人Ⅱ型肿瘤坏死因子受体-免疫球蛋白G1 Fc融合蛋白在糖基工程化毕赤酵母中的表达  

Expression of tumor necrosis factor receptor-immunoglobulin G1 Fc fusion protein in glycoengineered Pichia pastoris

作　　者：高新[1,2] 宋海峰[2] 林殿海[1] 杨小盼[2] 万德友[2] 陈枢青[1] 

机构地区：[1]浙江大学药学院,杭州310058 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《军事医学》2012年第11期843-846,共4页Military Medical Sciences

摘　　要：目的在糖基工程化毕赤酵母中表达人Ⅱ型肿瘤坏死因子受体-免疫球蛋白G1 Fc融合蛋白(tumor necrosis factor receptor-immunoglobulin G1 Fc fusion,TNFR-Fc),考察糖基工程化过程对其活性的影响。方法将编码TNFR-Fc的cDNA序列插入毕赤酵母表达载体pPIC6αA中,构建重组表达质粒pPIC6-TF。用表达质粒转染N-糖基化工程改造的毕赤酵母菌株GSB,在含有杀稻瘟菌素的YPD平板上进行筛选。然后再使用HRP标记的羊抗人IgG在醋酸-硝酸纤维素双层膜上进行斑点印迹(dot-blot)筛选。选择表达量较高的工程菌株TNFR-Fc/GSB57进行高密度培养,通过Protein A亲和层析从发酵上清中纯化融合蛋白,利用鼠L929细胞对融合蛋白的抗TNFα活性进行分析。结果通过筛选获得了表达人Ⅱ型肿瘤坏死因子受体-Fc融合蛋白的表达菌株,纯化后的TNFR-Fc融合蛋白拮抗TNFα的EC50高于野生型毕赤酵母表达产物,而低于哺乳动物细胞的表达产物。结论糖基化改造改善了人Ⅱ型肿瘤坏死因子受体-Fc融合蛋白的抗TNFα活性,但必须进行进一步的改造才可能获得与哺乳动物细胞表达产物相似的活性。Objective To express the tumor necrosis factor receptor-immunoglobulin G1 Fc fusion （TNFR-Fc） in Pichia pastoris and study the effect of N-glycoengineered process on the protein bioactivity. Methods The cDNA encoding TNFR-Fc fusion protein was subcloned into pPIC6αA vector to construct the expression plasmid pPIC6-TF that was trans- formed into the GSB, a glycoengineered P. pastoris strain. Positive colonies were selected by YPD culture containing blasti- cidin and further screened by induced expression on cellulose acetate and nitrocellulose membrane with HRP labeled human IgG. The TNFR-Fc/GSB57 strain, whose expression level was higher, was cultured in 10L fermentor. Purified TNFR-Fc was obtainedby Protein A affinity chromatography from the fermentation. Results Bioactivity analysis showed that the puri- fied protein could neutralize the cytotoxicity of 5 × 10^4U/L TNFα to L929 cells （ EC50 1.48 μmol/L）. Conclusion The activity of recombinant TNFR-Fc expressed in glycoengineered P. pastoris is higher than that of the wild type one, but still lower than that expressed in CHO. N-glycan should be further modified.

关 键 词：肿瘤坏死因子受体 毕赤酵母 糖基工程化过程 发酵 纯化 生物活性 

分 类 号：R341[医药卫生—基础医学]