【摘要】 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。更多还原

【Abstract】 Objective:To investigate the construction of eukaryotic expressing vector containing human full-length Tip30 gene(pAAV-TIP30) and observe its expression in the transfected hepatocellular carcinoma(HCC) cell line HepG2.Methods:Using the mRNA extracted from normal human liver tissue as a template,Tip30 gene was amplified through reverse transcription polymerase chain reaction(RT-PCR) method.The PCR products were digested with BamH I and Xba I,and cloned into eukaryotic expressing vector pAAV-MCS.The recombinant plasmids were then transfected into the HepG2 cells and the expression of TIP30 protein in the host cells was detected by Western blot analysis.Results:Full-length TIP30 gene was correctly ampli ed by RT-PCR method.The identity of the cloning of TIP30 gene in pAAV-MCS was confirmed by restrictive enzyme digestion and sequencing.Western blot showed that the protein expression of TIP30 increased in the HepG2 cells,after they were transfected with the recombinant plasmids.Conclusion:Recombinant plasmid pAAV-Tip30 has been cloned and transfected into HepG2 cells successfully,which can provide a basis for the further investigation of the effects of TIP30 gene on HCC and the underlying mechanisms as well.更多还原