一个遗传性白内障家系致病基因的突变检测及其致病机制研究

发布时间：2018-05-12 01:34

  本文选题：先天性白内障 + 基因突变　； 参考：《武汉大学》2010年博士论文

【摘要】： 背景：白内障是全球首要致盲眼病。其中,先天性白内障是一种常见的儿童眼病。环境和遗传因素是先天性白内障的两大病因,其中遗传因素在白内障的发生和发展中起着重要作用。随着分子遗传学技术的发展,越来越多的先天性白内障相关候选基因被定位和克隆。然而,先天性白内障的发病机制还研究得不是很清楚。本研究拟对一个先天性白内障家系进行致病基因的筛查及致病机制研究,以期找到先天性白内障相关的突变位点,揭示先天性白内障的发病机理。 方法：采用直接测序的方法对一个来自湖北省的常染色体显性遗传先天性白内障家系进行基因突变检测。应用生物信息学工具Expasy蛋白组学服务器(http://www.expasy.org)对aB-晶体蛋白及其突变体进行生物物理学预测。应用基因重组技术在大肠杆菌E. coli BL21(DE3)中表达野生型和R11H突变型aB-晶状体蛋白。应用圆二色光谱技术对野生型和R11H突变型aB-晶状体蛋白的结构进行分析；应用bis-ANS荧光探针结合实验分析aB-晶状体蛋白的表面疏水区域；分别以胰岛素和乙醇脱氢酶(ADH)为底物比较它们的分子伴侣活性。此外利用脂质体方法分别将野生型和R11H突变型CRYAB基因转入HeLa细胞和人晶状体上皮细胞(HLE),应用激光共聚焦显微镜和流式细胞仪对野生型和R11H突变型aB-晶状体蛋白的特征进行了比较分析。通过优化载体,诱导时间,诱导温度,诱导剂浓度等条件摸索aA-晶状体蛋白在大肠杆菌中的表达条件。 结果：候选基因直接测序在aB-晶状体蛋白基因(CRYAB)第一个外显子上发现了一个新的突变位点,该突变导致第11位密码子由精氨酸变成了组氨酸(R11H)。在家系中正常人和200例正常对照及40例老年性白内障患者中均未发现此突变,排除了多态性的可能。aB-晶状体蛋白计算机模型的构建和分析提示突变型aB-晶状体蛋白基因改变了该蛋白的结构及理化性质。R11H突变型和野生型aB-晶状体蛋白的近紫外和远紫外圆二色光谱呈现较大差异。bis-ANS荧光光谱实验显示突变型蛋白的表面疏水区域减少。而热稳定性测定表明R11H突变并没有改变蛋白的稳定性。分子伴侣活性测定表明突变型aB-晶状体蛋白的分子伴侣活性升高。激光共聚焦显微镜分析表明野生型和R11H突变型aB-晶状体蛋白都主要分布在细胞质里面,两者在细胞内的定位无明显差异。流式细胞术分析表明R11H突变型aB-晶状体蛋白能诱导晶状体上皮细胞的早期凋亡。利用pET-32a(+)重组表达菌株,在0.5 mM IPTG,37℃下诱导5 h成功表达并纯化得到aA-晶状体蛋白。 结论：在一常染色体显性遗传先天性白内障家系中发现了一个新的CRYAB基因突变(R11H)。突变型aB-晶状体蛋白基因改变了蛋白的结构,导致aB-晶状体蛋白的分子伴侣活性升高。同时突变基因能诱导晶状体上皮细胞的早期凋亡。这种结构与功能上的改变可能与白内障的发生相关。
[Abstract]:Background: cataract is the leading cause of blindness in the world. Congenital cataract is a common eye disease in children. Environmental and genetic factors are two major causes of congenital cataract, among which genetic factors play an important role in the occurrence and development of cataract. With the development of molecular genetics, more and more candidate genes related to congenital cataract have been located and cloned. However, the pathogenesis of congenital cataract is not well understood. In order to find the mutation sites of congenital cataract and reveal the pathogenesis of congenital cataract, this study aims to screen the pathogenetic gene and study the pathogenesis of congenital cataract in a family. Methods: an autosomal dominant congenital cataract pedigree from Hubei Province was detected for gene mutation by direct sequencing. Bioinformatics tool Expasy proteomics server http: / www.expasy.org) was used to predict the biochemistry of AB-crystal proteins and their mutants. Recombinant gene technique was used to express wild type and R11H mutant AB-crystallin in E. coli BL21DE3. Circular dichroism spectroscopy was used to analyze the structure of wild type and R11H mutant AB-crystallin, and bis-ANS fluorescence probe was used to analyze the surface hydrophobic region of AB-crystallin. The molecular chaperone activity of insulin and alcohol dehydrogenase (ADH) were compared. In addition, wild-type and R11H mutant CRYAB genes were transfected into HeLa cells and human lens epithelial cells by liposome method, respectively. Laser confocal microscopy and flow cytometry were used to detect the expression of AB-crystallin in wild type and R11H mutant. The characteristics are compared and analyzed. The expression conditions of AA-crystallin in Escherichia coli were studied by optimizing the carrier, inducing time, inducing temperature, concentration of inducer and so on. Results: a new mutation site was found in the first exon of AB-Cryptosin gene by direct sequencing. The mutation resulted in the transformation of the 11th codon from arginine to histidine R11H. This mutation was not found in normal subjects, 200 normal controls and 40 age-related cataract patients. The construction and analysis of the computer model of AB- crystallin indicated that the mutant gene changed the structure and physicochemical properties of the protein. R11H mutant and wild type of AB-crystallin were nearly purple. The external and far ultraviolet circular dichroism spectra showed great difference. Bis-ANS fluorescence spectra showed that the surface hydrophobic region of mutant protein decreased. The thermal stability test showed that the R 11 H mutation did not change the stability of the protein. The molecular chaperone activity test showed that the molecular chaperone activity of mutant AB- crystallin was increased. The results of laser confocal microscopy showed that both wild-type and R11H mutant AB-crystallin were mainly distributed in cytoplasm, but there was no significant difference in their localization in cells. Flow cytometry analysis showed that R11H mutant AB-crystallin could induce early apoptosis of lens epithelial cells. The recombinant strain pET-32a () was induced to express aA- crystallin at 0.5 mm IPTGG at 37 鈩，

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