可表达新型甲型H1N1流感病毒血凝素蛋白辅助病毒依赖型腺病毒载体的构建  

Construction and Identification of Helper-Dependent Adenoviral Vector Encoding Hemagglutinin Protein of Swin-Origin Influenza Virus A H1N1

作　　者：张梅[1] 江彦泽[2] 陈念华 付远辉[2] 乔伟[1,2] 王荷[2] 何金生[1,2] 

机构地区：[1]安徽医科大学免疫学教研室,合肥230032 [2]北京交通大学生命科学与生物工程研究院,北京100044 [3]山东省烟台第二中学,烟台264000

出　　处：《生物医学工程学杂志》2014年第1期157-160,共4页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30671965)

摘　　要：根据甲型H1N1流感病毒美国加利福尼亚毒株基因序列(A/California/07/2009(H1N1)),全基因合成甲型流感病毒血凝素(HA)编码基因,利用辅助病毒依赖型腺病毒载体(HDAd)骨架质粒pSC15B,构建可表达HA基因的HDAd/HA DNA分子载体,以磷酸钙法转染293Cre4细胞,与辅助病毒H14连续共感染,获得HDAd/HA载体,并大量制备和纯化,进行形态观察和体外感染的初步鉴定。透射电镜下观察HDAd/HA载体具有典型腺病毒形态;与辅助病毒H14共感染293细胞,RT-PCR检测HA基因有转录,显示成功构建HDAd/HA重组腺病毒载体,为甲型流感病毒体内免疫效果和免疫保护作用的研究奠定实验基础。In order to investigate immune protection against swine-origin influenza virus （S-OIV） A H1N1, the helper dependent adenovirus vector （HDAd） system was exploited to construct recombinant HDAd encoding hemaggluti- nin （HA）. The HA gene was synthesized and cloned to the HDAd backbone. Then, the HDAd/HA DNA molecules were transfected into 293Cre4 cells with calcium phosphate. The cells were infected by helper virus 16 hours after the transfection. The 293Cre4 cells were coinfeeted with HDAd/HA and the helper virus for large-scale preparation of HDAd/HA. The HDAd/HA was obtained and purified twice with CsCI density ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein was analyzed with RTPCR. Recombinant HDAd/HA expressing HA protein was successfully constructed which could pave the way for in vivo investigation on immunogenicity and efficacy against S-OIV A H1N1 infection.

关 键 词：辅助病毒依赖型腺病毒载体 甲型H1N1流感病毒 血凝素 

分 类 号：R373[医药卫生—病原生物学] Q78[医药卫生—基础医学]