小鼠TGFBI基因真核表达载体的构建和表达  

Construction and expression of eukaryotic expression vector of mouse TGFBI gene

作　　者：牛静宜[1] 陈鹏[1] 王晔[1] 谢立信[1] 王宜强[1] 

机构地区：[1]山东省眼科研究所山东省眼科学重点实验室-省部共建国家重点实验室培育基地,青岛266071

出　　处：《眼科研究》2009年第3期161-165,共5页Chinese Ophthalmic Research

基　　金：国家自然科学基金(30630476);973前期研究专项基金(2007CB516705)资助

摘　　要：目的构建小鼠TGFBI基因的真核表达载体,为研究角膜营养不良的发病机制奠定基础。方法提取BALB/cBy小鼠正常角膜组织总RNA,经反转录-PCR合成TGFBI cDNA,克隆入真核表达载体pcDNA3.1并测序验证。用不同剂量重组质粒pcDNA3.1-TGFBI转染NIH3T3细胞,通过SYBR荧光实时定量PCR和Western blot检测TGFBI在细胞中的表达。结果测序结果显示扩增到的TGFBI cDNA以正确序列和方式插入载体,实时定量PCR和Western blot结果显示TGFBI在NIH3T3细胞中表达增强。结论成功构建了小鼠TGFBI基因真核表达载体,并在细胞中进行表达,为进一步研究TGFBI在角膜内的生理、病理功能奠定基础。Objective It has been recognized that the mutation of over 30 types of transforming growth factor beta-induced gene （TGFBI） can induce corneal dystrophy,but its molecular mechanism is below understood.The goal of this study was to construct and express mouse TGFBI in eukaryotic expression vector for the further study on the effect of TGFBI in corneal biology and the pathogenesis of corneal dystrophy.MethodsThe eyeball from 5-week-old BALB/cBy mouse was utilized in this experiment.TGFBI cDNA was obtained by reverse transcription-PCR from total RNA extracted from mouse corneas and cloned into pcDNA3.1 vector.Recombinant plasmids were identified by restrictive digestion and direct sequencing.Different doses of pcDNA3.1-TGFBI plasmids were applied for transfection of cultured NIH3T3 cells.NIH3T3 mRNA and protein were harvested from transfected cells for real-time PCR（RT-PCR） analysis and Western blot assay respectively.ResultsRestriction digestion and sequencing indicated that TGFBI cDNA fragment was successfully inserted into the vector.Transfection of recombinant pcDNA3.1-TGFBI into NIH3T3 cells resulted in effective expression of TGFBI（about 90%）,showing the green fluorenscence under the fluorescence microscope.TGFBI mRNA from transfected pcDNA3.1-TGFBI cells was obviously increased by RT-PCR and TGFBI protein level followed the same pattern by Western blot.ConclusionRecombinant eukaryotic expression vector harboring murine TGFBI cDNA is obtained and efficiently expressed in NIH3T3 cells.The construction might be applied in studying the function and role of TGFBI in pathogenesis of corneal dystrophy.

关 键 词：TGFBI 构建 真核表达载体 角膜营养不良 

分 类 号：R772[医药卫生—眼科]