人源噬菌体抗体库的构建及抗D-二聚体抗体的获得

Construction of Hominine Antibody Library and Screening of the Hominine Anti-D-dimer Antibody

目前国际和国内筛选蛋白所用的技术主要是噬菌体展示技术和酵母双杂交技术。噬菌体展示技术在抗体的筛选方面已显示出良好的应用前景。本实验应用噬菌体展示技术展示出血栓病人的抗体,形成对血栓病人的抗体库。并从中淘选出能与血栓降解产物之一的 D-二聚体有特异结合作用的抗体 Fabp13。抗 D-二聚体的 Fab 在 XL1-Blue 中得到可溶表达。再利用抗 D-二聚体抗体进行竞争 ELISA 检验,证实 p13具有抗 D-二聚体的活性。从而我们筛选得到了抗 D-二聚体抗体 Fab p13。

Human phage-displayed Fab library was constructed by using phase surface display technology.We selected an Fab-phage specifically interacting with D-dimer from this library.Immunnglobulin heavy and light chains genes from human lymphocytes were amplified respectively by RT-PCR using family specific primers and signal-peptide sequences of immunoglobulin as semi-nested PCR primers.The PCR resulting gene fragments were cloned into phagemid vector pComb3H and the recombinant library of human immunoglobulin is generated with helper phage VCSM13.The human anti-D-dimer Fab-phage p13 is isolated via micropanning and then successfully expressed in E. coil XL1-Blue.The success of isolating human anti-D-dimer Fab proves the practicability of phage display system in human McAb preparation.