胞 ,经丙酮固定 ,制成SARS抗原片 ,建立了SARS荧光免疫方法 (IFA)。结果 用此抗原片检测多份血清 ,仅与SARS病人血清起反应 ,而与正常人及其它发热病人血清不起反应。结论 利用该方法制备抗原片 ,不需要P3实验室 ,操作简便 ,为诊断与研究SARS提供了方便而安全的方法。

Objective To establish an indirect immunofluorescence assay to detect the serum SARS associated coronavirus antibody specially using recombinant and avirulent SARS nucleocapsid antigen which is expressed in baculovirus system. Methods Nucleocapsid gene was obtained by reverse transcription and polymerase chain reaction (RT-PCR) from a patient with SARS. The recombinant virus was obtained by inserting nucleocapsid gene into baculovirus expression vector, which then infected Sf 9 cells. Infected cells were harvested and fixed by acetone to form reagent containing SARS antigen for the corresponding antibody detection. Results The recombinant SARS antigen reacted with the serum from SARS patients but didnt react with the serum from the other patient or normal people. Conclusions The technique described here has the advantage of simple preparing and a new resource of antigen for further research and diagnostic purposes.