【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。更多还原

【Abstract】 Objective To investigate the construction method of the recombinant plasmid of fibroblast growth factor receptor 4( FGFR4) and to identify the expression of its fusion protein. Methods Total RNA was extracted from Hep G2 cells. We obtained the full length of human FGFR4 coding sequence by RT-PCR,digested it by restriction enzyme and joined it with pc DNA3. 1 / Myc-His A to construct the pc DNA3. 1 / Myc-His A-FGFR4 recombinant plasmid. After the recombinant plasmid was identified by enzyme digestion and sequencing,the plasmid was transfected into HEK293 cells. The expression of fusion protein in HEK293 cells was detected by Western blotting. Results The coding sequence of human FGFR4 was successfully cloned into pc DNA3. 1 / Myc-His A. The pc DNA3. 1 / Myc-His A-FGFR4 vector was successfully constructed. After transfecting HEK293 cells,the stable expression of FGFR4 fusion protein with a molecular weight of 89 k D was detected. Conclusion The recombinant plasmid of FGFR4 full-length coding gene was successfully constructed and the FGFR4 protein was expressed in HEK293 cells.更多还原