本文選題：NGAL + 蛋白-蛋白相互作用　； 參考：《汕頭大學(xué)》2005年碩士論文

【摘要】：Lipocalins是一個細胞外蛋白家族,常作為疏水小分子轉(zhuǎn)運的載體,能與細胞表面的受 體相互作用。NGAL(neutrophil gelatinase-associated lipocalin)是lipocalins家族成員之一。 以往的研究表明,NGAL可能參與多種生命活動過程,包括胚胎發(fā)育、細胞分化、炎癥免 疫反應(yīng)、細胞凋亡、信號轉(zhuǎn)導(dǎo)、脂質(zhì)代謝、腫瘤的發(fā)生發(fā)展等。我們課題組曾研究發(fā)現(xiàn), NGAL在人永生化食管上皮細胞惡變過程中過表達,具有促進癌細胞侵襲功能,同時還可 能參與癌細胞的不良分化。目前,NGAL在細胞內(nèi)的具體生化作用機制還不明了。本文的 目的是從人類骨髓預(yù)轉(zhuǎn)化cDNA文庫中尋找與NGAL相互作用的蛋白,構(gòu)建蛋白-蛋白相 互作用圖譜,為揭示NGAL的生化作用機制提供一條新途徑。 主要研究內(nèi)容和方法: 一、利用酵母雙雜交技術(shù),篩選出與NGAL相互作用的目標蛋白,并給予驗證。 二、用免疫共沉淀方法,在體外和哺乳動物細胞內(nèi)驗證目標蛋白與NGAL的相互作用。 三、用熒光素酶雙報告系統(tǒng)進一步驗證目標蛋白與NGAL的相互作用。 主要研究結(jié)果: 一、酵母體內(nèi)一對一驗證66個文庫質(zhì)粒基因表達蛋白與NGAL的相互作用,得到40個陽 性克隆,測序,并在NCBI公共數(shù)據(jù)庫進行BLAST分析。最終獲得31個基因,11個 未知,20個已知。 二、構(gòu)建17對用于免疫共沉淀的質(zhì)粒,這些外源基因包括大T抗原和p53、NGAL及文庫 質(zhì)粒上的基因片段X等,分別插入到pCMV-HA和pCMV-Myc載體。在哺乳動物細 胞內(nèi)成功建立了免疫共沉淀技術(shù)平臺。 三、利用protein-A-agarose純化免疫兔血清,分離得到NGAL抗體。證明該抗體可以用于 免疫檢測或免疫共沉淀NGAL。 四、免疫共沉淀結(jié)果表明,NGAL和Galeetin-1之間可能存在弱相互作用。 五、運用哺乳動物細胞雙雜交系統(tǒng)研究發(fā)現(xiàn), 在哺乳動物細胞中NGAL可能以某種特殊 方式與Galectin-1相互作用。 結(jié)論:1獲得了31個可能與NGAL具有相互作用關(guān)系的候選蛋白。2驗證候選蛋白之一 galectin-1與NGAL之間的相互作用,提示初步陽性結(jié)果,但還需進一步證明。
[Abstract]:Lipocalins is a family of extracellular proteins , often used as a carrier for the transport of hydrophobic small molecules , capable of interacting with the surface of the cell








Body interaction . NGAL ( neutrophil elastase - associated lipocalin ) is one of the members of the lipocalins family .








Previous studies have shown that NGAL may participate in a variety of life - activity processes , including embryonic development , cell differentiation , inflammation , and the like .








We found that the disease response , cell apoptosis , signal transduction , lipid metabolism , tumor development , etc .








The NGAL is overexpressed in the malignant process of immortal esophageal epithelial cells , has the function of promoting the invasion of cancer cells , and also can








At present , the specific biochemical mechanism of NGAL in cells is unknown .








The aim is to find a protein interacting with NGAL from human bone marrow pre - transformed cDNA library and construct the protein - protein phase








Interaction patterns provide a new way for revealing the biochemical mechanism of NGAL .








Main research contents and methods :








1 . Using yeast two - hybrid technique , the target protein interacting with NGAL was screened and verified .








secondly , the interaction between the target protein and the NGAL is verified in vitro and in the mammalian cell by using an immune coprecipitation method .








Thirdly , the interaction between the target protein and NGAL was further verified by the luciferase double reporting system .








Key findings :








1 . In vivo one - to - one verification of the interaction between the expression protein of 66 library plasmid genes and NGAL in 66 library plasmids was verified , and 40 positive clones were obtained .








Sex cloning , sequencing , and BLAST analysis at the GenBank public database . Finally , 31 genes were obtained , 11








Unknown , 20 known .








II . Construction of 17 pairs of plasmids for immune codeposition , these foreign genes including large T antigens and p53 , NGAL and libraries








The gene fragments X and the like on the plasmid were inserted into pCMV - HA and pCMV - Myc vectors respectively .








The immune co - precipitation technology platform was successfully established in the cell .








and 3 , purifying the immunized rabbit serum by using protein - A - agarose to obtain the NGAL antibody .








immunodetection or immune co - precipitation of ngal .








Results showed that there might be a weak interaction between NGAL and Galegal - 1 .








5 . Using the mammalian cell double - hybrid system , it is found that NGAL in mammalian cells may be of some special significance








in that same way as galectin - 1 .








Conclusion : 1 candidate protein which may have an interactive relationship with NGAL is obtained . One of the candidate proteins is validated .








The interaction between galectin - 1 and NGAL suggests a preliminary positive result , but further evidence is required .

【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R341

【參考文獻】

相關(guān)期刊論文 前5條

1 熊華淇,許麗艷,李恩民,蔡唯佳,沈忠英,徐小虎;NGAL基因cDNA的克隆及其表達載體的構(gòu)建[J];腫瘤防治雜志;2001年05期

2 王朝陽,許麗艷,榮舉,韓溟,吉坤美,沈忠英,李恩民;食管癌相關(guān)基因NGAL四種融合表達載體的構(gòu)建及其蛋白表達產(chǎn)物的比較分析[J];腫瘤防治雜志;2003年10期

3 許麗艷,李恩民,熊華淇,蔡唯佳,沈忠英;NGAL基因在永生化食管上皮細胞惡性轉(zhuǎn)化中過表達的研究[J];生物化學(xué)與生物物理進展;2001年06期

4 林玨龍,許麗艷,李恩民,蔡唯佳,牛永東,方昆陽,熊華淇,沈忠英,曾毅;反義封閉NGAL基因表達對SHEEC食管癌細胞微絲骨架的影響[J];生物化學(xué)與生物物理進展;2004年05期

5 李恩民,許麗艷,蔡唯佳,熊華淇,沈忠英,曾毅;SHEEC食管癌細胞中NGAL基因的功能[J];生物化學(xué)與生物物理學(xué)報;2003年03期

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本文編號：1893128