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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 for Ecto-glycoprotein (GPdmucin) in the mucin domain and to obtain purified GPdmucin. Methods: The GPdmucin sequence was amplified from the full-length gene of the glycoprotein (GP) of Ebola virus and constructed into plasmids containing three different expression systems of pET32a, pFast Bac1 and pcDNA3.4. The prokaryotic, insect and lactation Animal expression system for expression, and the use of specific antibodies to identify the expression. Results: GPdmucin expressed in prokaryotic system was unstable and poorly active. In insect expression system, GPdmucin was expressed in insoluble form in cells. Using Expi293 transient protein expression system, GPdmucin was soluble in mammalian cells. Ni column affinity chromatography The purity of the obtained target protein is more than 90%, and has good binding activity with the specific antibody. CONCLUSION: The GPdmucin protein obtained from mammalian cell expression system will be used in the preparation of GPcl, GP antibody screening, vaccine efficacy evaluation and virus pathogenicity research.