重组复杂蛋白快速表达体系的建立

目的建立重组复杂蛋白快速表达体系。方法利用定点整合技术建立定点整合宿主细胞库,分别以X-Fc融合蛋白和人凝血因子Ⅷ(FⅧ)为研究对象进行定点整合转染和表达分析。结果通过定点整合转染建立了高表达的宿主细胞库;X-Fc融合蛋白和人FⅧ均在宿主细胞中获得了高表达,X-Fc融合蛋白的表达量为220 mg/L,人FⅧ在有血清培养条件下的酶活性为2 IU/ml,且细胞株表达稳定,易于扩大培养。结论已成功建立了基于CHO细胞定点整合的重组复杂蛋白快速表达体系,该体系在克隆效率和时间上具有很大优势,可实现蛋白的有效快速表达。

Establishment of Rapid Expression System for Recombinant Complex Protein

Objective To establish a rapid expression system for recombinant complex protein.Methods Targeted integration host cell bank was established by targeted integration technique,based on which targeted integration transfection and expression were analyzed by using X-Fc fusion protein and human coagulation factor Ⅷ(FⅧ) as models respectively.Results The host cell bank for high expression was established by targeted integration technique.Both X-Fc fusion protein and human FⅧ were highly expressed in host cells.The expression level of X-Fc fusion protein was 220 mg / L,and the enzyme activity of human FⅧ in serum-containing culture medium was 2 IU / ml.The host cell strain was suitable for stable expression of target protein and large-scale culture.Conclusion A rapid expression system for recombinant complex protein was successfully established based on the targeted integration of CHO cells,and showed advantages in cloning efficiency and time,by which the target protein might be expressed effectively and rapidly.