DDR2/Fc融合基因真核表达载体的构建及表达

Construction and expression of eukaryotic expression vector of fusion gene DDR2-Fc

目的:构建DDR2/Fc真核表达载体,使其在HEK293细胞中进行表达.方法:采用PCR技术,分别从大鼠脑组织、含人IgG1Fc全长互补DNA(cDNA)的质粒中扩增出盘状结构域受体2(DDR2)的DS区域、Fc段,以定向克隆的方法将其串联至真核表达载体pcDNA3.1(-)中,获得重组表达载体DDR2/Fc-pcDNA3.1(-);采用脂质体转染技术转染HEK293细胞;RT-PCR、免疫印迹检测融和蛋白的表达.结果:DNA测序和限制型酶切鉴定证明两段基因正确克隆至pcDNA3.1(-)的多克隆位点,细胞裂解物中检测到融合蛋白的表达,其分子质量与预期大小一致,该融合蛋白能正确表达于HEK293细胞中.结论:成功构建了DDR2/Fc融和表达载体,表达了融合蛋白.

AIM： To construct the eukaryotic expression vector of DDR2-Fc fusion gene and to investigate its expression in HEK293 cells. METHODS： The two cDNAs was amplified by PCR respectively from the rat brain tissue and the plasmid containing the full-length cDNA of human IgG1 Fc, and cloned to the eukaryotic expression vector pcDNA3. 1 （ - ） by directional cloning. The resultant recombinant plamid pcDNA3. 1 （ - ）/ DDR2-Fc was transfected into HEK293 cells with liposome trans- fection reagent. Then RT-PCR, Western Blot were used to detect the expression of the fusion protein. RESULTS： DNA sequencing and restriction enzyme digestion verified the correction of recombinant plasmid pcDNA3.1 （ - ）/DDR2-Fc. The expressed fusion protein was detected in the transfected HEK293 cells and the molecular weight of the protein was the same as we expected. CONCLUSION： The recombinant plasmid pcDNA3. 1 （ - ）/ DDR2-Fc was successfully constructed and the fusion protein DDR2/Fc was expressed in HEK293 cells.