神经粘附分子NCAM140基因RNA干扰质粒的构建与鉴定

Construction and Identification of RNA Interference Vector Targeting NCAM140 Gene

目的:构建有效的小鼠神经粘附分子(NCAM140)基因的RNA干扰(RNAi)质粒载体,为研究NCAM140参与的细胞信号通路转导、其生物学作用以及以NCAM140为靶点的基因治疗提供稳定转染的RNAi质粒。方法:使用基因序列软件设计、筛选符合公开文献筛选参数的4条靶序列以及1条阴性对照序列,由上海吉玛技术有限公司合成。与载体质粒pGPU6/GFP/Neo重组后,分别命名为pSi-nca1、pSi-nca2、pSi-nca3、pSi-nca4和pSi-control。转染大肠杆菌感受态细胞。选择阳性克隆进行DNA测序鉴定,Western blot方法进行干扰靶点的筛选。选取干扰效率最高的质粒转染MN9D细胞,普通光学显微镜分别计数同一视野细胞总数及GFP阳性细胞数,计算转染效率。结果:酶切和DNA测序结果证实shRNA正确插入pGPU6/GFP/Neo质粒;Western blot结果显示与空质粒对照组比较,pSi-nca4组细胞NCAM140表达明显下调,细胞转染效率为62%。结论:成功构建靶向小鼠NCAM140基因的RNAi质粒,为NCAM140参与的细胞信号通路的研究以及以NCAM140为靶点的基因治疗提供了稳定转染细胞的干扰质粒,为研究其生物学作用奠定了分子生物学基础。

Objective： To construct RNA interference plasmid targeting NCAM140, it provided stable RNA interference plasmid fur NCAM 140 study involved in cell signaling pathway, function of molecular biology and NCAM 140-targeting gene therapy. Methods： 4 gene sequences and 1 non-sence control gene sequence consistent with the screening features described in previous articles using open websites were designed, and the sequences were synthesized with the GenePharma Company. The sequences were amplified in Bacteri- tun coli after gene recombination with pGPU6/GFP/Neo plasmid, and were named as pSi-ncal, pSi-nca2, pSi-nca3, pSi-nca4 and pSi-control respectively. Those plasmids were transfected into E. coli competent cells and positive clones were selected for DNA se- quencing. After sequencing identification, the positive clones were transfected into MN9D cells. The GFP positive percentages （GFP pos- itive cells versus total number of MN9D cells） were used to determine the transfer efficiency of these plasmids, and the inhibitory effi- ciency of these plasmids were confirmed by western blot. Results： shRNA was inserted into plasmid pGPU6/GFP/Neo correctly con- tirmed by digestion and DNA sequencing. The protein level of NCAM140 in MN9D cells at 24 hr after transfected into pSi-nca4 Plasmid is depressed （P 〈0.001）. The most efficient plasmid was pSi-nca4. The pSi-nca4 transfection efficiency was 62 %. Conclusion： RNA in- terference plasmid targeting NCAM140 is successfully constructed, it provides stable RNA interference plasmid for the research of NCAM140 participating in cell signaling pathway, and offers favorable tool for the research of NCAM 140 biological effects and NCAM140-targeting gene therapy.