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目的:构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链可变区抗体库(VHH型抗体库),为抗H5N1的VHH抗体筛选奠定基础。方法:利用H5N1灭活疫苗免疫小羊驼,一定免疫时间后测定小羊驼外周血清中抗体中和活性,分离其外周淋巴细胞,利用RT-PCR方法得到VHH抗体片段。通过优化连接和电转化方法,将足量VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1,获得VHH抗体基因库;检测基因库库容以及多样性,并采用血凝抑制试验对噬菌体抗体库进行初步功能性鉴定。结果:利用H5N1灭活疫苗免疫小羊驼四次后,其外周血清中抗体血清抑制效价可达1∶2 560,构建的VHH抗体基因库库容可达3×108,随机挑选14个抗体基因克隆进行测序鉴定,结果显示均为独立克隆,表明所建抗体库多样性好。上述基因库经辅助噬菌体拯救后,得到抗H5N1的噬菌体VHH型抗体初级库,对初级库进行血凝抑制试验,结果呈阳性,表明初级库中存在具有潜在中和活性的抗H5N1抗体。结论:结果表明,已成功构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链抗体库,为进一步筛选抗H5N1禽流感的重链抗体打下良好基础,并为H5N1的早期临床诊断和治疗提供新的手段。
OBJECTIVE: To construct a llama immunization phage heavy chain variable region antibody library (VHH type antibody library) against H5N1 avian influenza virus and lay the foundation for the screening of VHH antibodies against H5N1. Methods: The alpaca was immunized with the H5N1 inactivated vaccine. After a certain immunization time, the antibody neutralization activity in the peripheral blood serum of llamas was determined. The peripheral lymphocytes were isolated and the VHH antibody fragments were obtained by RT-PCR. By optimizing the ligation and electrotransformation methods, a sufficient amount of VHH fragment was ligated into pCANTAB5E and then transformed into E. coli TG1 to obtain the VHH antibody gene pool. The gene pool capacity and diversity were tested and the phage antibody library was preliminarily tested by hemagglutination inhibition test Functional identification. Results: The antibody titer of peripheral blood serum of rabbits immunized with H5N1 inactivated four times was up to 1: 2506. The constructed VHH antibody library had a capacity of 3 × 108, and 14 antibody genes were randomly selected The clones were identified by sequencing. The results showed that all the clones were independent clones, indicating that the constructed antibody library has good diversity. After the above gene pool was rescued by the helper phage, a primary antibody library of phage VHH antibody against H5N1 was obtained, and the primary library was tested for hemagglutination inhibition. The result was positive, indicating that there is a potentially neutralizing anti-H5N1 antibody in the primary library. CONCLUSION: The results showed that the llama immunofluorescence antibody phage antibody against H5N1 strain has been successfully constructed, which laid a good foundation for further screening of heavy chain antibodies against H5N1 avian influenza virus and provided new clinical diagnosis and treatment for H5N1 s method.