Preparation And Identification Of The Monoclonal Antibody Against Four Canine B Cell Surface Markers

Single B cell monoclonal antibody technology is a method for rapid preparation of monoclonal antibody based on single-cell technology and single-cell RT-PCR technology,providing a faster method to obtain neutralizing antibody of Pathogens of sudden infectious diseases and difficult to prepare m Ab.One of the key for single B cell monoclonal antibody technology is the isolation of single B cells,a systematic B cell surface marker m Abs are necessary for the method.The reaserach of B cell surface molecule m Abs have been completed and the single B cell antibody technique has been widely used in human and mouse.Human single memory B cells or plasma cell can be effectively isolated by using monoclonal antibodies of CD19,CD27 and CD38.However,the mechanism of immune system response and the classification of related immune cells in dogs are rarely researched,and there is a lack of m Abs for a series of canine B cell surface markers.It has been found that the mechanism and characteristics of canine immune system and the function of CD molecules are highly similar to those of human.Therefore,canine CD19,CD27,CD38 and CD138 were selected to prepare its m Ab in this study,which is expected to lay a foundation for the platform of canine single B cell antibody technology to isolate canine single B cell.The study mainly includes the following aspects:1.Expression and purification of canine CD19,CD27,CD38 and CD138:The recombinant expression plasmids of CD19,CD27,CD38 and CD138 were constructed and and transfected into Expi CHO-STM cells and Expi293 cells independently to express the recombinant proteins,the recombinant proteins were identified by Western blot and SDS-PAGE and purified according to His affinity chromatography column to obtain canine CD19、CD27、CD38 and CD138.2.Preparation of canine CD19、CD27、CD38 and CD138 mAb:The recombinant proteins expressed by ExpiCHO-STM cells were used as mouse immune antigens,and the recombinant proteins expressed by Expi293 cells were used as screening antigens.Finally,10、3、12 and 2 strains of m Ab respectively targeting CD19、CD27、CD38 and CD138 were screened.The ascites m Ab ELISA titers was not less than 64 000.3.Identification of canine CD19,CD27,CD38 and CD138 m Ab:The stability of hybridoma cells was detected by cell passage,the specificity of m Ab was identified by IFA and Western-blot,and the biological function of each m Ab was verified by immunohistochemistry and flow cytometry.The stability analysis showed that the hybridoma cells could secrete m Ab stably for 30 generations;IFA and Western-blot indicating that each m Ab could specifically recognize the corresponding canine CD protein;and the immunohistochemical analysis showed that canine CD19 and CD27 molecules have respectively obtained one strain of m Ab that could be used to identify the cells expressing CD19 or CD27 in canine lymphoid tissue by immunohistochemistry.The results of flow cytometry showed that canine CD19,CD27,CD38 and CD138 molecules have respectively obtained two strains of m Ab,which can be used to detect the cell populations expressing related CD molecules in canine PBMCs by flow cytometry.In summary,this study successfully expressed and purified canine CD19,CD27,CD38 and CD138 proteins through eukaryotic expression cells,and the m Abs against four CD molecules were successfully prepared and systematically identified,which provided powerful tool for the study of canine B cell immune characteristics and laid a foundation for the isolation of single B cells on the platform of canine single B cell antibody technology.