發(fā)布時間：2018-04-25 21:07

  本文選題：AMP激活的蛋白激酶 + 大腸桿菌雙雜交　； 參考：《第三軍醫(yī)大學(xué)》2007年博士論文

【摘要】： AMP激活的蛋白激酶(AMPK)是真核生物中廣泛存在的一種蛋白激酶,主要協(xié)調(diào)代謝和能量的需要。一旦被激活,即可磷酸化下游靶蛋白,一方面關(guān)閉消耗ATP的合成代謝途徑,另一方面開啟產(chǎn)生ATP的分解代謝途徑,被稱為“細(xì)胞能量調(diào)節(jié)器”。AMPK除了通過調(diào)節(jié)參與糖、脂肪、蛋白質(zhì)代謝過程的酶類,還通過介導(dǎo)基因轉(zhuǎn)錄和翻譯過程,影響能量代謝,表明該酶系在維持能量穩(wěn)態(tài)過程中起重要作用。高原地區(qū)影響人們健康和勞動效率的最主要因素是缺氧。缺氧引起線粒體的氧化磷酸化功能障礙,造成能量產(chǎn)生減少。維持能量平衡現(xiàn)已成為減輕缺氧損傷的關(guān)鍵問題。我們推測AMPK可能是缺氧能量穩(wěn)態(tài)調(diào)節(jié)的一個重要因素。腦組織代謝率高,對氧和能量的需求量大但儲備低,這些特點決定了腦對缺氧和能量不足最敏感易受到損傷。因此,研究AMPK在腦內(nèi)的生物學(xué)效應(yīng)和調(diào)節(jié)機(jī)制,將為尋求高原低氧環(huán)境下的促習(xí)服措施提供理論和實驗依據(jù)。AMPK可能成為一個極具潛力的新型抗缺氧損傷的靶點。蛋白質(zhì)是生命活動的執(zhí)行者,生命活動內(nèi)在本質(zhì)的生物化學(xué)和/或催化活性在很大程度上受到蛋白質(zhì)相互作用的調(diào)節(jié)。因此,探究具有相互作用關(guān)系的蛋白對將有助于功能提示。為此,我們有必要尋找與AMPK相互作用的蛋白質(zhì)以促進(jìn)其功能的闡明。AMPK是一個由α、β、γ亞基形成的異源三聚體,α為催化亞基,β和γ為調(diào)節(jié)亞基。三種亞基存在不同的亞型,如α1和α2,β1和β2,γ1、γ2和γ3。α催化亞基作為AMPK的特征性結(jié)構(gòu),與該結(jié)構(gòu)域相互作用的蛋白質(zhì)在一定程度上代表了特異地與AMPK相互作用的分子。本研究選擇在腦神經(jīng)元中含量較高的α2亞基作為誘餌,利用大腸桿菌雙雜交系統(tǒng)篩選胎腦cDNA文庫尋找與其相互作用的蛋白,為進(jìn)一步了解AMPK在腦內(nèi)能量穩(wěn)態(tài)調(diào)節(jié)中的作用奠定基礎(chǔ),同時也為今后闡明AMPK在腦內(nèi)缺氧習(xí)服適應(yīng)中的作用機(jī)制提供新的思路。 本研究進(jìn)行了以下工作: 1.AMPKα2亞基編碼區(qū)片段的克隆及大腸桿菌雙雜交誘餌質(zhì)粒的構(gòu)建與鑒定采用PCR法擴(kuò)增AMPKα2亞基的編碼區(qū)序列,將其構(gòu)建到帶有λcI基因的克隆載體pBT上,與λcI基因形成融合基因,獲得pBT-AMPKα2重組質(zhì)粒,并保持目的基因的閱讀框與λcI基因的閱讀框一致。DNA測序結(jié)果表明,重組質(zhì)粒中AMPKα2編碼區(qū)的堿基序列和閱讀框均正確,pBT-AMPKα2重組質(zhì)粒(即誘餌質(zhì)粒)構(gòu)建成功。 2.誘餌質(zhì)粒的表達(dá)及自激活作用檢測 (1)將誘餌質(zhì)粒pBT-AMPKα2轉(zhuǎn)化入大腸桿菌XL1-Blue MR感受態(tài)細(xì)胞,在37℃培養(yǎng)條件下IPTG誘導(dǎo)重組融合蛋白的表達(dá)。提取大腸桿菌的全細(xì)胞裂解產(chǎn)物以及裂解上清和沉淀,SDS-PAGE檢測蛋白表達(dá),未見AMPKα2-λcI融合蛋白的表達(dá)。用Western blotting免疫印跡予以進(jìn)一步鑒定,結(jié)果顯示有AMPKα2-λcI融合蛋白的表達(dá),且存在可溶性蛋白和不溶性包涵體兩種形式,其中可溶性形式存在的AMPKα2-λcI融合蛋白易發(fā)生分解,產(chǎn)生AMPKα2和λcI蛋白;不溶性包涵體形式存在的AMPKα2-λcI融合蛋白不發(fā)生分解,穩(wěn)定存在。 (2)重組質(zhì)粒pBT-AMPKα2和空質(zhì)粒pTRG共轉(zhuǎn)化大腸桿菌XL1-Blue MR報告菌株,在no 3-AT非選擇性培養(yǎng)基平板及3-AT選擇性培養(yǎng)基平板上培養(yǎng),觀察菌落生長情況,并與陰性對照(即pBT空質(zhì)粒和pTRG-Gal11p共轉(zhuǎn)化大腸桿菌XL1-Blue MR報告菌株)比較。結(jié)果顯示,陰性對照組中的轉(zhuǎn)化子在no 3-AT平板上生長良好,而在3-AT平板上無菌落生長,表明3-AT培養(yǎng)板的質(zhì)量可靠;重組質(zhì)粒pBT-AMPKα2和空質(zhì)粒pTRG共轉(zhuǎn)化結(jié)果顯示,轉(zhuǎn)化子在no 3-AT平板上生長良好,而在3-AT平板上不生長,表明重組質(zhì)粒pBT-AMPKα2并無自主活化作用,可用于下一步的雙雜交篩選實驗。 3.cDNA文庫的擴(kuò)增及AMPKα2亞基相互作用蛋白的篩選和初步鑒定 (1)按文庫操作說明對cDNA文庫進(jìn)行擴(kuò)增,收獲的擴(kuò)增文庫經(jīng)梯度稀釋后涂板,隨機(jī)挑取10個菌落,抽提質(zhì)粒后進(jìn)行序列測定,結(jié)果顯示10個菌落測序的堿基序列均不相同,說明擴(kuò)增文庫未發(fā)生偏移,文庫多樣性仍保持。 (2)用CaCl_2法小量共轉(zhuǎn)化50ng pBT-AMPKα2重組質(zhì)粒和50ng pTRG文庫質(zhì)粒入100μl XL1-Blue MR大腸桿菌細(xì)胞,即先導(dǎo)文庫共轉(zhuǎn)化。根據(jù)先導(dǎo)文庫共轉(zhuǎn)化的結(jié)果估算篩選一定數(shù)量的文庫克隆所需的大規(guī)模共轉(zhuǎn)化反應(yīng)的數(shù)量。然后用CaCl_2法大量共轉(zhuǎn)化200ng pBT-AMPKα2重組質(zhì)粒和200ng pTRG文庫質(zhì)粒入500μl大腸桿菌XL1-Blue MR細(xì)胞。通過在3-AT選擇性篩選培養(yǎng)基上培養(yǎng),以及假定相互作用子的富集,結(jié)果獲得592個能夠表達(dá)HIS3報告基因的初篩克隆。 (3)將篩選得到的初篩克隆全部補(bǔ)綴于含有3-AT和鏈霉素(Strep)的選擇性篩選培養(yǎng)基上進(jìn)行第二次篩選,結(jié)果發(fā)現(xiàn)有20個克隆不能生長而被去除,其余克隆生長良好。這是利用HIS3和aadA報告基因的表達(dá)排除假陽性克隆。 (4)從第二次篩選得到的克隆中挑取最早長出的20個克隆進(jìn)行質(zhì)粒抽提,轉(zhuǎn)化大腸桿菌XL1-Blue MRF’Kan感受態(tài)細(xì)胞,轉(zhuǎn)化產(chǎn)物鋪于含有四環(huán)素(Ter)的LB瓊脂平板(LB-Ter),并通過LB-Ter瓊脂平板和含有氯霉素(Cam)的LB瓊脂平板(LB-Cam)進(jìn)行純化,對20個克隆進(jìn)行鑒定。結(jié)果顯示,有1個克隆是假陽性克隆,剩余19個是陽性侯選克隆。 (5)將陽性侯選克隆回復(fù)至大腸桿菌XL1-Blue MR報告菌株中進(jìn)行驗證。19個陽性侯選克隆對應(yīng)的pTRG靶質(zhì)粒分別與pBT-AMPKα2誘餌質(zhì)粒以及pBT空質(zhì)粒共轉(zhuǎn)化大腸桿菌XL1-Blue MR報告菌株,在3-AT選擇性篩選培養(yǎng)基上培養(yǎng),結(jié)果顯示有9個陽性侯選克隆具有與誘餌蛋白AMPKα2特異性結(jié)合的特性,為真陽性克隆。 (6)抽提陽性克隆質(zhì)粒,進(jìn)行測序,并將結(jié)果與GenBank數(shù)據(jù)庫進(jìn)行同源性比較分析,結(jié)果獲得7種AMPKα2的結(jié)合蛋白。它們分別是:磷酸果糖激酶、聚合泛素、細(xì)胞色素C氧化酶亞基I(COX I)、熱休克蛋白8(HSP8)、人類白細(xì)胞抗原B關(guān)聯(lián)性轉(zhuǎn)錄物3(BAT3)異構(gòu)體1、蛋白酪氨酸磷酸酶受體型D(Ptprd)、島-腦蛋白1(IB1),涉及糖酵解、蛋白質(zhì)降解、線粒體電子傳遞鏈以及凋亡調(diào)控等多條途徑,直接或間接參與能量調(diào)節(jié)。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R378

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