| Pneumococcal disease is one of serious public health problems in the world.It is also an important cause of morbidity and mortality among children and adults in China.Streptococcus pneumoniae infection can cause bacterial pneumonia,otitis media and meningitis and other diseases,its clinical treatment drugs are mainly antibiotics,however,the widespread use of antibiotics bring the drug resistance of pneumococcus.It is particularly necessary and urgent to use pneumococcal vaccines to prevent pneumococcal diseases or to use pneumococcal specific antibodies to treat patients with acute pneumococcal sepsis and to reduce bacterial resistance.Pneumolysin(PLY),a pore-forming toxin of Streptococcus pneumoniae,can be expressed by all Streptococcus pneumoniae strains,and it plays an important role in the inflammatory process of pneumococcal lung injury.In the preliminary research of the laboratory,the mouse-derived monoclonal antibody 2E5 mab targeting PLY was successfully screened and prepared,its biological activitives and protective effects on mice infected with Streptococcus pneumoniae were verified.This study used genetic recombination technology to humanize mouse-derived antibodies,explored the biological activities and characteristics of humanized antibodies,and lays a foundation for the treatment of Streptococcus pneumoniae disease.The PLY protein was expressed in E.coli prokaryotic system to verify its biological activity.The variable region of the mouse antibody was combined with the constant region of the human antibody to synthesize plasmids,and the human-mouse chimeric antibody was obtained by expression and purification in the eukaryotic system.We continued to humanize the four framework regions in the variable region of the humanmouse chimeric antibody,then obtained four heavy chains(2E5zumabVH0,2E5zumab-VH1,2E5zumab-VH2,2E5zumab-VH3)and a light chain(2E5zumab-VL0).The humanized antibody was obtained by expressing and purifying through the eukaryotic expression system.Elisa verified the antibody antigen affinity,Western Blot verified the specificity of the humanized antibody,anti-hemolysis experiment verified the anti-hemolytic activity of the antibody,animal bacteremia model in vivo to observe the survival rate of mice,HE staining to detect the degree of lung tissue damage,Real Time PCR Antibody thermal stability was determined and pharmacokinetics were studied.PLY protein has a strong hemolytic activity at 6.25 ng,the humanmouse chimeric antibody 2E5 ximab has high affinity with PLY protein,and has strong anti-hemolytic activity,which has a protective effect on Streptococcus pneumoniae-infected mice.Four humanized antibodies were obtained by cross-combination,2E5zumab-H0L0,2E5zumab-H1L0,2E5zumab-H2L0 and 2E5zumab-H3L0,among which the humanized antibody 2E5zumab-H3L0 has high affinity and specificity,and its antihemolytic activity was better than that of chimeric antibody.The efficacy of 2E5zumab-H3L0 in vivo was further verified,under the condition of equal dose,the efficacy of 2E5zumab-H3L0 in vivo was equal to or even better than that of chimeric antibody,and the survival rate of mice was improved.Administering 2E5zumab-H3L0 to the tail vein in advance could reduce the infiltration of inflammatory cells in lung tissue.2E5zumabH3L0 has a long half-life of 62.03 h,high structural stability,and Tm value of 74.647℃.In summary,this study humanized the constant region of the mouse antibody,and successfully obtained the human-mouse chimeric antibody2E5 ximab,the framework region of the murine variable region was further humanized to obtain a humanized antibody 2E5zumab-H3L0,which was successfully verified to have high affinity,biological activity and stability. |
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