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目的 :构建人源噬菌体展示单链抗体 (scFv)库 ,筛选抗狂犬病毒特异性、高亲和力的scFv。方法 :应用重组噬菌体抗体技术 ,从经狂犬病毒WISTARPM株疫苗免疫者的外周血淋巴细胞中 ,分离并构建scFv基因。将其克隆入噬粒载体pCANTAB 5E中 ,转化于大肠杆菌TG1,通过辅助噬菌体M13K0 7援救构建噬菌体单链抗体库。采用狂犬病毒Vero疫苗亲和富集法 ,淘选阳性重组噬菌体 ,经鉴定后对其进行序列分析。用竞争ELISA ,初步检测重组scFv的特异性抗原结合活性。结果 :成功地构建了库容量约为 7× 10 8抗狂犬病毒噬菌体scFv库 ,筛选到 1株新的抗狂犬病毒的scFv S12。结论 :噬菌体展示scFv库的成功构建及人源抗狂犬病毒特异性scFv的获得 ,为进一步研制抗狂犬病毒的高特异性、高亲和力的基因工程抗体奠定了基础
OBJECTIVE: To construct human phage display scFv library and screen for anti-rabies virus-specific and high-affinity scFv. Methods: The recombinant phage antibody was used to isolate and construct scFv gene from peripheral blood lymphocytes of rabies virus immunized with WISTARPM strain. Cloned into the phagemid vector pCANTAB 5E, transformed into E. coli TG1, and phage scFv library was constructed by the aid of helper phage M13K07. Rabies virus Vero vaccine affinity enrichment method, panning positive recombinant phage, after identification of its sequence analysis. The competitive ELISA was used to detect the specific antigen-binding activity of recombinant scFv. Results: A 7 × 10 8 anti-rabies virus phage scFv library was successfully constructed and a new anti-rabies virus scFv S12 was screened. Conclusion: The successful construction of phage display scFv library and the acquisition of human anti-rabies virus-specific scFv laid the foundation for the further development of anti-rabies virus with high specificity and high affinity.