【摘要】： hAph2基因是本實驗室大規(guī)模功能基因篩選中從人胎盤cDNA文庫中克隆獲得的一個新基因(GenBank Access No.AF258563),具有自主知識產(chǎn)權(quán),由于hAph2基因與小鼠Aph2(Abl-philin 2)基因在氨基酸水平具有96%同源性,因此將其命名為“human Aph2”基因(簡寫為hAph2)。該基因定位于人染色體10q24.1,有五種亞型:hAph2α,hAph2β,hAph2γ,hAph2δ和hAph2ε,編碼ZDHHC16(zinc finger,DHHC-type containing 16)蛋白,含有DHHC/zf-DHHC和DPG功能域,前者又稱為NEW1。本研究著重分析hAph2基因在COS-7細胞中的表達和hAPH2蛋白對細胞生長的影響。含有GFP標(biāo)簽的野生型hAph2α-GFP,hAph2β-GFP,hAph2γ-GFP和hAph2δ-GFP轉(zhuǎn)染COS-7細胞48小時后,采用流式細胞術(shù),western blotting及細胞免疫熒光觀察等方法對COS-7細胞研究分析發(fā)現(xiàn),hAph2基因在COS-7細胞中外源性表達有多聚體和單體兩種形式,而且hAPH2蛋白主要定位于核周細胞質(zhì),并且可誘導(dǎo)COS-7細胞發(fā)生凋亡。為深入探討hAPH2蛋白誘導(dǎo)COS-7細胞發(fā)生凋亡的機制,我們又構(gòu)建兩種突變體表達載體hAph2βΔDHHC-GFP和hAph2βΔDPG-GFP。我們主要運用western blotting技術(shù),檢測凋亡主要執(zhí)行者Caspases,MAPK超家族成員和其它凋亡相關(guān)蛋白。結(jié)果發(fā)現(xiàn),hAph2γ,hAph2δ,hAph2ΔDHHC和hAph2ΔDPG基因表達使得Caspases剪切活化體增多,JNK和Fas-FasL信號轉(zhuǎn)導(dǎo)途徑輕度活化。初步證實hAPH2蛋白誘導(dǎo)COS-7細胞發(fā)生凋亡的機制,以及DHHC和DPG這兩個主要功能域?qū)毎谋Ｗo作用。 我們還采用免疫共沉淀技術(shù)和熒光免疫細胞化學(xué)觀察方法,初步篩選與hAPH2β蛋白相互作用的蛋白,初步證實hAPH2β在肝癌SMMC-7721細胞中可與c-Ab1相互作用。
[Abstract]:HAph2 gene is a new gene (GenBank Access No.AF258563 cloned from human placental cDNA library in large scale functional gene screening in our laboratory. It has its own intellectual property rights, because hAph2 gene shares 96% homology with mouse Aph2 (Abl-philin 2) gene at amino acid level. Therefore, it is named "human Aph2" gene (abbreviated as hAph2). The gene is located on human chromosome 10q24.1 and has five subtypes: hAph2 偽 hAph2 尾 hAph2 緯 hAph2 未 and hAph2 蔚, which encode ZDHHC16 (zinc fingerDHHC-type containing 16) protein and contain DHHC/zf-DHHC and DPG functional domains, the former is also called NEW1. This study focused on the expression of hAph2 gene in COS-7 cells and the effect of hAPH2 protein on cell growth. The wild-type hAph2 偽 -GFPhAph2 尾 -GFPhAph2 緯 -GFP and hAph2 未 -GFP containing GFP tags were transfected into COS-7 cells for 48 hours. Western blotting and immunofluorescence were used to analyze the expression of hAph2 gene in COS-7 cells. It was found that there were two forms of heteropolymer and monomers in COS-7 cells, and hAPH2 protein was mainly located in perinuclear cytoplasm. And it can induce apoptosis of COS-7 cells. In order to investigate the mechanism of apoptosis induced by hAPH2 protein, we constructed two mutant expression vectors hAph2 尾 螖 DHHC-GFP and hAph2 尾 螖 DPG-GFP. We used western blotting technique to detect the members of Caspasesus MAPK superfamily and other apoptosis-related proteins. The results showed that the expression of hAph2 未 hAph2 螖 DHHC and hAph2 螖 DPG genes increased the number of Caspases shear activators and slightly activated the signal transduction pathway of JNK and Fas-FasL. The mechanism of apoptosis induced by hAPH2 protein and the protective effect of DHHC and DPG on COS-7 cells were preliminarily confirmed. We also screened the proteins interacting with hAPH2 尾 protein by immunocoprecipitation and fluorescence immunocytochemistry, and preliminarily confirmed that hAPH2 尾 could interact with c-Ab1 in SMMC-7721 cells of HCC.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前3條

1 ;Role of JNK activation in apoptosis:Adouble-edged sword[J];Cell Research;2005年01期

2 張鋒銳;李錦軍;王春艷;葛超;徐文;朱麗;陳燕;萬大方;顧健人;;新基因pp3774的亞細胞定位以及在肝癌細胞系和組織中的表達譜研究[J];腫瘤;2005年06期

3 張鋒銳;李錦軍;王春艷;朱麗;徐文;顧健人;;新基因pp3774抑制NIH/3T3細胞生長機制初探[J];腫瘤;2005年06期

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