绿色荧光蛋白与Rb94基因共表达载体的构建和表达第三军医大学学报.doc

增强型绿色荧光蛋白与Rb94基因共表达载体的构建和表达 郑敏明，周希瑗，王丽萍 （400010重庆，重庆医科大学附属第二医院眼科，眼科学重庆市市级重点实验室） [摘要] 目的 构建增强型绿色荧光蛋白与Rb94基因的真核共表达载体并鉴定其在真核细胞中的表达。方法 用PCR技术对cDNA-Rb质粒中的目的基因片段进行扩增，使用BamH Ⅰ和Sal Ⅰ进行双酶切，切胶回收DNA片段。将其连接入pEGFP-C1，构建pEGFP-C1/Rb94真核表达载体并再进行酶切鉴定。将重组质粒用脂质体转染法转染至人视网膜母细胞瘤细胞（HXO-Rb44）中，荧光显微镜下观察EGFP在细胞内的表达，Western blot检测Rb蛋白的表达情况，流式细胞仪检测细胞凋亡率。结果 重组克隆载体内的目的片段序列与Rb94基因序列完全一致。pEGFP-C1/Rb94酶切鉴定结果与预期结果一致。荧光显微镜下观察可见转染后的细胞有荧光出现。Western blot检测结果表明Rb94基因得到了有效表达。流式细胞仪检测结果显示，含Rb94基因的细胞凋亡率(21.17±0.45)%也明显高于其他组。结论 成功构建EGFP和Rb94真核共表达载体，并可在真核细胞中有效表达，Rb94对视网膜母细胞瘤细胞有明显抑制生长作用。 [关键词] Rb94基因；真核表达载体；人视网膜母细胞瘤细胞 Construction and expression of enhanced green fluorescent protein and Rb94 co-expression vector Zheng Minming, Zhou Xiyuan, Wang Liping(Chongqing Key Laboratory of Ophthalmology; Department of Ophthalmology, the Second Affiliated Hospital, Chongqing Abstract：Objective To construct a type of enhancement of EGFP and Rb94 co-expression vector, meanwhile, we can detect its expression in cultured eukaryocyte. Methods By means of PCR, we could get the amplification of Rb94 gene fragment with cDNA-Rb plasmid . The Rb94 gene fragment was digested with BamHI and SalI, sequentantly, what inserted into pEGFP-C1 that was cut with BamH Ⅰand Sal Ⅰ. The recombinant pEGFP-C1/Rb94 was identified with restriction analysis. The expression plasmid pEGFP-C1/Rb94 was transfected into HXO-Rb44 cell mediated by liposome reagent, simultaneously, we could make a observation of the expression of EGFP in cell by fluorescence microscopy, moreover, Rb protein was detected through Western blotting. The apoptosis rate of HXO-Rb44 cells was detected by flow cytometer. Result The sequence of the cloned DNA fragment is fully consistent with the sequence of Rb94 gene. The Rb94 gene was inserted into eukaryotic expression vector pEGFP-C1 correctly. The recombinant expression plasmid was successfully transferred into HXO-Rb44 observed by fluorescent microscopy and effective expression of Rb protein was also testified by Western