【作者】 祝成亮；

【导师】 周新； 刘芳；

【作者基本信息】 武汉大学 ， 临床检验诊断学， 2005， 硕士

【摘要】 载脂蛋白M是新近发现的载脂蛋白。在各种脂蛋白的亚类中,载脂蛋白M主要存在于人血浆高密度脂蛋白中,但在富含甘油三脂的脂蛋白和低密度脂蛋白中也发现了载脂蛋白M。在所有的载脂蛋白中,与其他载脂蛋白相比,载脂蛋白M只占很少一部分。载脂蛋白M存在于所有的哺乳动物的基因组中,但它仅在肝脏和肾脏表达,在其他的组织和器官不表达。载脂蛋白M位于6号染色体主要组织相容性抗原复合体Ⅲ区。 载脂蛋白M的功能尚不清楚,除了与高密度脂蛋白的合成有关外,它可能在胆固醇的代谢和转运中起到特殊的作用。载脂蛋白M有膜蛋白的功能,它可能与脂蛋白颗粒和来自内质网膜的磷脂蛋白相结合,然后分泌到血浆中,并参与运输疏水性的小分子复合物。 本研究通过克隆并高效表达小鼠载脂蛋白M基因,以探讨其生物学功能。通过提取小鼠肝脏总RNA,采用RT-PCR技术,扩增正常成年小鼠肝细胞载脂蛋白M全长cDNA,并重组于原核表达载体pGEX-KG中,转化大肠杆菌BL21。经IPTG诱导后,收集菌体蛋白,进行SDS-PAGE鉴定;获得小鼠载脂蛋白M全长cDNA,测序结果与已发表的基因序列一致,重组蛋白以包涵体的形式进行表达,经SDS-PAGE分析,在相对分子量54kDa处有一特异性的蛋白条带。本研究成功克隆了正常成年小鼠的载脂蛋白M基因,并在大肠杆菌BL21中得到高效表达,为进一步制备载脂蛋白M抗体创造了有利条件。更多还原

【Abstract】 Apolipoprotein M(ApoM) is a recently discovered human apolipoprotein. Among the various lipoprotein subclasses, ApoM is associated mainly with high density lipoprotein(HDL) in human plasma, but is also detected in triglyceride-rich lipoproteins(TGRLP) and low-density lipoprotein(LDL). In all of these lipoproteins, ApoM appeared to be a minor component as compared with the major apolipoproteins. The ApoM gene is found in all mammalian genomes. It is prominent in both liver and kidney but not in other organs. The synthesis of ApoM in liver may be associated with the production of HDL. The ApoM gene is located in the major histocompatibility complex Class III region on chromosome 6.The functions of ApoM are to be determined. Apart from the hypothetical involvement in synthesis of HDL, it is possible that ApoM severs a specific function in the metabolism and transfer of cholesterol. ApoM has the characteristics of a membrane intergral protein, and it is possible that ApoM is incorporated into the lipoptein particles together with phospholipids from the endoplasmic reticulum and then secreted to plasma. ApoM may transport hydrophobic low molecular weight compounds.To search for functions of ApoM gene, we clone and express mouse ApoM gene efficiently in the study. The total RNA was extracted from mouse liver, the complete cDNA of normal adult mouse ApoM was amplified by RT-PCR and recombined into pGEX-KG plasmid, then it was transformed into the competent expressive cells of E. coli BL21. After induced with IPTG, bacteria protein was collected for SDS-PAGE identification. The sequence of cloned mouse ApoM gene was identified with the reported one. Expressed fusion protein existed in the form of inclusion body. The protein band was 54 kDa appeared on SDS-GAGE gel. The mouse ApoM was cloned successfully and expressed efficiently in E. coli BL21 which create a favourable condition for preparation of anti- ApoM antidody.更多还原