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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。
OBJECTIVE: To express and purify Ebola virus trimeric glycoprotein (EBOV-GP) with glycosylation similar to that of mammalian cells, and to provide a basis for subunit vaccine research. Methods The EBOV-GPΔMLDΔTM gene encoding the full-length EBOV-GP gene and the deletion mucin-like domain (MLD) and the transmembrane region were cloned into pPICZ-αA vector by artificial synthesis of EBOV-GP gene. , Compared with EBOV-GP expressed in HEK-293T cells, digested with PNGaseF and EndoH to analyze their glycosylation, and purified by affinity chromatography and ion-exchange chromatography. The protein was sequenced by N-terminal sequencing Whether the signal peptide was cut off correctly during the process of protein translation modification and whether the trimer structure was formed by gel column analysis was also analyzed. Results The results of PNGaseF digestion showed that the relative molecular mass of EBOV-GP glycoprotein expressed by Saccharomyces cerevisiae and HEK-293T cells were consistent with the degree of N-glycosylation. EndoH digestion showed that the N-glycosylation of EBOV-GPΔMLDΔTM The modification is a complex glycosylation modification in the form of non-high mannose. After three-step purification, the target protein is N-terminal sequenced as the GP protein mature peptide sequence, and its own signal peptide is correctly excised. The gel column analysis shows that the purified protein Of the target protein exists as a trimer. Conclusion EBOV-GP with complex glycosylation modifications was prepared by glycoengineered yeast expression.