抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.

Construction and Characterization of Single Chain Fv Phage Display Library Against Tumor Necrosis Factor α

This study was to construct single chain Fv (scFv) library against tumor necrosis factor α (TNF-α) by phage display technique and to characterize the anti-TNF-α scFv clones selected from the library. Total RNA was extracted from the splenocytes of the BALB/c mice immunized with recombinant human TNF-α (rhTNF-α). Complementary DNA fragments of variable heavy (VH) and variable light (VL) chains of antibodies were prepared by RT-PCR and assembled into scFv with a flexible peptide linker (Gly3Ser)4. ScFv fragment was inserted into the phagemid vector pCANTAB 5E through SfiⅠ/NotⅠsites. The phagemides containing scFv cDNA were transformed into E.coli TG1 bacterial cells to generate scFv antibody library with a diversity of approximately 4.6×108. Three rounds of enrichment and panning were performed to select specific anti-TNF-α scFv from the library. The antigen binding activity was evaluated via ELISA assay. Sequencing analysis of one positive clone showed that the anti-TNF-α scFv was 774 bp, and coded 258 amino acids. The positive clone were transformed into E.coli HB2151 and induced with IPTG. The soluble scFv showed a molecular weight 28 kD analyzed by SDS-PAGE and Western blot. The affinity purified scFv exhibited the binding activity to rhTNF-α and could neutralize the cytolytic activity of rhTNF-α against L929 cells. In this work, a higher affinity anti-TNF-α scFv was obtained from scFv antibody library. These results laid the foundation for research of antibody for clinical immunization therapy.