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目的 :研制抗人肿瘤坏死因子 (rTNF α)嵌合抗体。方法 :以具有中和TNF α活性的鼠源性单抗 (Z12 )的轻、重链可变区基因 ,构建人 鼠嵌合抗体基因的表达载体 ,并转染COS7细胞。用RT PCR、ELISA、免疫印迹及体外中和rTNF α活性实验 ,分别对瞬时表达的抗rTNF α嵌合抗体 (CZ12 )的细胞培养上清进行检测。结果 :①瞬时转染后 ,细胞系经RT PCR检测有人 鼠嵌合抗体mRNA的转录。②ELISA和Westernblot检测表明 ,CZ12与TNF α产生特异性反应 ,并识别相对分子质量 (Mr)为 170 0 0的TNF α。③竞争抑制实验中 ,CZ12能竞争抑制Z12与rTNF α的特异性结合 ,④体外中和实验证实 ,CZ12能中和TNF α对L92 9细胞的毒性。结论 :成功地研制具有中和活性的人 鼠嵌合抗体CZ12。
Objective: To develop anti-human tumor necrosis factor (rTNF α) chimeric antibody. Methods: The light and heavy chain variable region genes of murine monoclonal antibody (Z12) with neutralizing TNFα activity were constructed. The chimeric antibody gene expression vector was constructed and transfected into COS7 cells. The cell culture supernatants of the transiently expressed anti-rTNF α chimeric antibody (CZ12) were detected by RT PCR, ELISA, Western blotting and in vitro neutralization of rTNF α activity, respectively. Results: ① After transient transfection, the mRNA expression of human chimeric antibody was detected by RT-PCR. ② ELISA and Western blot showed that CZ12 reacted specifically with TNFα and identified TNFα with relative molecular mass (Mr) of 170.0. ③ In competition inhibition experiment, CZ12 could competitively inhibit the specific binding of Z12 and rTNFα. ④In vitro neutralization experiments confirmed that CZ12 could neutralize the toxicity of TNFα to L92 9 cells. Conclusion: Human chimeric antibody CZ12 with neutralizing activity was successfully developed.