抗Aβ鼠源单克隆抗体的人源化改造及其表达鉴定

发布时间：2018-05-06 02:25

本文选题：阿尔茨海默病 + 人源化抗体　；参考：《基础医学与临床》2010年08期

【摘要】：目的将前期构建的抗Aβ人-鼠嵌合抗体进行可变区人源化改造,为抗Aβ抗体在临床人体中应用奠定基础。方法采用模板替换法对已构建的人-鼠嵌合抗体基因中的重、轻链可变区框架区进行人源化改造,构建抗Aβ人源化抗体的真核表达载体。用脂质体法将重、轻链共转染CHO细胞进行表达,采用ELISA法检测表达产物效价、表达量;同时用SDS-PAGE及Western blot检测表达产物分子质量;免疫组化法鉴定其生物学活性。结果重组改造后的抗Aβ人源化抗体基因重链约为1 500 bp,轻链约为750 bp,与预期一致。转染CHO细胞后获得表达,表达量为1.11 mg/L,抗体重链相对分子质量约为51 ku,轻链约为25 ku。ELISA结果显示能与Aβ特异性结合(细胞培养上清A值:2.24),与改造前的嵌合抗体比较效价基本相同。抗体人源化检测显示,只能被羊抗人抗体识别,不能被羊抗鼠抗体识别。免疫组化显示表达产物有结合Aβ抗原的活性。结论将抗Aβ人-鼠嵌合抗体进行可变区人源化改造后,基本保持了原嵌合抗体的生物学特性,为其今后应用于临床提供了可能性。
[Abstract]:Objective to humanize the previously constructed anti A 尾 human mouse chimeric antibody in order to lay a foundation for the clinical application of anti A 尾 antibody. Methods template replacement method was used to humanize the heavy and light chain variable frame region of the constructed human / mouse chimeric antibody gene, and the eukaryotic expression vector of anti A 尾 humanized antibody was constructed. CHO cells were cotransfected with heavy and light chains by liposome method. The titer and quantity of the expression products were detected by ELISA method. The molecular weight of the expressed products was detected by SDS-PAGE and Western blot. The biological activity of the expressed products was identified by immunohistochemical method. Results the heavy chain of anti-A 尾 humanized antibody gene was about 1 500 BP, and the light chain was about 750 BP, which was consistent with the expectation. After transfection of CHO cells, the expression amount was 1.11 mg / L, the relative molecular weight of antibody heavy chain was about 51 ku. and the light chain was about 25 ku.ELISA. The results showed that the cell culture supernatant A: 2.24 could bind to A 尾 specifically. The titer of chimeric antibody was basically the same as that of chimeric antibody before modification. Antibody humanization test showed that only sheep anti-human antibody can be recognized, not sheep anti-mouse antibody. Immunohistochemistry showed that the expressed product had the activity of binding A 尾 antigen. Conclusion after the humanization of anti-A 尾 human-mouse chimeric antibody, the biological characteristics of the original chimeric antibody were basically maintained, which provided the possibility for its clinical application in the future.
【作者单位】：中国医学科学院基础医学研究所北京协和医学院基础学院病理系;
【分类号】：R392

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