表达甲型H1N1流感病毒神经氨酸酶的重组腺病毒的构建及其免疫原性  

Construction and Immunogenicity of Recombinant Adenovirus for Expression of Neuraminidase of Influenza A(H1N1) Virus

作　　者：严敏[1] 孙茂盛[1] 王文举[1] 谢天宏[1] 李太华[1] 李鸿钧[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国生物制品学杂志》2011年第11期1264-1268,1277,共6页Chinese Journal of Biologicals

摘　　要：目的构建表达甲型H1N1流感病毒神经氨酸酶(Neuraminidase,NA)的重组腺病毒,并检测其免疫原性。方法从质粒pMD19T-simple-NA中扩增NA基因,克隆至穿梭质粒pShuttleCMV中,经同源重组获得重组腺病毒质粒,转染Ad-293细胞,包装出重组腺病毒Ad-NA,RT-PCR和免疫荧光法检测NA基因在Vero细胞中的转录和表达。CsCl密度梯度离心纯化重组腺病毒,免疫小鼠,ELISA法检测免疫小鼠血清中抗NA抗体滴度。结果重组腺病毒质粒经PacⅠ酶切鉴定表明带有目的基因的穿梭质粒已整合到腺病毒基因组中;NA基因在Vero细胞中成功转录和表达;重组腺病毒可刺激小鼠产生抗NA抗体,初免后4周,抗体水平达最高,为1∶100 000。结论成功构建了表达甲型H1N1流感病毒NA蛋白的重组腺病毒,其可刺激小鼠产生有效的免疫应答,为甲型H1N1流感病毒基因工程疫苗的研发奠定了基础。Objective To construct a recombinant adenovirus for expression of neuraminidase （NA） of influenza A （H1N1 ） virus. Methods NA gene was amplified from plasmid pMD19T-simple-NA and cloned into shuttle plasmid pShuttleCMV, based on which a recombinant adenovirus plasmid was constructed by homologous recombination and transfected to Ad-293 cells to package recombinant adenovirus Ad-NA, The transcription and expression of NA gene in Vero cells were determined by RT-PCR and IFA. Recombinant adenovirus Ad-NA was purified by density gradient centrifugation with cesium chloride, with which mice were immunized and determined for NA antibody titer in sera by ELISA. Results Digestion of recombinant adenovirus plasmid with Pac I proved that the shuttle plasmid containing the target gene was integrated into the genome of adenovirus. NA gene was successfully transcribed and expressed in Vero cells. Recombinant adenovirus Ad-NA stimulated the secretion of NA antibody in mice. The antibody level 4 weeks after primary immunization reached a peak value of 1 ： 100 000. Conclusion The recombinant adenovirus for expression of NA of influenza A （H1N1） virus was successfully constructed, which induced effective immune response in mice. It laid a foundation of development of recombinant influenza A （H 1Nl ） vaccine.

关 键 词：流感病毒A型 H1N1亚型 神经氨酸酶 重组腺病毒 免疫原性 

分 类 号：R373.13[医药卫生—病原生物学] R392-11[医药卫生—基础医学]