Yao Li-Hong, Fu Jin-Qi, Chen Ai-Jun, Liu Xiao-Yu, Xu Peng-Wei, Guo Jian-Qiang, Zhang Le-Cui
Institute for Viral Disease Control and Prevention, China CDC, Beijing 10052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2013 Apr;27(2):81-4.
To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.
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 rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids. After transfecting into sf9 cells, recombinant baculovirus rBac-NA was obtained. The rBac-NA genome was extracted and identified by PCR. NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA, Western Bolt and ELISA.
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.
Recombinant baculovirus 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.
构建携带甲型H1N1流感病毒神经氨酸酶（NA）基因的重组杆状病毒。
采用聚合酶链反应（PCR）扩增甲型H1N1流感病毒（A/PR/8/34）NA基因全长，将其插入pFastBacdual载体，构建重组杆状病毒转移载体pFBD-NA。将构建好的重组转移载体转化含有杆粒质粒的DH10B感受态细胞，获得重组穿梭载体rBacmid-NA。转染昆虫细胞sf9，获得重组杆状病毒rBac-NA。提取rBac-NA基因组进行PCR鉴定。通过间接免疫荧光法（IFA）、蛋白质免疫印迹法（Western Bolt）及酶联免疫吸附测定（ELISA）检测重组杆状病毒感染的sf9细胞表达的NA蛋白。
PCR结果证明成功构建了重组穿梭载体rBacmid-NA。IFA检测到NA蛋白，在感染细胞表面呈现强特异性绿色荧光。Western Blot结果显示，NA蛋白能被两种NA特异性多克隆抗体识别。ELISA结果显示重组NA蛋白与小鼠抗流感病毒（PR8）多克隆抗体有特异性反应，表明其具有较高的抗原性。
成功构建了表达甲型流感病毒NA蛋白的重组杆状病毒rBac-NA。该研究为进一步研究NA蛋白功能及新型流感疫苗的开发奠定了基础。
