目的:构建带Flag标签的α-烯醇酶(ENO1)基因以及带Myc标签的人白细胞抗原F相关转录物10(FAT10)基因的真核表达质粒载体,鉴定ENO1和FAT10在人肾上皮细胞株HEK293中的表达,检测类泛素蛋白FAT10是否共价修饰ENO1.方法:以人宫颈癌细胞系HeLa的cDNA为模板进行PCR,得到enol目的片段,与分别经EcoRV和SalⅠ酶切的pFlag-CMV载体连接得到重组质粒pFlag-CMV-eno1并进行鉴定测序;以人脑组织cDNA文库为模板进行PCR,得到fat10目的片段,与分别经EcoR Ⅰ和Xho Ⅰ酶切的pCMV-Myc载体连接得到重组质粒pCMV-Myc-fat 10并进行鉴定并测序.将重组质粒用脂质体法转染HEK293细胞,用Western印迹检测ENO1和FAT10蛋白的表达.将重组质粒共转染HEK293细胞,用免疫共沉淀检测FAT10对ENO1的修饰情况.结果:重组克隆载体内的eno1和fat10序列与GenBank报告的序列完全一致;转染HEK293细胞后,ENO1和FAT10蛋白过表达;FAT10能够共价修饰ENO1.结论:类泛素蛋白FAT10共价修饰ENO1,为肿瘤的发生、发展及迁移的研究提供了新的研究思路.%Objective:Through co-express two eukaryotic plasmids carrying Flag-tagged α-enolase(ENO1) gene and Myc-tagged F-related transporter 10(FAT10) gene to detect modification of ENO1 by FAT10 in HEK293 cell lines.Methods:The eno1 target fragment was obtained by PCR from the cDNA of HeLa cells and inserted into pFlag-CMV vector,and the constructed plasmid pFlag-CMV-eno1 was identified by sequencing.The fat10 fragment was obtained by PCR from human brain tissue cDNA library as template,and cloned into pCMV-Myc vector,and the constucted plasmid pCMV-Myc-fat10 was identified by sequencing.The two recombinant plasmids were co-transfected into HEK293 cell lines by liposome method,and Western blotting was used to detect the expression of ENO1 and FAT10 protein,and the co-immunoprecipitation method was used to detect whether ENO1 and FAT10 proteins could interact in cells.Results:The eno1 sequence and fat10 sequence in the recombinant cloning vector were identical with those reported data by GenBank.ENO1 and FAT10 proteins significantly expressed in HEK293 cell lines,and ENO1 was covalently modified by Ubiquitin protein FAT10.Conclusion:ENO1 is covalently modified by Ubiquitin protein FAT10 in HEK293 cell lines,which is a clue to study the development and immigration of tumor cells.
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