本文選題：NGAL　切入點：啟動子　出處：《汕頭大學》2005年碩士論文　論文類型：學位論文

【摘要】：中性粒細胞明膠酶相關脂質運載蛋白(neutrophil gelatinase associated lipocalin,NAGL)是脂質運載蛋白(lipocallin)家族的一個新的成員,是1993 年在人中性粒細胞中首先被發(fā)現(xiàn)的。同源分析發(fā)現(xiàn),NGAL與小鼠癌基因產(chǎn)物24 p3同源性很高;另外研究發(fā)現(xiàn),在結腸癌和乳腺癌等腫瘤組織、食管癌細胞系 和卵巢癌細胞系中NGAL的表達明顯增強。這提示NGAL可能是人類的一種新的癌 基因。然而迄今,NGAL在腫瘤組織細胞中的過表達調控機制尚不清楚。為此本 ‘ 研究聯(lián)合運用PCR、DNA重組、嵌套缺失以及雙熒光素酶報告基因檢測等技術對 NGAL啟動子區(qū)進行定位,同時利用生物信息學方法預測其反式作用因子,為全 面闡明NGAL表達調控機制提供基本數(shù)據(jù)。 內容與方法: 1.提取SHEEC細胞總DNA,作為模板,利用六對引物進行PCR反應,獲取NGAL5' 側翼區(qū)不同長度片段,-1431-+84、-1137-+84、-945-+84、-657-+84、-416-+84 和-152-+84等。將上述片段首先分別插入pGEM-Teasy,然后再亞克隆到 pGl3-Basic,構建熒光素酶報告基因檢測系列質粒pB1431、pB1137、pB945、 pB657、pB416和pB152,確定NGAL啟動子的大致分布區(qū)段。 2.聯(lián)合運用嵌套缺失和雙熒光素酶報告基因檢測等技術,進一步確定NGAL啟動 子的較確切位置。 3.運用生物信息學方法預測NGAL啟動子區(qū)功能組件及其反式作用因子,探討基 因表達調控機制。 結果: 1.與空載體pGL3-Basic相比,pB1431、pB1137、pB945、pB657、pB416和pB152 等的相對熒光素酶活性均明顯增強(P0.01或0.05),說明NGAL的啟動
[Abstract]:Neutrophil gelatinase associated. Lipocalin nagl is a new member of the lipocallin family of lipids. NGAL was first found in human neutrophils. Homology analysis revealed that NGAL and mouse oncogene products 24. P3 is highly homologous. In addition, esophageal cancer cell lines have been found in cancer tissues such as colon cancer and breast cancer. The expression of NGAL in ovarian cancer cell line and ovarian cancer cell line was significantly increased. This suggests that NGAL may be a new type of cancer in human. However, the mechanism of overexpression of NGAL in tumor cells is not clear. In combination with PCR DNA recombination, nested deletion and double luciferase reporter gene detection, The promoter region of NGAL was located and its trans action factor was predicted by bioinformatics. Surface elucidation of NGAL expression regulation mechanism provides basic data. Content and methods:. 1. The total DNA of SHEEC cells was extracted and used as template. Six pairs of primers were used for PCR reaction to obtain NGAL5'. Different length segments of flanking region, Ca-1431-84ON-1137-84ON-945-84ON-657-84ON-416-84... Insert the above fragments into pGEM-Teasyat first, then subclone the pGEM-Teasy. A series of plasmids pB1431, pB1137 and pB945 were constructed for luciferase reporter gene detection. PB657, pB416 and pB152 were used to determine the approximate distribution of NGAL promoter. 2. Combined use of nested deletion and double luciferase reporter gene detection to further determine NGAL priming. The exact position of the child. 3. Using bioinformatics method to predict the functional components of NGAL promoter region and its trans action factors, and to discuss the bases. Because of the mechanism of expression regulation. Results:. 1.Compared with the empty carrier pGL3-Basic, pB1431, pB1137, pB945, pB657, pB416 and pB152. The relative luciferase activity of P0.01 or 0.05% was significantly increased, indicating the initiation of NGAL.
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

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