中国医师杂志
中國醫師雜誌
중국의사잡지
JOURNAL OF CHINESE PHYSICIAN
2009年
7期
871-874
,共4页
袁娟%肖嵘%陆前进%李亚萍%杨心洁%汪峰%湛意%张桂英%张静%周英
袁娟%肖嶸%陸前進%李亞萍%楊心潔%汪峰%湛意%張桂英%張靜%週英
원연%초영%륙전진%리아평%양심길%왕봉%담의%장계영%장정%주영
膜糖蛋白类/生物合成%克隆,分子%DNA甲基化
膜糖蛋白類/生物閤成%剋隆,分子%DNA甲基化
막당단백류/생물합성%극륭,분자%DNA갑기화
Membrane glycopreteins/BI%Cloning,molecular%DNA methylation
目的 克隆穿孔素基因启动子区域(PRF1),并对PRF1进行体外区域性高甲基化,为探讨穿孔素基因调控序列高甲基化能否引起穿孔素表达下调奠定基础.方法 PCR扩增穿孔素基因启动子区域(PRF1)后,先将PRF1克隆到T载体,再定向亚克隆至报告基因栽体;然后对PRF1片段进行体外区域性高甲基化.结果 (1)克隆PRF1到报告基因载体pGL3-Basic后双酶切及测序鉴定结果正确.(2)对PRF1片段进行体外区域性高甲基化后鉴定显示PRF1片段区域性高甲基化完全.结论 成功克隆PRF1至报告基因载体pGL3-Basic并对PRF1片段进行体外区域性高甲基化,为今后研究奠定了基础.
目的 剋隆穿孔素基因啟動子區域(PRF1),併對PRF1進行體外區域性高甲基化,為探討穿孔素基因調控序列高甲基化能否引起穿孔素錶達下調奠定基礎.方法 PCR擴增穿孔素基因啟動子區域(PRF1)後,先將PRF1剋隆到T載體,再定嚮亞剋隆至報告基因栽體;然後對PRF1片段進行體外區域性高甲基化.結果 (1)剋隆PRF1到報告基因載體pGL3-Basic後雙酶切及測序鑒定結果正確.(2)對PRF1片段進行體外區域性高甲基化後鑒定顯示PRF1片段區域性高甲基化完全.結論 成功剋隆PRF1至報告基因載體pGL3-Basic併對PRF1片段進行體外區域性高甲基化,為今後研究奠定瞭基礎.
목적 극륭천공소기인계동자구역(PRF1),병대PRF1진행체외구역성고갑기화,위탐토천공소기인조공서렬고갑기화능부인기천공소표체하조전정기출.방법 PCR확증천공소기인계동자구역(PRF1)후,선장PRF1극륭도T재체,재정향아극륭지보고기인재체;연후대PRF1편단진행체외구역성고갑기화.결과 (1)극륭PRF1도보고기인재체pGL3-Basic후쌍매절급측서감정결과정학.(2)대PRF1편단진행체외구역성고갑기화후감정현시PRF1편단구역성고갑기화완전.결론 성공극륭PRF1지보고기인재체pGL3-Basic병대PRF1편단진행체외구역성고갑기화,위금후연구전정료기출.
Objective To construct a luciferase reporter gene vector of perforin promoter and methylate it in vitro. Methods The promoter of the human peffofin was amplified by PCR, cloned into pMD18-T vector and subcloned into pGL3-Basie vector, and then con-finned by restriction mapping and DNA sequencing. The regions of interest were excised with the appropriate restriction endonucleases, then it were methylated with methylase Sss I(M. SssI)and S-adenosymethioine(SAM), and methylation was confirmed by digestion with appropri-ate methylation sensitive enzyme AciI and agrose gel electrophoresis, and then the fragment was relegated back into the promoter-reporter constructor pGL3-Basic. Results The result of DNA sequencing showed that the sequence of cloned promoter was right. The result of diges-tion methylation with appropriate methylation sensitive enzyme showed that perforin promoter was completely methylated. Conclusion The promoter of perforin was successfully cloned and completely methylated in vitro, which provided an important basis for the study of transfec-tion.
