携带可调控人胰岛素基因的腺病毒载体的构建和鉴定

Construction and Identification of Recombinant Adenovirus Harboring the GIP-Human Insulin Gene

目的:构建携带葡萄糖依赖性促胰岛素多肽(GIP)启动子控制下的人胰岛素基因的腺病毒载体。方法:将GIP- hIns基因自pCDNA3-GIP-hIns质粒中酶切下来,克隆至切除CMV启动子的pCA13-△CMV质粒中,形成转移质粒 pCA13-GIP-hIns,然后与质粒pBHGE3共转染至293细胞株,在其中同源重组形成腺病毒颗粒。采用PCR方法对重组体进行鉴定,同时测定病毒滴度。体外转染STC-1细胞,并采用RT-PCR检测感染腺病毒的细胞内有无hIns mRNA 的转录。结果:由pCA13-GIP-hIns和pBHGD共转染至293细胞后,可得到阳性重组体Ad.GIP-hIns,经PCR检测表明已含有GIP-hIns基因。纯化所得腺病毒滴度约为1×1011 pfu／ml。RT—PCR证实在感染重组体腺病毒Ad．GIP— hIns的细胞中有相应mRNA的转录。结论:成功构建了携带GIP启动子控制下的人胰岛素基因的腺病毒载体,为进一步胰岛素基因治疗糖尿病的实验研究奠定了基础。

Objective. To construct a recombinant adenovirus harboring the human insulin gene regulated by the glucose--dependent insulinotropic polypeptide（GIP） promoter. Methods：GIP-hIns gene was cut from plasmid of pCDNA3 - GIP-hIns and cloned into plasmid pCA13 -△CMV, which the plasmid pCA13 with the CMV promoter deletion and formed transfer plas- mid of pCA13- GIP-hIns. The plasmid was co-transfected to 293 cells with plasmid pBHGE3 for package of recombinant adenovirus. The recombinant adenovirus vector was identified by PCR. The titer of the recombinant Ad was measured. A tumor-derived K-cell line STC-1 was transfected with Ad. GIP hIns in vitro, RT - PCR was used to proved target gene expression in infected cells. Results： Positive recombinant adenovirus was obtained from 293 cells, and FVCR test indicated that the recombinant Ad contained the GIP-hIns gene. The titer of purified Ad was 1 × 10^11 pfu/ml. Meanwhile, mRNA product of bins was detected in the infected cells by RT-- PCR. Conclusion： A recombinant adenovirus harboring the GIP-hIns was successfully constructed, and it provides a good tool for further research on gene therapy Of diabetes.