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 SalⅠand XbaⅠ.The validity of this newly constructed plasmid Flag-Smurf1 was confirmed first by double digestion with SalⅠand XbaⅠthen 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 SalⅠand 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.
作者
李莎莎
贺虹
崔冠一
欧丽娜
邓博
王梅林
LI Sha-sha;HE Hong;CUI Guan-yi;OU Li-na;DENG Bo;WANG Mei-lin(Medical College,Henan University of Science and Technology,Luoyang471023,China;Out-patientDepartment of Hospital No.988of PLA,Information Engineering University of PLA,Zhengzhou450002,China)
出处
《生物技术》
CAS
2019年第3期205-209,共5页
Biotechnology
基金
国家自然科学基金项目(31500631)
关键词
Smurf1
分子克隆
转染
免疫荧光
Smurf1
genetic engineering
transfection
immunofluorescence
