军事医学Issue(11)：875-878,4.DOI:10.7644/j.issn.1674-9960.2014.11.009
新型人源化抗CD20单克隆抗体的表达和活性检测
Expression and activity detection of novel humanized anti-CD20 monoclonal antibodies
摘要
Abstract
Objective To construct humanized monoclonal antibodies against CD 20 and check their affinity to CD 20 antigen and their anti-tumor activity.Methods Based on the computer model , human IgG1 candidates closest to rituximab in crystal structure were selected in the Protein Data Bank ( PDB) .With the selected human IgG 1 candidates as the frame , we modified and transplanted the complementarity determining region ( CDR) of rituximab .First,the target gene fragments were obtained by overlapping PCR.Then, the sequences of the light chains(L) and the heavy chains(H) were inserted in-to the pcDNA3.3 and pOptiVEC vectors.Next, the constructed clones were transfected into 293F cells through transient transfection.After a large-scale cell culture, the mAb was purified by affinity chromatography rProtein A column.The puri-ty and expression level of the humanized antibodies was tested by sodium dodecyl sulfate ( SDS)-polyacrylamide gelelectro-phoresis(PAGE).The affinity of the humanized antibodies to CD20 was assessed with Fortebio assay.Finally, the anti-tumor activity of the constructed antibodies was detected by checking the tumor growth inhibition of the nude mice transplan-ted with tumor .Results Three humanized monoclonal antibodies against CD 20 were expressed and purified successfully . In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25 ×103 and 55 ×103 , respectively.The band size of the antibodies matched the expected value.Fortebio assay revealed that the humanized antibodies could bind to CD20 with high affinity (rituximab:6.48 ×10 -9mol/L, L4H7:1.91 ×10 -9mol/L, L5H5:7.35 ×10 -10mol/L,and L5H7:1.91 ×10 -9mol/L).The tumor growth inhibition experiment showed that the anti-tumor activity of L5H7 mAb was better than that of rituximab .Conclusion Three humanized monoclonal antibodies against CD 20 have been successfully construc-ted and expressed.L5H7 mAb possesses high affinity for CD20 and a good ability to kill tumor cells.关键词
抗原,CD20/抗体,单克隆/人源化Key words
antigens,CD20/antibodies,monoclonal/humanization分类
生物科学引用本文复制引用
贾茹,靳彦文,李平,刘萱,高婷,刘子敬,曹诚..新型人源化抗CD20单克隆抗体的表达和活性检测[J].军事医学,2014,(11):875-878,4.基金项目
国家科技重大专项资助项目 （）
