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目的应用噬菌体展示技术构建抗人DR5(death receptor 5,DR5)单链抗体(single chain Fv,scFv)文库,从中筛选抗DR5 scFv。方法利用重组人DR5抗原免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸PCR将VH和VL基因拼接成scFv基因,以SfiⅠ位点定向插入PAK100噬菌粒载体,转化E.coli XL1-Blue,构建了库容为1×106的抗DR5单链抗体库。对抗体库进行5轮富集筛选后,phage-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。结果抗DR5 scFv基因序列长747 bp,编码249个氨基酸,具有和DR5结合的特异性。结论本文利用噬菌体抗体库筛选到了抗DR5 scFv,为研制临床免疫治疗的新型抗体奠定了实验基础。
Objective To construct anti-DR5 (sc5v5) single-chain Fv (scFv) library by phage display technique and screen for anti-DR5 scFv. Methods Mice were immunized with recombinant human DR5 antigen to amplify mouse VH and VL genes respectively. The VH and VL genes were spliced into scFv gene by overlap extension PCR, and inserted into PAK100 phagemid vector with SfiI site to transform into E.coli XL1-Blue, an anti-DR5 single-chain antibody library with a capacity of 1 × 106 was constructed. After 5 rounds of enrichment and screening of the antibody library, phage-ELISA was used to detect the antigen specificity of the positive clones. One positive clone was sequenced. Results The sequence of anti-DR5 scFv gene was 747 bp in length encoding a protein of 249 amino acids with the specificity of binding to DR5. Conclusion This study screened the anti-DR5 scFv using the phage antibody library and laid the experimental foundation for the development of novel antibodies for clinical immunotherapy.