H1N1亚型流感病毒M1和NA基因在昆虫杆状病毒系统中的共表达  

Co-expressing M1 and NA genes of infuenza H1N1 virus in Baculovirus expression system

作　　者：陈爱珺[1] 姚立红[1] 徐鹏卫[1] 张智清[1] 郭建强[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京100052

出　　处：《中华实验和临床病毒学杂志》2015年第1期29-31,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的 构建共表达H1N1亚型流感病毒M1和NA基因的重组杆状病毒.方法 以PCR扩增H1N1亚型流感病毒A/PR/8/34毒株M1和NA基因,构建含有M1、NA双基因的重组转移载体pFBD-M1-NA,将其转化DH10Bac,获得重组Bacmid-M1-NA穿梭载体.将Bacmid-M1-NA转染sf9昆虫细胞,在细胞内包装出重组杆状病毒rBac-M1-NA,空斑实验测定病毒滴度,PCR方法鉴定外源基因插入情况.间接免疫荧光（IFA）检测M1、NA基因的表达情况.结果 获得的重组杆状病毒滴度为1×10^7pfu/ml;PCR检测结果证实M1、NA基因成功整合到了重组杆状病毒基因组.IFA结果显示,rBac-M1-NA感染的sf9昆虫细胞成功表达了M1、NA基因.结论 利用Bac-to-Bac杆状病毒表达系统成功构建了共表达H1N1亚型流感病毒M1和NA基因的重组杆状病毒,为研究流感病毒样颗粒（VLPs）的形成机制以及新型流感疫苗研发奠定了基础.Objective To construct the recombinant baculovirus co-expressing M1 and]NA genes of influenza H1N1 virus.Methods The M1 and NA genes of influenza virus （A/PR8/8/34） were amplified by PCR and then inserted into the pFastBacdual （pFBD）donor plasmid to construct the recombinant pFBDM1-NA.pFBD-M1-NA was transformed into DH10Bac E.coli competent cells containing bacmid vector to construct the recombinant shuttle vector rBacmid-M1-NA.Sf9 cells were transfected with rBacmid-M1-NA and then the recombinant baculovirus rBac-M1-NA was obtained.The virus titer was tested by plaque formation,M1 and NA genes in genome were identified by PCR,and the expression of M1 and NA gene by IFA.Results PCR result proved that pFBD-M1-NA and rBacmid-M1-NA were constructed correctly.The recombinant baculovirus rBac-M1-NA titer was 1 × 10^7pfu/ml.IFA analysis showed that M1 and NA genes were expressed in sf9 cells infected with rBac-M1-NA.Conclusion The recombinant baculovirus coexpressing M1 gene and NA gene of infuenza H1 N1 virus was successfully established.

关 键 词：流感病毒 蛋白质类 神经氨酸酶 杆状病毒 

分 类 号：R511.7[医药卫生—内科学]