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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 :构建的真核表达载体获得了功能性表达
Objective: To construct and express chimeric antibodies against human TNFα. Methods: The light and heavy chain variable region genes of anti-TNFα murine McAb Z8 were inserted into the eukaryotic expression vector containing human kappa chain and IgG1 heavy chain constant region genes to construct the light and heavy chain separately expressed and co-expressed The expressed human chimeric antibody eukaryotic expression vector was transfected into eukaryotic cells and the activity of TNFα was detected by ELISA, RT PCR and Western blot. The chimeric antibody was tested for its ability to neutralize TNFα in vitro using L92 9 cells. Results: The results of ELISA showed that the chimeric antibody specifically bound to TNFα. PT PCR results showed that the human chimeric antibody mRNA was transcribed in the transfected cell line; the expression of human IgG protein was confirmed by Western blotting; This chimeric antibody neutralizes the toxicity of TNFα to L92 9 cells. Conclusion: The constructed eukaryotic expression vector obtained the functional expression