【摘要】 目的构建真核表达载体pIRES2-DsRed2-Hath1,并验证其在HEK293T细胞中的表达。方法从人的全血标本中提取DNA模板,经PCR获得Hath1-cDNA,将Hath1-cDNA和pIRES2-DsRed2质粒用限制性内切酶XhoⅠ、EcoRⅠ双酶切,酶切产物琼脂糖凝胶电泳,割胶纯化,前者回收1065bp片断,后者回收大片断,再将回收的2个片断用T4DNA连接酶连接,转化感受态DH5α细胞,挑选单克隆,提取质粒,将所提取的质粒双酶切鉴定并测序。再将构建成功的pIRES2-DsRed2-Hath1质粒通过脂质体LipofectamineTM2000转染HEK293T细胞,观测其转染情况和目的基因的表达情况。结果成功地构建了真核表达载体pIRES2-DsRed2-Hath1,该质粒能有效地转染HEK293T细胞,目的基因能有效表达。结论pIRES2-DsRed2-Hath1的构建为Hath1基因转染致聋小鼠的耳蜗的实验奠定了基础。更多还原

【Abstract】 [Objective] To construct pIRES2-DsRed2-Hath1, and to detect its expression in HEK293T. [Methods] At first, we purified nucleic acids from human whole blood. By PCR amplification, we got Hath1-cDNA from the purified template DNA. Then we digested the PCR product and the pIRES2-DsRed2 by double-enzyme respectively. By agarose gel electrophoresis, and we collected and purified the 1065bp PCR fragment of the former and the large fragment of the latter. Then the two purified products were ligated by T4 DNA ligase. Then the ligated product was transferred into DH5α E coli bacteria. After cultured overnight in LK plate, the clones were selected and the recombinant plasmids were purified, which were identified by double-enzyme digestion and sequencing. Then by cationic liposome, the pIRES2-DsRed2-Hath1 was transferred into HEK293T cell. Finally, we detected the expression of the interesting gene Hath1. [Results] The pIRES2-DsRed2-Hath1 was successfully constructed, and it could be effectively transferred into HEK293T cell by cationic liposome where Hath1 could properly express. [Conclusion] The construction of pIRES2-DsRed2-Hath1 lays the groundwork for the following experiments, such as Hath1 gene transfection in deaf cochlea.更多还原