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背景:为将鼠抗人cTnI单克隆抗体应用人体进行体内诊断或作为治疗心肌损伤的药物载体,必须降低鼠源性抗体的免疫源性,克服其人抗鼠抗体反应,需要对其进行人源化改造。目的:克隆鼠抗人cTnI单克隆抗体Fab段基因并进行序列分析。设计:单一样本研究。单位:一所医科大学附属医院的心血管病研究所。材料:本实验于2003-01/2004-05在南京医科大学第一附属医院心血管病研究所完成。分泌鼠抗人cTnI单克隆抗体的杂交瘤细胞株(JS200202)由南京医科大学第一附属医院心血管病研究所提供。方法:设计扩增鼠IgG重链Fd段及κ轻链引物,从分泌cTnI单抗的杂交瘤细胞中提取总RNA,RT-PCR扩增,对扩增产物进行分子克隆、测序及序列分析。主要观察指标:重链Fd段和κ轻链基因序列及其所属亚型。结果:重链和轻链引物分别扩增出一约700bp和800bpDNA片段。经序列分析,与已发表的鼠IgG基因序列对比,其核苷酸及其所推导的氨基酸序列符合鼠IgG1Fab段特征。在GenBank登录,登录号为AY484430(重链),AY484431(轻链);氨基酸序列登录号为AAR83243(重链),AAR83244(轻链)。结论:本实验室获得了完整的鼠源性抗人cTnI单克隆抗体Fab段基因,为鼠抗人cTnI单克隆抗体的人源化改造奠定了基础。
BACKGROUND: In order to use the human anti-human cTnI monoclonal antibody for in vivo diagnosis or as a drug carrier for the treatment of myocardial damage, it is necessary to reduce the immunogenicity of the murine antibody and overcome its human anti-mouse antibody response, Transformation. OBJECTIVE: To clone and sequence the murine anti-human cTnI monoclonal antibody Fab fragment. Design: Single Sample Study. Unit: A Cardiovascular Institute of the Affiliated Hospital of Medical University. Materials: The experiment was performed at the Institute of Cardiovascular Diseases, the First Affiliated Hospital of Nanjing Medical University from January 2003 to May 2004. The hybridoma cell line secreting murine anti-human cTnI monoclonal antibody (JS200202) was provided by the First Affiliated Hospital of Nanjing Medical University Cardiovascular Institute. Methods: The murine IgG heavy chain Fd fragment and kappa light chain primer were designed and amplified. The total RNA was extracted from the hybridoma cells secreting cTnI monoclonal antibody. The amplified product was amplified by RT-PCR and cloned, sequenced and sequenced. MAIN OUTCOME MEASURES: Fd and κ light chain genes of heavy chain and their subtypes. Results: The heavy chain and light chain primers amplified about 700bp and 800bp DNA fragments respectively. Sequence analysis showed that the nucleotide sequence and the deduced amino acid sequence of the deduced amino acid sequence were in accordance with the characteristics of murine IgG1 Fab fragment. GenBank accession numbers AY484430 (heavy chain), AY484431 (light chain); amino acid sequence accession numbers AAR83243 (heavy chain), AAR83244 (light chain). Conclusion: The full-length murine anti-human cTnI Fab fragment was obtained in our lab, which lays the foundation for humanized anti-human cTnI monoclonal antibody.