人Flt3配体基因的克隆及其在CHO细胞中的表达

Cloning of Human Flt3 Ligand cDNA and Its Expression in CHO Cells

目的克隆人Flt3配体(Flt3 LIg and,FL),并在CHO细胞中进行表达。方法从健康人外周血中提取总RNA,通过RT-PCR技术,扩增Flt3配体胞外区cDNA功能性片段,经DNA测序证实后,将得到的片段基因插入pcDNA3.0表达载体,构建了pcDNA3.0-FL真核表达载体;将重组质粒pcDNA3.0-FL转染CHO细胞,经G418筛选出阳性细胞克隆,扩大培养,提取细胞总蛋白,用SDS-PAGE分离并Western blotting检测到在相对分子质量约24 000处有一显色条带。结果FL胞外区cDNA被正确克隆到真核表达载体pcDNA3.0中并在CHO细胞中成功表达。结论成功构建了真核表达载体pcDNA3.0-FL并在CHO细胞中成功表达,为FL的相关研究和应用开发奠定了基础。

Objective To clone the functional fragment of FL gene and express it in CHO cells. Methods RTPCR was used to amplify the cDNA of the functional fragment of FL from the total RNA of human peripheral blood. After verification by sequencing, the DNA fragment was then cloned into expression plasmid of pcDNA3.0, and then expressed in CHO cells. Results The human FL cDNA was cloned into vector pcDNA 3.0. Conclusion The eukaryotic expression vectors pcDNA3.0-FL was successfully constructed and expressed in CHO cells. Our work lay the foundation for further research of FL.