人pcDNA3.1-TPX2真核表达载体的构建与表达

摘要

To construct recombinant eukaryotic expression vector carrying human TPX2 gene ( pcDNA3. 1-TPX2 ) and observe expression of TPX2 in esophageal carcinoma EC9706 cell. Methods: According to the coding region of TPX2 gene from GenBank, the PCR specific primers were designed with the help of Primer Premier 5. 0 software. And the full length of human TPX2 gene was amplified by PCR from Pqe-70-TPX2. After digested with restriction endonuclease Hind Ⅲ and Bam H Ⅰ , the TPX2 gene was inserted into eukaryotic expression vector pcDNA3. 1. Then the recombinant vector pcDNA3. 1-TPX2 was transformed into JM109, which would be identified by enzyme digestion and sequence analysis. And then the plasmid was transfected into EC9706 cell line by lipid transfection reagent. Expression of TPX2 gene was detected by Western blot. ResultS;The results of endonuclease digestion analysis and sequencing showed that TPX2 gene was successfully cloned into pcDNA3. 1 vector, and the TPX2 gene sequence was coincident with GenBank. Conclusion;The recombinant eukaryotic expression vector pcDNA3. 1-TPX2 has been constructed successfully, which provides basis for the further researches about TPX2 gene biological functions in tumor cells.%目的:构建TPX2的真核表达载体pcDNA3.1-TPX2,并观察其在食管癌EC9706细胞中的表达.方法:在pQE-70-TPX2原核表达载体基础上,根据GenBank上提供的TPX2基因序列及测序结果,采用Primer Premier 5.0软件设计扩增TPX2基因编码区的引物序列,经HindⅢ和BamHⅠ双酶切,与真核表达载体pcDNA3.1连接后转化感受态大肠杆菌JM109,并进行酶切分析和序列测定.将构建的质粒pcDNA3.1-TPX2体外转染EC9706细胞,进行稳定筛选.用Western blot鉴定基因的表达.结果:DNA测序和BLAST比对表明克隆的人TPX2基因序列正确,进一步酶切分析显示,成功构建了真核表达载体pcDNA3.1-TPX2,并在EC9706细胞中获得稳定表达.结论:TPX2基因真核表达载体的成功构建为后续研究TPX2基因在肿瘤细胞中的功能奠定了基础.