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

【摘要】：目的:研究人CD226基因表達(dá)調(diào)控的規(guī)律,確定CD226基因啟動子的位置,研究細(xì)胞因子、刺激劑和轉(zhuǎn)錄因子對其的調(diào)節(jié)作用。 方法:①從50ng/ml PMA刺激48小時的Jurkat細(xì)胞中提取mRNA,采用5′-RACE的方法經(jīng)過反轉(zhuǎn)錄和巢式PCR確定人CD226基因的轉(zhuǎn)錄起始點。②從GenBank中獲取CD226基因的上游調(diào)控序列,利用www.ifti.org中的轉(zhuǎn)錄因子掃描程序,分析可能存在的轉(zhuǎn)錄因子結(jié)合位點,用手工比對的方法剔除分析結(jié)果中可能存在的假陽性,篩選可能性比較大的轉(zhuǎn)錄因子進(jìn)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(shè)計引物,克隆了長約2kb的CD226基因上游調(diào)控序列。在此基礎(chǔ)上,制備了不同的截斷體,連入pGL3熒光素酶報告基因表達(dá)載體,轉(zhuǎn)染Jurkat細(xì)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(zhuǎn)染Jurkat細(xì)胞,用50ng/ml PMA和0.5μg/ml A23187分別刺激4小時和1小時,在轉(zhuǎn)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(diào)控作用。⑤把兩個啟動子間可能存在的負(fù)調(diào)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(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R392

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