军事医学
軍事醫學
군사의학
BULLETIN OF THE ACADEMY OF MILITARY MEDICAL SCIENCES
2014年
12期
932-935
,共4页
冀全博%徐小洁%张强%梁迎春%王涛%李玲%黄蓉%周丽英%史平安%王岩%叶棋浓
冀全博%徐小潔%張彊%樑迎春%王濤%李玲%黃蓉%週麗英%史平安%王巖%葉棋濃
기전박%서소길%장강%량영춘%왕도%리령%황용%주려영%사평안%왕암%협기농
细胞周期蛋白质依赖激酶类%基因,p53%真核表达
細胞週期蛋白質依賴激酶類%基因,p53%真覈錶達
세포주기단백질의뢰격매류%기인,p53%진핵표체
cyclin-dependent kinases%genes,p53%eukaryotic expression
目的：构建带Myc标签的细胞周期蛋白依赖性激酶7（cyclin－dependent kinase 7， CDK7）的真核表达载体，获得其表达产物，并验证该激酶与已知相互作用蛋白P53之间的相互作用。方法应用PCR技术从人乳腺文库中扩增出CDK7全长编码区基因，将其克隆到pXJ－40载体中；继而重组质粒转染人胚肾293 T细胞，以SDS－PAGE和Western印迹鉴定表达情况；免疫共沉淀检测Myc－CDK7与FLAG－p53的相互作用。结果双酶切和基因测序鉴定显示，Myc－CDK7真核表达质粒克隆构建成功；SDS－PAGE和Western印迹结果表明，Myc－CDK7转染人胚肾293T细胞后成功表达；免疫共沉淀显示，重组Myc－CDK7与已知相互作用蛋白P53在蛋白质水平上具有相互作用，证实其具有生物学活性。结论成功构建Myc－CDK7的真核表达载体，为进一步探讨Myc－CDK7对细胞周期的调控奠定了实验基础。
目的：構建帶Myc標籤的細胞週期蛋白依賴性激酶7（cyclin－dependent kinase 7， CDK7）的真覈錶達載體，穫得其錶達產物，併驗證該激酶與已知相互作用蛋白P53之間的相互作用。方法應用PCR技術從人乳腺文庫中擴增齣CDK7全長編碼區基因，將其剋隆到pXJ－40載體中；繼而重組質粒轉染人胚腎293 T細胞，以SDS－PAGE和Western印跡鑒定錶達情況；免疫共沉澱檢測Myc－CDK7與FLAG－p53的相互作用。結果雙酶切和基因測序鑒定顯示，Myc－CDK7真覈錶達質粒剋隆構建成功；SDS－PAGE和Western印跡結果錶明，Myc－CDK7轉染人胚腎293T細胞後成功錶達；免疫共沉澱顯示，重組Myc－CDK7與已知相互作用蛋白P53在蛋白質水平上具有相互作用，證實其具有生物學活性。結論成功構建Myc－CDK7的真覈錶達載體，為進一步探討Myc－CDK7對細胞週期的調控奠定瞭實驗基礎。
목적：구건대Myc표첨적세포주기단백의뢰성격매7（cyclin－dependent kinase 7， CDK7）적진핵표체재체，획득기표체산물，병험증해격매여이지상호작용단백P53지간적상호작용。방법응용PCR기술종인유선문고중확증출CDK7전장편마구기인，장기극륭도pXJ－40재체중；계이중조질립전염인배신293 T세포，이SDS－PAGE화Western인적감정표체정황；면역공침정검측Myc－CDK7여FLAG－p53적상호작용。결과쌍매절화기인측서감정현시，Myc－CDK7진핵표체질립극륭구건성공；SDS－PAGE화Western인적결과표명，Myc－CDK7전염인배신293T세포후성공표체；면역공침정현시，중조Myc－CDK7여이지상호작용단백P53재단백질수평상구유상호작용，증실기구유생물학활성。결론성공구건Myc－CDK7적진핵표체재체，위진일보탐토Myc－CDK7대세포주기적조공전정료실험기출。
Objective To construct the eukaryotic expression vector of cyclin-dependent kinase ( CDK) 7 labeled with Myc tag, obtain the expressed product , and identify its interaction with FLAG-P53 at the protein level .Methods Human CDK7 coding gene region amplified from the mammary cDNA library by PCR was inserted into the pXJ -40 vector.The recombinant plasmid Myc-CDK7 transfected into human 293T cell lines was investigated and examined by SDS-PAGE and Western blotting.In addition,assay was applied to determine the interaction between Myc-CDK7 and FLAG-p53.Results The coding region of CDK7 was successfully amplified by PCR and cloned into pXJ-40 vector, which was identified by double enzyme digestion and gene sequencing .Myc-CDK7 was successfully expressed in human 293T cell lines according to SDS-PAGE and Western blotting assay indicated that Myc-CDK7 could interact with P53 protein, which verified its known function .Conclusion The eukaryotic expression vector Myc-CDK7 is successfully obtained , which will contribute to further research on CDK 7-mediated cell cycle regulation .
