摘要
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 new eukaryotic expression vector in which aim gene is controlled under CEA promoter and CMV enhancer, and to evaluate in vitro its target expression in CEA positive cells. Methods The recombinant plasmid （CMVE-pCEA-IRES-EGFP） was obtained by gene recombination by which the promoter of plasmid pIRES-EGFP（pCMV） was replaced by CMVE-pCEA. PCR-generated CEA and CMV genes were verified by DNA sequence analysis. Electrophoretic analysis and endonuclease methods were used to verify CMVE-pCEA-IRES-EGFP. Through liposome medium A549 （CEA positive cell） and 16HBE （CEA negative cell） were transfected with CMVE-pCEA-IRES-EGFP respectively. The expression in two cell lines was evaluated by green fluorescent protein. Results Enzymatic digestion （VSP Ⅰ, VSP Ⅰ/Nhe Ⅰ） was used to confirm CMVE-pCEA-IRES-EGFP in which characteristic fragments （about405bp, 372bp,4110bp） were created by electrophoretic analysis. There were no significant differences between these fragments sizes with prolepsis. CEA promoter and CMV enhancer were proved to be correct by DNA sequence analysis. CMVE-pCEA could drive interesting gene target expression in CEA positive cells （A549）, and there was no expression in CEA negative cells （16HBE）. Conclusion The eukarvotic expression vector CMVE-pCEA-IRES-EGFP is successfully constructed, and its target can be expressed in CEA positive cells, which provides experimental evidence for the application of aim gene to the treatment for CEA positive malignant cancer.
出处
《现代临床医学生物工程学杂志》
2006年第6期484-487,共4页
Journal of Modern Clinical Medical Bioengineering
基金
国家自然科学基金资助项目(30471997)
