摘要
目的:构建VIM(Vimentin)基因的真核表达载体,以深入研究VIM的功能及其在相关疾病中的作用。方法:从人cDNA文库中,以RT-PCR方法扩增出1401 bp的VIM编码区片段,胶回收后连接入T载体,测序鉴定。再用Hind III和BamH I双酶切,将VIM编码区片段定向克隆到真核表达载体pcDNA3.1中,酶切鉴定重组质粒。将重组质粒转染NIH3T3细胞,分别以RT-PCR和Western Blot方法检测VIM的mRNA表达和蛋白表达。结果:将人VIM编码区基因成功克隆到真核表达载体pcDNA3.1中;继而转染NIH3T3细胞后,RT-PCR和Western Blot结果显示细胞可以表达VIM的mRNA和蛋白。结论:成功构建pcDNA3.1-VIM的真核表达载体,为进一步研究VIM基因的功能以及其在相关疾病中的作用奠定了基础。
Objective： To build eukaryotic expression plasmid of VIM （Vimentin） gene in order to investigate the function of VIM gene and its role in related diseases. Methods： VIM coding region of 1401 bp was amplified from human cDNA library by RT-PCR, cloned the gel-extracted fragment into T-vector, and then identified by sequencing. Thereafter, VIM CDS fragment was cut down by Hind III and BamH I, and was then sub-cloned into eukaryotic expression plasmid pcDNA3.1. The recombinant plasmid was identified with Hind III and BarnH I cutting then transfected into NIH3T3 cells via liposome. The expression of VIM was detected by RT-PCR for the mRNA transcription and by Western Blot for the protein expression. Results： VIM coding region fragment had been successfully cloned into eukaryotic expression plasmid pcDNA3.1. The recombinant plasmid was transfected into NIH3T3 cells, and the results by RT-PCR and Western Blot showed that those transfected cells could produce VIM mRNA and protein. Conclusions： Eukaryotlc expression plasmid pcDNA3.1-VIM had been successfully constructed, which will be helpful for the further investigation of the function of VIM gene and its role in related diseases.
出处
《现代生物医学进展》
CAS
2013年第9期1615-1617,1614,共4页
Progress in Modern Biomedicine
