CD20抗体亲和力和免疫原性的改造及表达

发布时间：2018-03-24 17:45

本文选题：CD分子　切入点：抗体　出处：《生物技术》2017年02期

【摘要】：[目的]构建较高亲和力较低免疫原性的CD20抗体。[方法]通过专利分析,进行高亲和力和人源化的设计改造,将抗体的轻链和重链分别克隆到pc DNA3.1/ZEO(+)、pc DNA3.1(+)真核表达载体中,共转染中国仓鼠卵巢细胞CHO-K1,通过RT-PCR检测mRNA的转录,夹心ELISA检测抗体表达量,流式细胞术检测细胞上清与CD20阳性细胞Raji的结合活性。[结果]得到了高亲和力的人源化CD20抗体的轻链和重链基因片段,并成功地构建了重组表达载体pc DNA3.1/ZEO(+)-CD20L和pc DNA3.1(+)-CD20H,并可在CHO-K1细胞中表达,通过RT-PCR证明基因在CHO细胞中成功表达,ELISA法测得培养上清的含量在0.45~4.72μg/m L之间,流式检测表明上清中的抗体可与Raji细胞结合。[结论]成功构建并表达筛选了17株单克隆抗体,15号的表达量最大,为4.72μg/m L,为下一步研究抗体的亲合力、免疫原性以及生物学功能打下基础。
[Abstract]:[method] [CD20] to construct high affinity antibody with low immunogenicity through patent analysis, design of high affinity and humanization, light chain and heavy chain antibody were cloned into PC DNA3.1/ZEO (+), PC (+) DNA3.1 eukaryotic expression vector, transfection in China hamster ovary cells CHO-K1, RT-PCR transcription through the detection of mRNA expression of sandwich ELISA for detection of antibody activity. Results combined with flow cytometry in cell supernatant and CD20 Raji positive cells were light chain humanized CD20 antibody with high affinity and heavy chain gene fragment, and successfully constructed the recombinant expression vector PC DNA3.1/ZEO (+) -CD20L and PC (DNA3.1 + -CD20H), and can be expressed in CHO-K1 cells, proven by RT-PCR gene was successfully expressed in CHO cells, the content of ELISA was measured in the supernatant between 0.45~4.72 g/m L and flow cytometry showed that the antibody in the supernatant With Raji cell binding. Conclusion: the successful construction and expression screening of 17 strains of monoclonal antibody, the expression of No. 15, 4.72 g/m L, for the next step of antibody affinity, lay the foundation for immunogenicity and biological function.

【作者单位】：聊城大学药学院;利津县中心医院药剂科;聊城大学农学院;
【基金】：国家高技术研究发展计划(863计划)项目(“动物细胞大规模批次流加培养关键技术及产品研发”,No.2012AA02A306)
【分类号】：R733.1

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