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目的 克隆小鼠慢性视网膜变性 /盘膜边缘蛋白基因 ,并构建其重组质粒载体。方法 以正常小鼠视网膜 RNA为模板 ,采用 RT- PCR方法扩增出 c DNA目的片段 ,PCR扩增 ,克隆至 p Bluescript II KS(+ )质粒 ,进行限制性内切酶 Bam H I和测序分析 ;再克隆到带有 CMV启动子的 pc DNA3质粒 Bam H I位点 ,Bam H I和 Eco R I限制性内切酶及测序证实。结果 限制性内切酶分析及测序证实 p Bluescript II KS(+ )质粒和 pc DNA 3质粒中插入的 1.2 kb片段 ,与小鼠慢性视网膜变性 /盘膜边缘蛋白基因全部编码区相吻合。结论 获得小鼠慢性视网膜变性 /盘膜边缘蛋白基因的全部编码区序列 ,并构建到带有CMV启动子的 pc DNA3载体中 ,为进一步研究打下基础。[眼科新进展 2 0 0 1;2 1(1)∶ 7- 11]
Objective To clone the mouse chronic retinal degeneration / membrane edge protein gene and construct its recombinant plasmid vector. Methods The normal mouse retinal RNA was used as a template to amplify the c DNA target fragment by RT-PCR. The amplified fragment was amplified by PCR and cloned into p Bluescript II KS (+) plasmid. The restriction endonuclease Bam HI and sequencing analysis were performed. Then cloned into the pcDNA3 plasmid Bam HI site with the CMV promoter, and confirmed by Bam HI and Eco RI restriction enzymes and sequencing. Results The restriction endonuclease analysis and sequencing confirmed that the 1.2 kb fragment inserted into p Bluescript II KS (+) plasmid and pcDNA 3 plasmid was consistent with the coding region of mouse chronic retinal degeneration / membrane border protein gene. Conclusion The coding sequence of mouse chronic retinal degeneration / membrane edge protein gene was obtained and cloned into pcDNA3 vector with CMV promoter, which laid the foundation for further study. [Progress in Ophthalmology 2001; 2 1 (1): 7-11]