埃博拉病毒截短型糖蛋白GPdmucin的表达与纯化  

Expression and Purification of a Truncated Form of Ebola Virus Glycoprotein

作　　者：范鹏飞[1] 迟象阳[1] 宋小红[1] 房婷[1] 吴诗坡[1] 陈旖[1] 王潇霖 张冠英[1] 于长明[1] 陈薇[1] FAN Peng-Fei CHI Xiang-Yang SONG Xiao-Hong FANG Ting WU Shi-Po CHEN Yi WANG Xiao-Lin ZHANG Guan-Ying YU Chang-Ming CHEN Wei(Laboratory of Vaccine and Antibody Engineering, Beijing Institute of Biotechnology, Beijing 100071, China)

机构地区：[1]军事医学科学院生物工程研究所疫苗与抗体工程研究室,北京100071

出　　处：《生物技术通讯》2017年第1期50-57,共8页Letters in Biotechnology

基　　金：国家科技重大专项(2014ZX09J14301)

摘　　要：目的:确定黏蛋白区缺失的埃博拉病毒包膜糖蛋白(GPdmucin)的最佳表达系统,并获得纯化的GPdmucin。方法:从埃博拉病毒的包膜糖蛋白(GP)全长基因上扩增GPdmucin序列,构建至pET32a、pFast Bac1和pcDNA3.4三种不同表达系统的质粒中,分别在原核、昆虫和哺乳动物表达系统中进行表达,并用特异抗体鉴定表达情况。结果:原核系统表达的GPdmucin不稳定,活性差;在昆虫表达系统中,GPdmucin在细胞内以不溶形式表达;利用Expi293瞬时蛋白表达系统,GPdmucin在哺乳动物细胞中可溶性表达,Ni柱亲和层析获得的目的蛋白纯度达90%以上,且与特异抗体具有很好的结合活性。结论:获得哺乳动物细胞表达系统表达的GPdmucin蛋白,将用于GPcl的制备、GP抗体筛选、疫苗效果评价及病毒致病机理的研究。Objective： To determine the optimal expression system of a truncated envelope glycoprotein（GPdmu-cin） of Ebola virus, and to obtain purified GPdmucin. Methods： The GPdmucin sequences were amplified fromfull-length gene of glycoprotein（GP） and cloned into pET32 a, pFastBac1 and pcDNA3.4 vectors, then they weretransfected into prokaryotic, insect and mammalian expression systems, respectively. The expression of GPdmucinwas identified through Western blotting. Results： The GPdmucin expressed in prokaryotic cells was unstable, andhad poor biological activity. In the insect expression system, insoluble GPdmucin was expressed intracellularly. Sol-uble GPdmucin was produced in Expi293 mammalian transient protein expression system, and the purity of whichwas over 90% after purification by Ni affinity chromatography. The biological activity of GPdmucin was confirmed-with specific antibodies in ELISA. Conclusion： GPdmucinhas been expressed in mammalian expression systems,and it can be used in production of GPcl, screening of GP specific antibodies, evaluation of vaccine efficacy andresearch on viral pathogenesis.

关 键 词：埃博拉病毒 截短型糖蛋白 哺乳动物表达系统 生物学活性 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]