hAph2基因影响细胞生长主要功能域的筛选
发布时间：2018-07-28 13:36
【摘要】： hAph2基因是本实验室大规模功能基因筛选中从人胎盘cDNA文库中克隆获得的一个新基因(GenBank Access No.AF258563),具有自主知识产权,由于hAph2基因与小鼠Aph2(Abl-philin 2)基因在氨基酸水平具有96%同源性,因此将其命名为“human Aph2”基因(简写为hAph2)。该基因定位于人染色体10q24.1,有五种亚型:hAph2α,hAph2β,hAph2γ,hAph2δ和hAph2ε,编码ZDHHC16(zinc finger,DHHC-type containing 16)蛋白,含有DHHC/zf-DHHC和DPG功能域,前者又称为NEW1。本研究着重分析hAph2基因在COS-7细胞中的表达和hAPH2蛋白对细胞生长的影响。含有GFP标签的野生型hAph2α-GFP,hAph2β-GFP,hAph2γ-GFP和hAph2δ-GFP转染COS-7细胞48小时后,采用流式细胞术,western blotting及细胞免疫荧光观察等方法对COS-7细胞研究分析发现,hAph2基因在COS-7细胞中外源性表达有多聚体和单体两种形式,而且hAPH2蛋白主要定位于核周细胞质,并且可诱导COS-7细胞发生凋亡。为深入探讨hAPH2蛋白诱导COS-7细胞发生凋亡的机制,我们又构建两种突变体表达载体hAph2βΔDHHC-GFP和hAph2βΔDPG-GFP。我们主要运用western blotting技术,检测凋亡主要执行者Caspases,MAPK超家族成员和其它凋亡相关蛋白。结果发现,hAph2γ,hAph2δ,hAph2ΔDHHC和hAph2ΔDPG基因表达使得Caspases剪切活化体增多,JNK和Fas-FasL信号转导途径轻度活化。初步证实hAPH2蛋白诱导COS-7细胞发生凋亡的机制,以及DHHC和DPG这两个主要功能域对细胞的保护作用。 我们还采用免疫共沉淀技术和荧光免疫细胞化学观察方法,初步筛选与hAPH2β蛋白相互作用的蛋白,初步证实hAPH2β在肝癌SMMC-7721细胞中可与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 伪 hAph2 尾 hAph2 纬 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 伪 -GFPhAph2 尾 -GFPhAph2 纬 -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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346
本文编号：2150318
[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 伪 hAph2 尾 hAph2 纬 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 伪 -GFPhAph2 尾 -GFPhAph2 纬 -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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346
【参考文献】
相关期刊论文 前3条
1 ;Role of JNK activation in apoptosis:Adouble-edged sword[J];Cell Research;2005年01期
2 张锋锐;李锦军;王春艳;葛超;徐文;朱丽;陈燕;万大方;顾健人;;新基因pp3774的亚细胞定位以及在肝癌细胞系和组织中的表达谱研究[J];肿瘤;2005年06期
3 张锋锐;李锦军;王春艳;朱丽;徐文;顾健人;;新基因pp3774抑制NIH/3T3细胞生长机制初探[J];肿瘤;2005年06期
，本文编号：2150318
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2150318.html
最近更新
教材专著
