本文選題：糖基工程酵母 + 埃博拉病毒　； 參考：《軍事醫(yī)學(xué)》2017年05期

【摘要】：目的用新型糖基工程酵母表達(dá)并純化得到具有類似哺乳動物細(xì)胞糖基化修飾的埃博拉病毒三聚體糖蛋白(EBOV-GP),為其亞單位疫苗研究提供基礎(chǔ)。方法人工合成EBOV-GP基因,將編碼全長EBOV-GP基因和編碼缺失黏蛋白樣域(MLD)與穿膜區(qū)的EBOV-GPΔMLDΔTM基因分別克隆到pPICZ-αA載體上,電轉(zhuǎn)化糖基工程酵母,與在HEK-293T細(xì)胞中表達(dá)的EBOV-GP進行比較,利用PNGaseF和EndoH酶切分析其糖基化修飾,利用親和層析與離子交換層析純化目的蛋白,蛋白質(zhì)N端測序分析其在蛋白翻譯修飾過程中是否正確切除了信號肽,同時利用凝膠柱分析是否能形成三聚體結(jié)構(gòu)。結(jié)果 PNGaseF酶切結(jié)果顯示,用糖基工程酵母和HEK-293T細(xì)胞表達(dá)的EBOV-GP糖蛋白相對分子質(zhì)量大小與N-糖基化程度均一致,EndoH酶切顯示EBOV-GPΔMLDΔTM的N-糖基化修飾是非高甘露糖形式的復(fù)雜型糖基化修飾;經(jīng)三步純化得到的目的蛋白,N端測序顯示為GP蛋白成熟肽序列,其自身信號肽被正確切除;凝膠柱分析顯示純化得到的目的蛋白以三聚體形式存在。結(jié)論用糖基工程酵母表達(dá)制備了具有復(fù)雜型糖基化修飾的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- 偽 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.
【作者單位】： 安徽大學(xué)健康科學(xué)研究院;軍事醫(yī)學(xué)科學(xué)院生物工程研究所微生物工程研究室;
【基金】：國家科技重大專項重大新藥創(chuàng)制資助項目(2014ZX09J14105-07C-001) 國家自然科學(xué)基金資助項目(81673339) 北京市自然科學(xué)基金資助項目(7152112)
【分類號】：R392

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