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目的 探讨 β 葡萄糖醛酸苷酶 (βG)基因对人膀胱癌T2 4细胞的转染及表达的可行性。方法 采用基因工程技术构建带 βG基因的真核表达载体 ,利用脂质体介导法在体外将 βG基因导入人膀胱癌T2 4细胞 ,利用mRNA打点杂交、原位杂交、免疫组织化学、Westernbloting方法检测βG基因的转录和表达情况。 结果 成功克隆出含人 βG基因全长的真核表达载体 pcDNA3 .1 βG ,以阳离子脂质体LipofectAMINE为载体将βG基因转染T2 4细胞后 ,经 40 0mg/L的G41 8筛选后可形成抗性克隆 ;βG基因打点杂交和原位杂交结果显示转基因细胞中有 βGmRNA的高表达 ;免疫组织化学和Westernbloting证实转基因细胞中有 βG蛋白的强阳性高表达。结论 βG基因可被成功转染入人膀胱癌细胞并能有效表达
Objective To investigate the feasibility of transfection and expression of β-glucuronidase (βG) gene in human bladder cancer T2 4 cells. Methods The eukaryotic expression vector with βG gene was constructed by genetic engineering. ΒG gene was introduced into human bladder carcinoma T2 4 cells by liposome-mediated method. The mRNA expression was detected by dot blot hybridization, in situ hybridization, immunohistochemistry and Western blotting Detection of βG gene transcription and expression. Results The full-length eukaryotic expression vector pcDNA3 .1G containing human βG gene was successfully cloned, and the βG gene was transfected into T2 4 cells with LipofectAMINE as a carrier, and then was screened by 4041mg / L G41 8 Resistant clone; βG gene RBI and in situ hybridization results showed high expression of βG mRNA in transgenic cells; strong positive expression of βG protein in transgenic cells was confirmed by immunohistochemistry and Western blotting. Conclusion The βG gene can be successfully transfected into human bladder cancer cells and can be efficiently expressed