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。
Objective Humanize the frame regions in variable regions of the murine monoclonal antibody against Alzheimer＇s disease for potential clinical application.Methods We humanized the frame regions of the heavy chain and the light chain in variable regions using template-replacement method.Eukaryotic expression vectors were constructed by inserting humanized antibody genes of the heavy and light chains into vectors.Then the eukaryotic expression vectors were co-transfected into CHO cells using liposome fusion method.The tests included the titer and expression using ELISA,molecular weight using SDS-PAGE and Western blot and biological activity tested by immunohistochemistry.Results The heavy chain of the recombinant antibody was about 1 500 bp and the light chain was about 750 bp,which was in accordance with our anticipation.The expression of the product was 1.11 mg/L.The molecular weight of the humanized antibody was about 51 ku（heavy chain） and 25 ku（light chain）.The expressed product can bind Aβ antigen specifically tested by ELISA（A value of the humanized antibody： 2.24）,keeping the same as that of the chimeric antibody basically.The expressed product can only be recognized by goat-anti-human antibody rather than goat-anti-mouse antibody.The results also showed that the expressed product canbind Aβ antigen specifically tested by immunohistochemistry.Conclusion The humanized antibody provides a possibility for its clinical application for Alzheimer＇s disease in the future.
出处
《基础医学与临床》
CSCD
北大核心
2010年第8期862-867,共6页
Basic and Clinical Medicine
