【摘要】 目的:利用HEK293细胞内同源重组法构建带有绿色荧光蛋白报告基因的人血管能抑素(canstatin)重组腺病毒载体,扩增并纯化重组腺病毒颗粒.方法:PCR法扩增canstatin全长基因,克隆至pGEM-T载体,经酶切和测序证实后,亚克隆到穿梭质粒pAd5-CMV中,获得穿梭质粒pAd5-CMV/canstatin.用PacI酶单独酶切穿梭质粒pAd5-CMV/canstatin和腺病毒E3区带有绿色荧光蛋白表达元件的腺病毒骨架载体,采用标准的磷酸钙方法共转染HEK293细胞.7~10d后收获细胞裂解物,在HEK293细胞中扩增,病毒颗粒经氯化铯密度梯度离心纯化后,分光光度计检测重组病毒滴度.结果:测序证实pEGM-T/canstatin基因片段与GenBank公布的can-statin基因序列一致.重组腺病毒质粒转染293细胞后24h观察到绿色荧光,病毒纯化后制备出高滴度重组腺病毒(病毒滴度为2.0×1015pt/L).结论:成功构建了表达canstatin基因的重组腺病毒载体并制备了重组腺病毒颗粒,为研究该基因在肿瘤治疗中的作用奠定了基础.更多还原

【Abstract】 AIM:To construct recombinant adenovirus vector containing an enhanced green fluorescent protein reporter gene for expressing human canstatin by homologous recombination method in human embryo kidney(HEK) 293 cells,and further to propagate and purify the virus particles. METHODS: Canstatin gene was amplified by PCR method and cloned into the pEGM-T vectors, resulting in pEGM-T/canstatin, which was identified by enzyme digestion and sequencing. The canstatin gene was then sub-cloned into the shuttle plasmid pAd5-CMV, resulting in a shuttle vector pAd5-CMV/canstatin. The shuttle vector pAd5-CMV/canstatin and the backbone vector containing the coding sequence of enhanced green fluorescent protein in adenovirus E3 region were linearizated through the digestion of PacⅠ enzyme independently, then these products were co-transfected into HEK293 cells using standard calcium phosphate method. The cell lysates were harvested 7 to 10 d post transfection, and further propagated in HEK293 cells. The virus particles were purified by centrifugation in CsCl gradients, and virus concentration was determined under spectrophotometry. RESULTS: The sequencing result showed the gene fragment in pEGM-T/canstatin vector was almost the same as that published on GenBank. The green fluorescence was observed in HEK293 cells after 24 h transfection. The recombinant adenovirus of high titration was obtained after purification (viral titer was about 2.0×1015pt/L). CONCLUSION: A recombinant adenovirus vector for expression of canstatin gene is successfully constructed, and the virus particles are prepared, which lays a foundation for studying a role of canstatin in cancer gene therapy.更多还原