EBOV三聚体糖蛋白在新型糖基工程酵母中的表达与纯化

Expression and purification of Ebola virus trimeric glycoprotein based on novel glycoengineered Pichia pastoris

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 mammalian glycosylation modified trimeric Ebola virus trimeric glyeoprotein （EBOV-GP） using novel glycoengineered Pichia pastoris. Methods The EBOV-GP and EBOV-GPAMLDATM genes were cloned into the pPICZ-αA vector, electrochemically converted to glycoengineered Pichia pastoris, and compared with EBOV-GP expressed in HEK-293T cells. Glyeosylation was analyzed by PNGaseF and EndoH digestion, while the target protein was purified by affinity chromatography and ion exchange chromatography. N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimerie structure was formed. Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree. EndoH digestion showed that the N-glycosylation modification of EBOV-GPAMLDATM was in a non-high mannose form. N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised. Gel column analysis showed that the purified protein was in a trimerie form. Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.