PAB-Gal4-DBD-HA-Twist重组质粒的制备

Construction of PAB-Gal4-DBD-HA-Twist Recombinant Plasmid

目的:构建人Twist基因的表达载体pAB-Gal4-DBD-HA-Twist,为研究其转录调控机制提供实验工具。方法:重组克隆质粒pcDNA-Flag-Twist用含有特异BamHⅠ和HindⅢ酶切位点的引物进行聚合酶链反应;对含有两种酶切位点的目的 Twist基因片段和pAB-Gal4-DBD-HA载体进行酶切、分离、回收纯化、连接后转入感受态DH5α中,细菌培养,菌落聚合酶链反应、测序鉴定。结果:鉴定结果显示,Twist基因正确克隆到PAB-Gal4-DBD-HA表达载体,并且转入了大肠杆菌中。结论:成功构建了人twist基因PAB-Gal4-DBDHA-Twist表达载体,为进一步研究其表达、调控、作用机制奠定基础。

To construct PAB-Gal4-DBD-HA-twist recombinant expression plasmid,providing experimental tools to study the transcription regulation.Method：The recombinant plasmid pcDNA-Flag-Twist was detected by polymerase chain reaction（PCR）using primers containing specific BamHⅠand HindⅢrestriction enzyme site.For the purpose of the Twist gene and pAB-Gal4-DBD-HA vector containing two kinds of enzyme cutting sites were studied by enzyme digestion,separation,recovery and purification,connected and transformed into competent DH5 alpha,they were detected by bacterial culture,colony polymerase chain reaction and sequencing identification.Result：The identification results showed that the twist gene recombinant plasmid was successfully constructed and transformed in E.coli.Conclusion：Full-length twist is subcloned into pAB-Gal4-DBD-HA vector successfully,and lay the foundation to further study its expression,regulation and mechanism.