本文選題：NGAL　切入點(diǎn)：啟動(dòng)子　出處：《汕頭大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：中性粒細(xì)胞明膠酶相關(guān)脂質(zhì)運(yùn)載蛋白(neutrophil gelatinase associated lipocalin,NAGL)是脂質(zhì)運(yùn)載蛋白(lipocallin)家族的一個(gè)新的成員,是1993 年在人中性粒細(xì)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(jié)腸癌和乳腺癌等腫瘤組織、食管癌細(xì)胞系 和卵巢癌細(xì)胞系中NGAL的表達(dá)明顯增強(qiáng)。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(xì)胞中的過(guò)表達(dá)調(diào)控機(jī)制尚不清楚。為此本 ‘ 研究聯(lián)合運(yùn)用PCR、DNA重組、嵌套缺失以及雙熒光素酶報(bào)告基因檢測(cè)等技術(shù)對(duì) NGAL啟動(dòng)子區(qū)進(jìn)行定位,同時(shí)利用生物信息學(xué)方法預(yù)測(cè)其反式作用因子,為全 面闡明NGAL表達(dá)調(diào)控機(jī)制提供基本數(shù)據(jù)。 內(nèi)容與方法: 1.提取SHEEC細(xì)胞總DNA,作為模板,利用六對(duì)引物進(jìn)行PCR反應(yīng),獲取NGAL5' 側(cè)翼區(qū)不同長(zhǎng)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(gòu)建熒光素酶報(bào)告基因檢測(cè)系列質(zhì)粒pB1431、pB1137、pB945、 pB657、pB416和pB152,確定NGAL啟動(dòng)子的大致分布區(qū)段。 2.聯(lián)合運(yùn)用嵌套缺失和雙熒光素酶報(bào)告基因檢測(cè)等技術(shù),進(jìn)一步確定NGAL啟動(dòng) 子的較確切位置。 3.運(yùn)用生物信息學(xué)方法預(yù)測(cè)NGAL啟動(dòng)子區(qū)功能組件及其反式作用因子,探討基 因表達(dá)調(diào)控機(jī)制。 結(jié)果: 1.與空載體pGL3-Basic相比,pB1431、pB1137、pB945、pB657、pB416和pB152 等的相對(duì)熒光素酶活性均明顯增強(qiáng)(P0.01或0.05),說(shuō)明NGAL的啟動(dòng)
[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(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346

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