抗HER2人源化单克隆抗体在毕赤酵母中的表达及其产物分析

Expression and Characterization of Humanized Anti-HER2 Monoclonal Antibody in Pichia pastoris

目的:研究抗人表皮生长因子受体2(HER2)人源化单克隆抗体能否在毕赤酵母中表达,并对表达产物进行结构分析和活性测定。方法:合成抗HER2人源化单克隆抗体的全基因,构建轻重链双表达盒表达载体,转入毕赤酵母中;通过表型筛选和诱导表达实验得到抗体表达工程菌,对表达产物进行分离纯化和活性测定。结果:表达产物存在于表达上清中,摇瓶表达水平达到53.7±2.9mg/L;毕赤酵母表达的抗体的轻重链能够自发通过二硫键装配成正确的抗体结构,其中重链发生了N-糖基化;酵母表达的抗HER2人源化单克隆抗体可以抑制高表达p185erbB2肿瘤细胞SKBR-3的生长,半数有效浓度为0.17mg/L。结论:实现了抗HER2人源化单克隆抗体在毕赤酵母中的表达,为毕赤酵母表达系统成为抗体等人用剂量较大的复杂型糖蛋白的大规模、低成本制备平台提供了基础。

Objective： To study whether the humanized anti-HER2 monoclonal antibody could be expressed in Pichia pastoris, and to investigate the structure and the activity of the expressed products. Methods： Gene of humanized anti- HER2 monoclonal antibody was synthesized. Then the expression vector with both the heavy and the light chains expres- sion cassette was constructed and transformed into P.pastoris by electroporation. Through phenotype selection and inducing assay, the expression engineering strain was obtained. Finally, the expressed products were purified and their activity was analyzed. Results： The engineered antibodies were expressed in soluble form by secreting into culture medium. The expres- sion level of the antibody protein in flask reached 53.7＋2.9 mg/L. The light and heavy chains of antibody which were ex- pressed in P.pastoris could assemble the right antibody structure through disulfide bonds spontaneously, and the heavy chain has N-linked glycosylation. Humanized anti-HER2 monoclonal antibody expressed in P.pastor/s can inhibit cell pro- liferation overexpressed of p185~s2, and the EC50 of the antibody protein to the SKBR-3 cell was 0.17 mg/L. Conclusion： The humanized anti-HER2 monoclonal antibody was expressed in P.pastoris, and this will be basis for a platform of large- scale preparation and low-cost production of complex therapeutic glycoproteins which were used in clinical patients widely.