毕赤酵母表达gp96-scFv抗体及生物活性测定  

Expression of anti-gp96 scFv fragment in Pichia pastoris and identification of its biological activity

作　　者：桂明明[1] 武慧英[2,3] 孙璐[2,3] 徐亚星[2,3] 赵报[2] 李鑫[2,3] 李长菲[2] 王希东[1] 孟颂东[2] 

机构地区：[1]新疆农业大学农学院,新疆乌鲁木齐830052 [2]中国科学院微生物研究所中国科学院病原微生物与免疫学重点实验室,北京100101 [3]中国科学院大学,北京100049

出　　处：《生物工程学报》2014年第4期595-604,共10页Chinese Journal of Biotechnology

基　　金：北京自然科学基金(No.KQA201105260010A);深圳市科技创新委员会项目(No.CYC201012290024A)资助~~

摘　　要：旨在利用毕赤酵母分泌表达gp96-scFv抗体,纯化后得到能特异性结合gp96抗原的小分子抗体片段(scFv)。根据gp96-scFv抗体基因序列,合成gp96-scFv抗体基因序列,将gp96-scFv抗体序列克隆到毕赤酵母表达质粒pPICZα-A,线性化的重组表达载体电转化到毕赤酵母X33,甲醇诱导目的蛋白表达,通过亲和层析法纯化目的蛋白,并以SDS-PAGE和Western blotting进行鉴定。通过Western blotting、Immunofluorescence、ELISA、FACS方法对gp96-scFv抗体的生物活性进行了检测。结果成功地构建了分泌表达抗gp96蛋白scFv抗体的毕赤酵母菌,每升毕赤酵母菌培养上清经纯化可获约50 mg gp96-scFv抗体,所获抗体其分子量大约为15 kDa,具有与gp96抗原特异性结合的活性。本研究通过毕赤酵母菌成功表达了gp96-scFv抗体,生物活性Western blotting、Immunofluorescence、ELISA、FACS分析表明该抗体能特异性结合gp96。Secretory anti-gp96 scFv fragment was expressed in Pichia pastoris to obtain a small molecule antibody that specifically recognizes heat shock protein gp96. The gp96-scFv fragment gene was synthesized and cloned to Pichia pastoris expression plasmid pPICZ^-A. Pichia pastoris X33 was electroporated with the linearized recombinant expression vector, and expression of gp96-scFv fragment was induced by methanol. The His-tagged recombinant protein was then purified by affinity chromatography and analyzed with SDS-PAGE and Western blotting assays. The biological activities of recombinant gp96-scFv fragment were determined by Western blotting, Immunofluorescence, ELISA and FACS assays. The gp96-scFv fragment was expressed successfully in Pichia pastoris. About 50 mg of recombinant protein could be purified from 1 liter of the Pichia pastoris culture supernatant. Its molecular weight was about 15 kDa. The gp96-scFv fragment could specifically bind to gp96 protein by Western blotting, immunofluorescence, ELISA and FACS analyses. Pichia pastoris-expressed gp96-scFv fragment specifically recognizes gp96 protein, which could be used for Western blotting, Immunofluorescence, ELISA and FACS analyses.

关 键 词：gp96-scFv抗体 毕赤酵母 GP96 分泌表达 亲和力 特异性 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]