PTEN基因亚克隆和pTRE-PTEN反应质粒构建

Subcloning of PTEN gene and construction of pTRE-PTEN responsive plasmids

目的 亚克隆野生型与张力蛋白在 10号染色体同源缺失的磷酸酶 (PTEN)基因 ,构建四环素反应性元件调控的与张力蛋白在 10号染色体同源缺失的磷酸酶反应质粒 (pTRE PTEN)。方法以pBP PTEN质粒为模板 ,应用pfuDNA聚合酶 ,多聚酶链反应法 (PCR)扩增PTENcDNA。将扩增的PTENcDNA与三磷酸脱氧腺苷 (dATP)反应 ,然后将产物与pGEM T Easy载体直接连接 ;连接产物转化大肠杆菌DH5α,蓝白筛选 ,挑选白色克隆作鉴定测序。将测序正确的pGEM T Easy/PTEN和带潮霉素筛选标记的四环素反应性元件质粒 (pTRE2Hyg)分别双酶切 ,前者回收 1.2kb的PTEN片段 ,后者回收线性化的pTRE2Hyg片段 ,将两者连接成 pTRE PTEN表达重组体 ,转化大肠杆菌DH5α ,抽提重组体质粒 ,经NotⅠ和SalⅠ酶切鉴定阳性克隆含有大小约 6 .4kb和 1.2kb的两条带。结果 成功地从 pBP PTEN质粒中亚克隆人PTENcDNA ,并构建了pTRE PTEN反应质粒。结论 pTRE PTEN反应质粒的构建为建立pTet on PTEN

Objective To subclone human cDNA of gene of phosphatase and tensin homology deleted on chromosome ten (PTEN) and construct plasmids of tetracycline(Tet) responsive element,which regulates and controls the expression of PTEN (pTRE PTEN).Methods Utilizing pfuDNA polymerase and modeling pBP PTEN plasmids, PTENcDNA was amplified by polymerase chain reaction(PCR), and PTENcDNA was added “A” by the reaction with deoxyadenosine triphosphate(dATP). The purified products of PTENcDNA were ligated with pGEM T easy vector, and were transformed into DH5α colibacilli. The white clones were selected and the plasmids were purified,which were further identified by double enzyme digestion and were sequenced. Sequence proofed pGEM T Easy/PTEN and plasmids of Tet responsive element with the selection marker of hygromycine(pTRE2Hyg) were digested by double enzyme respectively and the purified 1.2Kb PTEN fragment of the former and the purified linear fragment of the latter were ligated and transferred into DH5α colibacilli. The clones were randomly selected and recombinant plasmids were purified, which were identified by double enzyme digestion. Results PTENcDNA was successfully subcloned and recombinant pTRE PTEN responsive plasmids were constructed . Conclusion Construction of recombinant pTRE PTEN responsive plasmids settles the basis for the establishment of pTet on PTEN U87MG glioma cell line.