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目的制备1株人源性的β-淀粉样蛋白(Aβ)特异性单链抗体(scFv),并进行性能鉴定。方法利用噬菌体展示技术,用20个AD病人的外周血B细胞首次构建了AD患者的单链可变区噬菌体抗体库。以Aβ1-40为靶,挑选出77个阳性抗体克隆,从中筛选出一株抗Aβ单链抗体H1,对其重链和轻链可变区基因进行测序分析。通过表达载体pET28a(+)对目的蛋白进行大量表达,并利用Western-blot对单链抗体H1的免疫活性进行鉴定。结果成功构建出AD患者的单链可变区噬菌体抗体库,库容量为1.5×106。从库中筛选并表达出一株抗Aβ1-40的单链抗体H1,DNA测序结果证实了其抗体的基因结构及氨基酸序列。Western-blot检测证实该抗体可特异性结合Aβ1-40。结论利用噬菌体抗体库技术可成功制备Aβ肽特异性单链抗体,为AD患者的临床诊断和被动免疫治疗提供了一种新的可供选择的途径。
Objective To prepare a human β-amyloid (Aβ) specific single chain antibody (scFv) and to characterize it. Methods Phage display technique was used to construct single-chain variable region phage antibody library of AD patients with peripheral blood B cells of 20 AD patients. Aβ1-40 was selected as the target, and 77 positive antibody clones were selected. One anti-Aβ single chain antibody H1 was screened out, and the heavy chain and light chain variable region genes were sequenced. The expressed protein pET28a (+) was used to express a large amount of the target protein, and the immunological activity of the single chain antibody H1 was identified by Western-blot. Results The single-chain variable region phage antibody library of AD patients was successfully constructed with a capacity of 1.5 × 106. A single anti-Aβ 1-40 scFv H1 was screened from the library and sequenced. DNA sequencing confirmed the gene structure and amino acid sequence of the antibody. Western-blot analysis confirmed that the antibody can specifically bind to Aβ1-40. Conclusion The phage antibody library technology can be successfully prepared Aβ peptide specific single chain antibody for the clinical diagnosis of AD patients and passive immunotherapy provides a new alternative route.