大容量人天然噬菌体单链抗体库的构建(1)
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[摘要] 目的：构建大容量和具有良好多样性的人天然噬菌体单链抗体库。方法：从人外周血分离淋巴细胞，提取mRNA后用RT-PCR技术采用新设计的引物扩增VH 和VL 基因片段，两者分别与Linker连接后，采用改进的重叠延伸PCR法将VH基因和VL基因连接成人单链抗体(scFv)基因(VH-Linker-VL片段)，继而连接到噬菌粒载体pCANTAB5E中，连接产物通过电转化方法转入大肠杆菌TG1，构建噬菌体scFv抗体库。结果：所有VH、VL亚类基因都得到了扩增和有效的连接，经电转化E. coli TG1和噬菌体的超感染，构建了总库容达到了6×108的单链抗体库。结论：成功地构建了一个多样性良好的人源天然噬菌体抗体库, 可用于制备具有应用前景的人源抗体。
[关键词] 单链抗体(scFv)；噬菌体抗体库；重叠延伸拼接
[中图分类号] R392-33 [文献标识码]A[文章编号]1673-7210(2008)05(a)-011-03
Construction of a large human natural phage single-chain antibody library
SUN Chun-yan
(Guangdong Food and Drug Vocational College,Guangzhou510520，China)
[Abstract] Objective: To construct a human natural phage single -chain antibody (scFv) library with diversity.Methods:Total RNA was extracted from the peripheral blood lymphocytes of the non-immunized healthy donors, VH and VL genes were amplified by RT-PCR and were assembled to form scFv by overlap PCR and cloned into phagemid pCANTAB5E, and then transformed into E ......
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