Construction And Expression Of Hepatitis B Surface Antigen Antibody And Detection Of Its Activity

Background:New infectious diseases can easily cause outbreaks due to their strong infectivity.Antibodies,as one of the strategies for artificial active immune prevention and treatment,can be used for emergency prevention and treatment of patients.The fully human monoclonal antibody(mAb)has many advantages like high specificity,low toxicity and side effects,and mass production.During the outbreak of disease,it is very important to quickly screen the fully human monoclonal antibodies for the development of neutralizing antibody vaccines or antibody drug preparation.The preparation of fully human monoclonal antibodies usually uses peripheral blood mononuclear cells(PBMCs)in the recovery phase of the patient.But the content of virus-specific plasma cells in the recovery phase of the patient is very low,which is difficult to capture.The method of inducing memory B lymphocytes to differentiate into antigen-specific plasma cells in PBMCs in vitro can obtain a large number of plasma cells for antibody gene extraction,which provides a research basis for the preparation of fully human monoclonal antibodies.Objective:The purpose of this study is to establish a method of inducing the proliferation and differentiation of pathogen-specific plasma cells in peripheral blood memory B lymphocytes of patients with pathogen infection in vitro,with a view to obtaining a large number of antigen-specific plasma cells for the isolation of antibody genes.It provides a research basis for the preparation of high-affinity,neutralizing,fully human monoclonal antibody.Methods:In this study,healthy volunteers who had been vaccinated with hepatitis B vaccine for more than 3 years were used to isolate PBMCs using density gradient centrifugation,using TLR7/8 agonist R848 or TLR9 agonist CpG DNA 2006 in combination with different cytokines(IL-2,IL-4,IL-10)to induce activation and evaluate the activation effect.Cell survival status were detected by trypan blue staining to count the activated cells.The total IgG or/and HBsAg antigen-specific IgG secretion in the cell culture supernatant were detected by an enzyme-linked immunosorbent assay(ELISA).The number of total antibody secreting cells(ASCs)or/and HBsAg antigen-specific ASCs in the activated cells were assessed by Enzyme-linked immunospot assay(ELISPOT).The induction schemes are: different concentrations of R848 and IL-2 combined to determine the optimal concentration of R848;R848+IL-2 combined with different cytokines(IL-4,IL-10)to determine the effect of cytokines on R848 induced activation;CpG DNA 2006 and CD40 antibodies combined with cytokines IL-2,IL-4 and/or IL-10 to determine the CpG DNA 2006 activation optimal combination.Results:1.The results of ELISA and ELISPOT show that the combination of R848+IL-2 can effectively induce the expansion and differentiation of B lymphocytes into plasma cells when the concentration of R848 is 1-2.5?g/ml,and the earliest on the fifth day after activation ASCs can be detected;cytokines IL-4 and IL-10 have no significant effect on the method of R848+IL-2 combined induction of B cell activation.2.The results of the combination of CpG DNA 2006 and different cytokines proved that under the action of anti-CD40 antibodies,the combined use of CpG DNA 2006 and cytokines IL-2,IL-4 and IL-10 can better induce B lymphocytes differentiate into ASCs;3.Compare the activation effects of the two methods using R848+IL-2(R848 group)or CpG DNA 2006+CD40 antibody IL-2,IL-4,IL-10(CpG group),and find that R848 group can be more effective,the detection rate of ELISPOT plasma cells is at least 8 times higher than that of CpG group;4.After inducing PBMCs of healthy volunteers vaccinated with hepatitis B vaccine using R848+IL-2 method,ELISA can detect HBsAg-specific IgG in the cell supernatant and ELISPOT can detect HBsAg-specific plasma cells.Conclusion:The two methods that R848+IL-2 or under the action of anti-CD40 antibody,CpG DNA 2006 combined with the cytokines IL-2,IL-4,IL-10 can induce the proliferation and differentiation of memory B lymphocytes in PBMCs.By the method of using R848+IL-2,antigen-specific antibody secreting cells can be obtained from several milliliters of peripheral blood in patients during recovery for analysis and sorting,which might provide effective method for the preparation of neutralizing antibodies.Background:Hepatitis B is an infectious disease caused by Hepatitis B virus(HBV)infection that seriously endangers human health.China is a high incidence area of HBV infection.Because there is currently no effective treatment for patients with chronic hepatitis B,it often leads to unsustainable disease progression and increases the risk of complications.The high-efficiency immunoglobulin used in clinic can neutralize the invading virus and protect the body from viral infection.However,due to the problems of limited blood source and high preparation cost,the application is limited.Hepatitis B therapeutic vaccine refers to the addition of surface antigen antibodies on the basis of a certain proportion of vaccines prepared using only antigens,and its research has now entered clinical phase III,confirming its effectiveness.The construction of fully human monoclonal antibodies with high-affinity hepatitis B surface antigens makes it possible to produce humanized antibodies on a large scale,providing a research basis for the clinical treatment of hepatitis B and the development of therapeutic vaccines.Objective:Different fully human monoclonal antibodies of hepatitis B surface antigen are constructed and expresses,then some of them were transformed into chimeric murine.Then,explore their purity and the ability to specific combine with surface antigens.Establishment bacterial display technology to screen hepatitis B surface antigen epitopes,providing a basis for the preparation of monoclonal antibodies and the development of hepatitis B therapeutic vaccines.Methods:1.Construction and identification of recombinant plasmids.Using PCR method to obtain the antibody light and heavy chain variable region genes and human/mouse Ig G light and heavy chain constant region genes,then use the overlap PCR method to connect the variable region and the constant region of the light and heavy chains respectively.It was inserted into the eukaryotic expression vector Pc DNA3.4 and verification of vectors accuracy through enzyme digestion reaction and gene sequenced.2.293 F system expresses antibody protein.Transfect 293 F cells with the correct plasmid,the target antibodies protein can be secreted into the cell culture supernatant.Purify protein with Protein G affinity chromatography column and concentrate with PEG20000 to obtain both higher purity and concentration antibody protein.3.Detection of antibody purity.SDS-PAGE gel stained by Coomassie brilliant blue to detect the purity of antibody protein.4.Detection of antibody activity and specific binding capacity.Using enzyme-linked immunosorbent assay(ELISA)to detect the activity of antibody protein and its specific binding capacity to hepatitis B surface antigen;using immunohistochemical method to detect chimeric mouse antibody ability to bind hepatitis B surface antigen in samples from patients with HBV-related HCC.5.Screening of hepatitis B antigen epitopes.Establishment bacterial display technology to screen antigen epitope and use the prepared antibodies to display the full length of hepatitis B surface antigens,and perform preliminary identification of epitopes.Results:1.Successfully constructed 5 pairs of fully humanized hepatitis B surface antigen monoclonal antibody light and heavy chain expression plasmids,namely HBs Ab-h116,HBs Ab-h1444,HBs Ab-h1425,HBs Ab-h1453,HBs Ab-h477,two of them were HBs Ab-h116 and HBs Ab-h477 were transformed into chimeric murine antibodies and plasmids were constructed.Two theoretical bands were obtained by agarose gel electrophoresis after the digestion recombinant plasmids.Comparing the sequence results showed that the inserted sequences were correct.2.The antibody was successfully expressed using 293 F cells,purified using Protein G affinity chromatography column.After Coomassie brilliant blue staining using SDS-PAGE gel,7 pairs of antibodies showed two clear bands of light and heavy chains.The relative molecular weight about 27 KD and 55 KD respectively,which proves that the antibody purity is high;3.The ELISA and immunohistochemistry confirmed that the antibodies had specific binding ability to hepatitis B surface antigen.4.Successfully established bacterial display technology,constructed a full-length bacterial display APEX vector containing the hepatitis B surface antigen ADR/AYW subtypes,using flow cytometry to confirm that the prepared HBs Ab-h116 and HBs Ab-h1444 can react more significantly with the ADR subtype.Conclusion:Five pairs of fully human hepatitis B surface antigen antibodies and two pairs of chimeric murine antibody light and heavy chain expression plasmids were successfully constructed and expressed in 293 F eukaryotic cells.The purity and specificity of the purified antibody were verified,which confirmed its specific binding ability to hepatitis B surface antigen.Successful establishment of bacterial display technology and preliminary screening of antigen epitopes,confirming that the prepared HBs Ab-h116 and HBs Ab-h1444 can have a more obvious reaction with the full-length of ADR subtype.Our experiment provides a research basis for the preparation of monoclonal antibodies and the development of hepatitis B therapeutic vaccines.