PcDNA3.1-Tum-5基因真核表达载体的构建

The Construction of PcDNA3.1-Tum-5 Eukaryotic Expression Vectors

目的构建携带Tum-5基因真核表达载体PcDNA3.1-Tum-5。方法利用巢式PCR扩增人胚肾细胞株HEK293 cDNA获得Tum-5基因,并利用DNA回收试剂盒进行回收;同时扩增、提取pcDNA3.1质粒,并将其与Tum-5基因分别进行EcoRV和XhoI双酶切,回收相应条带,通过T4DNA连接酶进行连接,转化DH5α感受态细胞,获得阳性克隆。获得质粒DNA后行KpnI和XhoI双酶切,克隆并测序酶切产物。结果巢式PCR扩增出的产物与目的基因大小相同;重组质粒经双酶切电泳分析,与预期结果一致;对连接点两端进行测序,结果显示接头两端序列连接正确,插入序列与载体读码框架相匹配。结论本方法可以成功构建人Tum-5基因表达载体,为进一步研究Tum-5基因功能提供了理论依据。

Objective To construct PcDNA3.1-Tum-5 eukaryotic expression vector carrying Tum-5 genes.Methods Tum-5 genes were constructed from human embryonic kidney cell line HEK293 cDNA by using nested PCR and DNA extraction kit.PcDNA3.1 plasmid was amplified and extracted simultaneously.The Tum-5 genes and PcDNA3.1 plasmid received EcoRV and XhoI double digestions respectively,recovery corresponding bands were connected by T4DNA ligase,transformed DH5α competent cells,and obtained positive clones.Plasmid DNA received KpnI and XhoI double digestions,digestion products were cloned and sequenced.Results Production amplified by nested PCR had the same size with the target genes;the analytic results of recombinant plasmid by double digestion electrophoresis were consistent with the expected results.Both ends of the connection points were sequenced,the results showed the connection of sequences were correct at both ends,the inserted sequences matched with vector reading frame.Conclusion Tum-5 gene expression vector is constructed successfully in this way.This lays the foundation for the further study of Tum-5 gene function.