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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 construct a eukaryotic expression vector of human carcinoembryonic antigen (CEA) small molecule chimeric antibody Rch24 F (ab ’) 2 and to achieve high yield of expression in Chinese Hamster Ovary Cell (CHO) , The biological activity of Rch24 F (ab ’) 2 was identified. Methods: The eukaryotic expression vector of Rch24 F (ab ’) 2 was constructed by DNA recombination technique and transfected into CHO-dhfr- cells. The expression of MTX was amplified and amplified by sandwich enzyme-linked immunosorbent assay (ELISA) , And Rch24 F (ab ’) 2 was purified from cell culture supernatant by affinity purification. Molecular weight was identified by SDS-PAGE. The expression of Rch24 F (ab’) 2 was identified by Western blot, direct ELISA and immunofluorescence staining Of human and antigen binding activity. Results: The eukaryotic expression vector of anti-human CEA chimeric antibody Rch24 F (ab ’) 2 was constructed successfully. After high-yielding clone 5C4 was screened by MTX, the yield of antibody was 32.3μg / ml. Results The secreted Rch24 F (ab ’) 2 molecules were mainly secreted correctly and had a molecular weight of about 110 kD. West-ern blot, direct ELISA and immunocytochemistry showed that the expressed Rch24 F (ab’) 2 containing human antibody was constant Region and specifically binds to CEA expressed by tumor cells. CONCLUSION: The chimeric antibody Rch24 F (ab ’) 2, which has good biological activity, has been successfully expressed in eukaryotic CHO cells.