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.coliTG1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。
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 pCANTABSE, and then transformed into E. coli TG1 by electroporation to construct a human natural phage scFv antibody library. Results：VH gene families and VL gene families were successfully amplified. A large human antibody library containing 6×10^8 clones was created after rescuing the recombinant phagemids from the transformed E. coli TGI cells.Condusion：A human scFv antibody library is successfully constructed finally. In the future, we can try to obtain antibody by panning purposely using related antigens.
出处
《中国医药导报》
CAS
2008年第13期11-13,共3页
China Medical Herald
