鼠抗人cTnI单克隆抗体Fab段基因克隆和序列分析

Cloning and DNA sequence analysis of anti-cTnI murine antibody Fab fragment

目的:克隆鼠抗人cTnImAb Fab段基因并进行序列分析.方法:设计扩增鼠IgG重链Fd段及κ轻链引物,从分泌cTnI mAb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.结果:重链和轻链引物分别扩增出一约700bp和800bp DNA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cTnImAb Fab段基因,为鼠抗人cTnI的人源化改造奠定了基础.

AIM： To obtain the gene of murine anti-cTnI Fab fragment and analyse the nucleotide and deduced amino acid sequences. METHODS ： By RT-PCR method, the cDNA of anti- cTnI murine antibody Fab fragment was amplified from the hybridoma cells. Cloning and subsequent sequence analysis of the Fab fragment were performed. The deduced amino acid sequence was compared and analysed with previously published sequences. RESULTS： A band of approximate 700 and 800 base pairs was 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 present in the murine IgG1 Fab fragment （GenBank accession NO AY484430, AY484431 ; Protein Bank accession NO AAR83243, AAR83244 ）. CONCLUSION： The cloning and sequencing of a complete murine anti-cTnI Fab fragment provides a basis for the production of the chimeric anti-eTnI antibody.