H3、H5、H7、H9亚型流感病毒通用多表位核酸疫苗构建及表达研究  

Construction and expression of multiple epitopes DNA vaccines of influenza A virus H3579 subtypes

作　　者：陈义锋[1] 金宁一[1] 贾雷立[1] 鲁会军[1] 田明尧[1] 南文龙[1] 马鸣潇[1] 刘成宏[1] 沈国顺[1] 马海利[1] 陈晓月[1] 

机构地区：[1]军事医学科学院十一所全军基因工程重点实验室,长春130062

出　　处：《中华微生物学和免疫学杂志》2007年第12期1136-1138,共3页Chinese Journal of Microbiology and Immunology

基　　金：863计划项目(2006AA10A205);教育部重点项目(306006);吉林省科技发展重大项目(20065020);长春市科技计划项目(04-02GG217)

摘　　要：目的构建表达A型流感病毒多种表位的核酸疫苗,并研究其在幼仓鼠肾细胞(BHK细胞)内表达产物的生物学活性。方法筛选流感病毒主要抗原(HA、NA、NP、M)表位基因,以生物信息学软件优化其排列结构,合成流感通用的复合多表位基因(Epi),以H5、H7亚型流感病毒HA融合表达基因为骨架,将多表位基因Epi插入构建H7HA-Epi-H5HA阅读盒,定向克隆至pVAX1,构建抗A型流感H3579亚型的pVAX1-H7HA-Epi-H5HA。最后将该重组质粒转染BHK细胞,提取细胞总RNA,以RT-PCR方法检测目的基因;以间接免疫荧光试验(IFA)初步测其表达产物抗原性。结果RT-PCR检测到目的基因;IFA检测到特异性荧光存在。结论本试验所构建的重组质粒pVAX1-H7HA-Epi- H5HA,在真核细胞内均获了有效地转录和表达;而且其表达产物均能与相应的抗体特异性结合,证明了其具有一定的生物学活性和抗原性,有望成为抗多种A型流感病毒的通用核酸疫苗。Objective To construct DNAvaccines which express influenza A viruses multiple epitopes, and to study the bioactivity of the expression products in BHK cells. Methods Selecting the main antigen （HA, NA, NP, M） dominant epitopes of influenza A viruses, a sequence was synthesized containing multiple epitopes of influenza A viruses. A box co-expressing H7H5HA and Epi gene was contructed by inserting the Epi gene in HTHSHA ORF. The box was then cloned into pVAX1. To detect the mRNA of the gene of interest the whole RNA of the BHK cells transfected with the reconstructed plasmids pVAXl, pVAX1-H7HA-Epi-HSHA by liposome method were extracted. IFA was also utilized to detect the bioactivity of the expression products. Results The gene of interest was identified by RT-PCR, and the antibody specificity of expression products was detected by IFA. Conclusion The reconstructed pLasmid could express product efficiently, and the product can specially recognized by the corresponding antibodies. This proved the reconstructed eukaryotic expression vector pVAX1-H7HA- Epi-H5HA might be a candidate vaccine strains for influenza A viruses.

关 键 词：表位 流感病毒 核酸疫苗 

分 类 号：R392[医药卫生—免疫学]