先天性核性白内障家系致病基因的克隆及在真核细胞中的表达  

Clone and expression of the candidate genes in eukaryotic cell of a congenital inherited nuclear cataract in vitro

作　　者：郑建秋[1] 刘平[1] 傅松滨[2] 

机构地区：[1]哈尔滨医科大学第一临床医学院附属眼科医院,150001 [2]哈尔滨医科大学,150001

出　　处：《眼科研究》2009年第8期664-667,共4页Chinese Ophthalmic Research

基　　金：黑龙江省博士后基金(LRB-05-451);黑龙江省青年科学基金(QC07C93)资助

摘　　要：目的克隆1个先天性核性白内障家系致病基因-突变型GJA8(mGJA8),研究其编码的蛋白质在真核细胞系中的表达和定位。方法从研究发现的先天性核性白内障家系患者基因组中扩增突变型mGJA8,克隆入载体pGEF-T,然后酶切,定向克隆到真核表达质粒pEGFP-N1中,构建pEGFP-N1-mGJA8重组表达质粒,然后用限制性内切酶消化和DNA测序鉴定,最后通过脂质体包埋转染法,用pEGFP-N1-mGJA8和pEGFP-N1质粒转染COS-7细胞,荧光显微镜观察其在COS-7细胞内的表达,采用细胞免疫组织化学法验证蛋白表达。结果经双酶切和DNA序列测定,证实致病基因mGJA8重组质粒构建成功,荧光显微镜观察在COS-7细胞上致病基因mGJA8蛋白表达,细胞免疫组织化学法显示致病基因表达蛋白在COS-7细胞中特异性表达。结论成功克隆出该家系的致病基因mGJA8,并且完成了该致病基因在体外真核细胞中的特异性表达,为进一步研究该家系的发病机制奠定基础。Objective Congenital inherited cataract is a sort of eye disease of childhood which can cause loss of visual acuity. Genetic diagnosis and treatment is the main trend to prevention and treatment of congenital inherited cataract. This study was to clone the sequence of mutation type GJA8 gene（mGJA8） from a congenital inherited nuclear cataract family and study its expression and location in eukaryotic cell lines in vitro. Methods The periphery blood samples of 16 members were collected in this family,including 7 members with congenital inherited cataract. The mGJA8 gene was amplified from this family＇ s DNA by PCR. The DNA was cloned into plasmid pGEM-T and then was digested by restriction enzyme. The amplified products were cloned into eukaryotic expression plasmid pEGFP-N1 for construction of recombinant plasmid pEGFP-N1-mGJA8. The accuracy of pEGFP-NI-mGJA8 was confirmed by restriction enzyme digestion and DNA sequencing. Final pEGFP-N1- mGJA8 and pEGFP-N1 plasmids were transfected into COS-7 cell lines by lipofectin. The expression and localization of pEGFP-N1-mGJA8 and pEGFP-N1 fusion protein were detected under the fluorescence microscope. The two fusion proteins were identified by immunohistochemical staining. Results Confirmed by restriction enzyme digestion and DNA sequencing,recombinant plasmid pERFP-NI-mGJA8 was constructed correctly. The pEGFP-N1-mGJA8 and pEGFP-N1 fusion protein was expressed in transfected COS-7cells,showing the green fluorescence, pEGFP-N1- mGJA8 fusion protein showed the positive reaction in cells for Biotin SP-HRP,presenting the brown staining. Biotin SP-HRP was absent response in pEGFP-N1. Conclusion The mGJA8 gene is cloned successfully,and the pEGFP-N1-mGJA8 fusion protein can be expressed in COS-7 ceils, which offers a foundation for further study of the mechanism of this congenital inherited nuclear cataract family.

关 键 词：先天性核性白内障 GJA8 突变型 质粒 Cx50 

分 类 号：R776.1[医药卫生—眼科] R394[医药卫生—临床医学]