【摘要】 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。更多还原

【Abstract】 To screen the monoclonal antibody with high affinity to vWF-Al fragment, phage display library was constructed from mouse immunized with this fragment to lay a foundation for its clinical usage. The heavy chain and light chain variable region (VH and NL ) gene repertoire of immunoglobulin were amplified from mRNA of spleen cells by RT-PCR, and joined by a DNA linker encoding peptide as a single chain Fv (ScFv )fragment with overlap extension. These fragments were cloned into phage vector pHENl and the phage display library was constructed. The affinity selection and ELISA assay were adopted for the identification of specific phage antibody to vWF-Al. Results showed that the high affinity ScFv was obtained after 5 rounds of panning, with its DNA sequence conforming to that of mouse antibody. It concludes that the phage display library of repertoire single chain anti-vWF-Al antibody was successfully constructed and the single chain antibody with high affinity to vWF-Al was screened.更多还原