articles
吴慕胜
2017, 41(5): 0-0.
[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蛋白的基因，将编码全长EBOV-GP的基因和编码缺失MLD和穿膜区的EBOV-GP∆MLD∆TM蛋白基因分别克隆到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的切除了信号肽，同时利用凝胶柱分析是否能够形成三聚体结构。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糖蛋白分子量大小与N-糖基化程度都一致，EndoH酶切显示EBOV-GP∆MLD∆TM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的形式存在。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埃博拉病毒三聚体糖蛋白。
[关键词]糖基工程酵母，埃博拉病毒，三聚体糖蛋白，糖基化修饰，表达纯化
Expression and Purification of Ebola virus trimeric Glycoprotein based on Novel Glycoengineered Pichia pastoris
Wu Mu-sheng1, 2, Gong Xin2, Chang Shao-hong2, Liu Bo2, Wu Jun2
（1 Institute of Health Sciences，Anhui University，Hefei 230601，China：2 Laboratory of Microorganism Engineering，Institute of Biotechnology，Academy of Military Medical Science，Beijing 100071，China）
Co-corresponding author，Liu Bo，E-mail：liubo7095173@163.com；Wu Jun，Email：junwu1969@163.com
[Abstract] Objective: Expression in Novel Glycoengineered Pichia pastoris and purified mammalian glycosylation modified trimeric Ebola virus glycoprotein (EBOV-GP). Methods: The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector, electrochemically converted to Glycoengineered Pichia pastoris. Compared with EBOV-GP expressed in HEK-293T cells, the glycosylation was analyzed by PNGaseF and EndoH digestion, 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 whether the trimeric structure was formed by gel column analysis. Results：The results of PNGaseF showed that EBOV-GP protein expressed by Glycoengineered Pichia pastoris and HEK-293T cells had the same molecular weight and N-glycosylation degree. EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in the 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 the form of trimer. Conclusion: We successfully obtained Ebola virus trimeric glycoproteins with a complex glycosylation modification based on Glycoengineered Pichia pastoris.
[Key words] Glycoengineered Pichia pastoris, Ebola virus, Trimeric glycoprotein, Glycosylation modification, Expression and Purification
