论文部分内容阅读
目的构建重组人α-半乳糖苷酶A(GLA)真核表达载体,并在中华仓鼠卵巢细胞CHO中表达重组的人GLA。方法利用RT-PCR方法从人肝癌细胞中克隆人GLA cDNA,并构建到哺乳动物细胞表达载体pcDNA3.1/myc-His A中。将重组质粒转染CHO细胞并用G418筛选。用丁酸钠诱导G418抗性细胞,检测培养上清中的GLA活性,筛选高表达GLA的细胞株。采用HisTrapTMFF纯化培养上清中的GLA蛋白并利用Western印迹进行鉴定。结果成功构建了哺乳动物细胞表达载体pcDNA3.1/myc-His A-GLA,并在中华仓鼠卵巢细胞CHO中表达,得到高表达量的单克隆(蛋白比活为1935 U/mg)。纯化后的培养上清在50×103(Mr)附近出现单一条带,通过Western印迹确定为GLA蛋白。结论成功构建了真核表达载体pcDNA3.1/myc-His A-GLA,获得了具有生物活性的重组人GLA,为临床上治疗法布莱病奠定了一定基础。
Objective To construct recombinant human α-galactosidase A (GLA) eukaryotic expression vector and express recombinant human GLA in Chinese hamster ovary CHO. Methods Human GLA cDNA was cloned from human hepatocellular carcinoma cells by RT-PCR and cloned into mammalian expression vector pcDNA3.1 / myc-His. Recombinant plasmids were transfected into CHO cells and screened with G418. G418-resistant cells were induced by sodium butyrate, the activity of GLA in culture supernatants was detected, and the cell lines with high GLA expression were selected. The GLA protein in the culture supernatant was purified using HisTrapTMFF and identified by Western blotting. Results The mammalian cell expression vector pcDNA3.1 / myc-His A-GLA was successfully constructed and expressed in Chinese hamster ovary CHO. The monoclonal antibody with a high specific expression (1935 U / mg protein) was obtained. The purified culture supernatant showed a single band around 50 × 103 (Mr) and was identified as GLA protein by Western blotting. Conclusion The eukaryotic expression vector pcDNA3.1 / myc-His A-GLA has been successfully constructed and a recombinant human GLA with bioactivity has been obtained. It has laid a solid foundation for the treatment of Fabre disease.