β淀粉样肽单克隆抗体可变区基因的5′RACE扩增及序列分析  

Cloning and Analysis of the Sequence of Variable Region in an Anti-Amyloid β-Peptide Monoclonal Antibody Through 5'RACE

作　　者：王鑫[1] 张莹[1] 何金生[1] 

机构地区：[1]北京交通大学生物科学与技术研究所,北京100044

出　　处：《生物技术通讯》2010年第2期192-195,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2006AA02A247)

摘　　要：目的:克隆并分析抗β淀粉样肽单克隆抗体轻链与重链可变区基因。方法:从分泌抗β淀粉样肽单克隆抗体的杂交瘤细胞株A8中提取总RNA,根据恒定区序列设计基因特异性引物,通过5′RACE法扩增抗体的轻链和重链可变区基因,测定并分析可变区基因序列,并克隆入pMD18-T载体。结果:重链可变区基因序列全长450bp,编码150个氨基酸残基;轻链可变区基因序列全长429bp,编码143个氨基酸残基。在GeneBank中对氨基酸序列进行比对分析,二者均符合小鼠IgG可变区基因的特征。根据Kabat法则对A8抗体轻链和重链可变区氨基酸序列基因进行分析并确定了3个抗原互补决定区(CDR)、4个框架区(FR)和信号肽。结论:通过5′RACE法得到了抗β淀粉样肽单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构,以及对该抗体进行人源化改造奠定了基础。Objective：To clone,sequence and analyze the genes of light chain variable region（VL） and heavy chain variable region（VH） of monoclonal antibody（mAb） against amyloid β-peptide（Aβ）.Methods：Total RNA was extracted from hybridoma cell A8 which secrete mAb against Aβ.Gene specific primers were designed to bind to the high conservative nucleotide sequence of constant regions of IgG.Then VH and VL genes were amplified using RACE technique.The PCR products were sequenced,analyzed and inserted into pMD18-T vector.Results：The VH gene contained 450 bp and encoded 150 amino acid residues,and the VL gene contained 429 bp and encoded 143 amino acid residues.They were homologous with the sequences of variable region of mouse IgG,which were published in GenBank.According to Kabat＇s method there were 4 FRs,3 CDRs and a leader sequence in the VH and VL genes respectively.Conclusion：The successful sequencing of the VH and VL genes of anti Aβ mAb will become experimental basis for potential study of the mAb＇s 3D structure and construction of chimeric antibody.

关 键 词：Β淀粉样肽 单克隆抗体 可变区序列 5’RACE 

分 类 号：Q78[生物学—分子生物学]