人Smurf1基因真核表达质粒的构建和表达

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英文篇名：Eukaryotic expression vector construction of human Smurf1 gene and its expression analysis 作者：李莎莎 ; 贺虹 ; 崔冠一 ; 欧丽娜 ; 邓博 ; 王梅林 英文作者：LI Sha-sha;HE Hong;CUI Guan-yi;OU Li-na;DENG Bo;WANG Mei-lin;Medical College,Henan University of Science and Technology;Out-patient Department of Hospital No.988 of PLA,Information Engineering University of PLA; 关键词：Smurf1 ; 分子克隆 ; 转染 ; 免疫荧光 英文关键词：Smurf1;;genetic engineering;;transfection;;immunofluorescence 中文刊名：SWJS 英文刊名：Biotechnology 机构：河南科技大学医学院;解放军第988医院信息工程大学门诊部; 出版日期：2019-06-20 出版单位：生物技术 年：2019 期：v.29;No.172 基金：国家自然科学基金项目(31500631) 语种：中文; 页：SWJS201903001 页数：5 CN：03 ISSN：23-1319/Q 分类号：7-11

摘要

[目的]构建人Smurf1基因的真核表达载体并检测其表达和细胞亚定位。[方法]PCR法从人胚肾细胞HEK-293T中扩增Smurf1基因的ORF序列,将其插入到带Flag标签的p CMV5真核表达载体,插入位点为限制性内切酶SalⅠ和XbaⅠ之间。酶切并测序鉴定该质粒的正确性。将成功构建的质粒Flag-Smurf1转染HEK-293T细胞和Hela细胞,Western Blotting检测Smurf1蛋白的表达情况;转染Mv. 1. Lu细胞,免疫荧光检测Smurf1蛋白在细胞中的亚定位。[结果]成功扩增出Smurf1基因的ORF序列;连入p CMV5中获得了质粒Flag-Smurf1;酶切鉴定得到2. 2 kb大小的片段,测序结果显示连入的是Smurf1基因的c DNA序列正确。Western Blotting结果显示该质粒可在HEK-293T细胞和Hela细胞中表达,表达大小约为80 k Da,符合预期。免疫荧光实验结果显示过表达的Smurf1主要定位在细胞膜上。[结论]成功构建了带Flag标签的人Smurf1基因的真核表达质粒,该质粒可在细胞中顺利表达。
        [Objective] To construct a eukaryotic expression vector carrying the ORF region of human E3 ligase Smurf1 gene and to validate its expression and sub-cellular localization.[Method]Smurf1 ORF region was amplified from HEK-293 T cell by PCR and then cloned into a modified eukaryotic expression vector p CMV5 containing Flag tag using restriction endonuclease SalⅠ and XbaⅠ. The validity of this newly constructed plasmid Flag-Smurf1 was confirmed first by double digestion with SalⅠ and XbaⅠ then by DNA sequencing. Plasmids Flag-Smurf1 was transfected into HEK-293 T cells or Hela cells,Western Blotting was employed to determine Smurf1 protein expression. Flag-Smurf1 was transfected into Mv. 1. Lu cells,immunofluorescence were employed to determine its sub-cellular localization.[Result] The ORF region of Smurf1 gene was successfully amplified from HEK-293 T cells,then cloned into vector p CMV5 and get the recombinant plasmid Flag-Smurf1. A length of 2. 2 kb fragment was obtained by double digestion with SalⅠ and XbaⅠ. DNA sequencing analysis verified its accuracy.Western Blotting showed this Flag-Smurf1 plasmid expressed well in HEK-293 T and Hela cells,the size of the band was about 80 k Da,as expected. Immunofluorescence revealed the plasma membrane sub-cellular localization of over-expressed Smurf1 protein.[Conclusion]Human E3 ligase Smurf1 eukaryotic expression vector Flag-Smurf1 was successfully constructed and expressed well in cells. Over-expressed Smurf1 protein localized on the plasma membrane.

引文

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