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目的从人源化噬菌体抗体库中筛选到能高亲和性、特异结合新甲型H1N1流感病毒的单链抗体,为分析H1N1病毒抗原中和表位的活性位点和人源化治疗单抗奠定基础。方法将细胞培养的新型H1N1病毒毒株,经超速离心浓缩纯化后,获得纯的新甲型H1N1病毒,以新甲型H1N1病毒为靶蛋白,以亲和结合为原理,筛选噬菌体scFv抗体文库,经过3轮筛选富集后,随机挑选了96个噬菌体克隆扩增培养,ELISA法挑选能特异性、高亲和性结合目的蛋白的噬菌体克隆。结果经过3轮富集性的亲和筛选,分别从96个噬菌体克隆中挑选到了4株能特异结合新甲型H1N1病毒,而不结合季节性H1N1病毒的单链抗体分子,且PCR扩增都得到了长为368、527和935bp的轻链、重链和轻链-连接片段-重链的基因片段。结论从噬菌体抗体库中筛选到的特异结合新甲型H1N1病毒的单链抗体片段,可为进一步研发新甲流的H1N1快速筛选试剂和人源性治疗抗体奠定基础,也可为鉴定新甲型H1N1病毒中的抗原决定簇提供结构信息。
OBJECTIVE: To screen single-chain antibodies with high affinity and specificity for the new influenza A (H1N1) virus from humanized phage antibody library. To analyze the active sites of neutralizing epitopes of H1N1 virus and humanized monoclonal antibody Lay the foundation. Methods The purified novel H1N1 virus strain was purified by ultracentrifugation to obtain pure new type A H1N1 virus. The phage scFv antibody library was screened by the principle of affinity binding using the novel type A H1N1 virus as target protein. After 3 rounds of screening and enrichment, 96 phage clones were randomly selected for expansion culture, and phage clones capable of binding specific proteins with high affinity to the target protein were selected by ELISA. Results After three cycles of enrichment affinity screening, four strains of single-chain antibodies that specifically bind to the new H1N1 virus but not the seasonal H1N1 virus were selected from 96 phage clones, respectively, Gene fragments of the light chain, the heavy chain, and the light chain-linker fragment-heavy chain were obtained at 368, 527 and 935 bp in length. Conclusion The single-chain antibody fragment specific to the novel H1N1 virus screened from the phage antibody library can lay a foundation for the further development of the H1N1 rapid screening reagent for the new influenza A virus and the human therapeutic antibody, Epitopes in the H1N1 virus provide structural information.