CEA单链抗体表达质粒构建及产物纯化

Expression Vector Construction and Prodoct Purification of Single-Chain Antibody with Specificity for Carcinoembryonic Antigen in E.coli

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。

Objective Research in construction of anti-carcinoembryonic antigen (CEA) antibody gene, expression in E.coli and purification of the expressed product. Methods The heavy and light chain variable region (V H and V k) genes encoding the murine monoclonal antibody E 7B 10 with specificity for CEA were linked into single-chain antibody gene with 45 oligenucleotide (Gly 4Ser) 3 by using recombinant PCR, and was cloned into the expression vector pET-22b(+). Results The construction was expressed in E.coli with high level soluble fusion protein tailed with six additional histidine residues at its C-terminus, reaching 10% of the total bacteria protein, and characterized by SDS-PAGE, and Western blot. The histidine-tagged ScFv was purified with nickeliminodiacetic acid affinity chromatography. 100ml induction culture had 0.8mg ScFv-(His) 6 fusion proteins. Its purifity reached 90%. Conclusion The purified ScFv's affinity constant binding to CEA reached 5.4× 10 7/M , lower its parental monoclonal antibody E 7B 10 2.7×10 9/M.