H1N1亚型流感病毒NA基因在昆虫杆状病毒系统中的表达及鉴定

Expression of neuraminidase gene of influenza virus HIN1 in baculovirus-expression system

目的构建含有H1N1亚型流感病毒NA基因的重组杆状病毒。方法用PCR方法扩增甲型H1N1亚型流感病毒（A／PR8／34）全长神经氨酸酶基因，并将其连接于pFastBacdual载体，构建杆状病毒转移载体pFBD-NA。转化含有Bacmld的DHl0B感受态细胞后，获得重组穿梭载体rBacmid—NA。将其转染昆虫细胞sf9，获得重组病毒rBac—NA。提取重组杆状病毒rBac—NA的DNA并使用PCR方法检测；采用间接免疫荧光，SDS-PAGE，WesternBlot鉴定以及ELISA方法检测所表达的NA蛋白。结果经PCR鉴定所构建质粒rBacmid—NA正确；使用间接免疫荧光检测可见特异性绿色荧光位于感染细胞的表面，WesternBolt检测可与小鼠抗PR8株多克隆抗体和兔抗甲型流感病毒NA多克隆抗体发生特异性免疫反应；经ELISA检测，NA蛋白可被鼠抗流感病毒（PR8）多克隆抗体特异性识别。结论上述结果证明，我们获得了表达流感病毒NA蛋白的重组杆状病毒，为进一步研究NA蛋白的功能和新型流感疫苗的开发奠定了基础。

Objective To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus. Methods Full-length NA gene of Influenza virus H1N1 （A/PR/8/34） was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA. Recombinant shuttle vectors rBacmld-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sD cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected si9 cells was determined by IFA, Western Bolt and ELISA. Results PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed. NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells. NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot. ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus （PR8）, indicating high antigenicity. Conclusion Recombinant baculovirns rBac-NA that expresse NA protein of influenza virus was successfully constructed. This work provides a basis for further study on NA protein function and novel influenza vaccine development.