前列腺素D合成酶在男性生殖中的基础及应用研究

发布时间：2018-02-07 11:01

  本文关键词： 前列腺素D合成酶 单克隆抗体 单链抗体 α-葡萄糖苷酶　出处：《南京师范大学》2005年博士论文　论文类型：学位论文

【摘要】：Lipocalin型前列腺素D合成酶(Lipocalin-type Prostagladln D Synthase,L-PGDS)亦称β-trace protein,是机体组织屏障的主要成分,属Lipocalin家族。Lipocalins为一组分子量较小的分泌性蛋白质,它们的共同特性是可结合和运输亲脂/疏水分子,L-PGDS为此家族中唯一有酶活性的蛋白质。L-PGDS主要分布于哺乳动物的中枢神经系统和雄性生殖器官,并分泌至脑脊液、血清、精液、尿液、羊水等体液中。L-PGDS是一种双功能蛋白,除催化PGD_2产生外,亦可结合和运输亲脂/疏水分子。该蛋白具有广泛的生理学作用,它不仅与睡眠诱导、体温调节、感受伤害、气味应答有关,亦是一种生育相关蛋白,可能参与精子的发生和成熟。研究显示,对于一些神经系统疾病、肾脏疾病、心血管疾病、生殖系统疾病等,L-PGDS已成为一个有用的辅助诊断指标。 虽然L-PGDS的性质、结构已经基本研究清楚,但是它在雄性生殖系统中的组织分布和细胞内的定位、在雄性生殖系统内的具体功能、代谢过程、作用机制以及与相关疾病的具体关系仅限于假设性的推测,尚缺乏有力的实验来确证。本研究的焦点集中在L-PGDS在男性生殖中的基础研究以及在体内的生理作用功能和病理作用机制的初步探讨等方面。 实验研究以本研究室构建的重组表达质粒pPIC9/htL-PGDS为基础,GglⅡ将重组质粒pPIC9/htL-PGDS线性化后,电穿孔法将其导入巴斯德毕赤氏酵母Gs115。菌落PCR对酵母转化菌进行鉴定后,甲醇诱导重组蛋白的表达,结果得到一株L-PGDS表达阳性克隆,Mr约为27 000,与理论值完全一致。培养上清经SDS-PAGE电泳后,进行薄层扫描分析,Mr为27 000的L-PGDS的含量约占酵母培养上清中总蛋白含量的18%。Bradford法测定培养上清的总蛋白含量约为150 mg/L,因此,重组L-PGDS的表达量可达27mg/L左右。再通过镍离子金属亲和层柱对重组L-PGDS进行了纯化,并用糖苷酶F对其进行了消化。结果表明,通过Picflia Pastoris系统表达的重组蛋白除Mr为27 000的主带外,尚存在Mr为24 000、21 000的两条弱带,这两条弱带为糖基化不完全所致。纯化蛋白
[Abstract]:Lipocalin type prostaglandin D synthase Lipocalin-type Prostagladln D. Synthase L-PGDS, also known as 尾 -trace protein, is the main component of the body's tissue barrier. It belongs to the Lipocalin family. Lipocalins is a group of secretory proteins with small molecular weight. Their common feature is that they can bind and transport lipophilic / hydrophobic molecules, L-PGDS, the only protein in the family that has enzyme activity, mainly distributed in the central nervous system and male reproductive organs of mammals, and secreted to cerebrospinal fluid, serum, semen, L-PGDS is a bifunctional protein in urine, amniotic fluid and other fluids, which not only catalyzes the production of PGD_2, but also binds and transports lipophilic / hydrophobic molecules. Smell response is also a procreation related protein that may be involved in spermatogenesis and maturation. Studies have shown that for some neurological diseases, kidney diseases, cardiovascular diseases, L-PGDS has become a useful marker for diagnosis of reproductive system diseases. Although the properties and structures of L-PGDS have been basically studied, the tissue distribution and intracellular localization of L-PGDS in the male reproductive system, the specific function and metabolic process in the male reproductive system, The mechanism of action and the specific relationship with related diseases are limited to hypothetical speculation. The focus of this study is on the basic research of L-PGDS in male reproduction and the preliminary study on physiological function and pathological mechanism of L-PGDS in vivo. On the basis of the recombinant expression plasmid pPIC9/htL-PGDS constructed in our laboratory, the recombinant plasmid pPIC9/htL-PGDS was linearized by GGL 鈪，

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