人LIGHT-Fc融合蛋白在毕赤酵母中的表达及鉴定  

Expression and identification of human LIGHT-Fc fusion protein in Pichia pastoris

作　　者：郝文丽[1] 赵勇 任华[1] 江文正[1] 

机构地区：[1]华东师范大学生命科学学院,上海200241 [2]上海实验动物研究中心,上海201203

出　　处：《中国生物制品学杂志》2015年第8期791-794,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81072459);教育部新世纪优秀人才支持计划(NCET-12-0179);上海市"科技创新行动计划"实验动物领域科技支撑项目(14140904200)

摘　　要：目的利用酵母表达系统制备人LIGHT-Fc融合蛋白。方法利用基因工程方法构建含人LIGHT胞外段基因和人Ig G4 Fc基因的重组质粒p PIC9K-LIGHT-Fc,经SalⅠ线性化后电转化感受态酵母菌GS115,PCR鉴定重组转化子。将阳性重组转化子经甲醇诱导表达后,RT-PCR法检测目的基因的转录水平,SDS-PAGE及Western blot法进行表达产物的鉴定。结果表达质粒p PIC9K-LIGHT-Fc经酶切及测序鉴定,证明构建正确。10个重组子均为Mut+型阳性转化子,经甲醇诱导后可扩增出特异性目的基因片段。表达的重组LIGHT-Fc融合蛋白相对分子质量约45 000,可与鼠抗人LIGHT多克隆抗体发生特异性结合。结论人LIGHT-Fc融合蛋白在毕赤酵母中已成功获得了表达,为进一步开展其生物学功能的研究奠定了基础。Objective To prepare human LIGHT-Fc fusion protein by using yeast expression system. Methods The recombinant plasmid p PIC9K-LIGHT-Fc containing the gene of outer membrane domain of human LIGHT and human Ig G4 Fc was constructed by genetic engineering technology, linearized with Sal Ⅰ and electrotransformed to competent yeast GS115. The recombinant transformants were identified by PCR, of which the positive ones were induced with methanol. The transcription level of target gene was determined by RT-PCR, while the expressed product was identified by SDS-PAGE and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid p PIC9KLIGHT-Fc was constructed correctly. All the ten recombinants were Mut＋transformants, from which a specific target gene fragment was amplified after induction with methanol. The expressed fusion protein LIGHT-Fc, with a relative molecular mass of about 45 000, showed specific binding to mouse anti-human LIGHT polyclonal antibody. Conclusion Fusion protein LIGHT-Fc was expressed successfully in P. pastoris, which laid a foundation of further study on its biological function.

关 键 词：LIGHT 毕赤酵母 基因表达 

分 类 号：Q786[生物学—分子生物学] R392-33[医药卫生—免疫学]