人源性抗HBsAg Fab抗体的发酵生产研究  

Production of Recombinant Humanized anti-HBsAg Fab Antibody by Fermentation

作　　者：邓宁[1] 向军俭[1] 陈文吟[2] 熊盛[3] 王珣章[4] 粟宽源[2] 

机构地区：[1]暨南大学生命科学技术学院,广州510632 [2]解放军第458医院传染病研究中心,广州510602 [3]暨南大学药学院,广州510632 [4]中山大学生物防治国家重点实验室,生物医药中心,广州510175

出　　处：《生物工程学报》2004年第5期800-804,共5页Chinese Journal of Biotechnology

基　　金：广州市科技局重点项目 (No .2 0 0 3J1 C0 171);国家自然科学基金项目 (No .40 3 760 3 0) ~~

摘　　要：为了适应工业生产的需要 ,利用fed batch方法 ,重组人源性抗HBsAgFab抗体酵母工程菌在 30L发酵罐中进行了高密度发酵 ,发酵最适温度 30℃ ,pH值范围 5 0～ 5 3,溶氧范围 2 0 %～ 30 %。发酵液OD6 0 0 值达到 30 0时开始诱导 ,甲醇最佳诱导浓度为 1 0mL L。重组人源性抗HBsAgFab抗体经离子交换层析纯化 ,纯化产品经SDS PAGE、Westernblot进行分析和ELISA方法进行活性测定。结果显示 ,重组Fab抗体在Fed batch发酵系统中可高效表达 ,经过 1 92h的发酵生产 ,重组人源性抗HBsAgFab抗体的表达量可达 4 1 2mg L。发酵上清经过离子交换层析纯化 ,获得纯度为 95 %的重组Fab抗体 ,该Fab抗体经ELISA分析具有较高的HBsAg抗原亲和力和特异性。结果证实可以通过高密度发酵毕赤酵母工程菌来高效生产重组人源性抗HBsAgFab抗体 ,为后续的工业化生产应用奠定了基础。In order to produce recombinant human anti HBsAg Fab antibody in Pichia pastoris , the recombinant yeast was fermented using fed batch system in a 30 L bioreactor. The fermentation temperature was 30℃, the pH was 5 0～5 3, and the DO was 20%～30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS PAGE and western blot and ELISA. When the absorbance ( OD 600 ) of broth reach 300 at the end of fed batch phase, the induced phase was initiated. The results showed that recombinant human anti HBsAg Fab antibody was high level expressed in recombinant Pichia pastoris using a fed batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti HBsAg Fab antibody could be high level produced by fed batch fermentations in Pichia pastoris . Which can be efficiently used in industrial production.

关 键 词：乙型肝炎病毒 HBsAgFab 抗体 发酵 毕赤酵母 生物制品 表面抗原 

分 类 号：TQ464[化学工程—制药化工]