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目的 进行肝癌双自杀基因逆转录病毒转移体系的建立及鉴定, 为后续将其转染肝癌细胞、并进行抗肝癌细胞株的功能实验和功效研究奠定基础.方法 采用基因工程的方法, 将pWZLneoC-DglyTK质粒转化到 DH5α 感受态菌后, 提取质粒 DNA, 并采用酶切和 PCR 及 A260 nm 和 A260 nm/A280 nm吸光度测定等方法进行分析鉴定; 再将其质粒DNA用脂质体介导转染PA317 细胞, 然后经G418 筛选阳性克隆, 进行扩增培养PA317/CD+TK细胞, 即成功构建含大肠杆菌胞嘧啶脱氨酶( E. coli CD)和单状疱疹病毒胸苷激酶( HSV-tk)的肝癌双自杀基因逆转录病毒转移体系.结果 转化的阳性菌株可在含Amp的琼脂上生长; 质粒经1%琼脂糖凝胶电泳后可见有质粒 DNA 存在; 经单酶切和双酶切后, 分别显示相应的电泳条带; PCR鉴定可见有扩增出的CD和TK阳性条带, 测定DNA浓度为5. 25 μg/μl, 纯度为1. 81.同时, 两种质粒转入PA317 细胞48 h后荧光显微镜下可观察细胞内有绿色荧光产生, 其转染效率约为20% ~30% ; CD、 TK基因PCR分别扩增, 已整合到PA317 细胞基因组上, 其基因PCR产物的相应处分别有一特异性条带; 将RNA逆转录后进行PCR扩增, CD、 TK基因在 PA317 细胞中得到表达, 其基因RT-PCR产物的相应处也分别有一特异性条带; 另外, 透射电镜下观察到培养液上清液中病毒颗粒散在或聚集成堆, 呈圆球形, 边缘呈波状, 直径约100 nm.结论 采用上述方法并通过分析鉴定结果证实,已成功构建出肝癌双效自杀基因逆转录病毒转移体系, 这将为今后该体系对人类肝癌细胞系的 HepG2 及联合肝癌双效自杀基因前体药物并结合天然抗癌药物共济增效及代谢协同杀伤肝癌细胞奠定治疗物质和方法学基础.“,”Objective To establish and identify the double suicide gene retrovirus transfer system for cancer gene therapy. Methods The pWZLneoCDglyTK plasmid was transformed into DH5α competent bacteria. Plasmid DNA was extracted and analyzed by restriction enzyme digestion, PCR, and measurement of absorbance at A260nm and A260nm/A280nm. The plasmid DNA was then transfected into PA317 cells followed by selection of positive clones by G418 for expansion of cultured PA317/CD+TK cells. Re-sults The transformed positive strains could be grown on Amp-containing agar. The plasmid DNA could be detected by 1% agarose gel electrophoresis, and the corresponding electrophoresis bands were shown after single digestion and double digestion. The electro- phoresis of PCR product revealed bands of CD and TK. The plasmid DNA concentration was determined to be 5. 25 μg/μl and the pu-rity was 1. 81. After transfection of plasmids into PA317 cells for 48 h, green fluorescence was observed in the cells under the fluores-cence microscope. The transfection efficiency was approximately 20% -30% . The electrophoresis of PCR product revealed that CD and TK genes were integrated into the PA317 cell genome. RT-PCR showed that CD and TK genes expressed in PA317 cells. Trans-mission electron microscopy showed virus particles in the culture supernatant. Conclusion Retrovirus transfer system of double sui-cide gene has been successfully constructed.