Construction of enhanced green fluorescent protein and Rb94 co-expression vector and its expression

郑敏明; 周希瑗; 王丽萍

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Construction of enhanced green fluorescent protein and Rb94 co-expression vector and its expression

更新时间：2026-01-09

- Construction of enhanced green fluorescent protein and Rb94 co-expression vector and its expression
- Journal of Army Medical University Vol. 32, Issue 15, Pages: 1625-1627(2010)
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  CLC： R394-33;R394.3;R739.72
- Published：2010
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郑敏明,周希瑗,王丽萍.增强型绿色荧光蛋白与Rb94基因共表达载体的构建和表达[J].陆军军医大学学报(原第三军医大学学报),2010,32(15):1625-1627.

　Zheng Minming,Zhou Xiyuan,Wang Liping.Construction of enhanced green fluorescent protein and Rb94 co-expression vector and its expression[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):1625-1627.
郑敏明,周希瑗,王丽萍.增强型绿色荧光蛋白与Rb94基因共表达载体的构建和表达[J].陆军军医大学学报(原第三军医大学学报),2010,32(15):1625-1627. DOI：

　Zheng Minming,Zhou Xiyuan,Wang Liping.Construction of enhanced green fluorescent protein and Rb94 co-expression vector and its expression[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(15):1625-1627. DOI：

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BamHⅠ和SalⅠ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基因的细胞凋亡率为(21.17±0.45)%，明显高于其他组（P0.05）。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。

Abstract

Objective To construct the enhanced green fluorescent protein (EGFP) and Rb94 co-expression vector and identify its expression in cultured eukaryocytes. Methods PCR was performed to amplify Rb94 gene fragments from cDNA-Rb plasmids. Rb94 gene fragments

digested with BamHⅠand SalⅠ

were harvested and inserted into pEGFP-C1 to construct the pEGFP-C1/Rb94 expression vector. Recombinant plasmid pEGFP-C1/Rb94 was transfected into human retinoblastoma (HXO-Rb44) cells with liposome. Its expression was observed under a fluorescence microscope. Expression of Rb protein was detected by Western blotting. Apoptotic rate of HXO-Rb44 cells was detected by flow cytometry. Results The sequence of cloned DNA fragments was fully consistent with that of the Rb94 gene. The Rb94 gene was inserted into the eukaryotic expression vector

pEGFP-C1. The recombinant expression plasmid was successfully transfected into HXO-Rb44 cells. Fluorescent microscopy showed that fluorescence occurred in HXO-Rb44 cells. Western blotting demonstrated the expression of Rb protein. Flow cytometry revealed that the apoptotic rate of HXO-Rb44 cells containing the Rb94 gene was significantly higher than that of other cells. Conclusion A recombinant eukaryotic co-expression vector of EGFP and Rb94 can be successfully constructed

which is effectively expressed in HXO-Rb44 cells. The Rb94 gene can effectively inhibit the growth of HXO-Rb44 cells.

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