摘要
目的:构建抗人癌胚抗原(carcinoembryonic antigen,CEA)小分子嵌合抗体Rch24 F(ab')2真核高效表达载体并在中国仓鼠卵巢细胞(Chinese Hamster Ovary Cell,CHO)中实现高产量表达,对Rch24 F(ab')2的生物学活性进行鉴定。方法:采用DNA重组技术构建Rch24 F(ab')2真核高效表达载体,转染CHO-dhfr-细胞,通过提高MTX浓度进行基因扩增表达,夹心ELISA法测定抗体产量,筛选高表达细胞株,采用亲和纯化的方法从细胞培养上清中纯化Rch24 F(ab')2;采用SDS-PAGE鉴定分子量,采用Western blot、直接ELISA和细胞免疫荧光鉴定所表达的Rch24 F(ab')2的人源性和抗原结合活性。结果:成功构建了抗人CEA小分子嵌合抗体Rch24 F(ab')2的真核高效表达载体,MTX加压扩增表达后筛选获得高产克隆5C4,其抗体产量为32.3μg/ml;结果显示所分泌的主要为正确组装的Rch24 F(ab')2分子,分子量约110kD;West-ern blot、直接ELISA、细胞免疫荧光结果证明所表达的Rch24 F(ab')2含人抗体的恒定区,并特异地与肿瘤细胞表达的CEA结合。结论:成功地在真核细胞CHO中高效表达了具有良好的生物学活性的抗人CEA小分子嵌合抗体Rch24 F(ab')2。
Objective： To express anti- carcinoembyronic antigen chimeric antibody F （ab＇）2 at high -level in the Chinese Hamster Ovary Cell and to identify the characterization of Rch24 F（ab＇） 2. Methods： The eukaryotic high -level expression vectors of Rch24 F（ ab＇）2 were constructed by DNA recombination and inducted into CHO - dhfr - cells. High - level expression of Rch24 F （ab＇）2 was achieved after cells adapted to methotrexate , and high -yielding clones of Rch24 F（ab＇）2 were screened by sandwich ELISA. Rch24 F（ab＇）2 was purified by affinity chromatography, which was identified by direct ELISA, W.estern blot , SDS- PAGE. The specific binding activity of Rch24 F（ab＇） 2 to membrane CEA expressed on tumor cells surface was detected by immunofluorescence. Results：It was successful to complete the construction of eukaryotic high- level expression vectors of Rch24 F（ ab＇）2. The expression level of 5C4, a stable clone secreting Rch24 F （ab＇）2, achieved a high -level expression of 32.3 ug/ml following stepwise MTX amplification. Rch24 F（ab＇） 2 was confirmed to have expected molecular mass of approximately 110 kD by SDS- PAGE, which contained the human antibody constant re.gion and specifically targeted to CEA expressed on tumor cells surface by Western blot, direct ELISA and immunofluorescence. Conclusion： Anti - human CEA small molecular chimeric antibody Rch24 F（ab＇） 2 was expressed at high level in CHO cells, and the antibody possessed a good biological activity to specifically target CEA expressed on tumor cells surface.
出处
《中国卫生检验杂志》
北大核心
2013年第6期1367-1370,共4页
Chinese Journal of Health Laboratory Technology
基金
"重大新药创制"科技重大专项(2011ZX09102-001-28)
"重大新药创制"科技重大专项(2011ZX09401-030)
中国医学科学院肿瘤医院基本科研业务费项目(2011JKB24)
关键词
CEA
小分子嵌合抗体
高效表达
CHO
CEA
Small molecular chimeric antibody
High - level expression
CHO
