携带hTERT-P2A-EGFP基因真核表达质粒的构建和鉴定

摘要

目的:构建真核表达质粒hTERT-P2A-EGFP,探讨其在HEK293FT细胞中的表达和转染效率.方法:利用pBABE-puro-hTERT和pRRLSIN-cPPT-MSCV-EGFP质粒构建重组质粒.以该质粒为模板采用PCR法获取hTERT、P2A和EGFP基因,采用重叠PCR法获得目的片段hTERT-P2A-EGFP,经酶切后目的片段与真核表达载体pRRLSIN-cPPT-MSCV-EGFP连接,得到并鉴定含有hTERT-P2A-EGFP基因的重组质粒.经脂质体介导重组质粒转染到HEK293FT细胞,荧光显微镜观察绿色荧光蛋白(GFP)在细胞中的表达.结果:PCR检测目的基因hTERT、P2A和EGFP的片段长度分别为3 400、110和720 bp.酶切后目的基因片段hTERT-P2A-EGFP约4 300 bp.测序分析,目的基因1 547位点发生了突变.利用定点突变技术成功诱变,再次测序后目的基因序列与 GenBank 公布的基因序列完全一致.重组质粒转染HEK293FT细胞后在荧光显微镜下可以观察到GFP.流式细胞术检测,重组质粒转染HEK293FT细胞的效率为44.8%.结论:成功构建携带目的基因hTERT-P2A-EGFP的重组质粒,且可用于细胞转染.%Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.