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目的:探讨人Tip30全长基因的真核表达载体pAAV-TIP30的构建,并观察其转染肝癌细胞株HepG2后的表达。方法:以正常人肝组织mRNA为模板,用RT-PCR方法扩增Tip30,利用限制性内切酶BamH I和Xba I,将Tip30基因克隆到真核表达载体pAAV-MCS中,并经酶切和测序鉴定。重组质粒转染HepG2细胞,运用Western blot法检测Tip30蛋白表达。结果:RT-PCR方法正确地扩增出全长Tip30基因。限制性内切酶酶切和测序结果证实Tip30基因克隆完全正确。重组质粒转染肝癌细胞系HepG2后,Western blot证实Tip30蛋白表达增高。结论:成功构建了真核表达重组质粒pAAV-TIP30并转染HepG2细胞,为进一步研究Tip30基因对肝癌的影响及其机制打下了基础。
Objective: To investigate the construction of eukaryotic expression vector pAAV-TIP30 of human Tip30 full-length gene and observe its expression after transfection into HepG2 hepatocellular carcinoma cell line. Methods: The Tip30 gene was cloned into the eukaryotic expression vector pAAV-MCS using restriction endonucleases BamH I and Xba I using the mRNA of human liver tissue as a template. Tip30 was amplified by RT-PCR. And sequencing identification. Recombinant plasmids were transfected into HepG2 cells and the expression of Tip30 protein was detected by Western blot. Results: The full-length Tip30 gene was amplified by RT-PCR. Restriction endonuclease digestion and sequencing confirmed that the Tip30 gene was cloned correctly. Recombinant plasmids transfected HepG2 cells, Western blot confirmed Tip30 protein expression increased. CONCLUSION: The eukaryotic expression plasmid pAAV-TIP30 was successfully constructed and transfected into HepG2 cells, which laid the foundation for the further study of the effect of Tip30 on hepatocellular carcinoma and its mechanism.