人Endostatin cDNA的克隆及其在HT1080细胞中的表达

Cloning and expression of human Endostatin cDNA in HT1080 cells

目的 :构建能表达人Endostatin的真核表达质粒。方法 :设计引物从成人肝cDNA文库中通过PCR扩增到EndostatincDNA ,并在 5′端融合编码人胰岛素信号肽序列 ,经测序证实后连接到真核表达载体pcDNA3.1(- )中。将携带基因的真核表达载体质粒pcDNA3.1(- ) SEND转染HT10 80细胞后用G4 18筛选获得克隆 ,采用RT PCR和West ern blot检测Endostatin的表达。结果 :测序结果表明克隆的人EndostatincDNA完全正确 ,并且在其 5′端融合编码人胰岛素信号肽序列 ;RT PCR显示转染后的HT10 80细胞可以有效地转录EndostatinmRNA ;Western blot检测到转染后的HT10 80细胞上清有相应的蛋白质 ,大小为 2 0kD左右。结论 :含有人胰岛素信号肽序列的人EndostatincDNA重组质粒pcDNA3.1(- ) SEND转染HT10 80细胞后可以有效的表达目的蛋白并能分泌到细胞外 。

Objective To construct recombination eukaryotic expression vector pcDNA3.1(-)/SEND. Methods Human Endostatin cDNA was obtained from the human liver cDNA library by PCR, and a fragment comprising human insulin signal peptide coding sequence fused to the human Endostatin cDNA was produced by PCR. After being verified by sequence, the recombination eukaryotic expression vector pcDNA3.1(-)/SEND was constructed. The plasmid pcDNA3.1(-)/SEND was transferred into HT1080 cells and positive clones were obtained after being selected by G418. The expression of Endostatin was verified by RT PCR and Western blot. Results The human Endostatin cDNA and human insulin signal peptide coding sequence fused to the human Endostatin cDNA were verified by sequence; RT PCR indicated the transcription of Endostatin in the transfected HT1080 cells and Western blot detected that the Endostatin protein, about 20kD in size, in the supernatant of HT1080 cells was transfected by pcDNA3.1(-)/SEND. Conclusion The expressed Endostatin protein was secreted into the supernatant of HT1080 cells transfected by pcDNA3.1(-)/SEND.