摘要
目的构建A546D突变型TGFBI基因真核表达载体和体外转染体系,为研究野生型及A546D突变型TGFBI基因的功能提供实验基础。方法应用重叠延伸PCR法定点诱变,构建A546D突变型TGFBI基因真核表达载体,以阳离子脂质体介导体外转染技术瞬时转染人角膜上皮(human corneal epithelium,HCE)细胞,Western Blot法检测转染后培养基中HCE细胞分泌的TGFBI蛋白的表达。结果重组质粒总长约7.5 kb,由HindⅢ、XbaⅠ双酶切鉴定产生5.4 kb和2.1 kb两条片段者为克隆正确质粒。测序鉴定证实了TGFBI基因编码区完整插入到载体pcDNA3.1的多克隆位点中,突变载体的第1685位碱基由C替换为A,使其编码的第546个氨基酸由Ala突变为Asp即A546D突变。野生型及A546D突变型载体转染入HCE细胞后,TGFBI蛋白有效表达。结论成功构建了A546D突变型TGFBI基因真核表达载体,为TGFBI基因相关性角膜营养不良致病机制的研究奠定了基础。
Objective To construct A546D mutant TGFBI gene eulmtyoUe expression vector and transfection system in vitro in order to study the function of wild and mutant TGFBI. Methods The mutant site was induced by SOE- PCR. The A546D mutant TGFBI plasmid was transfected into human corneal epi- thelial cells by cationic liposomes. The expression of TGFBI protein in medium was examined by Western Blot analysis. Results Enzyme digestion analysis of the recombinant plasmid （7.5 kb length） showed two fragments （5.4 kb and 2.1 kb） by Hind I]I and Xba I. Sequencing result showed that the coding region of TGFBI exactly inserted to the pcDNA3.1 vector. And sequencing revealed a C-→A mutation in the 1685 base of recombinant plasmid. This single-nucleotide change was predicted to introduce an alanine to substitute an aspartic acid （A546D）. TG- FBI protein was efficiently expressed in HCE transfected with the recombinant plasmid. Cone｜us｜on The constructed eukaryote expression plasmid pcDNA3~ 1-TGFBI-A546D can efficiently express in human corneal epithelial cells in vitro. It provides an experimental basis for further study on the pathogenesis of TGFBI related corneal dystrophies.
出处
《眼科新进展》
CAS
北大核心
2015年第6期525-528,共4页
Recent Advances in Ophthalmology
基金
黑龙江省博士后科研启动基金(编号:LBH-Q13126)
黑龙江省卫生厅科研基金(编号:2012-554)
哈医大一院科研基金(编号:2011BS017)~~
