摘要
Abstract
Objective:To engineer a monoclonal antibody against TP53 and explore its application in clinical pathology diagnosis.Methods:The variable region genes encoding the heavy and light chains of the TP53 monoclonal antibody derived from hybridoma were cloned into the pGEM-T vector.Sequencing analysis was subsequently performed to identify effective heavy and light chain sequences for cloning into the pFUSEss-CHIg-mG2B,pFUSE2ss-CLIg-mk,and pcDNA3.4 vectors.Eukaryotic expression of the antibody was achieved utilizing the ExpiCHO-S™ Expression System,and the TP53 recombinant antibody was purified through a protein a column.Finally,a preliminary evaluation of the TP53 recombinant antibody's application,alongside a commercialized antibody was conducted using Western blot and IHC techniques.Results:Successful expression and purification of the TP53 recombinant antibody were achieved.Liquid chromatography-mass spectrometry analysis confirmed the molecular weights of the TP53 light chain and heavy chain to be approximately 25 kDa and 53 kDa,respectively.ELISA titer analysis revealed a high titer of 1∶2 180 000,while affinity measurements yielded a Ka value of 6.6×1010 L·mol-1,indicating strong binding affinity.Western Blot analysis demonstrated consistent expression patterns between the TP53 recombinant antibody and the commercialized TP53 antibody.IHC results demonstrated that both antibodies recognized the same target,located within the cell nucleus,across multiple cancer types,including human renal cancer,human breast lobular adenocarcinoma,human sigmoid colon adenocarcinoma,and mouse liver cancer.Notably,the TP53 recombinant antibody exhibited superior immunohistochemical detection efficacy compared to the commercialized TP53 antibody in human well-differentiated squamous cell carcinoma,human hepatocellular carcinoma,and human breast cancer.Conclusion:This study successfully engineered a monoclonal antibody against TP53 and provided preliminary data supporting its potential application in clinical pathological diagnosis.关键词
TP53单克隆抗体/抗体,单克隆/基因工程抗体技术/载体构建/免疫组织化学Key words
TP53 monoclonal antibody/Antibody,monoclonal/Genetically engineered antibody technology/Vector construction/Immunohistochemistry分类
医药卫生
