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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 the anti-H7N9 hemagglutinin Fab antibody selected from the whole-human Fab phage antibody library to express the full-length anti-H7N9 hemagglutinin neutralizing antibody Ig G and to verify its effect on H7N9 avian influenza Neutralizing activity of the virus. Methods: The whole-phage Fab phage antibody library was screened by H7N9 avian influenza virus hemagglutinin to construct the heavy and light chain expression vector of allogeneic Ig G antibody and co-transfected into 293 Free Style (293F) cells to express and purify Ig G antibody . The immunological activity of the antibody was detected by ELISA and Western blot. The activity of neutralizing activity was detected by micro neutralization test and chicken embryo prevention and protection test. The hemagglutination inhibition test was used to determine the binding of hemagglutinin subunit. Results: One full human anti-H7N9 hemagglutinin Fab antibody was successfully screened and the Ig G eukaryotic expression vector was constructed. The whole IgG antibody was obtained with the correct light chain sequence and specific binding to H7N9 hemagglutinin. Neutralization titration was 15.6μg / m L with good neutralization activity against H7N9 avian influenza virus. Chicken prevention and protection experiments showed that the amount of antibody at 200μg up to 100% protection rate. The hemagglutination inhibition test shows that it binds the HA2 subunit of hemagglutinin. CONCLUSION: The recombinant whole-human anti-H7N9 hemagglutinin Ig G antibody can specifically identify the hemagglutinin of H7N9 avian influenza virus and has a significant neutralization effect on H7N9 avian influenza virus. In order to further develop the antibody against H7N9 avian influenza virus, Flu prevention and treatment of antibody drugs lay the foundation.