重组人源抗HBsAg单链抗体的纯化及亲和常数测定  

Purification of an anti-HBsAg scFv and measurement of its affinity constant

作　　者：熊盛[1] 任向荣[2] 严兴[1] 唐永红[2] 郑业华[2] 粟宽源[2] 余宙耀[2] 姚汝华[1] 

机构地区：[1]华南理工大学生物工程系,广东广州510640 [2]解放军第458医院全军传染病中心,广东广州510602

出　　处：《中国病理生理杂志》2002年第9期1069-1073,共5页Chinese Journal of Pathophysiology

基　　金：广州市科技局重大攻关项目 (99- 2 - 0 10 - 0 1)

摘　　要：目的 :对融合 6×His标签的人源抗乙型肝炎表面抗原单链抗体的包涵体进行纯化和复性 ,并对复性产物的抗原结合性质进行鉴定。方法 :工程菌表达的包涵体裂解后 ,金属螯合亲合层析纯化 ,然后以尿素透析、盐酸胍透析和金属螯合亲和层析柱原位复性 3种方法进行复性 ;复性产物以免疫亲和层析精制 ,非竞争酶免疫法测定亲和常数结果 :盐酸胍透析复性产物的比活性最高 ,蛋白回收率为 (6 1 0 8± 1 4 5 ) % ;精制后的重组单链抗体的亲和常数为 (2 30± 0 32 )× 10 7L/mol。结论 :本株单链抗体可以应用优化的透析复性技术 ,在体外高效复性 ,其抗原结合性质不受N末端纯化标签的影响。AIM: To purify and refold the inclusion body of a human anti-HBsAg scFv with a 6×His tag, and to determine the affinity constant of the purified recombinant product.METHODS: Solubilizing in buffers containing urea or guanidine hydrochloride (GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis against urea or GuHCl, at the same time, Ni 2+ charged chelate column was utilized for in situ refolding. The affinity constant of the refolded scFv, polished by immune-affinity chromatography, was determined by non-competitive ELISA. RESULTS: The refolded scFv with highest specific bioactivity was produced by dialysis against GuHCl. Under this condition, the recovery of target protein reached (61.08±1 45)%. The affinity constant of the polished scFv was confirmed to be(2.30±0.32) ×10 7 L/mol. CONCLUSION: The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro . The antigen-binding property of this recombinant scFv is not affected by the purification tag fused to the N terminal of the protein.

关 键 词：乙型肝炎 抗体 包涵体 抗体亲和力 

分 类 号：R512.62[医药卫生—内科学]