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目的构建由癌胚抗原(CEA)启动子控制报道基因增强型绿色荧光蛋白(EGFP)表达的重组表达质粒CMVE-pCEA-IRES-EGFP。了解CEA启动子靶向驱动的含目的基因的重组质粒的效率。方法构建重组质粒CMVE-pCEA-IRES-EGFP,以PCR,DNA测序及酶切进行鉴定。通过脂质体介导重组质粒CMVE-pCEA-IRES-EGFP体外分别转染A549细胞(CEA阳性)和16HBE(CEA阴性)细胞,进行绿色荧光蛋白检测分析基因表达的靶向性。结果CMVE-pCEA-IRES-EGFP分别用VSPⅠ,VSPⅠ/NheⅠ酶切后电泳分别出现大小约405bp,372bp和4110bp的片断,与预计各片断大小相符。以CMVE-pCEA-IRES-EGFP为模板PCR扩增得到CMV和CEA片段,分别连于T载体(pMD18-T)并测序,结果正确。嵌合启动子CMVE-pCEA能特异地驱动下游报告基因在CEA阳性肺癌细胞株绿色荧光表达阳性,而CEA阴性细胞株16HBE表达阴性。结论成功构建了嵌合启动子CMVE-pCEA驱动的CMVE-pCEA-IRES-EGFP重组质粒,并能在转染的CEA阳性细胞株中表达,为靶向启动双基因表达载体构件提供了实验依据。
Objective To construct a recombinant plasmid CMVE-pCEA-IRES-EGFP whose expression of enhanced green fluorescent protein (EGFP) was controlled by CEA promoter. Understand the efficiency of CEA promoter-targeted driven recombinant plasmids containing the gene of interest. Methods The recombinant plasmid CMVE-pCEA-IRES-EGFP was constructed and identified by PCR, DNA sequencing and restriction enzyme digestion. A549 cells (CEA positive) and 16HBE (CEA negative) cells were transfected with liposome-mediated recombinant plasmid CMVE-pCEA-IRES-EGFP respectively, and green fluorescent protein (EGFP) was used to detect the gene expression target. Results The fragments of about 405bp, 372bp and 4110bp in CMVE-pCEA-IRES-EGFP were digested with VSPⅠ and VSPⅠ / NheⅠrespectively, which was consistent with the expected size of each fragment. The CMV and CEA fragments were amplified by PCR using CMVE-pCEA-IRES-EGFP as a template and sequenced with the T vector (pMD18-T) respectively. The results were correct. The chimeric promoter CMVE-pCEA could specifically drive the downstream reporter gene to express green fluorescence in CEA positive lung cancer cell lines, while the negative CEA negative cell line 16HBE expression. CONCLUSION CMVE-pCEA-IRES-EGFP recombinant plasmid driven by chimeric promoter CMVE-pCEA was successfully constructed and expressed in the transfected CEA positive cell lines, which provided an experimental basis for targeting the initiation of double gene expression vector components.