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目的:构建表达新城疫病毒血凝素-神经氨酸酶(Hemagglutinin-neuramidinase,HN)基因的重组腺病毒,为进一步研究HN基因对消化道肿瘤的抑制作用及分子机制建立基础。方法:用BamHⅠ和XbaⅠ双酶切质粒pVHN,将获得的HN基因片段连接入穿梭质粒pacAd5CMVK-NpA,构建含HN基因的穿梭质粒pacAd5-HN。利用PacⅠ单酶切对pacAd5-HN和腺病毒基因组质粒(pAd5)进行线性化处理后应用脂质体介导法共转染AAV-293细胞,分别利用蚀斑纯化和RT-PCR、Western blot等方法对重组病毒进行筛选和鉴定,并测定所获得重组病毒的滴度。结果:所构建重组病毒可有效表达HN基因,表达产物分子量约为63kD,重组病毒滴度为108~1010PFU/ml。结论:成功构建含HN基因的重组腺病毒,所获得重组病毒的滴度可以满足体内外实验要求。
Objective: To construct recombinant adenovirus expressing Newcastle disease virus Hemagglutinin-neuramidinase (HN) gene and lay a foundation for further study on the inhibitory effect and molecular mechanism of HN gene on gastrointestinal tumors. METHODS: The plasmid pVHN was double digested with BamHⅠand XbaⅠ. The HN gene fragment was ligated into the shuttle plasmid pacAd5CMVK-NpA to construct the shuttle plasmid pacAd5-HN containing the HN gene. Plasmid PacAd5-HN and adenovirus plasmid pAd5 were linearized with restriction enzyme PacⅠ and then co-transfected into AAV-293 cells by liposome-mediated method. Plasmid purification and RT-PCR, Western blot Methods The recombinant virus was screened and identified, and the titer of recombinant virus was determined. Results: The constructed recombinant virus could efficiently express HN gene. The molecular weight of the expressed product was about 63 kD and the recombinant virus titer was 108-1010 PFU / ml. Conclusion: The constructed recombinant adenovirus containing HN gene successfully obtained the titer of recombinant virus, which can meet the in vitro and in vivo experimental requirements.