抗人卵巢癌×抗人CD3×抗CD28V_H单链三特异抗体的胞内可溶表达、纯化及体外活性测定

Soluble expression, purification and bioactivity assay of anti-ovarian carcinoma×anti-CD3×anti-CD28V_H single chain trispecific antibody

目的 研究抗人卵巢癌 (ovariancarcinoma ,oc)×抗人CD3×抗CD2 8VH 单链三特异抗体(singlechaintrispecificantibody,scTsAb)在大肠杆菌中的可溶表达与纯化及纯化后产物的活性测定 ,从而为其应用于卵巢癌治疗的临床研究打下基础。方法 将已构建的scTsAb表达载体转化大肠杆菌BL2 1(DE3)Star菌株 ,采用低温 (30℃ )、低剂量IPTG(0 .2mmol L)诱导 ,进行胞内可溶表达。根据抗卵巢癌三特异抗体 (ocTsAb)等电点较高 (pI9.0 ) ,而菌体蛋白大多为酸性蛋白的特点 ,利用DEAE弱阴离子交换层析(pH8.0 )进行一步纯化 ,并利用ELISA及FACS的方法检测纯化后抗卵巢癌三特异抗体的活性。结果 (1)SDS PAGE鉴定低温诱导时可溶比例达到 5 6 %。 (2 )绝大多数菌体蛋白被DEAE层析柱吸附 ,而抗卵巢癌三特异抗体在穿透液中流出 ,SDS PAGE检测纯度达到 90 %。 (3)ELISA结果显示纯化后的抗卵巢癌三特异抗体与重组CD2 8纯抗原 ,Jurkat(CD3+ )细胞膜提取抗原 ,SKOV3细胞膜提取抗原均有特异性结合。 (4 )FACS结果证明纯化后的抗卵巢癌三特异抗体与Jurkat(CD3+ )活细胞、SKOV3活细胞有特异性结合。结论 低温诱导胞内可溶表达的抗人卵巢癌×抗人CD3×抗CD2 8VH 单链三特异抗体经弱阴离子交换层析一步纯化后仍保持原有免疫学活性 。

Objective To investigate the soluble expression and to purify a recombinant multifunctional single chain trispecific antibody (scTsAb) which contains anti-ovarian carcinoma(oc) scFv, anti-CD3 scFv and V H domain of anti-CD28 antibody and in vitro bioactivity of the purified scTsAb. Methods The plasmid of scTsAb was transformed into E.coli strain BL21(DE3) Star. The scTsAb was over-expressed as a soluble protein when the E.coli was induced in a low concentration of IPTG (0.2mmol/L) and cultured at a low temperature (30℃). The scTsAb was purified by DEAE anion exchange chromatography. ELISA and FACS mensurate in vitro bioactivity of the purified scTsAb. Results (1) The soluble expression ratio is about 56%. (2) The scTsAb flow through DEAE chromatography and the purity is over 90%. (3) The purified scTsAb shows strong binding activities to the antigen of ovarian carcinoma cells, recombinant CD28 pure antigen and to CD3 molecules on Jurkat cells. (4) The purified antibody tightly binds to Jurkat cells and SKOV3 cells. Conclusion The soluble scTsAb maintaines its inherent bioactivity after purification by DEAE anion exchange chromatography. These results provide a technical guarantee to produce abundant sample for further bioactivity experiments and in vivo experiments. They also provide the reference standard for expression, purification and production of similar genetical engineered antibodies.