Preparation Of Monoclonal Antibody And Nanobody Against Human Cytomegalovirus PP65 Antigen

Objective:1.Prepare monoclonal antibody against human cytomegalovirus?human cytomegalic virus,HCMV?PP65 antigen,and establish an immunohistochemical assay for HCMV PP65 antigen.2.The nanobody against PP65 antigen of HCMV was prepared and their reactivity with PP65 antigen was preliminarily identified.Methods:1.Induce PQE30-PP65 recombinant plasmid to express PP65 antigen and immunize BALB/c mice.Monoclonal antibody was obtained by cell fusion of spleen cells in mice with positive serum antibody and myeloma cells?SP2/0?,HTS screening,expanded culture and affinity purification.The subtype,titer and specificity of the prepared monoclonal antibody were determined and it was used for the slide immunohistochemistry in clinical samples.2.In order to obtain HCMV PP65 specific nanobodies,the recombinant PP65protein was used as the target antigen to screen the constructed non-immune Phage Library,eight PP65 specific nanobodies sequences were screened by indirect ELISA and constructed into pClod low-temperature induced prokaryotic expression vector for expression and purification.The reaction of the prepared nanobodies with PP65 antigen was confirmed by indirect ELISA.Results:1.The PQE30-PP65 recombinant plasmid was transformed into E.coli M15competent cells.After IPTG was added to induce expression,SDS-PAGE identification showed that there were obvious bands near 26-33kDa in the experimental group,which was consistent with the expected size,indicating that the recombinant plasmid was successfully transformed and could express the target protein.2.After western-blot identification,a clear band of HRP labeled anti-His monoclonal antibody can be seen near 26-33kDa,which indicated that the protein is a target protein with his tag and can be purified by nickel column.3.The PQE30-PP65 recombinant plasmid was expressed in a large amount,the bacteria was ultrasonically crushed,and inclusion bodies were purified by nickel column affinity chromatography.The analysis of 12%SDS-PAGE showed that a single band was visible around 26-33kDa,which was consistent with the expected,indicating that the target protein with higher purity was obtained after purification.4.BALB/c mice were immunized with PP65 antigen,and the mice with the highest serum titer were selected toenhance the immunity again.The hybridoma cells were prepared from the spleen cells of immunized mouse.After positive hybridoma cell screening and 3 times of subcloning,8 strains of anti HCMV PP65 specific monoclonal antibody were screened.5.The purity of 8 purified monoclonal antibodies was identified.After 12%SDS-PAGE detection,found the heavy chain of the monoclonal antibody could be seen at about 50kDa,and the light chain could be seen at about 25kDa,which indicated that the purified monoclonal antibody had high purity.6.The monoclonal antibody immunoglobulin subclass identification kit was used to identify antibody subclasses by indirect ELISA.After identification,the heavy chain subclasses of the 8 antibodies were IgG type 1 antibodies,and the light chains were?chains.7.It was confirmed that the titers of the 8 monoclonal antibodies were above:1:1024000 detected by indirect ELISA.8.The specificity of 8 monoclonal antibodies was detected,PP65 protein and other4 different unrelated proteins were tested by ELISA,respectively.Found that OD_450-630absorption value of 8 monoclonal antibodies reacting with PP65 protein was significantly higher than other unrelated proteins.All of the 8 monoclonal antibodies can specifically recognize PP65 protein,but have low reactivity with other proteins,showing good specificity.9.Eight purified monoclonal antibodies detected clinical positive and negative blood samples by immunohistochemistry proves that 6 of them can accurately define positive and negative infection.10.PP65 was used as the screening antigen,and after four rounds of elutriation of the non-immune library,eight specific antigen-specific nanobodies were screened.11.Insert the gene sequence corresponding to 8 nanobodies into the procaryotic expression vector of pCold I,and identify the expression of 8 nanobodies.The results of SDS-PAGE and werstern-blot showed that the 8 nanobodies were consistent with the expected molecular weight and could bind to the anti his antibody of HRP labeled mice,indicating that 8 nanobodies were successfully expressed.12.Eight nanobodies were induced and expressed in large quantities,purified by nickel column affinity chromatography,replaced and concentrated by ultrafiltration centrifuge tube,and identified by SDS-PAGE.The results showed that 8 nanobodies with high purity were successfully obtained.13.The reactivity of 8 nanobodies with PP65 protein was detected by indirect ELISA.It can be seen that two of them have good reactivity with PP65 antigen.Conclusion:1.Eight monoclonal antibodies against PP65 antigen of HCMV were prepared,and six of them were able to accurately identify the infection of HCMV by slide immunohistochemistry.The results provide materials for the development of HCMV infection detection.2.Two nanobodies were screened and prepared from the constructed phage non immune library,which laid the foundation for the next step to prepare specific high penetrating vector for the treatment of HCMV infection.