抗TNF-α人-鼠嵌合抗体的构建及表达

Construction and expression of human mouse chimeric antibody directed against TNF-α

目的 :构建和表达抗TNF α人 鼠嵌合抗体。方法 :将抗TNF α鼠单抗Z8的轻、重链可变区基因插入到含有人κ链和IgG1重链恒定区基因的真核细胞表达载体中 ,分别构建轻、重链分开表达和共同表达的人 鼠嵌合抗体真核表达载体 ,转染真核细胞 ,通过ELISA、RT PCR、免疫印迹检测TNF α的活性。用L92 9细胞检测嵌合抗体体外中和TNF α的活性。结果 :ELISA鉴定结果表明该嵌合抗体与TNF α特异性结合 ;PT PCR结果显示转染后细胞系有人 鼠嵌合抗体mRNA的转录 ;免疫印迹结果证实有人IgG蛋白的表达 ;体外中和实验证明此嵌合抗体能中和TNF α对L92 9细胞的毒性。结论

Objective:Construction and expression of TNF α human mouse chimeric antibody CZ8.Methods:The light and heavey chain variable genes were inserted into the chimeric antibody expression vectors and transfected into SP 2/0 or CHO cells.The expression of anti TNF α human mouse chimeric antibody was tested by ELISA、RT PCR and Western blot.The in vitro neutralization assay was used to test the biological activity of chimeric antibody.Results:Anti TNF α human mouse chimeric antibody was expressed by transfected CHO cells with a single vector that contained both light chain and heavy chain genes.ELISA analysis showed the TNF α chimeric antibody was specific for TNF α.The transcription of human mouse chimeric light and heavy chain mRNAs were proved by RT PCR.Protein expression of both κ and H chain was further verified by Western blot.The in vitro neutralization assay demonstrated that the chimeric antibody could neutralize the cyctoxicity of TNF α.Conclusion:Human mouse chimeric antibody CZ8 was successfully expressed in eukaryotic cells.