EBOV三聚体糖蛋白在新型糖基工程酵母中的表达与纯化

发布时间：2018-05-29 04:36

本文选题：糖基工程酵母 + 埃博拉病毒　；参考：《军事医学》2017年05期

【摘要】：目的用新型糖基工程酵母表达并纯化得到具有类似哺乳动物细胞糖基化修饰的埃博拉病毒三聚体糖蛋白(EBOV-GP),为其亚单位疫苗研究提供基础。方法人工合成EBOV-GP基因,将编码全长EBOV-GP基因和编码缺失黏蛋白样域(MLD)与穿膜区的EBOV-GPΔMLDΔTM基因分别克隆到pPICZ-αA载体上,电转化糖基工程酵母,与在HEK-293T细胞中表达的EBOV-GP进行比较,利用PNGaseF和EndoH酶切分析其糖基化修饰,利用亲和层析与离子交换层析纯化目的蛋白,蛋白质N端测序分析其在蛋白翻译修饰过程中是否正确切除了信号肽,同时利用凝胶柱分析是否能形成三聚体结构。结果 PNGaseF酶切结果显示,用糖基工程酵母和HEK-293T细胞表达的EBOV-GP糖蛋白相对分子质量大小与N-糖基化程度均一致,EndoH酶切显示EBOV-GPΔMLDΔTM的N-糖基化修饰是非高甘露糖形式的复杂型糖基化修饰;经三步纯化得到的目的蛋白,N端测序显示为GP蛋白成熟肽序列,其自身信号肽被正确切除;凝胶柱分析显示纯化得到的目的蛋白以三聚体形式存在。结论用糖基工程酵母表达制备了具有复杂型糖基化修饰的EBOV-GP。
[Abstract]:Objective to express and purify Ebola virus trimer glycoprotein (EBOV-GPN) with glycosylation modification of mammalian cells by a novel glycosylated yeast, and to provide a basis for the study of its subunit vaccine. Methods EBOV-GP gene was synthesized and cloned into pPICZ- 伪 A vector, respectively, encoding full-length EBOV-GP gene and EBOV-GP 螖 MLD 螖 TM gene encoding missing mucin-like domain. The expression of EBOV-GP in HEK-293T cells was compared with that in HEK-293T cells. The glycosylation modification of the protein was analyzed by PNGaseF and EndoH, the target protein was purified by affinity chromatography and ion exchange chromatography. The N-terminal sequence of the protein was sequenced to determine whether the signal peptide was removed correctly in the process of protein translation modification. At the same time, the gel column was used to analyze whether the trimer structure could be formed. Results the results of PNGaseF digestion showed that, The relative molecular weight of EBOV-GP glycoprotein expressed by glycosylengineering yeast and HEK-293T cells was consistent with the degree of N-glycosylation. The N-glycosylation modification of EBOV-GP 螖 MLD 螖 TM was not a complex glycosylation modification in the form of high mannose. The N-terminal sequence of the purified protein showed that it was a mature peptide sequence of GP protein, and its signal peptide was removed correctly, and the gel column analysis showed that the purified protein existed in the form of trimer. Conclusion EBOV-GP with complex glycosylation modification was prepared by glycosylated yeast expression.
【作者单位】：安徽大学健康科学研究院;军事医学科学院生物工程研究所微生物工程研究室;
【基金】：国家科技重大专项重大新药创制资助项目(2014ZX09J14105-07C-001) 国家自然科学基金资助项目(81673339) 北京市自然科学基金资助项目(7152112)
【分类号】：R392

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