寻找一种诊治心肌损伤的新方法:鼠抗人cTnI单抗Fab段基因克隆和序列分析(英文)

A novel method to evaluate myocardial injury:cloning of anti-cTnI murine antibody Fab fragment and DNA sequence analysis

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:To apply mouse anti-human cTnI monoclonal antibody as the drug vector in the treatment and diagnosis of myocardial injury,it is important to degrade the immunity of murine antibody and overcome human anti mouse reaction.Humanization has been applied as an attempt to resolve this problem.OBJECTIVE:To clone murine anti cTnI Fab fragment and analyse the nucleotide and deduced amino acid sequences.DESIGN:Single sample study.SETTING:An institute of cardiovascular disease under a medical university affiliated hospitalMATERIALS:The study was conducted in the Institute of Cardiovascular Diseases,First Affiliated Hospital of Nanjing Medical University from January 2003 to May 2004.The hybridoma cell line JS200202 which secrets the anti cTnI monoclonal antibody was provided by Institute of Cardiovascular Disease,First Affiliated Hospital of Nanjing Medical University.METHODS:IgG heavy chain primers and κ light chain primers of amplified mouse were designed.Total RNA was extracted from hybridoma cells which secrete cTnI.Reverse transcription polymerase chain reaction(RT PCR) was amplified.Cloning and subsequent sequence analysis of the Fab fragment was performed.The deduced amino acid sequence was compared and analysed with previously published sequences.MAIN OUTCOME MEASURES:Heavy chain Fd segment and κ light chain gene sequence and its subgroups.RESULTS:A band of approximate 700 and 800 base pairs were amplified using IgG heavy chain primers and κ light chain primers respectively.Sequence analysis indicated that the deduced amino acid sequences were in consistent with the characterization of the amino acid in the murine IgG1 Fab fragment(GenBank accession NO AY484430,AY484431;Protein Bank accession NO AAR83243,AAR83244).CONCLUSION:A complete murine anti cTnI Fab fragment was obtained in this study,which may provide basis for the production of the chimeric anti cTnI antibody.