糖基工程酵母表面呈现抗体库的构建及抗真菌β甘露糖型抗体的筛选  

Preparation by Glyco-Engineered Pichia pastoris and Antibody Library and Screening Against β-Mannose by Candida albicans

作　　者：房泽轩 刘波[1] 巩新[1] 唱韶红[1] 吴军[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2016年第3期326-330,370,共6页Letters in Biotechnology

基　　金：国家自然科学基金(31200082);北京市自然科学基金(7152112)

摘　　要：目的:通过构建糖基工程酵母表面呈现免疫抗体库,筛选与毕赤酵母表达的禽流感血凝素HA7结合的特异性抗体。方法:利用糖基工程毕赤酵母细胞膜锚定蛋白Sed将人Ig G的Fc段锚定在酵母表面,然后用毕赤酵母表达的HA7免疫小鼠,从小鼠脾脏细胞中扩增抗体轻重链可变区基因构建抗体转化锚定了Fc的糖基工程酵母,构建表面展示免疫抗体库,通过免疫磁珠法筛选与毕赤酵母表达的HA7特异性结合的抗体表达酵母,分析筛选该抗体的特异性结合靶点。结果:构建了多态性良好的抗体表达质粒库,转化糖基工程酵母后筛选得到特异性结合HA7的抗体呈现酵母,Western印迹分析发现该抗体只与糖基化的HA7结合而不与切除糖链后的HA7结合,说明抗体结合区域在HA7的糖链上,进一步实验发现该抗体不与α甘露糖型和哺乳动物复杂型糖型结合,通过β甘露糖苷酶切研究发现抗体结合靶点为酵母表达HA7糖链上的β甘露糖。结论:利用糖基工程酵母构建抗体库,筛选得获得了抗真菌β甘露糖的抗体,动物实验发现抗体具有中和活性。Objective：Through the construction of glycosylation engineering yeast surface display antibody library,found a kind of antibody against the β-mannose which was particular in fungi.Methods：By using the cell membrane anchored protein Sed,the Fc segment of IgG was anchored on the yeast surface.Immunize the mice of HA7 expressed by Pichia pastoris,amplification of antibody variable region gene from the mice spleen cells and construct the full-long antibody,and translate to the Sed yeast,we have constructed the glycosylation engineering yeast surface display antibody library.After screening the library,we found a kind of antibody that combine with the HA7 protein.Results：The good polymorphism of the antibody expression plasmid library has been constructed,by screening the library we found a kind of antibody that against the β-mannose which can combine with the HA7 protein,Western blot demonstrated that the antibody was particular against the β-mannose.Conclusion：By constructing and screening the glycosylation engineering yeast surface display antibody library,we found a kind of antibody against the β-mannose which was particular in fungi.Animal experiments demonstrated that the antibody has neutralizing activity in vivo.

关 键 词：糖基工程酵母抗体库 免疫磁珠筛选 β甘露糖抗体 中和活性 

分 类 号：R392.1[医药卫生—免疫学] Q78[医药卫生—基础医学]