小鼠EVL基因的克隆和真核表达载体的构建  

Clone of EVL and Construction of EVL Eukaryotic Expression Plasmid

作　　者：邹海[1] 韩骅[1] 高方[2] 曹秀丽[2] 徐骁[2] 

机构地区：[1]第四军医大学基础部医学遗传学与发育生物学教研室,陕西西安710032 [2]第四军医大学基础部神经生物学教研室,陕西西安710032

出　　处：《现代生物医学进展》2012年第24期4606-4610,共5页Progress in Modern Biomedicine

基　　金：国家重点基础研究发展规划(973项目)资助(001CB509906);国家自然科学基金资助项目(30200148)

摘　　要：目的:构建小鼠EVL(Ena/VASP like)基因的真核表达载体,为深入研究EVL的功能奠定基础。方法:采用PCR方法,从小鼠cDNA文库中,扩增出1245bp的EVL编码区片段,经电泳、胶回收后连接入pMD-18T载体中,测序鉴定正确。用BamH I和HincII双酶切,定向克隆EVL编码区片段到真核表达载体pcDNA3.1中,用限制性内切酶酶切鉴定重组质粒正确后。将重组质粒转染入HELA细胞中,以RT-PCR检测EVL的mRNA的表达,以Western Blot检测EVL蛋白的表达。结果:酶切鉴定结果显示小鼠EVL编码区基因被成功克隆入真核表达载体pcDNA3.1中;RT-PCR和Western Blot结果以及免疫荧光染色显示Hela细胞中有EVL的mRNA和蛋白的表达。结论:成功获得pcDNA3.1-EVL的真核表达载体,为进一步深入研究EVL蛋白的功能奠定了基础。Objective： To generate eukaryotie expression plasmid of EVL （Ena/VASP like） gene, to lay foundation for further inv- estigation of the function of EVL gene. Methods： EVL coding region of 1272/1245bp was amplified from mouse eDNA library by PCR, and was then cloned into pMD-18T-vector after being electrophoresed and gel-extracted. After identifying the T-EVL plasmid by se- quencing, EVL CDS fragment was cut down by double digestion with BamH I and Hinc II, and then was subcloned into eukaryotie ex- pression plamid pcDNA3.1. The recombinant plasmid was identified with restriction enzymes and was transfected into HELA cells via lipo- some mediation. The expression of EVL was detected by RT-PCR for the mRNA transcription and by Western Blot for the protein expres- sion. Results： Identification by restriction enzymes showed that EVL coding region fragment had been successfully cloned into eukaryotie expression plasmid pcDNA3.1. After transfected into HELA cells, the recombinant plasmid could produce EVL mRNA and protein indi- cated by RT-PCR results and Western Blot results. Conclusion： Eukaryotic expression plasmid pcDNA3.1-EVL was successfully cloned, which is helpful for a further investigation of the function of EVL protein.

关 键 词：EVL蛋白 基因克隆 真核表达 

分 类 号：Q95-3[生物学—动物学] Q75