鼠源抗B型肉毒毒素Fab抗体库的构建及初步筛选

Construction and Screening of a Phage Display Library of Repertoire Fab Antibody from Mouse Immunized with BoNTB/Hc

目的:应用重组噬菌体抗体库技术制备抗B型肉毒毒素Fab抗体。方法:用重组B型肉毒毒素重链C端片段(BoNTB/Hc)免疫BALB/c小鼠,从其脾淋巴细胞扩增免疫球蛋白Fd段和κ链基因,克隆至表达载体pComb3中,并将抗体Fab段表达于噬菌体表面,建立容量为5.96×106cfu的噬菌体抗体库。以BoNTB/Hc为抗原对所建抗体库进行4轮亲和筛选,获得与B型肉毒毒素特异性结合的克隆,并进行序列测定。结果:构建了抗B型肉毒毒素Fab抗体库,筛选出特异性克隆1个。结论:从鼠源噬菌体免疫抗体库中初步获得了特异性抗B型肉毒毒素的Fab抗体。

Objective： To construct mouse Fab against botulinum neurotoxin type B（BoNTB） through phage-displayed antibody technology. Methods： The recombinant carboxy-terminal end of BoNTB/Hc containing the major determinants responsible for specific toxin was produced. After mice were immunized with BoNTB/Hc, the spleens were harvested, and Fab phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. After transformation, library of 5.69×10^6 members was obtained. To isolate antibodies with high affinity, the library was selected on BoNT/Hc. Results showed that the high affinity Fab was obtained after 4 rounds of panning, with its DNA sequence conforming to that of mouse antibody. Results： The Fab phage antibody against BoNTB was constructed successfully, and one positive clone was obtained. Conclusion： Specific genetic antibody against BoNTB can be made from phage-displayed antibody library.