【摘要】 目的为提高小分子抗体表达量,利用酵母表达系统表达二硫键稳定的人源性抗bFGF双链抗体(ds-Diabody)并研究其生物学活性。方法将ds-Diabody基因构建到酵母表达载体中获得重组质粒pPICZαA-ds-Diabody,经BglⅡ线性化电转至毕赤酵母GS115中,甲醇诱导表达。表达产物经SDS-PAGE及Western blot鉴定,并进行镍离子亲和层析和阴离子交换层析纯化。间接ELISA检测其抗原结合活性,CCK8法和划痕实验检验其肿瘤抑制作用。结果成功构建人源性抗bFGF ds-Diabody酵母表达载体,并获得4株高表达酵母工程菌,经1%甲醇诱导96 h表达量即可恒定,表达量可达158 mg/L。SDS-PAGE及Western blot结果显示,目的蛋白大小约Mr35 000左右。通过两步纯化方案,目的蛋白的纯度可达95%以上。ELISA结果显示纯化的ds-Diabody可与bFGF特异性结合。CCK8结果显示,纯化的ds-Diabody可剂量依赖性地抑制人肺癌细胞株A549的增殖,最大抑制率为43.4%。划痕实验表明ds-Diabody可以抑制肿瘤细胞的迁移。结论研究结果表明人源性抗bFGF ds-Diabody在毕赤酵母中可获得高效表达,且具有很好的生物学活性。更多还原

【Abstract】 In order to improve the expression of human anti-bFGF ds-Diabody in Pichia pastoris and study theirs biological activity, ds-Diabody gene was transfected into expression vector pPICZαA for constructing recombinant plasmid pPICZαA-ds-Diabody, which was then linearized with Bgl II and electroporated into Pichia pasporis strain GS115. The transformants were induced by methanol for ds-Diabody expression. Then, the expression products were purified by affinity chromatography of Ni-Seproase 6 FF and ion exchange chromatography of DEAE Sepharose FF. The results of SDS-PAGE and Western-blot showed that anti-bFGF ds-Diabody was highly expressed and the yield was about 158 mg/L. The target protein was purified from the expression products and the purity was more than 95%. The results of ELISA showed that the purified ds-Diabody could bind to bFGF with improved binding activity; the results of CCK-8 showed that the purified ds-Diabody could inhibit the proliferation of A549 cells in a dose-dependent manner in vitro, and the inhibitive rate is 43.4%. Furthermore, wound-healing assay showed that dsDiabody could inhibit the migration of A549 cells. All the results demonstrated that the anti-bFGF ds-Diabody with good biological activity can be highly expressed in Pichia pastoris.更多还原