抗人禽流感病毒H5N1人源化单链抗体噬菌体文库的构建和筛选

摘要

Objective To construct and screen the phage antibody library for seFv fragment from recovery phase patient infected with H5N1 virus.Methods Total RNA was extractod from peripheral blood lymphocytes from a patient infected with H5N1 virus and was transformed to cDNA by reverse transcription.The variable domains of heavy and light chains were amplified by PCR with a set of specific primer of human IgG.The scFv gene fragments linked VH and VL with synthesized scFv linker were cloned into phagemid pCANTAB5E vector.Then the recombined phagemids were electroperated into E.coli TG1.The recombinant rate of scFv phage antibody library Was identified by enzyme digestion analysis and PCR.And the library repertoire Was determined by titration the PFU of library.Results The phage antibody library of scFv which specific binding to the proteins of H5N1 virus Was generated from the peripheral blood of the recovery phase patient infected with H5N1 virus and the repertoire of library was 3.75×104.Conclusion Phage antibody library of humanized scFv fragment which specific binding to the proteins of H5N1 virus has been constructed and screened successfully,which lay the foundation for further study of quick detection method and medicihal purpose.%目的 利用抗人禽流感病毒H5N1 IgG抗体阳性的人禽流感康复患者外周血淋巴细胞,构建人源化Fv段单链抗体(seFv)噬菌体文库,并筛选与禽流感病毒相关蛋白有结合活性的scFv抗体文库.方法 提取人外周血淋巴细胞总RNA,逆转录成cDNA,以其为模板,利用家族特异性IgG基因的引物,扩增重链和轻链的可变区基因,并用合成的连接子将轻链和重链基因连接成单链抗体片段后,重组到噬菌粒载体pCANTAB5E中.将重组噬菌粒载体电转化大肠杆菌TG1,酶切和PCR鉴定抗体库的重组率,通过测定噬菌体抗体库的滴度计算抗体库的库容,用特异性禽流感病毒相关蛋白筛选表达的单链抗体.结果 构建了源于人禽流感康复患者血清的scFv抗体文库,库容为3.75×104;筛选出与禽流感病毒相关蛋白有结合活性的scFv抗体文库.结论 成功构建了抗人禽流感病毒H5N1的人源scFv噬菌体抗体库,并筛选出特异性结合人禽流感病毒相关蛋白的单链抗体,为进一步制备快速检测试剂和治疗研究提供了基础数据.