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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 expressing the influenza A (H1N1) virus neuraminidase (NA) and test its immunogenicity. Methods NA gene was amplified from plasmid pMD19T-simple-NA and cloned into shuttle plasmid pShuttleCMV. Recombinant adenovirus plasmid was obtained by homologous recombination and transfected into Ad-293 cells. The recombinant adenovirus Ad-NA was packaged and RT-PCR The transcription and expression of NA gene in Vero cells were detected by immunofluorescence. CsCl density gradient centrifugation purified recombinant adenovirus, mice were immunized with anti-NA antibody titers in serum of the immunized mice. Results Restriction endonuclease digestion of the recombinant adenovirus plasmid showed that the shuttle plasmid with the target gene was integrated into the adenovirus genome. The NA gene was successfully transcribed and expressed in Vero cells. The recombinant adenovirus stimulated the mouse to produce anti-NA antibody. After 4 weeks of free, antibody levels reached the highest, 1: 100 000. Conclusion The recombinant adenovirus expressing the NA protein of influenza A (H1N1) virus was successfully constructed, which can stimulate the mice to produce an effective immune response and lay a foundation for the development of the genetically engineered A (H1N1) virus vaccine.