抗人Endoglin胞外段单链抗体噬菌体表面呈现文库的构建/筛选及鉴定

Construction,Preliminary Selection and Identification of Phage Display Library of Extracellular Region of Recombinant Anti-Human Endoglin scFv Antibody

目的构建抗人Endoglin胞外段的特异性单链抗体(single chain variable fragment,scFv)噬菌体表面呈现文库。方法用重组人Endoglin(recombinant human Endoglin,rhEndoglin)胞外段蛋白免疫Balb/c小鼠,提取脾脏组织mRNA,经RT-PCR分别扩增出VH(heavy chain variable region)、VL(light chain variable region)基因,经Linker连接成scFv基因,再把scFv基因重组到噬菌粒载体pCANTAB5E,转化到大肠杆菌TG1,经辅助噬菌体M13KO7拯救后建成噬菌体呈现文库。用rhEndoglin对文库进行5轮吸附-洗脱-富集panning淘筛,选择ELISA结果最强的克隆提取质粒并测序。结果经过筛选,随机挑取94个克隆进行ELISA检测,37个克隆呈阳性。序列分析表明,该scFv DNA全长738bp。其中VH基因366bp,编码122个氨基酸残基,VL基因327bp,编码109个氨基酸残基,它们之间通过(Gly4Ser)3组成的15肽连接。结论成功构建了抗人Endoglin胞外段scFv噬菌体表面呈现文库,并筛选获得一个能与Endoglin特异结合的重组噬菌体克隆。

Objective To construct the phage display library of extracellular region of anti-rhEndoglin scFv antibody, Methods Balb/c mice were immunized by the extracellular region of rhEndoglin and their mRNAs of the splenic cells were extracted. VH and VL were amplified by RT-PCR, which were ligated into scFv DNA through a linker, then the scFv DNA was recombined into pCANTAB5E vector, The recombinant plasmid was transformed to E, coli TG1, The construction of phage display library was finished after rescuing the transformed TG1 by Helper phage M13KO7, Five rounds of absorb-elute-enrich panning against rhEndoglin were done by phage-ELISA, The positive recombinant phage clones after phage-ELISA were used to extract the plasmids and the plasmids were sequenced. Results After panning, 94 clones from the library were selected randomly and 37 positive clones were obtained. The result of sequencing showed that the scFv DNA was 738 bp, of which VH encoding 122 amino acid residues was 366 bp and VL encoding 109 amino acid residues was 327 bp. VH and VL were ligated by a peptide consisting of 15 amino acid residues in （Gly4 Ser）3. Conclusion The phage display library of the extracellular region of anti-rhEndoglin scFv was successfully constructed, from which a recombinant phage clone that could specifically bind with rhEndoglin was obtained by panning.