人源抗H7N9血凝素中和抗体IgG的制备及鉴定

Preparation and characterization of a full human neutralizing IgG antibody against H7N9 hemagglutinin

目的 :将从全人源Fab噬菌体抗体库筛选获得的抗H7N9血凝素Fab抗体基因进行基因工程改造,以表达全分子抗H7N9血凝素中和抗体Ig G,并验证其对H7N9型禽流感病毒的中和活性。方法:用H7N9型禽流感病毒血凝素筛选全人源Fab噬菌体抗体库,构建全分子Ig G抗体重、轻链表达载体,共转染293 Free Style(293F)细胞,表达并纯化Ig G抗体。应用ELISA及Western blot实验检测抗体免疫学活性,微量中和实验及鸡胚预防保护实验检测其中和活性,血凝抑制试验判断其结合血凝素亚基。结果:成功筛选出1株全人源抗H7N9血凝素Fab抗体基因并构建Ig G真核表达载体。获得的全分子Ig G抗体重、轻链序列正确,并可特异性结合H7N9血凝素。微量中和实验证明其对H7N9型禽流感病毒有良好中和活性,中和滴度为15.6μg/m L。鸡胚预防保护实验显示抗体用量为200μg时可达100%保护率。血凝抑制试验表明其结合血凝素HA2亚基。结论:制备的重组全人源全分子抗H7N9血凝素Ig G抗体可特异性识别H7N9型禽流感病毒血凝素,对H7N9型禽流病毒有显著的中和作用,为进一步研发用于禽流感防治的抗体药物奠定基础。

Objective：To genetically engineer an anti-H7N9 hemagglutinin Fab gene obtained by screening a full human Fab phage antibody library and to express it by antibody engineering, as well as verify its neutralizing activity to H7N9 avain influenza viruses. Methods： We screened a full human Fab phage antibody library using H7N9 avain influenza virus hemagglutinin and constructed IgG heavy and light chain expression vectors. Both vectors were transfected into 293 FreeStyle （293F） cells and the IgG antibody was expressed and purified. Then, the immunological activity was detected by ELISA assay and Western blotting assay, and the neutralizing activity was detected by microneutralization assay and prophylactic protection test in embryonated eggs. The hemagglutinin binding subunit was speculated by hemagglutination inhibition test. Results： A full human anti-H7N9 hemagglutinin Fab gene was successfully obtained and the IgG eukaryotic expression vectors were constructed. The sequences of both the heavy and the light chains of the antibody were correct and the antibody could bind to HTN9 hemagglutinin specifically. The antibody showed obvious neutralizing activity against HTN9 avain influenza viruses in microneutralization assay, and the neutralizing titer was 15.6 μg/mL. A dose of 200 μg IgG conferred a 100% survival rate in embryonated eggs. Hemagglutination inhibition test indicated that the IgG may bind the HA2 subunit of hemagglutinin. Conclusion： The reconstructed full human anti-H7N9 hemagglutinin IgG antibody could specifically recognize H7N9 avain influenza hemagglutinin and could obviously neutralize the vires, and laid a solid foundation for the further development of antibody drugs for the prophylaxis and treatment of avain influenza.