【作者】 汪军梅；

【导师】 解用虹；

【作者基本信息】 天津医科大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 脂蛋白脂肪酶(lipoprotein lipase,LPL)是血浆脂蛋白代谢的关键酶,其可将乳糜微粒和极低密度脂蛋白中的甘油三酯水解为甘油和游离脂肪酸供组织利用。载脂蛋白CⅡ(Apolipoprotein CⅡ,ApoCⅡ)作为脂蛋白脂肪酶的必需激活因子,可激活LPL,在脂类代谢中发挥着重要作用。ApoCⅡ结构、水平、功能异常和LPL基因变异会使脂蛋白脂肪酶(LPL)活性降低,导致脂蛋白代谢紊乱,发生异常脂蛋白血症。异常脂蛋白血症是最常见的代谢性疾病之一,也是动脉粥样硬化的重要危险因子。 本实验分为两部分,第一部分为探讨中国人群LPL基因常见突变的种类、频率以及其可能产生的影响,我们利用聚合酶链反应-单链构象多态性(PCR-SSCP)分析技术对203例人群LPL基因转录起始位点上游140bp以内的调节区和外显子4(包括内含子-外显子交界区)进行了突变的筛查。在203例样品中检出1例发生在外显子4扩增区域的可疑突变,通过PCR产物直接测序,证实为内含子3受位剪接位点上游6bp的C→T转换突变杂合子,未检出其他突变。目前该突变仅发现于日本和中国,尚未读到其他国家的相关报告。结合国内外相关文献和分子流行病学调查,以及关于突变基因结构和作用机制的讨论,显示该突变可能是中国人高甘油三酯血症的遗传易患因素之一。与以往国内外报道该突变仅见于高甘油三酯血症患者不同,本次在一名血脂正常人员检出LPL基因内含子3C→T突变,对探讨该突变的可能影响具有一定的参考价值。 第二部分为小鼠载脂蛋白CⅡ的克隆和表达。首先从小鼠肝组织提取总RNA,采用逆转录—聚合酶链式反应(RT-PCR)方法扩增获得小鼠ApoCⅡ基因编码序列,将小鼠载脂蛋白CⅡ编码基因片段重组入质粒pUC18,得到重组克隆载体pUC18-mouseApoCⅡ(pUC18-mApoCⅡ),并进行DNA序列测定。更多还原

【Abstract】 Lipoprotein lipase (LPL) is a key enzyme of lipid metabolism, its major function is the hydrolysis of the core triglycerides of circulating chylomicrons and very low density lipoproteins. Apolipoprotein CⅡ is required for the enzymatic activity of LPL. The major deficiency of LPL and Apolipoprotein CⅡ can cause hypertriglyceridemia. Hypertriglyceridemia is the important risk factor of many diseases , such as, atherosclerosis and pancreatitis et al..This experiment is divided into two parts. Firstly, to screen the LPL gene for mutations in 203 Chinese population and to study the possible effects of the mutations on plasma lipids, the LPL gene (exon 4 and regulatory sequence) was examined by PCR-SSCP analysis. The PCR products showing abnormal pattern on SSCP were sequenced using dideoxy chain termination method. One mutation was found in exon 4 amplified fragment of a person with normolipidemia by PCR-SSCP and identified to be a single transition (C→T) at six bp upstream from acceptor splicing site of intron 3 by DNA-sequencing. none of the other mutations was found in this study. The transition (C→T) mutation in intron 3 of the LPL gene was generally considered to be one of the genetic risks for hyperglyceridemia. Up to day, all this mutation in references was only found in hypertriglyceridemia, so the discovery of the mutation in person with normolipidemia may be certainly valuable to study the possible effects of themutation.Secondly, the encoding gene of Apolipoprotein C II was amplified byRT-PCR using total RNA derived from mouse liver tissues as template. The amplified fragment was recombined into pUC18 plasmid. Recombinant vector pUC18-mouse ApoCⅡ(pUC18-mApoCⅡ) was obtained and then sequenced.The gene encoding for apolipoprotein CII obtained from pUC18-mApoC II was inserted into fusion expressing vector pGEX-3X. The sequencing of Apolipoprotein CII in recombined plasma pUC18-mApoClIand pGEX-mouseApoC II (pGEX-mApoC II) was the same to GenBank sequence. Furthermore, recombinant vector pGEX-mApoC II was transformed into E.coli DH5a. After the culturing conditions were optimized, fusion protein GST-mApoC II was obtained. Fusion proteins was purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. The fusion protein was characterized by SDS-PAGE and Western blotting, hi this experiment, the mouse Apolipoprotein CII was cloned and expressed successfully. The production of mouse ApoC II by genetic engineering may provide a nessasary basis to prepare antibodies of ApoC II and to study the possibility and effects of treatment of ApoC II to patients with hypertriglyceridemia.更多还原