抗DR5单链抗体的构建与表达

目的:构建抗DR5单链抗体的真核表达载体,以实现真核表达。方法:RT-PCR法从分泌抗DR5抗体的杂交瘤细胞中合成第一链cDNA,用通用引物钓取单克隆抗体的轻、重链可变区基因。经测序和BLAST比对,证实为一株新的鼠源性抗体基因。利用overlapping PCR方法构建单链抗体真核表达载体pCMV-express-scFv,Lipofectamine2000法转染宿主细胞293T,72 h后ELISA法检测细胞培养上清,检测抗体表达。结果:经序列鉴定和BLAST比对证实克隆的抗体轻、重链可变区基因为新鼠源抗体基因。ELISA实验结果显示抗体有效表达在细胞培养上清中。结论:成功得到抗体轻、重链可变区基因,实现了抗DR5单链抗体的真核表达,为进一步的研究和开发奠定了基础。

Construction and expression of anti-DR5 scFv antibody

Objective: To construct anti-DR5 antibody scFv eukaryotic expression vector and identify its expression.Methods: The variable region gene of from hybridoma cells was amplified which secretes anti-DR5 monoclonal antibody with universal primer by PCR and cloned into pGEM-T-Easy vector for sequence analysis.The expression vector pCMV-express-scFv was constructed by DNA recombinant method and transfected the hosts cells 293T by lipofectine 2000.The culture supernatant of 293T cells was collected after cultured 72 h.The scFv antibody was identified by ELISA method.Results: The variable region sequence of monoclonal antibody was certified as a new mouse derived antibody gene.The scFv antibody by eukaryotic expression was achieved.Conclusion: The light and heavy chain variable region genes was obtained and scFv antibody was expressed successfully,which laid a good foundation for further research.