鼠源抗B型肉毒毒素单链抗体噬菌体文库的构建筛选及抗体免疫学活性的初步研究

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

摘要:

B型肉毒毒素重链C-端片段(BoNTB/Hc)经金属螯和层析法纯化后免疫Balb/c小鼠，从其脾淋巴细胞中提取总RNA，反转录成cDNA，用抗体可变区混合引物进行全套抗体重、轻链可变区基因的扩增，体外随机装配成单链抗体(scFv)。将其克隆至pCANTAB5E中，构建单链抗体噬菌体抗体库。结果表明经过4轮“吸附-洗脱-扩增”的富集过程，筛选获得高亲和力的克隆。序列测定符合抗体可变区结构特点。

关键词:B型肉毒毒素， scFv， 噬菌体抗体库

Abstract:

To produce antibodies capable of neutralizing botulinum neurotoxin type B (BoNT/B), We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed. The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv (scFv) DNA fragment. These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed. Results showed that the high affinity scFv was obtained after 4 rounds of panning, with its DNA sequence conforming to that of mouse antibody.

Key words:Botulinum neurotoxin type B,scFv，Phage display library