摘要
目的:利用基因工程抗体技术制备TP53重组抗体,并进行临床病理诊断应用的初步探究。方法:将杂交瘤来源的TP53单克隆抗体重链及轻链可变区基因分别与pGEM-T载体连接,测序分析并选择有效重链及轻链序列分别与pFUSEss-CHIg-mG2B、pFUSE2ss-CLIg-mk及pcDNA3.4载体连接。利用ExpiCHO-S™Expression System进行真核表达,经Protein A柱纯化获得TP53重组抗体。最后将TP53重组抗体和商品化TP53抗体进行蛋白质免疫印迹(Western blot,WB)和免疫组织化学(Immunohistochemistry,IHC)应用的初步探究。结果:通过抗体表达纯化获得TP53重组抗体,液相色谱-质谱测定结果显示TP53轻链/重链分子量分别为25 kDa和53 kDa,ELISA效价为1∶2180000,亲和力测定Ka值为6.6×10^(10) L·mol^(-1)。WB结果显示TP53重组抗体与商品化TP53抗体表达结果一致。IHC结果显示TP53重组抗体和商品化TP53抗体识别靶标均定位于细胞核,在人肾癌、人乳腺叶状腺癌、人乙状结肠腺癌及小鼠肝癌中的检测结果一致,且TP53重组抗体在人高分化鳞癌、人肝细胞癌和人乳腺癌中的免疫组化检测效果优于商品化的TP53抗体。结论:本研究成功制备了小鼠抗人TP53重组抗体,并为临床病理诊断提供初步的数据支持。
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∶2180000,while affinity measurements yielded a Ka value of 6.6×10^(10) 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 di
作者
蒋道梅
姚新欣
杨琴
张鹏
JIANG Dao-mei;YAO Xin-xin;YANG Qin;ZHANG Peng(Institute of Innovative and Translational Medicine,Gannan Medical University,Ganzhou,Jiangxi 341000;Department of Cardiovascular Medicine,Huanggang Central Hospital,Huanggang,Hubei 438000)
出处
《赣南医学院学报》
2024年第6期571-579,共9页
JOURNAL OF GANNAN MEDICAL UNIVERSITY
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
TP53 monoclonal antibody
Antibody,monoclonal
Genetically engineered antibody technology
Vector construction
Immunohistochemistry
