【作者】 苏娇；

【导师】 何淑雅；

【作者基本信息】 南华大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 脆性X综合征(Fragile X syndrome, FraX)是最常见的遗传性智力低下疾病之一。临床表现多样,主要表现为中到重度的智力低下,常伴有巨睾、特殊面容、多动症、癫痫发作,大量患者脑电图存在特征性弥漫性瞬间慢波发放等临床病理表现。超过99%的FraX是由位于Xq27.3的脆性X智力低下基因1(fragile X mental retardation 1,FMR1)5′端非编码区(CGG)n三核苷酸重复序列的异常扩增及CpG岛的异常甲基化引起FMR1编码产物脆性X智力低下蛋白(fragile X mental retardation protein,FMRP)的表达下降或缺失所导致。分别定位于3q28及17p13.1的脆性X相关基因1/2(fragile X related gene 1/2,FXR1/2)被发现与FMR1具有高度的序列及结构相似性,三者统称为脆性X基因家族(FXR gene family)。其编码的蛋白不仅可以与自身结合形成同源二聚体,也可与家族中其他蛋白结合以异源二聚体形式存在。FXR1P系列相互作用蛋白的筛选以及验证将在一定程度上有助于了解FXR1P的调节机制及其基本功能,进而明确FXR1P与脆性X综合征发病机制的关系。本研究利用酵母双杂交技术研究FXR1P相互作用蛋白,首先构建酵母双杂交系统诱饵质粒pGBKT7-FXR1,并将其转化酵母菌AH109,与转化了Matchmaker人胎脑pGADT7-cDNA文库质粒的酵母菌Y187进行配合(Mating),通过一系列报告基因的表达检测筛选阳性克隆,将经酶切鉴定后阳性克隆质粒AD/Library转化酵母菌Y187,再与诱饵质粒pGBKT7-FXR1酵母菌进行一对一小交配验证蛋白质的相互作用,真阳性克隆的AD/Library质粒测序及生物信息学分析,共得到了七种可能与FXR1P相互作用的蛋白:Btf,SAFB,CMAS,FTH1,CSH1,GOLGA4,HSD17B1;对其中的两种与凋亡相关的蛋白(Btf和SAFB)进行了免疫共沉淀实验,构建真核表达载体pCMV-FXR1-Myc,pCMV-Flag-FXR1, pCMV-Btf-Myc, pCMV-SAFB-Myc,哺乳动物细胞内的免疫共沉淀验证FXR1P与Btf、SAFB的相互作用,结果表明FXR1P能够与Btf相互作用,而与SAFB的相互作用免疫共沉淀未检测到。根据实验结果提示Btf是FXR1P蛋白调控网络的新成员,在神经、肌肉等组织生长发育过程中发挥一定的作用。FXR1P可能参与细胞凋亡的发生过程。更多还原

【Abstract】 Screening and Identification of FXR1P interacting proteins Fragile X syndrome is one of the most common form of inherited mental retardation, which have a diversity of clinical manifestation. The main is mental retardation range from moderate to severe, and associated with giant testis, particular face, minimal brain dysfunction, epileptic attack.The check of electroencephalogram present typical and diffused moment slow wave releasing and some other clinical pathological features. Research shows that more than 99% of the FraX are caused by the expression decrease or absence of FMRP(fragile X mental retardation protein), the coding products of FMR1(fragile X mental retardation 1) located at Xq27.3,which is caused by the abnormal amplification of the unstable CGG repeat sequence in the 5’untranslated region of FMR1 and the subsequent methylation of its CpG island. FXR1/2 (fragile X related gene 1/2) located in 3q28 and 17p13.1 respectively were found have considerable similarity with FMR1 in sequence and structure, so they were called fragile X gene family. Any two of the three genes can exist in the form of homodimers or heterodimers. The screening and identification of the series of interaction proteins of FXR1P will be helpful for the clarification of the function of FXR1,understand the regulatory mechanism of FXR1P,and then to comprehend the relationship between FXR1P and FraX.The FXR1P interacting proteins were researched by the yeast two hybrid system. Firstly ,we constructed bait plasmid pGBKT7-FXR1, and mated the transformed bait plasmid pGBKT7-FXR1 in yeast AH109 with the yeast Y187 transformed of Matchmaker human fetal brain pGADT7-cDNA library plasmid. Screened positive clones through detection the express of a series of report genes, and identified the positive AD/library plasmids by enzymatic digestion and then validated the protein-protein interaction by retransformed the AD/library plasmids to yeast Y187 and applied one-to-one small scale mating with yeast AH109 transformed bait plasmid pGBKT7-FXR1. seven proteins which may interact with FXR1P were acquired, namely Btf,SAFB,CMAS,FTH1,CSH1,GOLGA4,and HSD17B1; Later, the interaction of two screend proteins associated with apoptosis with FXR1P was checked with co-immunopricipitation(Co-IP) experiment. Four eukaryon expression vectors, pCMV-FXR1-Myc, pCMV-Flag-FXR1, pCMV-Btf-Myc, pCMV-SAFB-Myc were constructed, and the interaction of FXR1P and Btf, SAFB were validated by Co-IP in the mammals cells. We found FXR1P can interact with Btf, but the interaction between FXR1P and SAFB was not checked. Thus we concluded that Btf is a new member of the FXR1P-related regulation network and play a role in the development process of nervous and muscle system.FXR1P may involeved in the progress of cell apoptosis.更多还原