人FGFR4基因重组质粒的构建及融合蛋白的表达  

Construction of recombinant plasmid of human FGFR4 gene and expression of fusion protein

作　　者：刘彩红[1] 张婷婷[2] 纪书婷[1] 

机构地区：[1]哈尔滨医科大学大庆校区医学检验与技术学院,黑龙江大庆163319 [2]大庆油田总医院

出　　处：《山东医药》2015年第26期14-16,共3页Shandong Medical Journal

基　　金：黑龙江省卫生计生委科研课题(2014-429)

摘　　要：目的探讨成纤维细胞生长因子4(FGFR4)重组质粒的构建方法,并检测FGFR4融合蛋白的表达。方法从Hep G2细胞中提取总RNA,采用RT-PCR方法扩增FGFR4的全长编码序列,双酶切后与pc DNA3.1/MycHis A载体连接,构建pc DNA3.1/Myc-His A-FGFR4重组质粒。重组质粒经双酶切和测序鉴定后瞬时转染人胚肾HEK293细胞,采用Western blot法检测FGFR4融合蛋白的表达。结果 FGFR4编码序列被成功克隆至pc DNA3.1/Myc-His A质粒中,成功构建了pc DNA3.1/Myc-His A-FGFR4载体。转染HEK293细胞后检测到FGFR4融合蛋白的稳定表达,分子量约为89 k D。结论成功构建了FGFR4全长编码基因重组质粒,并检测到转染后FGFR4蛋白在HEK293细胞中的表达。Objective To investigate the construction method of the recombinant plasmid of fibroblast growth factor re-ceptor 4（FGFR4） and to identify the expression of its fusion protein.Methods Total RNA was extracted from HepG2 cells.We obtained the full length of human FGFR4 coding sequence by RT-PCR, digested it by restriction enzyme and joined it with pcDNA3.1/Myc-HisA to construct the pcDNA3.1/Myc-HisA-FGFR4 recombinant plasmid.After the recom-binant plasmid was identified by enzyme digestion and sequencing, the plasmid was transfected into HEK293 cells.The ex-pression of fusion protein in HEK293 cells was detected by Western blotting.Results The coding sequence of human FG-FR4 was successfully cloned into pcDNA3.1/Myc-HisA.The pcDNA3.1/Myc-HisA-FGFR4 vector was successfully con-structed.After transfecting HEK293 cells, the stable expression of FGFR4 fusion protein with a molecular weight of 89 kD was detected.Conclusion The recombinant plasmid of FGFR4 full-length coding gene was successfully constructed and the FGFR4 protein was expressed in HEK293 cells.

关 键 词：成纤维细胞生长因子4 质粒 转染 

分 类 号：Q291[生物学—细胞生物学]