本文選題：酵母雙雜交 + ATP結(jié)合盒超家族轉(zhuǎn)運(yùn)蛋白��； 參考：《第三軍醫(yī)大學(xué)》2006年碩士論文

【摘要】： ATP結(jié)合盒超家族轉(zhuǎn)運(yùn)蛋白[The ATP-binding cassette (ABC) transporter superfamily]屬于一類分布廣泛、數(shù)目眾多的跨膜轉(zhuǎn)運(yùn)體系。它們可以轉(zhuǎn)運(yùn)代謝產(chǎn)物、離子、糖、氨基酸、脂類、固醇以及藥物等多種底物通過細(xì)胞內(nèi)、外膜。一些特定的ABC轉(zhuǎn)運(yùn)蛋白在多藥耐性的腫瘤細(xì)胞系或癌癥過表達(dá),與臨床耐藥密切相關(guān)。這些基因的突變可能直接和間接導(dǎo)致多種遺傳疾病。而目前關(guān)于ATP結(jié)合盒超家族轉(zhuǎn)運(yùn)蛋白具體轉(zhuǎn)運(yùn)機(jī)制以及功能還未完全了解。關(guān)于其與細(xì)胞內(nèi)蛋白的相互作用研究甚少。規(guī)模化進(jìn)行蛋白質(zhì)相互作用研究可能發(fā)現(xiàn)其更精確的轉(zhuǎn)運(yùn)調(diào)節(jié)機(jī)制并揭示新的功能。 蛋白質(zhì)-蛋白質(zhì)相互作用是一切生命活動的基礎(chǔ),揭示相互作用規(guī)律是認(rèn)識生命奧秘的必經(jīng)之路。酵母雙雜交技術(shù)是目前應(yīng)用最成功的大規(guī)模研究蛋白質(zhì)相互作用的技術(shù)平臺,規(guī)�；湍鸽p雜交技術(shù)不是常規(guī)實(shí)驗(yàn)室酵母雙雜交技術(shù)在誘餌數(shù)量上的簡單增加,需要解決大量的技術(shù)問題。本文以ATP結(jié)合盒超家族轉(zhuǎn)運(yùn)蛋白為研究對象,摸索規(guī)�；湍鸽p雜交研究蛋白質(zhì)相互作用的技術(shù)條件,優(yōu)化各關(guān)鍵技術(shù)環(huán)節(jié),完善可用于規(guī)模化蛋白相互作用研究的酵母雙雜交技術(shù)平臺;發(fā)現(xiàn)20對高可信的蛋白相互作用。結(jié)果對采用酵母雙雜交技術(shù)規(guī)模化研究蛋白質(zhì)相互作用提供可借鑒的經(jīng)驗(yàn),發(fā)現(xiàn)的相互作用有助于揭示ABC蛋白的轉(zhuǎn)運(yùn)機(jī)制,并為其新功能的認(rèn)識提供重要的功能線索。 本文采用酵母雙雜交技術(shù),選取具有重要功能,代表6類亞家族的14種ATP結(jié)合盒超家族轉(zhuǎn)運(yùn)蛋白ABCA1、ABCA5、ABCA6、ABCB1、ABCB11、ABCB4、ABCC1、ABCC13、ABCC2、ABCC3、ABCC5、ABCC6、ABCF1、ABCG8的胞質(zhì)區(qū)(C端或者N端)為誘餌,篩選成人肝cDNA文庫,得到67種不同的相互作用。在對相互作用數(shù)據(jù)的初步分析中,除去不符合讀框和常見假陽性,經(jīng)過回轉(zhuǎn)驗(yàn)證,得到20對可信度較高的相互作用。這些相互作用提示ATP結(jié)合盒超家族轉(zhuǎn)運(yùn)蛋白可能有許多新的功能,同時也揭示可能存在共同的轉(zhuǎn)運(yùn)機(jī)制。如發(fā)現(xiàn)ABCC2與NF-κB1的相互作用,提示ABBC2可能作為一個受體,參與NF-κB信號通路的激活和調(diào)節(jié)。 用GO(gene ontology)進(jìn)行功能注釋,發(fā)現(xiàn)相互作用蛋白中有參與物質(zhì)轉(zhuǎn)運(yùn)過程的,
[Abstract]:ATP-binding cassette transporter (ATP-binding cassette) transporter superfamily is a widely distributed and numerous transmembrane transporter system. They transport metabolites, ions, sugars, amino acids, lipids, steroids and drugs through the intracellular and outer membranes. Some specific ABC transporters are overexpressed in multidrug resistant tumor cell lines or cancer, which is closely related to clinical drug resistance. Mutations in these genes can lead directly and indirectly to multiple genetic diseases. However, the mechanism and function of ATP binding cassette superfamily transporters have not been fully understood. Little research has been done on the interaction between intracellular proteins and them. A large-scale study of protein interactions may reveal more precise mechanisms of transport regulation and reveal new functions. Protein-protein interactions are the basis of all life activities. Revealing the law of interaction is the only way to understand the mystery of life. Yeast two-hybrid technology is the most successful platform for studying protein interaction on a large scale at present. Large-scale yeast two-hybrid technology is not a simple increase in the number of decoys of yeast two-hybrid technology in conventional laboratory. A large number of technical problems need to be solved. In this paper, ATP-binding cassette superfamily transporters were used to explore the technical conditions for the study of protein interaction in large-scale yeast two-hybrid, and to optimize the key technical links. To improve the yeast two-hybrid technology platform for the study of large-scale protein interaction, 20 pairs of highly credible protein interactions were found. Results the experience of using yeast two-hybrid technique to study protein interaction on a large scale was provided, and the discovered interaction was helpful to reveal the transport mechanism of ABC protein. In this paper, yeast two-hybrid technique was used to select 14 ATP-binding box superfamily transporter proteins ABCA1, ABCA6, ABCB1, ABCB11, ABCC4, ABCC13, ABCC3, ABCC5, ABCA1, ABCA5, AB@@ The adult liver cDNA library was screened and 67 different interactions were obtained. In the preliminary analysis of the interaction data, 20 pairs of interactions with high reliability were obtained by gyrological verification, except for non-conforming reading frames and common false positives. These interactions suggest that ATP-binding cassette superfamily transporters may have many new functions and that there may be a common transport mechanism. If the interaction between ABCC2 and NF- 魏 B1 is found, ABBC2 may be a receptor involved in the activation and regulation of NF- 魏 B signaling pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R341

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 李丹丹;hALR相互作用蛋白的篩選與鑒定[D];第二軍醫(yī)大學(xué);2005年

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本文編號：2002067