人ACAT1基因的rs10753191位點SNP分析及可變剪接報告質粒構建

生物工程論文編號:SW110  論文字數:8645,頁數:24

目  錄
中文摘要 I
英文摘要 III
目錄 V
1. 緒論 1
 1.1  ACAT研究的歷史背景 2
 1.2  ACAT基因的克隆及ACAT家族的發現 3 
 1.3  ACAT-1基因的組織結構 4
 1.4  ACAT的組織細胞分布與生物活性 4
2. 實驗部分 7
 2.1  材料及試劑 7
 2.2  實驗方法 7
3. 結果與討論 12
 3.1  結果  12
 3.2  討論 15
4．總結與展望 16
致謝 18
參考文獻 19

 摘 要：�；�輔酶A:膽固醇酰基轉移酶(ACAT)是細胞內唯一催化游離的膽固醇和脂肪酸合成膽固醇酯的酶，在膽固醇的吸收轉運與代謝平衡調控中起非常重要的作用。ACAT基因家族包含ACAT1和ACAT2兩個基因。ACAT1廣泛的存在于幾乎各種組織細胞中，調控細胞內的膽固醇代謝平衡；而ACAT2則在肝腸細胞中特異性表達，主要參與外源飲食和肝合成的膽固醇吸收酯化和載脂蛋白的裝配。人類一些重要疾病如：高膽固醇血癥、引起冠心病和中風的動脈粥樣硬化、以及神經系統的阿爾海默氏疾病、消化系統的結石病等，都與ACAT密切相關。因此，ACAT已成為分子篩藥的重要靶蛋白。
 人�；�輔酶A:膽固醇酰基轉移酶-1（ACAT-1）mRNA序列來自兩條不同的染色體（1q25和7q31.3），含各自啟動子P1和P7。 通過計算機單核苷酸多態性（SNP）分析，發現1號染色體對應的genomic DNA（Exon1～Exon16）中Exon10的1032位置上有一個外顯子剪接增強位點（ESE），將其C突變成T后，發現SR蛋白不能與ESE結合而發生選擇性剪接。有統計表明正常人Exon10 的1032位點大多為C，而冠心病（CHD）病人大多為T。本論文在此基礎上展開，首先，從不同來源的CHD血樣中克隆包涵Exon10的genomic DNA，測序分析1032位點的堿基。再利用Tetra-primer ARMS-PCR方法進行基因型分析。在成功構建人ACAT1 genomic DNA（Exon8～11）質粒的基礎上再利用Nested PCR將Exon10 1032位點上的C突變成T，確保只有這一個點發生突變。
關鍵詞：ACAT-1基因,DNA克隆,基因型分析,Nested PCR,點突變

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