抗口蹄疫病毒phage-scFv及可溶性scFv的构建

利用重组DNA技术在抗口蹄疫病毒单克隆抗体1C 7 VH基因和VL基因之间导入一段连接肽(G ly4Ser)3],采用重叠延伸拼接法,经聚合酶链反应(PCR)扩增获得scF v基因。将scF v基因克隆至噬菌粒pCANTAB 5E载体,转化E.coli TG 1,构建噬菌体抗体文库。用M 13KO 7辅助噬菌体挽救及固相口蹄疫病毒(FMDV)抗原对噬菌体抗体文库的三轮“吸附-洗脱-扩增”的淘洗,筛选出scFv阳性克隆。将阳性克隆转化E.coli BH 2151,通过异丙基硫代-β-D-半乳糖苷(IPTG)诱导可溶性scFv蛋白的表达。酶联免疫吸附实验(EL ISA)检测表明:scFv克隆表达的phage-scFv及可溶性scFv与FMDV亲和力高,特异性强。

Construction of Phage-scFv and Soluble scFv against Foot and Mouth Disease Virus

The anti-foot-and-mouth disease virus(FMDV) single-chain Fv(scFv) gene was constructed by DNA recombinant technology.The 1C7V_H and V_L genes were assembled into scFv gene with a linker sequence(Gly_4Ser)_3 by splicing overlop extension and scFv gene was amplified by PCR.The scFv gene was subcloned into phagemid vector pCANTAB 5E.The phage scFv displaying library was constructed by male Escherichia coli TG1 transfected with resultant phagemid pCANTAB 5E-scFv.The scFv positive clone was obtained after the phage scFv displaying library was rescued by M13KO7 and biopanned with FMDV antigen for 3 rounds.Escherichia coli BH2151 was transfected with the positive clone and scFv protein was expressed by Escherichia coli BH2151 through the inducement of IPTG.The results of ELISA show that phage-scFv and soluble scFv,which were expressed by the selected scFv gene,have high affinity and specificity to FMDV.