NGAL启动子区的克隆与初步鉴定

发布时间：2018-03-08 17:58

本文选题：NGAL　切入点：启动子　出处：《汕头大学》2005年硕士论文　论文类型：学位论文

【摘要】：中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase associated lipocalin,NAGL)是脂质运载蛋白(lipocallin)家族的一个新的成员,是1993 年在人中性粒细胞中首先被发现的。同源分析发现,NGAL与小鼠癌基因产物24 p3同源性很高;另外研究发现,在结肠癌和乳腺癌等肿瘤组织、食管癌细胞系和卵巢癌细胞系中NGAL的表达明显增强。这提示NGAL可能是人类的一种新的癌基因。然而迄今,NGAL在肿瘤组织细胞中的过表达调控机制尚不清楚。为此本 ‘ 研究联合运用PCR、DNA重组、嵌套缺失以及双荧光素酶报告基因检测等技术对 NGAL启动子区进行定位,同时利用生物信息学方法预测其反式作用因子,为全面阐明NGAL表达调控机制提供基本数据。内容与方法: 1.提取SHEEC细胞总DNA,作为模板,利用六对引物进行PCR反应,获取NGAL5' 侧翼区不同长度片段,-1431-+84、-1137-+84、-945-+84、-657-+84、-416-+84 和-152-+84等。将上述片段首先分别插入pGEM-Teasy,然后再亚克隆到 pGl3-Basic,构建荧光素酶报告基因检测系列质粒pB1431、pB1137、pB945、 pB657、pB416和pB152,确定NGAL启动子的大致分布区段。 2.联合运用嵌套缺失和双荧光素酶报告基因检测等技术,进一步确定NGAL启动子的较确切位置。 3.运用生物信息学方法预测NGAL启动子区功能组件及其反式作用因子,探讨基因表达调控机制。结果: 1.与空载体pGL3-Basic相比,pB1431、pB1137、pB945、pB657、pB416和pB152 等的相对荧光素酶活性均明显增强(P0.01或0.05),说明NGAL的启动
[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.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【相似文献】