真核重组表达质粒pTT5-CTP-PTEN的构建及其在HEK 293-6E细胞中的表达

Construction of recombinant eukaryotic expression vector pTT5-CTP-PTEN and its expression in HEK 293-6E cells

目的构建真核重组表达质粒pTT5-CTP-PTEN,并在HEK 293-6E细胞中进行表达。方法合成融合基因CTP-PTEN,定向克隆至真核表达载体pTT5中,构建重组表达质粒pTT5-CTP-PTEN,转化感受态E.coli DH5α,提取质粒,进行双酶切及测序鉴定。在Lipofectamine 2000的介导下,将鉴定正确的重组表达质粒转染至HEK 293-6E细胞,Western blot法检测细胞中融合蛋白CTP-PTEN的表达。结果经双酶切及测序鉴定,证明重组表达质粒pTT5-CTPPTEN构建正确。转染48 h后,HEK 293-6E细胞中可见相对分子质量约49 700的CTP-PTEN融合蛋白条带。结论成功构建了重组质粒pTT5-CTP-PTEN,并于HEK 293-6E细胞中表达了CTP-PTEN融合蛋白。

Objective To construct recombinant eukaryotic expression vector pTT5-CTP-PTEN and express in HEK 293-6E cells. Methods Fusion gene CTP-PTEN was synthesized and cloned into eukaryotic expression vector pTT5. The constructed recombinant plasmid pTT5-CTP-PTEN was transformed to E.coli DH5α. The recombinant plasmid was extracted,identified by restriction enzyme digestion and DNA sequencing, and transfected to HEK 293-6E cells in mediation of Lipofectamine 2000. The expressed fusion protein was identified by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pTT5-CTP-PTEN was constructed correctly. CTP-PTEN fusion protein band,with a relative molecular mass of about 49 700, was observed in HEK 293-6E cells 48 h after transfection. Conclusion Recombinant plasmid pTT5-CTP-PTEN was successfully constructed, and CTP-PTEN fusion protein was successfully expressed in HEK 293-6E cells.