【摘要】 载脂蛋白apoA Ⅰ是高密度脂蛋白中最主要的蛋白 ,在胆固醇代谢及动脉粥样硬化等疾病的发生和控制中有重要作用。为了建立大量生产和制备该蛋白的方法 ,昆虫杆状病毒表达系统被用来高效表达了两种形式的人apoA Ⅰ。不带有信号肽序列的proapoA Ⅰ蛋白表达后主要在细胞内 ,但在感染的晚期也有相当量的重组蛋白进入细胞培养液中。用蛇磷脂酶A2抑制因子α亚基信号肽序列引导表达的apoA Ⅰ蛋白在感染早期可以被分泌到培养液中。在此基础上 ,通过PhenylSepharose疏水柱层析 ,从感染细胞的培养液中初步分离纯化了成熟的apoA Ⅰ蛋白。这一结果为apoA Ⅰ的进一步研究和应用奠定了基础。更多还原

【Abstract】 Apolipoprotein A-Ⅰ is the major apolipoprotein in high-density lipoprotein known to have a wide range of physiological functions, the best-studied one of which is in regulating cholesterol metabolism and preventing arteriosclerosis. Human blood has been the only source of this protein. To facilitate further research and application, it is essential to produce it through genetic engineering. In the current research, the baculovirus-insect cell system was used to overexpress human apolipoprotein A-Ⅰ. Two recombinant baculoviruses were constructed. The first one expressed a pro form of apoA-Ⅰ lacking native signal peptide. The recombinant protein was found to remain mainly inside cells in the early phase of infection, while being largely excreted to the medium late in infection. The second one used a heterologous signal peptide, snake phospholipase A2 inhibitor α subunit signal peptide, to lead the secretion of mature apoA-Ⅰ. In contrast to the first virus, recombinant apoA-Ⅰ was found in the culture medium at the early phase of virus infection. The mature apoA-Ⅰ was purified from culture medium using Phenyl Sepharose hydrophobic interaction chromatography (HIC) and eluted with water and Propylene. This work shows that snake phospholipase A2 inhibitor α subunit signal peptide can be used to secret human apoA-Ⅰ in insect cells, but the efficiency of its secretion is limited when the expression level is high.更多还原