SREBP-1c基因重组腺病毒的构建及其在犊牛原代肝细胞中的表达

Construction of SREBP-1c recombinant adenovirus and its expression in calf hepatocytes cultured in vitro

将携带有化学合成的SREBP-1c基因的质粒先用XbaⅠ酶切,Klenow补平突出末端,纯化回收后再用HindⅢ酶切,切胶回收目的基因片段;并将其克隆至用EcoRⅤ/HindⅢ酶切回收的pShuttle-EGFP-CMV(-)TEMP穿梭载体上。经酶切鉴定后,重组穿梭质粒和腺病毒pAdxsi质粒分别用I-CeuⅠ+I-SceⅠ双酶切处理,T4DNA连接酶连接,获得重组质粒pAdxsi-GFP-SREBP1c,酶切鉴定后经Pac I酶切线性化转染HEK293细胞,经包装扩增后用TCID50测定病毒滴度。以重组腺病毒感染犊牛原代肝细胞,采用qRT-PCR和Western blot检测细胞中SREBP-1cmRNA和蛋白的表达量。结果显示,重组腺病毒酶切鉴定正确,滴度可达1.2×1010,腺病毒感染犊牛原代肝细胞中SREBP-1cmRNA和蛋白的表达显著升高。结果表明,本试验成功构建了SREBP-1c基因过表达重组腺病毒,且SREBP-1c在犊牛原代肝细胞中高度表达。

Plasmid carrying SREBP-lc gene of chemical synthesis was first digested by Xba I , blunted with Klenow,and then recycled after Hind Ill digestion. These recycled target gene frag- ments were then inserted into the EcoR V/Hind I]I sites of adenovirus shuttle vector pShuttle-EG- FP-CMV（-） TEMP. After digestion assessment,recombinant shuttle plasmid and adenovirus plasmid pAdxsi that were digested by I-Ceu I and I-Sce I were connected with T4DNA ligase to obtain recombinant plasmid pAdxsi-GFP-SREBPlc assessed via digestion. The pAdxsi-GFPSREBPlc was linearized by Pac I and transfected into HEK293 cells via liposome. The recombinant adenovirus was packaged and amplified in HEK293 cells. After purifying,virus titer was determined by TCID50. Hepatocytes were infected by the recombinant adenovirus. The mRNA and protein expression of SREBP-lc gene were detected by qRT-PCR and western blotting. The recombinant adenovirus plasmid pAdxsi-GFP-SREBPlc was constructed correctly by restriction enzyme analysis, and the virus titer reached 1. 2 × 10^10. The mRNA and protein expression of SREBP-lc were significantly increased in the hepatocytes transduced with recombinant adenovi- rus. The recombinant adenovirus plasmid carrying SREBPlc gene was constructed successfully.And the expression of SREBP-lc was markedly increased in the hepatocytes.