摘要
目的:克隆结缔组织生长因子(CTGF)及制备表达CTGF基因的重组腺病毒.方法:体外克隆出CTGF并将该基因采用定向克隆的方法克隆进入穿梭质粒pAdTrack-CMV中,进一步采用同源重组方法将该质粒和腺病毒质粒Adeasy-1连接,然后用脂质体介导的方法导入包装细胞HEK293中产生表达目的基因的重组腺病毒,收集病毒液感染HCT116wt骨软骨肉瘤细胞,通过荧光显微镜观察GFP和Western Blot检测CTGF蛋白的表达.结果:经限制性内切酶检测和GFP表达证实成功地构建了携带该基因的重组腺病毒载体并制备出高滴度重组病毒,重组腺病毒转染后可使HCT116wt细胞CT-GF蛋白表达明显增加.结论:成功构建了携带CTGF基因的重组腺病毒载体Ad5-CTGF,为探讨CTGF的作用机制、功能及与相关疾病的发生、发展上的进一步研究打下基础.
AIM：To clone connective tissue growth factor（ CT- GF） gene and construct the recombinant adenovirus vector containing mouse CTGF. METHODS： We cloned the target gene in vitro and inserted it into the shuttle vector of pAdTrack-CMV by directed cloning, and combined it with adenovirus skeleton vector Adeasy-1 by homologous recombination. Finally, we transfected the product into package cell HEK293 by Lipofectamine and generated the recombinant adenovirus expressing the target gene. After infection, the expression of CTGF was detected by Western Blotting. RESULTS： As proved by the restriction enzyme digestion assay and observation of GFP expression, recombinant adenoviral vector carrying CTGF gene was constructed successfully. The CTGF expression in HCTll6wt cells infected by the virus was much higher than the controls. CONCLUSION： The recombinant adenovirus vector containing mouse CTGF is constructed successfully, which lays a real good foundation for further research on CTGF gene.
出处
《第四军医大学学报》
北大核心
2008年第20期1829-1833,共5页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30500602)
关键词
结缔组织生长因子
克隆
分子
重组
遗传
腺病毒科
connective tissue growth factor
cloning, molecular
recombination, genetici
adenoviridae
