本文選題：CD226 + 啟動子�。� 參考：《第四軍醫(yī)大學》2005年博士論文

【摘要】：目的:研究人CD226基因表達調控的規(guī)律,確定CD226基因啟動子的位置,研究細胞因子、刺激劑和轉錄因子對其的調節(jié)作用。 方法:①從50ng/ml PMA刺激48小時的Jurkat細胞中提取mRNA,采用5′-RACE的方法經過反轉錄和巢式PCR確定人CD226基因的轉錄起始點。②從GenBank中獲取CD226基因的上游調控序列,利用www.ifti.org中的轉錄因子掃描程序,分析可能存在的轉錄因子結合位點,用手工比對的方法剔除分析結果中可能存在的假陽性,篩選可能性比較大的轉錄因子進行功能研究。③從正常人外周血中提取基因組DNA,以GenBank中獲得的序列為模板設計引物,克隆了長約2kb的CD226基因上游調控序列。在此基礎上,制備了不同的截斷體,連入pGL3熒光素酶報告基因表達載體,轉染Jurkat細胞48小時后檢測熒光素酶活性,確定CD226的啟動子的位置。④將含有CD226基因啟動子序列的報告基因載體轉染Jurkat細胞,用50ng/ml PMA和0.5μg/ml A23187分別刺激4小時和1小時,在轉染48小時后檢測熒光素酶活性,觀察PMA和A23187對CD226基因啟動子的調控作用。⑤把兩個啟動子間可能存在的負調控元件(NRE)通過PCR的方法,克隆到CMV啟動子的下游,DNA測序正確后將CMV-NRE
[Abstract]:Aim: to study the regulation of human CD226 gene expression, determine the position of CD226 gene promoter, and study the regulatory effects of cytokines, stimulators and transcription factors.Methods MRNAs were extracted from Jurkat cells stimulated by 50ng/ml PMA for 48 hours. The upstream regulatory sequence of CD226 gene was obtained from GenBank by reverse transcription and nested PCR by 5'-RACE.Using the transcription factor scanning program in www.ifti.org, the possible transcription factor binding sites were analyzed, and the false positives in the analysis results were eliminated by the method of manual comparison.Functional study on the screening of transcription factors. 3 the genomic DNAs were extracted from the peripheral blood of normal subjects. Using the sequences obtained from GenBank as template primers, the upstream regulatory sequence of CD226 gene was cloned.On this basis, different truncation bodies were prepared and inserted into pGL3 luciferase reporter gene expression vector. After 48 hours of transfection into Jurkat cells, luciferase activity was detected.The reporter gene vector containing the promoter sequence of CD226 gene was transfected into Jurkat cells. The promoter was stimulated with 50ng/ml PMA and 0.5 渭 g/ml A23187 for 4 hours and 1 hour, respectively. The luciferase activity was detected after 48 hours of transfection.Observation of the regulatory effect of PMA and A23187 on the promoter of CD226 gene ..5 the possible negative regulatory element between the two promoters was cloned into the downstream of the CMV promoter by PCR, and the CMV-NRE was sequenced correctly.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R392

【相似文獻】

相關期刊論文 前10條

1 馬凌,曹廣文;組織特異性轉錄調控序列及在腫瘤基因治療研究中的應用[J];國外醫(yī)學.免疫學分冊;1997年03期

2 郭亞兵,戴煒,盧橋生,侯金林,馮筱榕,駱抗先;重型乙型肝炎病毒C基因啟動子變異[J];中華傳染病雜志;1999年02期

3 齊永;高江平;李琦;洪寶發(fā);伍志強;趙亞力;;阿司匹林對基質金屬蛋白酶9啟動子活性的影響[J];中國臨床藥理學與治療學;2005年10期

4 王軍利;張惠中;白萬勝;劉麗;卞卡;成詩銀;;喉癌組織中TSLC1基因啟動子過甲基化及mRNA表達[J];第四軍醫(yī)大學學報;2007年07期

5 武圣明,鄒竹英,周向軍,蔡龍榮,溫肇榮;人αB干擾素在Saccharomyce cerevisiae中的合成和分泌[J];第二軍醫(yī)大學學報;1988年04期

6 張玉彬;吳梧桐;王e，

本文編號：1756359