【摘要】：目的 Np95/ICBP90-like RING finger protein(NIRF)是新近發(fā)現(xiàn)的一種核蛋白，其包括802個氨基酸殘基，蛋白分子內(nèi)部含有UBL(ubiquitin-like domain)、PHD、YDG/SRA和RING finger結(jié)構(gòu)域。隨著其研究的深入，NIRF功能已涉及細(xì)胞增殖調(diào)節(jié)、蛋白多聚泛素化降解、細(xì)胞癌變進(jìn)程控制以及更多未知的領(lǐng)域。已有研究報道，NIRF能與Cdk2-cyclin E復(fù)合體和let-7a結(jié)合，過表達(dá)的NIRF能阻止細(xì)胞周期于G1期。另外NIRF還具有泛素化酶E3的作用，即泛素化和自我泛素化功能，表明NIRF介導(dǎo)重要的蛋白降解途徑。p53是細(xì)胞內(nèi)重要的抑癌基因，p53的功能活動被認(rèn)為是細(xì)胞對細(xì)胞異常、損傷、壓力的一種應(yīng)激反應(yīng)，通過啟動自我修復(fù)程序抑制細(xì)胞分裂誘導(dǎo)其進(jìn)入衰老程序，或啟動細(xì)胞程序性死亡清除異變的細(xì)胞。已有研究報道NIRF能與p53相互作用, NIRF本身也是一個高度調(diào)節(jié)蛋白，在細(xì)胞正常的生理狀態(tài)下發(fā)揮泛素化E3連接酶的作用結(jié)合p53并將其降解，但NIRF與p53結(jié)合的蛋白結(jié)合域目前尚不清楚。本文研究證明NIRF能與p53結(jié)合成復(fù)合體參與泛素化蛋白降解途徑，并測定出NIRF與p53結(jié)合的區(qū)域，為下一步研究蛋白泛素化降解和腫瘤的治療提供實驗基礎(chǔ)和依據(jù)。 方法 （1）采用DMEM培養(yǎng)基培養(yǎng)HEK293細(xì)胞。培養(yǎng)基中補(bǔ)充含10%小牛血清和雙抗。 （2） RIPA蛋白裂解液收集HEK293細(xì)胞總蛋白。 （3）細(xì)胞轉(zhuǎn)染按照轉(zhuǎn)染試劑說明說進(jìn)行，轉(zhuǎn)染48小時之后收獲細(xì)胞，細(xì)胞收獲前10小時添加蛋白酶體抑制劑MG-132。 （4）將NIRF突變體在細(xì)胞內(nèi)全部表達(dá)。在HEK293細(xì)胞內(nèi)，分別轉(zhuǎn)染四個突變體質(zhì)粒，同時轉(zhuǎn)染pCMV-3×FLAG空質(zhì)粒和pCMV-FLAG-NIRF全長質(zhì)粒，作為陰陽性對照．細(xì)胞裂解液經(jīng)Western印跡，anti-NIRF, anti-FLAG和anti-β-Actin抗體檢測分析。 （5）用免疫蛋白共沉淀技術(shù)和蛋白質(zhì)印跡分析技術(shù)鑒定NIRF與p53結(jié)合的蛋白結(jié)合域。HEK293細(xì)胞分別同時轉(zhuǎn)染p53和各突變體質(zhì)粒，細(xì)胞裂解液經(jīng)p53抗體IP純化，產(chǎn)物經(jīng)WB分析。NIRF全長能與p53穩(wěn)定結(jié)合，同時轉(zhuǎn)染NIRF全長質(zhì)粒作為陽性對照。 結(jié)果 結(jié)果顯示，所有的突變體都能在細(xì)胞中表達(dá)，并且兩種抗體檢測結(jié)果完全一致。在免疫共沉淀試驗中，，ΔUBL、ΔYDG/SRA和ΔRING缺失突變體蛋白依然能被p53沉淀出，而ΔPHD卻未能被p53沉淀出。 結(jié)論 本研究表明，NIRF通過PHD區(qū)域與p53形成復(fù)合體.該復(fù)合體可能參與蛋白分選、蛋白降解、DNA修復(fù)以及細(xì)胞凋亡等一系列重要的細(xì)胞活動，從而形成與細(xì)胞增殖相關(guān)的新的信號通路，在腫瘤的發(fā)生發(fā)展中可能發(fā)揮著某種程度上作用。
[Abstract]:Objective Np95/ICBP90-like RING finger protein (NIRF) is a newly discovered nuclear protein, which contains 802 amino acid residues and contains UBL (ubiquitin-like domain), PHD,YDG/SRA and RING finger) domains. With the development of its research, the function of NIRF has been involved in the regulation of cell proliferation, the degradation of protein polyubiquitin, the control of cell carcinogenesis and more unknown fields. It has been reported that NIRF can bind to Cdk2-cyclin E complex and let-7a, and overexpression of NIRF can block cell cycle in G1 phase. In addition, NIRF also has the function of ubiquiding enzyme E3, that is, ubiquitization and self-ubiquification, which indicates that NIRF mediates important protein degradation pathway. P53 is an important tumor suppressor gene in cells, and p53 function is thought to be abnormal and damaged by cells. A stress response by initiating a self-repair program that inhibits cell division and induces it to enter the aging process, or activates programmed cell death to clear the metamorphosis of the cells. It has been reported that NIRF can interact with p53, and NIRF itself is a highly regulated protein, which acts as a Ubiquitin E3 ligase to bind and degrade p53 in normal physiological state of cells. However, the binding domain of NIRF to p53 protein is unclear. In this paper, it is proved that NIRF can bind to p53 complex to participate in the degradation pathway of ubiquitin, and the region of NIRF binding to p53 is determined, which provides the experimental basis and basis for further study on the degradation of protein ubiquitin and the treatment of tumor. Methods (1) HEK293 cells were cultured on DMEM medium. The medium was supplemented with 10% calf serum and double antibodies. (2) the total protein of HEK293 cells was collected by RIPA protein lysate. (3) Cell transfection was carried out according to the instructions of transfection reagent. After 48 hours of transfection, the cells were harvested and the proteasome inhibitor MG-132. was added 10 hours before harvest. (4) all the NIRF mutants were expressed in the cells. Four mutants were transfected into HEK293 cells, and pCMV-3 脳 FLAG empty plasmids and pCMV-FLAG-NIRF full-length plasmids were transfected respectively. The cell lysate was detected by Western blot, anti-NIRF, anti-FLAG and anti- 尾-Actin antibodies. (5) the protein binding domains of NIRF and p53 were identified by immunoprecipitation and Western blot analysis. HEK293 cells were transfected with p53 and mutant granulocytes respectively, and the cell lysate was purified by p53 antibody IP. The product was analyzed by WB. The full length of NIRF could bind to p53 stably, and the full-length plasmid of NIRF was transfected as positive control at the same time. The results showed that all the mutants could be expressed in the cells, and the results of the two antibodies were identical. In immunoprecipitation assay, 螖 UBL, 螖 YDG/SRA and 螖 RING deletion mutants could still be precipitated by p53, while 螖 PHD could not be precipitated by p53. Conclusion this study suggests that NIRF forms a complex with p53 via the PHD region. The complex may be involved in a series of important cellular activities, such as protein sorting, protein degradation, DNA repair and apoptosis, thus forming a new signal pathway related to cell proliferation. It may play a role in the occurrence and development of tumor to some extent.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 段昌柱;蒲淑萍;TSUTOMU MORI;HIDEO KOCHI;邱宗蔭;;NIRF對P53蛋白泛素化作用的研究[J];生物化學(xué)與生物物理進(jìn)展;2006年02期

2 金芳敏;錢冠華;楊艷;段昌柱;;人NIRF蛋白與HBV核心蛋白相互作用的初步研究[J];生命科學(xué)研究;2011年01期

3 楊艷;金方敏;錢冠華;段昌柱;;NIRF蛋白的原核表達(dá)及純化[J];中國生物制品學(xué)雜志;2011年01期

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