【摘要】 目的:从人源性噬菌体抗体库中筛选抗人D二聚体单克隆抗体并进行可溶性表达。方法:以人D二聚体标准品为抗原对所构建的噬菌体抗体库进行两轮“吸附-洗脱-扩增”筛选,从中筛选出抗人D二聚体的噬菌体Fab抗体,并在大肠杆菌细胞中进行可溶性表达。结果:经4次电转化构建了库容为2. 8×108cfu的抗体库,滴度为4. 1×1014PFU/mL,Fab基因重组率为46%。经过淘筛后携带抗人D二聚体抗体的特异性噬菌体得到了明显的富集。用试剂盒以ELISA法检测对人D二聚体的结合活性,得到抗人D二聚体单克隆抗体。筛选出的阳性克隆成功在大肠杆菌细胞中可溶性表达筛选出的阳性克隆在大肠杆菌细胞中可溶性表达。结论:人D二聚体标准品对抗体库的淘筛,富集了表面呈现抗人D二聚体的单克隆抗体的噬菌体,成功构建了人源性抗D二聚体噬菌体抗体,并成功在大肠杆菌细胞中可溶性表达。更多还原

【Abstract】 Objective: Selecting Fab phage antibodies to D dimer from the phage antibody library and solutibility expressed in E.coli XL1-blue. Methods: After two rounds of panning with D dimer calibrator,antibodies against D dimer were screened.And then isolated human anti-D dimer Fab with ELISA.Results: A human combinatorial antibody library consisting of 2.8×10 8cfu in dependent clones was generated with a titer of 4.1×10 14 PFU/mL.Forty six percent of the clones contained Fab genes.Two rounds of selection against D dimer showed specific enrichment of phage antibodies. ELISA verified that the positive phage antibodies were specific against D dimer. The positive phage antibody was solutibility expressed in E.coli XL1-blue.Conclusions:Our work on constructing human immunoglobulin combinatorial library and selecting anti-D dimer Fab phage antibodies would be beneficial to the further study on the production of soluble Fab antibodies.更多还原