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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。
According to the gene sequence of the type A H1N1 influenza virus of the United States of America (A / California / 07/2009 (H1N1)), the gene encoding the influenza A virus hemagglutinin (HA) gene was synthesized using the helper virus-dependent adenoviral vector HDAd) backbone plasmid pSC15B to construct HDAd / HA DNA molecule vector which can express HA gene. The recombinant plasmid was transfected into 293Cre4 cells by calcium phosphate method and co-infected with helper virus H14 to obtain HDAd / HA vector, which was prepared and purified in large quantities Observation and initial identification of in vitro infections. The results of transmission electron microscopy showed that HDAd / HA vector possessed the typical adenovirus morphology. Co-infection of 293 cells with helper virus H14 resulted in the transcriptional analysis of HA gene by RT-PCR. The results showed that HDAd / HA recombinant adenovirus vector was successfully constructed, The study of effects and immunoprotection laid the foundation for the experiment.