Preparation And Detection Of PSMA(Glutamate Carboxypeptidase 2)Monoclonal Antibody

Objective:Prostate cancer(PCa)is one of the most common malignant tumors and the second leading cause of cancer in Western men.For many years,the treatment of advanced prostate cancer is mainly based on the regulation of androgen receptor-controlled proliferation pathways,and the inevitable final progress is castration-resistant prostate cancer(CRPC),and the treatment of CRPC has been lacking in more effective treatments..The breakthrough development of immunology in recent years has also provided new inspiration for the treatment of prostate cancer.Prostate-specific membrane antigen(PSMA)is a popular target in current research,and its characteristics of targeting prostate cancer have been agreed.Therefore,a new monoclonal antibody capable of targeting prostate cancer cells has been prepared whether it is for the treatment of prostate cancer or The diagnosis has important significance.In this lesson,we plan to prepare a full-length recombinant protein of PSMA and immunize mice with this protein to finally obtain a brand new monoclonal antibody that can target PSMA.Methods:1.Prepare PSMA recombinant protein by gene recombination technology and eukaryotic cell transient transfection technologyAfter extracting the total RNA from LNCaP cells,cDNA is synthesized by reverse transcription PCR technology,and the designed primers are used to synthesize the FOLH1 gene from the cDNA,and the expression vector is connected to the FOLH1 gene by homologous recombination technology to synthesize a plasmid.Cells amplify the plasmid in large quantities,and the plasmid is transferred into mammalian cells in suspension culture by transient transfection technology to express a large amount of PSMA recombinant protein,which is purified by affinity chromatography,and the PSMA recombinant protein is detected by SDS-PAGE electrophoresis and other experiments To identify whether the target protein is successfully expressed.2.Preparation of PSMA monoclonal antibodyImmunize BALB/c mice with PSMA recombinant protein,take out the mouse spleen and fused with SP2/0 cells to prepare hybridoma cells when the serum titers of the mice reach the standard,select positive hybridoma cells by ELISA,and use the hybridization method The cells were subcloned to obtain hybridoma cells expressing a single antibody,which were then injected into the abdominal cavity of mice to prepare ascites antibodies,which were purified by affinity chromatography techniques to obtain monoclonal antibodies.3.Identify the specificity and internalization ability of the prepared monoclonal antibodyThe specificity of the monoclonal antibody to PSMA recombinant protein was detected by Western blot,the specificity of the monoclonal antibody to PSMA-expressing prostate cancer cells was detected by flow cytometry,and the specificity and internalization ability of the monoclonal antibody to PSMA-expressing prostate cancer cells were detected by cellular immunofluorescence.Results:1.Prepare PSMA recombinant protein by gene recombination technology and eukaryotic cell transient transfection technologyThe DNA gel electrophoresis showed a clear band above 2000bp.After sequencing,the results showed that there were only two base mutations,which proved that the FOLH1 gene was successfully expressed.SDS-PAGE electrophoresis and Coomassie brilliant blue staining were used to detect the PSMA recombinant protein.The results showed that a more obvious band appeared in the 80kDa-115kDa region,indicating that the PSMA recombinant protein was successfully expressed.2.Preparation of PSMA monoclonal antibodyThe final titers of 3 mice after 5 immunization tests by ELISA all reached more than 128000,indicating successful immunization.3.Identify the specificity and internalization ability of the prepared monoclonal antibodyWestern blot experiments showed a clear band at the electrophoresis of PSMA recombinant protein,indicating that the monoclonal antibody can specifically recognize the PSMA recombinant protein.By flow cytometry,the fluorescence intensity of prostate cancer cells expressing PSMA was significantly higher than that of the negative control group,while the fluorescence intensity of prostate cancer cells not expressing PSMA was almost the same as that of the negative control group.This indicates that the monoclonal antibody can specifically recognize prostate cancer cells expressing PSMA.Through cellular immunofluorescence detection,PSMA-expressing prostate cancer cells can clearly observe the fluorescence signal,but PSMA-expressing prostate cancer cells can hardly observe the fluorescence signal,and the PSMA-expressing prostate cancer cells can also observe obvious fluorescence.The signal indicates that the antibody is not only specific but also internalized into the cell.Conclusion:In this study,the whole gene of FOLH1 was prepared by gene recombination technology,and the PSMA recombinant protein was expressed in mammalian cells by connecting the gene with eukaryotic vector,and the mice were immunized with the protein.Finally,a new monoclonal antibody targeting PSMA was obtained,and the detection by Western blot demonstrated that the monoclonal antibody could specifically recognize PSMA protein.Flow cytometry demonstrated that the antibody could specifically recognize PSMA-expressing prostate cancer cells and did not recognize prostate cancer cells that did not express PSMA.By cellular immunofluorescence detection,it was demonstrated that the monoclonal antibody not only recognized prostate cancer cells expressing PSMA,but also internalized into the cell interior.This monoclonal antibody is of great significance for the diagnosis and treatment of prostate and basic experiments.