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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(P<0.05)。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。
Objective To construct eukaryotic co-expression vector of enhanced green fluorescent protein and Rb94 gene and identify its expression in eukaryotic cells. Methods The target gene fragment in cDNA-Rb plasmid was amplified by PCR and double-digested with BamHⅠ and SalⅠ. The DNA fragments were excised by gel filtration. Then ligated into pEGFP-C1 to construct pEGFP-C1 / Rb94 eukaryotic expression vector and identified by restriction enzyme digestion. The recombinant plasmids were transfected into human retinoblastoma cells (HXO-Rb44) by lipofectamine. The expression of EGFP in cells was observed by fluorescence microscopy. The expression of Rb protein was detected by Western blot. The expression of Rb protein was detected by flow cytometry Detect the rate of apoptosis. Results The sequence of the target fragment in the recombinant cloning vector was identical to the sequence of Rb94 gene. The results of pEGFP-C1 / Rb94 restriction analysis were consistent with the expected results. Fluorescence microscopy showed transfected cells with fluorescence appeared. Western blot results showed that Rb94 gene was effectively expressed. Flow cytometry results showed that the rate of apoptosis of Rb94-containing cells was (21.17 ± 0.45)%, which was significantly higher than that of other groups (P <0.05). Conclusion The eukaryotic co-expression vector of EGFP and Rb94 was successfully constructed and expressed efficiently in eukaryotic cells. Rb94 significantly inhibited the growth of retinoblastoma cells.