人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本，通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因，将ScFv克隆入噬粒载体，通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性，用5种不同抗原对其进行筛选，均获得了特异性噬菌体抗体的不同富集，表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。

Construct and Characterization of Human Na(i)ve ScFv Phage Display Library

Phage display antibody library is an important technology for generating therapeutic antibody.It was reported that the construction and characterization of a large human antibody phage display library. Total RNA was extracted from peripheral blood lymphocytes of 20 healthy donors. VH and VL gene were amplified by RT-PCR. The ScFv (single chain variable fragment) gene were assembled by overlap PCR, and cloned into a phagemid vector by electroporation of E.coli TG1. A large naive human ScFv phage antibody library containing 1.3×109 antibody members was constructed by repetitive electroporation about 300 times. The quality of the library was examined by sequence analysis and selection against 5 different protein antigens. Sequence analysis of the randomly picked 40 clones showed this library had good diversity. Panning of the 5 different antigens all resulted in successful isolation of antigen specific ScFv antibodies. The results indicate that a large nave human ScFv phage antibody library with good diversity was constructed successfully.