NGAL相互作用蛋白的筛选与鉴定

发布时间：2018-05-15 16:46

本文选题：NGAL + 蛋白-蛋白相互作用　；参考：《汕头大学》2005年硕士论文

【摘要】：Lipocalins是一个细胞外蛋白家族,常作为疏水小分子转运的载体,能与细胞表面的受体相互作用。NGAL(neutrophil gelatinase-associated lipocalin)是lipocalins家族成员之一。以往的研究表明,NGAL可能参与多种生命活动过程,包括胚胎发育、细胞分化、炎症免疫反应、细胞凋亡、信号转导、脂质代谢、肿瘤的发生发展等。我们课题组曾研究发现, NGAL在人永生化食管上皮细胞恶变过程中过表达,具有促进癌细胞侵袭功能,同时还可能参与癌细胞的不良分化。目前,NGAL在细胞内的具体生化作用机制还不明了。本文的目的是从人类骨髓预转化cDNA文库中寻找与NGAL相互作用的蛋白,构建蛋白-蛋白相互作用图谱,为揭示NGAL的生化作用机制提供一条新途径。主要研究内容和方法: 一、利用酵母双杂交技术,筛选出与NGAL相互作用的目标蛋白,并给予验证。二、用免疫共沉淀方法,在体外和哺乳动物细胞内验证目标蛋白与NGAL的相互作用。三、用荧光素酶双报告系统进一步验证目标蛋白与NGAL的相互作用。主要研究结果: 一、酵母体内一对一验证66个文库质粒基因表达蛋白与NGAL的相互作用,得到40个阳性克隆,测序,并在NCBI公共数据库进行BLAST分析。最终获得31个基因,11个未知,20个已知。二、构建17对用于免疫共沉淀的质粒,这些外源基因包括大T抗原和p53、NGAL及文库质粒上的基因片段X等,分别插入到pCMV-HA和pCMV-Myc载体。在哺乳动物细胞内成功建立了免疫共沉淀技术平台。三、利用protein-A-agarose纯化免疫兔血清,分离得到NGAL抗体。证明该抗体可以用于免疫检测或免疫共沉淀NGAL。四、免疫共沉淀结果表明,NGAL和Galeetin-1之间可能存在弱相互作用。五、运用哺乳动物细胞双杂交系统研究发现, 在哺乳动物细胞中NGAL可能以某种特殊方式与Galectin-1相互作用。结论:1获得了31个可能与NGAL具有相互作用关系的候选蛋白。2验证候选蛋白之一 galectin-1与NGAL之间的相互作用,提示初步阳性结果,但还需进一步证明。
[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 .

【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

【参考文献】