大肠杆菌双杂交筛选AMPKα2相互作用蛋白

发布时间：2018-04-25 21:07

本文选题：AMP激活的蛋白激酶 + 大肠杆菌双杂交　；参考：《第三军医大学》2007年博士论文

【摘要】： AMP激活的蛋白激酶(AMPK)是真核生物中广泛存在的一种蛋白激酶,主要协调代谢和能量的需要。一旦被激活,即可磷酸化下游靶蛋白,一方面关闭消耗ATP的合成代谢途径,另一方面开启产生ATP的分解代谢途径,被称为“细胞能量调节器”。AMPK除了通过调节参与糖、脂肪、蛋白质代谢过程的酶类,还通过介导基因转录和翻译过程,影响能量代谢,表明该酶系在维持能量稳态过程中起重要作用。高原地区影响人们健康和劳动效率的最主要因素是缺氧。缺氧引起线粒体的氧化磷酸化功能障碍,造成能量产生减少。维持能量平衡现已成为减轻缺氧损伤的关键问题。我们推测AMPK可能是缺氧能量稳态调节的一个重要因素。脑组织代谢率高,对氧和能量的需求量大但储备低,这些特点决定了脑对缺氧和能量不足最敏感易受到损伤。因此,研究AMPK在脑内的生物学效应和调节机制,将为寻求高原低氧环境下的促习服措施提供理论和实验依据。AMPK可能成为一个极具潜力的新型抗缺氧损伤的靶点。蛋白质是生命活动的执行者,生命活动内在本质的生物化学和/或催化活性在很大程度上受到蛋白质相互作用的调节。因此,探究具有相互作用关系的蛋白对将有助于功能提示。为此,我们有必要寻找与AMPK相互作用的蛋白质以促进其功能的阐明。AMPK是一个由α、β、γ亚基形成的异源三聚体,α为催化亚基,β和γ为调节亚基。三种亚基存在不同的亚型,如α1和α2,β1和β2,γ1、γ2和γ3。α催化亚基作为AMPK的特征性结构,与该结构域相互作用的蛋白质在一定程度上代表了特异地与AMPK相互作用的分子。本研究选择在脑神经元中含量较高的α2亚基作为诱饵,利用大肠杆菌双杂交系统筛选胎脑cDNA文库寻找与其相互作用的蛋白,为进一步了解AMPK在脑内能量稳态调节中的作用奠定基础,同时也为今后阐明AMPK在脑内缺氧习服适应中的作用机制提供新的思路。本研究进行了以下工作: 1.AMPKα2亚基编码区片段的克隆及大肠杆菌双杂交诱饵质粒的构建与鉴定采用PCR法扩增AMPKα2亚基的编码区序列,将其构建到带有λcI基因的克隆载体pBT上,与λcI基因形成融合基因,获得pBT-AMPKα2重组质粒,并保持目的基因的阅读框与λcI基因的阅读框一致。DNA测序结果表明,重组质粒中AMPKα2编码区的碱基序列和阅读框均正确,pBT-AMPKα2重组质粒(即诱饵质粒)构建成功。 2.诱饵质粒的表达及自激活作用检测 (1)将诱饵质粒pBT-AMPKα2转化入大肠杆菌XL1-Blue MR感受态细胞,在37℃培养条件下IPTG诱导重组融合蛋白的表达。提取大肠杆菌的全细胞裂解产物以及裂解上清和沉淀,SDS-PAGE检测蛋白表达,未见AMPKα2-λcI融合蛋白的表达。用Western blotting免疫印迹予以进一步鉴定,结果显示有AMPKα2-λcI融合蛋白的表达,且存在可溶性蛋白和不溶性包涵体两种形式,其中可溶性形式存在的AMPKα2-λcI融合蛋白易发生分解,产生AMPKα2和λcI蛋白;不溶性包涵体形式存在的AMPKα2-λcI融合蛋白不发生分解,稳定存在。 (2)重组质粒pBT-AMPKα2和空质粒pTRG共转化大肠杆菌XL1-Blue MR报告菌株,在no 3-AT非选择性培养基平板及3-AT选择性培养基平板上培养,观察菌落生长情况,并与阴性对照(即pBT空质粒和pTRG-Gal11p共转化大肠杆菌XL1-Blue MR报告菌株)比较。结果显示,阴性对照组中的转化子在no 3-AT平板上生长良好,而在3-AT平板上无菌落生长,表明3-AT培养板的质量可靠;重组质粒pBT-AMPKα2和空质粒pTRG共转化结果显示,转化子在no 3-AT平板上生长良好,而在3-AT平板上不生长,表明重组质粒pBT-AMPKα2并无自主活化作用,可用于下一步的双杂交筛选实验。 3.cDNA文库的扩增及AMPKα2亚基相互作用蛋白的筛选和初步鉴定 (1)按文库操作说明对cDNA文库进行扩增,收获的扩增文库经梯度稀释后涂板,随机挑取10个菌落,抽提质粒后进行序列测定,结果显示10个菌落测序的碱基序列均不相同,说明扩增文库未发生偏移,文库多样性仍保持。 (2)用CaCl_2法小量共转化50ng pBT-AMPKα2重组质粒和50ng pTRG文库质粒入100μl XL1-Blue MR大肠杆菌细胞,即先导文库共转化。根据先导文库共转化的结果估算筛选一定数量的文库克隆所需的大规模共转化反应的数量。然后用CaCl_2法大量共转化200ng pBT-AMPKα2重组质粒和200ng pTRG文库质粒入500μl大肠杆菌XL1-Blue MR细胞。通过在3-AT选择性筛选培养基上培养,以及假定相互作用子的富集,结果获得592个能够表达HIS3报告基因的初筛克隆。 (3)将筛选得到的初筛克隆全部补缀于含有3-AT和链霉素(Strep)的选择性筛选培养基上进行第二次筛选,结果发现有20个克隆不能生长而被去除,其余克隆生长良好。这是利用HIS3和aadA报告基因的表达排除假阳性克隆。 (4)从第二次筛选得到的克隆中挑取最早长出的20个克隆进行质粒抽提,转化大肠杆菌XL1-Blue MRF’Kan感受态细胞,转化产物铺于含有四环素(Ter)的LB琼脂平板(LB-Ter),并通过LB-Ter琼脂平板和含有氯霉素(Cam)的LB琼脂平板(LB-Cam)进行纯化,对20个克隆进行鉴定。结果显示,有1个克隆是假阳性克隆,剩余19个是阳性侯选克隆。 (5)将阳性侯选克隆回复至大肠杆菌XL1-Blue MR报告菌株中进行验证。19个阳性侯选克隆对应的pTRG靶质粒分别与pBT-AMPKα2诱饵质粒以及pBT空质粒共转化大肠杆菌XL1-Blue MR报告菌株,在3-AT选择性筛选培养基上培养,结果显示有9个阳性侯选克隆具有与诱饵蛋白AMPKα2特异性结合的特性,为真阳性克隆。 (6)抽提阳性克隆质粒,进行测序,并将结果与GenBank数据库进行同源性比较分析,结果获得7种AMPKα2的结合蛋白。它们分别是:磷酸果糖激酶、聚合泛素、细胞色素C氧化酶亚基I(COX I)、热休克蛋白8(HSP8)、人类白细胞抗原B关联性转录物3(BAT3)异构体1、蛋白酪氨酸磷酸酶受体型D(Ptprd)、岛-脑蛋白1(IB1),涉及糖酵解、蛋白质降解、线粒体电子传递链以及凋亡调控等多条途径,直接或间接参与能量调节。
[Abstract]:AMP activated protein kinase (AMPK) is a protein kinase that exists widely in eukaryotes, which mainly coordinates metabolic and energy needs. Once activated, it can phosphorylate downstream target proteins. On the one hand, it closes the anabolic pathway that consumes ATP. On the other hand, it opens the metabolic pathway of ATP, which is called the "cell energy regulator".A. In addition to regulating enzymes involved in sugar, fat, and protein metabolism, MPK also affects energy metabolism by mediating gene transcription and translation processes, indicating that the enzyme system plays an important role in maintaining energy homeostasis. The most important factor affecting people's health and labor efficiency in plateau areas is hypoxia. Hypoxia causes oxidative phosphoric acid in mitochondria. Acidification dysfunction leads to a decrease in energy. Maintaining energy balance has now become a key problem in reducing hypoxia injury. We speculate that AMPK may be an important factor in the regulation of hypoxia energy homeostasis. High metabolism rate of brain tissue, large demand for oxygen and energy and low reserve, which determine the most sensitive brain to hypoxia and energy shortage Therefore, the study of the biological effects and regulatory mechanisms of AMPK in the brain will provide a theoretical and experimental basis for the search for the measures of acclimatization in the environment of high altitude hypoxia..AMPK may become a potential new target for anti hypoxia injury. Protein is the executor of life activities and the intrinsic nature of life activities. Learning and / or catalytic activity is largely regulated by protein interaction. Therefore, exploring proteins that interact with each other will contribute to functional hints. To this end, it is necessary to find proteins interacting with AMPK to promote their function..AMPK is a hetero trimer formed by alpha, beta, and gamma, alpha Catalytic subunits, beta and gamma are regulated subunits. Three subunits have different subtypes, such as alpha 1 and alpha 2, beta 1 and beta 2, gamma 1, gamma 2, and gamma 3. a catalytic subunit as the characteristic structure of AMPK, and the proteins interacting with the domain represent the molecules of the interaction with AMPK to a certain extent. This study chose higher content in brain neurons. The alpha 2 subunit is used as a bait to screen the cDNA Library of fetal brain by using the two hybrid system of Escherichia coli to find the proteins that interact with them. It lays the foundation for further understanding the role of AMPK in the regulation of energy homeostasis in the brain. It also provides a new way of thinking on the mechanism of AMPK in the adaptation to hypoxia in the brain in the future.
The following work has been carried out in this study:
Cloning of the 1.AMPK alpha 2 subunit coding region fragment and the construction and identification of the double hybrid decoy plasmid of Escherichia coli, PCR method was used to amplify the coding region of AMPK alpha 2 subunit. It was constructed on the clone carrier pBT with the lambda cI gene and formed a fusion gene with the lambda cI gene to obtain the pBT-AMPK a 2 weight group plasmid and maintain the reading frame of the target gene and the lambda cI. The consistent.DNA sequencing results of the gene reading frame showed that the base sequence and reading frame of the AMPK alpha 2 coding region of the recombinant plasmid were all correct, and the recombinant plasmid of pBT-AMPK alpha 2 (the decoy plasmid) was successfully constructed.
Expression of 2. decoy plasmids and detection of their self activation
(1) the decoy plasmid pBT-AMPK alpha 2 was transformed into the XL1-Blue MR receptive cell of Escherichia coli, and the expression of recombinant fusion protein was induced by IPTG at 37 C. The whole cell lysis product of Escherichia coli and the cleavage supernatant and precipitate were extracted, the expression of the protein was detected by SDS-PAGE, and the expression of the AMPK alpha 2- lambda cI fusion protein was not found. Western blotting was exempting from the expression of the fusion protein. The results showed that there were two forms of AMPK alpha 2- lambda cI fusion protein, soluble protein and insoluble inclusion body, in which the soluble AMPK alpha 2- cI fusion protein was easily decomposed, produced AMPK alpha 2 and lambda cI protein, and the AMPK alpha 2- lambda cI fusion protein in the insoluble inclusion body form did not occur. Birth decomposition, stable existence.
(2) the recombinant plasmid pBT-AMPK alpha 2 and empty plasmid pTRG CO transformation of the Escherichia coli XL1-Blue MR reported strain were cultured on the no 3-AT non selective medium plate and 3-AT selective medium plate, and the colony growth was observed and compared with the negative control (i.e. pBT empty plasmid and pTRG-Gal11p CO transformation Escherichia coli XL1-Blue MR reported strain). The transformants in the negative control group grew well on the no 3-AT plate, but were aseptic on the 3-AT plate, which showed that the quality of the 3-AT culture plate was reliable. The CO transformation results of the recombinant plasmid pBT-AMPK alpha 2 and empty plasmid pTRG showed that the transformant grew well on the no 3-AT plate and did not grow on the 3-AT flat plate, indicating that the recombinant plasmid pBT-AMPK a 2 was found. No independent activation can be used for the next two hybrid screening experiments.
Amplification of 3.cDNA library and screening and preliminary identification of AMPK alpha 2 subunit interacting protein
(1) the cDNA library was amplified according to the operation of the library. The amplified library was diluted after the gradient dilution, and then the 10 colonies were randomly selected and the plasmid was extracted. The results showed that the sequences of the 10 colony sequences were all different, indicating that the amplification library did not shift and the library diversity remained.
(2) a small amount CO transformation of 50NG pBT-AMPK alpha 2 recombinant plasmid and 50NG pTRG library plasmid into 100 micron XL1-Blue MR Escherichia coli cells, that is, the pilot library CO transformation. The number of Large-Scale CO conversion reactions needed for a certain number of library clones was estimated according to the results of the CO transformation of the pilot library. Then, a large amount of CaCl_2 method was used to convert a large amount of 2. 00ng pBT-AMPK alpha 2 recombinant plasmids and 200ng pTRG library plasmids into 500 l Escherichia coli XL1-Blue MR cells. 592 primary clones expressing HIS3 reporter genes were obtained by selectively screening the culture medium on 3-AT and assuming the enrichment of the interacting subgroups.
(3) the screened clones were all mended in the selective screening medium containing 3-AT and streptomycin (Strep). The results showed that 20 clones could not grow and were removed, and the other clones were well grown. This was the exclusion of false positive clones from the expression of HIS3 and aadA reporter genes.
(4) from the second screened clones, the earliest 20 clones were extracted by plasmid extraction, transformed into Escherichia coli XL1-Blue MRF 'Kan receptive cells, and the transformation products were spread in LB agar plate (LB-Ter) containing tetracycline (Ter), and purified by LB-Ter agar plate and LB agar plate containing chloramphenicol (Cam), to 20. The results showed that 1 clones were false positive clones, and the remaining 19 were positive candidate clones.
(5) the positive candidate clones were returned to the Escherichia coli XL1-Blue MR report strain to verify that the pTRG target plasmids corresponding to.19 positive candidate clones were transformed into the pBT-AMPK alpha 2 decoy plasmids and pBT empty plasmids to co convert the Escherichia coli XL1-Blue MR reported strain, and were cultured on the 3-AT selective screening culture medium. The results showed that there were 9 positive candidates. It has a specific binding characteristic to bait protein AMPK alpha 2, which is a true positive clone.
(6) the positive cloned plasmids were extracted and sequenced, and the results were compared with the GenBank database. The results obtained the binding proteins of 7 AMPK alpha 2. They were phosphoric acid fructose kinase, polymerized ubiquitin, cytochrome C oxidase subunit I (COX I), heat shock protein 8 (HSP8), and human leukocyte antigen B associated transcriptional 3 (BAT3). Structure 1, protein tyrosine phosphatase is D (Ptprd), Isle brain protein 1 (IB1), involving glycolysis, protein degradation, mitochondrial electron transfer chain and apoptosis regulation, which are directly or indirectly involved in energy regulation.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378

【参考文献】