重组人G—CSF免疫小鼠全套单链抗体噬菌体表面展示文库的构建

Construction of a phage display library of repertoire single-chain Fv antibodies from mouse immunized with recombinant human granulocytecolony-stimulating factor (G-CSF)

利用全套噬菌体抗体表面展示技术，绕过杂交瘤技术，从重组人G-CSF免疫的小鼠脾淋巴细胞中提取总RNA，反转录成cDNA后，用抗体可变区PCR混合引物进行全套抗体重、轻链可变区（VH和VL）基因的扩增。经重叠延伸反应，在体外随机装配成单链抗体（ScFv）。将其克隆至噬菌粒载体pCANTAB5E中，电转化含SupE的E.Coli菌株，以辅助噬菌体M13K07超感染噬菌粒文库，构建成全套ScFv表面展示文库。为利用亲和富集筛选技术，获得具有G-CSF结合活性的完整重组噬菌粒克隆奠定了基础。

Uaing Phage display tecboCIUe we COnStrUcted a library Of repertoire inununogloballn from mouse inunedzed with recombinant human granulOCytecolony-sit inulating factor (abC S F ). The heaVy -chat n and hap pa light-c ha- in v art able region bine (YH and VL ) repertobo of irmunoglobulin were amPAned individually horn the spleen cell mRNA by RT-PCR and joined by a DNA linker encoding Peptide (GGGGS ) 3 as a single-chain Fv (SCFv ) DNA fragment with overlap extension.These framents were cloned into the phagendd PCANTAB SE and expressed as a fusion protein with phage M 13 P Ill in E. coli W 1. In the presence Of helper phage M13KO7, the ScFv fuSion protein were displayed on the surface of re combest phages.