人ACAT1基因的rs10753191位点SNP分析及可变剪接报告质粒构建

论文编号:SW110  论文字数:8645,页数:24

 摘 要：酰基辅酶A:胆固醇酰基转移酶(ACAT)是细胞内唯一催化游离的胆固醇和脂肪酸合成胆固醇酯的酶，在胆固醇的吸收转运与代谢平衡调控中起非常重要的作用。ACAT基因家族包含ACAT1和ACAT2两个基因。ACAT1广泛的存在于几乎各种组织细胞中，调控细胞内的胆固醇代谢平衡；而ACAT2则在肝肠细胞中特异性表达，主要参与外源饮食和肝合成的胆固醇吸收酯化和载脂蛋白的装配。人类一些重要疾病如：高胆固醇血症、引起冠心病和中风的动脉粥样硬化、以及神经系统的阿尔海默氏疾病、消化系统的结石病等，都与ACAT密切相关。因此，ACAT已成为分子筛药的重要靶蛋白。
 人酰基辅酶A:胆固醇酰基转移酶-1（ACAT-1）mRNA序列来自两条不同的染色体（1q25和7q31.3），含各自启动子P1和P7。 通过计算机单核苷酸多态性（SNP）分析，发现1号染色体对应的genomic DNA（Exon1～Exon16）中Exon10的1032位置上有一个外显子剪接增强位点（ESE），将其C突变成T后，发现SR蛋白不能与ESE结合而发生选择性剪接。有统计表明正常人Exon10 的1032位点大多为C，而冠心病（CHD）病人大多为T。本论文在此基础上展开，首先，从不同来源的CHD血样中克隆包涵Exon10的genomic DNA，测序分析1032位点的碱基。再利用Tetra-primer ARMS-PCR方法进行基因型分析。在成功构建人ACAT1 genomic DNA（Exon8～11）质粒的基础上再利用Nested PCR将Exon10 1032位点上的C突变成T，确保只有这一个点发生突变。
关键词：ACAT-1基因,DNA克隆,基因型分析,Nested PCR,点突变

Human ACAT gene rs10753191 site SNP genotyping and construction of alternative splicing plasmid

Abstract: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an intracellular enzyme catalyzing the formation of cholesteryl esters from cholesterol and long-chain fatty-acyl-coenzyme A. ACAT plays an important role in cholesterol absorption and transportation, as well as in cellular cholesterol homeostatic regulation. ACAT gene family consists of Acat1 and Acat2. In adult human tissues, ACAT1 protein is found in almost all of the cells and tissues examined, and believed to be crucial for maintaining cholesterol homeostasis.  On the other hand, ACAT2 is selectively expressed in liver and small intestine, mainly involving in the dietary cholesterol absorption and assembly of apoB-containing lipoprotein.  ACATs slao play important roles in some serious human diseases including atherosclerosis, Alzheimer’s disease (AD) and gallstone.  For these reasons, ACAT has been considered as one of major pharmaceutical targets for developing CE-lowering, anti-atherosclerosis and/or anti-AD drugs.
 Human Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) cDNA K1 is distributed over two different chromosomes (chromosome 1 and 7) with two separate promoters, designated as P1 and P7.Analyzing SNPs by computer and there is an ESE at Exon10 1032.If mutatiom the C to the T , and the SR proteid will not combine on the ESE then alternative splicing happen.There is statistic shows that normal people most with the C and DHC patients with the T.My work from here.First,Klone genomic DNA inclute Exon10 from DHC patients’blood lipid.Second, sequence and analyse 1032 site.Third,use Tetra-primer ARMS-PCR to study genotyping SNPs.Nested PCR to do mutation on the basic of constructing plasmids with human ACAT1 genomic DNA.
Keywords：ACAT-1gene,DNA klone,genotyping,Nested PCR,mutation

目  录
中文摘要 I
英文摘要 III
目录 V
1. 绪论 1
 1.1  ACAT研究的历史背景 2
 1.2  ACAT基因的克隆及ACAT家族的发现 3 
 1.3  ACAT-1基因的组织结构 4
 1.4  ACAT的组织细胞分布与生物活性 4
2. 实验部分 7
 2.1  材料及试剂 7
 2.2  实验方法 7
3. 结果与讨论 12
 3.1  结果  12
 3.2  讨论 15
4．总结与展望 16
致谢 18
参考文献 19