重组人α-半乳糖苷酶A在CHO细胞中的表达及鉴定

Expression and identification of recombinant human alpha-galactosidase A in Chinese hamster ovary cells

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 a recombinant eukaryotic expression plasmid of alpha-galactosidase A（GLA） and express GLA in Chinese hamster ovary cells（CHO）.Methods Human GLA cDNA was cloned from human liver cancer cells by RT-PCR and subcloned into mammalian expression systems pcDNA3.1/myc-His A.The recombinant plasmid was transfected into CHO cells and the positive clones were selected by G418.After the G418 resistant CHO cells were induced by sodium butyrate,GLA activity of the supernatant was determined.The recombinant GLA was purified by HisTrapTM FF and identified by Western blot.Results The mammalian expression vector pcDNA3.1/myc-His-GLA was constructed and transfected into CHO cells.A transfectant with high GLA expression level was obtained（the protein specific activity was 1935 U/mg）.There was an unique band near 50×103（Mr） in the purified supernatant and was identified as GLA protein by Western blot.Conclusion The recombinant eukaryotic expression plasmid of GLA is constructed and recombinant human GLA with biological activity is obtained.The recombinant human GLA can be used for Fabry′s disease treatment in the future.