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目的单链抗体基因的构建并在大肠杆菌中进行表达以及表达产物的纯化研究。 方法采用重组PCR技术将已获得的抗癌胚抗原 (CEA)鼠源单克隆抗体E7B10 重、轻链可变区基因 (VH、Vk)经 (Gly4 Ser) 3 编码的寡核苷酸拼接成单链抗体基因 ,并在大肠杆菌中表达了单链抗体C端融合 6×His蛋白 ,表达产物经Ni2 +-IDASepharose 6B亲和柱纯化。 结果表达的融合蛋白在胞内主要以可溶性存在 ,其表达量达到菌体总蛋白的 10 % ,SDS -PAGE和Western印迹图谱显示表达产物分子量为 2 7kd ,与其基因编码蛋白的理论推算值相符。表达产物经Ni2 +-IDASepharose 6B亲和柱纯化 ,纯度可达 90 %以上 ,得率为 0 .8mg/ 10 0ml。 结论纯化后的融合蛋白具有CEA结合活力 ,其亲和常数为 5 .4× 10 7/M(抗体结合浓度 ) ,略低于其亲本单抗E7B10 2 .7× 10 9/M。
Construction of the target single-chain antibody gene and expression in E. coli and purification of the expression product. Methods The oligodeoxynucleotides encoded by (Gly4 Ser) 3 were cloned from the anti-carcinoembryonic antigen (CEA) murine monoclonal antibody E7B10 heavy and light chain variable region genes (VH, Vk) obtained by the recombinant PCR technique. The chain antibody gene was expressed in E. coli, and the 6-His protein was fused to the C-terminal of the single-chain antibody. The expressed product was purified by Ni2 +-IDASepharose 6B affinity column. Results The expressed fusion protein was mainly soluble in the cell and its expression amount reached 10% of the total bacterial protein. SDS-PAGE and Western blotting showed that the molecular weight of the expressed product was 27 kd, which was consistent with the theoretically calculated value of the protein encoded by the gene. The expressed product was purified by a Ni2 +-IDASepharose 6B affinity column with a purity of more than 90% and a yield of 0.8 mg/100 ml. Conclusion The purified fusion protein has CEA binding activity, and its affinity constant is 5. 4×10 7 /M (antibody binding concentration), which is slightly lower than its parental monoclonal antibody E7B10 2 .7×10 9/M.