导　　师： 杨祥良;刘木根

学科专业： 070301

授予学位： 硕士

作　　者： ;

机构地区： 华中科技大学

摘　　要： 视网膜色素变性/(RP/)是一组以感光细胞进行性丧失为表现的遗传异质性疾病。1998年由一个西班牙的研究组,通过对常染色体隐性视网膜色素变性家系/(ARRP/)进行连锁分析,将该家系的致病基因定位于2q31-2q33,并命名为RP26。后在两个无血缘关系的RP西班牙家系中发现该位点的新克隆基因CERKL存在相同的纯合突变,认为该基因是一个新的视网膜色素变性致病基因。目前有关CERKL的文献报道只有5篇,对其正常的生理功能,以及突变后所引起视网膜色素变性/(RP/)的分子机制都不清楚。为了研究该基因在体内的生物学功能,揭示其在视网膜中的代谢途径,本研究克隆了CERKL基因全长,应用酵母双杂交系统筛选与CERKL相互作用的蛋白质,以期通过这一线索获得CERKL基因的功能信息,为研究CERKL致病机理提供分子基础。 实验首先通过巢式PCR方法从HeLa细胞cDNA中扩增得到CERKL基因全长,然后将其作为目的基因构建筛选时所用的诱饵载体pGBKT7-CERKL。转化酵母AH109细胞,检查细胞毒性和自激活作用。再将pGBKT7-CERKL和来源于人胰腺的cDNA文库质粒顺序转化酵母AH109细胞中,通过营养缺陷型培养基进行阳性克隆的筛选。随后将所得的560个克隆通过多次营养筛选、酶切鉴定、α-gal分析、一对一酵母双杂交系统的回复性实验排除假阳性克隆。最后借助数据库中的BLAST工具对候选阳性克隆的测序结果进行同源性分析,确定可能发生相互作用的蛋白质。经过筛选和多重验证,最后确定了2种相关蛋白,分别为CTRC/(Homo sapiens chymotrypsin C preproprotein/)、EIF3S9/(Homo sapiens eukaryotic translation initiation factor 3 subunit 9/)。 总之,本研究在世界上首次发现2种可能与CERKL相互作用的蛋白质,对这些蛋白的最终确认,以及对它们与CERKL之间相互作用机制的进一步研究,必将为我们认识这一基因的结构与功能,阐明其� Retinitis pigmentosa /(RP/) is a genetically heterogeneous disorder characterized by progressive loss of photoreceptors by apoptosis. In 1998, a Spanish group performed linkage analysis in a large nuclear Spanish family with autosomal recessive retinitis pigmentosa, and fine mapped the RP26 locus in this family to a 2.5-Mb region on chromosome 2q31.2-q32.3. They identified a novel gene, CERKL, in the RP26 critical linkage region and found a homozygous mutation in exon 5 of the gene in two Spanish families. However, the physiological functions of CERKL in retina and molecular mechanism by which CERKL mutation causing RP remains unclear. There have been only 5 publications on CERKL reported until now. In order to study biology function of CERKL and reveal its pathway in the retina, we cloned the full-length CERKL gene and screened the proteins interacted with CERKL by using Yeast Two-Hybrid System. We firstly cloned the full-length CERKL gene using nested PCR from HeLa cell cDNA library. Secondly, the Yeast Two-Hybrid System /(Clontech/) was used to screen a human pancreas GAL4-AD-cDNA library with human CERKL cDNA fused to GAL4-BD in plasmid pGBKT7 as bait. Then we transformed the pGBKT7-CERKL into yeast AH109 to examine the toxicity and self-activation of pGBKT7-CERKL plasmid. After that, pGBKT7-CERKL and the library plasmids were transformed sequentially into yeast strain AH109. The transformants were plated on SD agar to screen for interacting partners according to the manufacturer’s instructions. 560 preliminary positive clones were further checked by phenotypes retesting, clones sorting andα-galactosidase activity detecting to exclude the false positive clones. Plasmids recovered from positive clones were amplified by PCR for the inserts and sequenced. The sequencing results of the candidate clones were analyzed using the BLAST database to find the potential interaction of proteins. Two proteins, CTRC/(Homo sapiens chymotrypsin C preproprotein/) and EIF3S9/(Homo sapiens eukaryotic translation i

关 键 词： 视网膜色素变性 酵母双杂交 蛋白质相互作用

领　　域： [生物学]