本文關(guān)鍵詞： 前列腺素D合成酶 單克隆抗體 單鏈抗體 α-葡萄糖苷酶　出處：《南京師范大學(xué)》2005年博士論文　論文類型：學(xué)位論文

【摘要】：Lipocalin型前列腺素D合成酶(Lipocalin-type Prostagladln D Synthase,L-PGDS)亦稱β-trace protein,是機體組織屏障的主要成分,屬Lipocalin家族。Lipocalins為一組分子量較小的分泌性蛋白質(zhì),它們的共同特性是可結(jié)合和運輸親脂/疏水分子,L-PGDS為此家族中唯一有酶活性的蛋白質(zhì)。L-PGDS主要分布于哺乳動物的中樞神經(jīng)系統(tǒng)和雄性生殖器官,并分泌至腦脊液、血清、精液、尿液、羊水等體液中。L-PGDS是一種雙功能蛋白,除催化PGD_2產(chǎn)生外,亦可結(jié)合和運輸親脂/疏水分子。該蛋白具有廣泛的生理學(xué)作用,它不僅與睡眠誘導(dǎo)、體溫調(diào)節(jié)、感受傷害、氣味應(yīng)答有關(guān),亦是一種生育相關(guān)蛋白,可能參與精子的發(fā)生和成熟。研究顯示,對于一些神經(jīng)系統(tǒng)疾病、腎臟疾病、心血管疾病、生殖系統(tǒng)疾病等,L-PGDS已成為一個有用的輔助診斷指標(biāo)。 雖然L-PGDS的性質(zhì)、結(jié)構(gòu)已經(jīng)基本研究清楚,但是它在雄性生殖系統(tǒng)中的組織分布和細(xì)胞內(nèi)的定位、在雄性生殖系統(tǒng)內(nèi)的具體功能、代謝過程、作用機制以及與相關(guān)疾病的具體關(guān)系僅限于假設(shè)性的推測,尚缺乏有力的實驗來確證。本研究的焦點集中在L-PGDS在男性生殖中的基礎(chǔ)研究以及在體內(nèi)的生理作用功能和病理作用機制的初步探討等方面。 實驗研究以本研究室構(gòu)建的重組表達質(zhì)粒pPIC9/htL-PGDS為基礎(chǔ),GglⅡ?qū)⒅亟M質(zhì)粒pPIC9/htL-PGDS線性化后,電穿孔法將其導(dǎo)入巴斯德畢赤氏酵母Gs115。菌落PCR對酵母轉(zhuǎn)化菌進行鑒定后,甲醇誘導(dǎo)重組蛋白的表達,結(jié)果得到一株L-PGDS表達陽性克隆,Mr約為27 000,與理論值完全一致。培養(yǎng)上清經(jīng)SDS-PAGE電泳后,進行薄層掃描分析,Mr為27 000的L-PGDS的含量約占酵母培養(yǎng)上清中總蛋白含量的18%。Bradford法測定培養(yǎng)上清的總蛋白含量約為150 mg/L,因此,重組L-PGDS的表達量可達27mg/L左右。再通過鎳離子金屬親和層柱對重組L-PGDS進行了純化,并用糖苷酶F對其進行了消化。結(jié)果表明,通過Picflia Pastoris系統(tǒng)表達的重組蛋白除Mr為27 000的主帶外,尚存在Mr為24 000、21 000的兩條弱帶,這兩條弱帶為糖基化不完全所致。純化蛋白
[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，

本文編號：1494221