CMV早期转录增强子对人癌胚抗原启动子效率的作用研究

Study on the Effect of Human CMV Early Transcriptional Enhancer on Human CEA Promoter

为了探讨人巨细胞病毒(CMV)早期转录增强子用于癌胚抗原(CEA)阳性肿瘤的靶向基因治疗的意义,我们参考有关文献利用PCR法分别克隆了369 bp的人CEA启动子和531 bp的CMV早期转录增强子,构建了相应的pGL4.10荧光素酶报告基因载体,与内参照pGL4.74质粒共转染CEA阳性的人肠癌细胞LoVo、HT-29、SW620、肺癌细胞A549、乳腺癌细胞MCF7和CEA阴性的肠癌细胞SW480、宫颈癌细胞HeLa、人肺成纤维细胞LL47,利用双荧光素酶检测系统检测分析了这些细胞中CEA重组启动子的效率。结果表明,CMV增强子能够明显增强CEA启动子在CEA阳性细胞中的效率(提高7.46至70.16倍),但是也能提高CEA启动子在CEA阴性细胞中的效率(提高24.01至76.40倍)。因此上述CMV增强子虽然可以大大提高CEA启动子的效率,但是对特异性有影响,在将其用于CEA阳性肿瘤基因治疗时还需采用其它保证特异性的手段。

In order to explore the probability of Cytomegalovirus（CMV） early transcriptional enhancer in targeted gene therapy for carcinoembryonic antigen（CEA） positive cancers,CEA promoter（369 bp） and CMV early transcriptional enhancer（531 bp） were cloned by PCR using genomic DNA of human colon carcinoma SW620 cells and pcDNA3.1（＋） plasmid as template,respectively according to literatures.CEA promoter and CMV early transcriptional enhancer were subcloned into luciferase reporter vector pGL4.10 to obtain pGL4.10-CEA and pGL4.10- CMV-CEA,and co-transfected with transfection-efficiency normalization vector pGL4.74 into CEA-positive human colon carcinoma LoVo,HT-29,SW620,lung carcinoma A549 cells,breast carcinoma MCF7 cells and CEA-negative colon carcinoma SW480 cells,cervix carcinoma HeLa cells and lung fibroblast LL47 cells.The transcription initiation efficiencies of CEA promoter in these cells were analyzed by Dual Luciferase Assay system and relative luciferase unit（RLU） was used to evaluate the expression efficiency.The results showed that CMV early transcriptional enhancer up-regulated CEA promoter transcriptional activity significantly by 7.46 to 70.16 folds in the above CEA-positive cancer cells,however,it also up-regulated CEA promoter transcriptional activity in CEA-negative Hela and fibroblast cells by 24.01 to 76.40 folds.Therefore,the above CMV enhancer can greatly up-regulate CEA promoter transcriptional activity,but may attenuate the specificity of CEA promoter.The side effect of the nonspecificity of CEA promoter with CMV early transcriptional enhancer should be considered for targeted gene therapy of CEA-positive cancer.Combination of other ways such as tissue-specific transcription factor-binding sites in the upstream regulatory region are needed for maintaining the specificity of CEA promoter when utilizing CMV early transcriptional enhancer in targeted gene therapy of CEA-positive cancer.