Tip30真核表达载体的构建及其在HepG2细胞中的表达  

Construction of TIP30 expression vector and its expression in HepG2 cells

作　　者：李浩[1] 吴金术[1] 龚连生[2] 潘一峰[2] 王渊璟[2] 刘勤[2] 王宪伟[3] 

机构地区：[1]湖南省人民医院肝胆外科,湖南长沙410002 [2]中南大学湘雅医院卫生部肝胆肠外科中心,湖南长沙410008 [3]中南大学湘雅医院普通外科,湖南长沙410008

出　　处：《中国普通外科杂志》2012年第1期58-62,共5页China Journal of General Surgery

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。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, q-he PCR products were digested with BamH I and Xba I, and cloned into eukaryotic expressing vector pAAV-MCS, qlae 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 amplified 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 successfully, which can provide a basis for the further the underlying mechanisms as well. has been cloned and transfected into HepG2 cells investigation of the effects of TIP30 gene on HCC and

关 键 词：癌 肝细胞 基因表达 转染 真核表达载体 

分 类 号：R735.7[医药卫生—肿瘤]