锌螯合物抗体可变区的克隆鉴定、真核表达及重组抗体的三维模建

筛选得到能分泌抗锌(Ⅱ)-二乙烯三胺五乙酸( Zn(H)-DTPA)的单克隆抗体杂交瘤细胞株2E8E3.经测定,此株细胞分泌IgM,轻链K型抗体.为了得到该抗体可变区序列,分别在小鼠抗体的信号肽区域和抗体的第一恒定区半胱氨酸残基(Cys)附近选择合适引物,通过RT-PCR方法,得到重(VH)、轻(V1)链可变区序列.将序列进行BLAST分析、SignalP分析、IMGT-V/QUEST分析,结果显示,克隆到的VH-2E8E3( 465 bp)和VL-2E8E3( 417 bp),序列与小鼠μ重链,κ轻链抗体可变区序列具有同源性(相似度＞97％),前端序列符合信号肽特征,抗体骨架区( Framework region,FR)及第一恒定区均含有保守半胱氨酸(Cys)残基.在此基础上,构建了重组抗体的真核表达载体,转染293T细胞并用间接ELISA法初步验证其具有表达活性.对重组抗体进行了三维模拟,并推测了主要氨基酸残基.试验结果表明,克隆到了正确的2E8E3抗体可变区序列并初步鉴定为有活性.

Cloning, Identification and Eukaryotic Expression of Variable Region of Monoclonal Antibodies Against Chelated Zinc and Three Dimensional Modeling of Recombinant Antibody

Hybridoma cellines which secrete monoclonal antibody against heavy metals chelators were selected in our lab.2E8E3 is the monoclonal antibody against zinc(II)-SCN-DTPA chelator(Zn(II)-DTPA).Primers were designed and synthesized to contain the signal peptide,whole variable region segments and partial constant region including Cys.VH-2E8E3(465 bp) and VL-2E8E3(417 bp) were cloned by RT-PCR method.After sequencing,the DNA sequences were analyzed by BLAST,SignalP analysis and IMGT-V/QUEST analysis.The results showed that the VH-2E8E3 and VL-2E8E3 were homologous with murine antibody μ chain and κ chain respectively.The leader sequences were with character translated to be the signal peptide,the specific-site-cysteines in antibody were also in the clonal DNA sequence.The expression plasmid vector in eukaryotic cells was constructed and used for transfecting 293T cell lines.The recombinant antibody protein expressed in transected 293T cell lines was active proved by ELISA.The three dimentional modeling of recombinant antibody were got by Swiss-Pdb Viewer.The important amino acids were also selected according to this model.