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

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。

Abstract:

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 pPICZα-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.