人源Fab噬菌体抗体库的构建与抗精氨酸加压素抗体的筛选

Construction of a human nave Fab library and screening of phage antibody against arginine vasopressin

目的:构建人源天然Fab噬菌体抗体库,筛选抗精氨酸加压素特异性抗体并进行初步鉴定。方法:从18位健康成人的外周血淋巴细胞,提取总RNA。利用RT-PCR扩增人Fab抗体基因片段,将其克隆至噬菌粒载体pComb3XSS内,构建人源天然Fab噬菌体抗体库。以固相化的精氨酸加压素为靶抗原对抗体库进行五轮筛选后,随机挑取50个单克隆进行phage-ELISA检测,阳性克隆行DNA测序分析。结果:成功构建库容为2.4×108的噬菌体抗体库,从中筛选到6株阳性克隆能够与精氨酸加压素特异性结合,其中阳性值最高的C4克隆行DNA测序分析证实其重链属IgG亚类,轻链为λ链。结论:构建大容量人源天然Fab抗体库,从中获得了特异性抗精氨酸加压素人源Fab抗体片段,有望为临床上精氨酸加压素的快速检测技术的建立奠定基础。

Objective：To construct a nave human Fab phage display library,screen and identify arginine vasopressin Fab antibody from the library.Methods：Total RNA was extracted from peripheral blood lymphocytes of 18 healthy donors,and the light chain and heavy chain Fd genes were amplified by RT-PCR.Then the amplification products were sequentially cloned into phagemid vector pComb3XSS to construct a human Fab phage antibody library.The insertion of the light chain or heavy chain Fd genes were identified by cutting with endonucleases and PCR amplification.Arginine vasopressin was used as target antigen to pan the original Fab antibody library.After five rounds of panning were carried out,fifty randomly selected clones were assayed by phage-ELISA analysis.The positive clones were analyzed by DNA sequencing.Results：A large human Fab phage antibody library consisting of 2.4×108 members was successfully constructed.After having been panned by AVP,we obtained six positive clones which had specificity and binding reactivity towards AVP.The C4 clone was analyzed and showed that its heavy chain belonged to IgG subvariety and its light chain to λ family.Conclusion：We successfully constructed a large human Fab phage antibody library and isolated the specific human anti-AVP Fab antibodies,which provided a solid foundation for the establishment of rapid detection method of arginine vasopressin in future research.