二硫键稳定的人源性抗bFGF双链抗体的酵母表达及鉴定  

Construction and expression of a human ds-Diabody against bFGF in Pichia pastoris

作　　者：张晋霞[1,2] 姜浩武[1,2] 亢中奎[1,2] 潘磊[1,2] 王金胜[1,2] 刘羿辰 邓宁[1,2] 

机构地区：[1]暨南大学生命科学技术学院 [2]暨南大学抗体工程研究中心广东省分子免疫与抗体工程重点实验室,广州510632

出　　处：《免疫学杂志》2014年第6期550-559,共10页Immunological Journal

基　　金：中央高校基本科研业务费专项资金(21612112);国家自然科学基金(81372281);国家863项目(2009AA02Z112)

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。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 eleetroporated 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 ds- Diabody 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 Pichiapastoris.

关 键 词：bFGF ds—Diabody 毕赤酵母 肺癌细胞 

分 类 号：R392.11[医药卫生—免疫学]