Cx50 V64G突变的生物信息学分析及其真核表达载体的构建

目的:构建pcDNA3.1/Cx50 V64G真核表达载体,用生物信息学软件分析V64G突变对人缝隙连接蛋白Cx50的影响。方法:经PCR获得Cx50基因片段,重组至真核表达载体pcDNA3.1/Amp( )中,经PCR,酶切和序列测定方法鉴定重组质粒。结果:获得了具有V64G突变的Cx50的编码基因,并成功构建了其真核表达载体。并证明64位缬氨酸在不同物种的Cx50及在人的多种缝隙连接蛋白是高度保守的区域,与白内障的发生高度相关。结论:pcDNA3.1/Cx50 V64G真核表达质粒的成功构建和鉴定为进一步研究白内障机理奠定了基础。

Bioinformatics analysis and construction of eukaryotic expression plasmid of Cx50 V64G mutation

AIM : To construct and analyze eukaryotic expression plasmid inserted by Cx50 with V64G mutation through bioinformatics software.METHODS: The full coding domain sequence of Cx50 with V64G mutation was acquired from the blood of patients with cataract and was cloned into pcDNA3.1 /Amp (+).The constructed plasmid was identified with PCR , enzyme digestion and sequencing. The analysis of Cx50 with V64G mutation was performed with bioinformatics software.RESULTS : Cx50 with V64G mutation was successfully amplified and its eukaryotic expression plasmid was constructed. Valine-64 is well conserved in the first extracellular loop of connexin 50 in different species and also in different human α -type gap junctional proteins.CONCLUSION : The successive reconstruction and verification of eukaryotic expression plasmid containing Cx50 with V64G mutation established the foundation for further studying the mechanism of cataract.