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为构建抗人vWF-A1的全套单链抗体噬菌体表面展示文库,从中筛选与vWF-Al高亲合力的单链抗体,为其临床应用奠定基础。从vWF-A1免疫小鼠的脾淋巴细胞中提取总RNA,纯化mRNA并反转录成cDNA后,用抗体可变区混合引物扩增全套轻、重链可变区基因,经重叠延伸反应,在体外装配成单链抗体(scFv),将其克隆至噬菌体载体pHENl中,构建单链抗体噬菌体抗体库。对抗体库进行亲合筛选后,ELISA法鉴定抗vWF-A1单链抗体。结果经过5轮“吸附-洗脱-扩增”的富集过程,phage-ELISA筛选到与vwF-A1有高亲和力的克隆。序列测定符合抗体可变区结构特点。成功构建了全套抗vWF-A1单链抗体噬菌体展示文库,并筛选出具有结合vWF-A1功能的单链抗体。
To construct a full-length single-chain antibody phage display library of anti-human vWF-A1, scFv with high affinity for vWF-Al was screened for its clinical application. Total RNA was extracted from splenic lymphocytes of vWF-A1-immunized mice. After purification of mRNA and reverse transcribed into cDNA, a complete set of light and heavy chain variable region genes were amplified by mixed variable region antibody. After overlapping extension reaction, After being assembled into single-chain antibody (scFv) in vitro, it was cloned into the phage vector pHENl to construct a single-chain antibody phage antibody library. After affinity screening of antibody library, anti-vWF-A1 single-chain antibody was identified by ELISA. Results After 5 rounds of “adsorption-elution-amplification” enrichment, phage-ELISA screened clones with high affinity to vwF-A1. Sequencing was in accordance with the structure of antibody variable region. A complete set of anti-vWF-A1 scFv phage display library was successfully constructed and single-chain antibodies with binding function of vWF-A1 were screened out.