冠状病毒HCoV-229E S1蛋白片段的原核表达及鉴定

发布时间：2018-01-13 01:29

本文关键词：冠状病毒HCoV-229E S1蛋白片段的原核表达及鉴定　出处：《佳木斯大学》2008年硕士论文　论文类型：学位论文

更多相关文章： 冠状病毒HCoV-229E S1蛋白 原核表达

【摘要】： 目的:人冠状病毒(HCoV-229E)属Ⅰ型冠状病毒,是引起人类上呼吸道感染的病原,常引起人的普通感冒。其基因组RNA是一不分节段的正链单股RNA,长约27 kb,已知编码11种蛋白,其中包括4种结构蛋白,分别是刺突蛋白(S)、包膜蛋白(M)、小包膜蛋白(E)和核衣壳蛋白(N)。S蛋白三聚体形成突起,伸出包膜表面,是病毒的主要表面抗原成分,具有受体结合和膜融合活性,在组织嗜性、细胞融合和毒力等方面起重要作用。S蛋白由S1和S2两个亚基组成,其中S1形成成熟蛋白的球状部分,包括受体结合区(RBD)和高变区(HVR),而S2相对保守,包括一段内在的融合肽和两段疏水的七次重复区域(HR1和HR2)。这些区域对于病毒的侵入、细胞与细胞间的融合具有重要意义。本研究将靶蛋白锁定为S1蛋白片段(S1 417—547位氨基酸),克隆获得冠状病毒HCoV-229E S1基因片段(S1蛋白1249-1642位核苷酸),在原核表达系统大肠杆菌中获得高效表达,对表达蛋白加以纯化,初步研究其抗原活性。方法:根据Genbank提供的HCoV-229E株S基因序列(DQ243973)人工合成冠状病毒HCoV-229E S1蛋白特异性基因片段,并克隆入pET-21a原核表达载体,构建克隆载体pET-21a-S1,通过PCR鉴定。重组载体转化宿主菌BL21(DE3),经IPTG高效诱导表达得到重组蛋白,用金属螯合亲和层析纯化,并通过SDS-PAGE电泳及Western blot对表达的重组蛋白进行鉴定。结果:获得了主要以包涵体形式存在的目的蛋白,Western blot鉴定其为S1基因片段蛋白。结论:成功构建了HCoV-229E S1蛋白的表达载体,并在BL21(DE3)中得到了高效表达,为下一步表达蛋白免疫原性及疫苗抗病毒保护性测定打下了基础。
[Abstract]:Objective: human coronavirus (HCoV-229E) belongs to type I coronavirus and is the cause of human upper respiratory tract infection. Human common cold. Its genome RNA is a non-segmented positive strand single-stranded RNAs, about 27 kb in length, which is known to encode 11 proteins, including 4 structural proteins. The nucleocapsid protein (NV) and the nucleocapsid protein (NV) are the main surface antigen components of the virus, which are formed protuberances and protruded on the surface of the envelope respectively, such as the spiny protein (S), the envelope protein (M), the small envelope protein (E) and the nucleocapsid protein (NV). It has receptor-binding and membrane fusion activity, and plays an important role in tissue eosinophilia, cell fusion and virulence. S protein is composed of S1 and S2 subunits, among which S1 forms the globular part of mature protein. These include receptor binding region (RBD) and hypervariable region (HVRN), while S2 is relatively conserved. These include an intrinsic fusion peptide and two hydrophobic seven repeats, HR1 and HR2. These areas invade the virus. The fusion between cells and cells is of great significance. In this study, the target protein was locked into the S1 protein fragment S417-547 amino acid). The HCoV-229E S1 gene fragment of coronavirus was cloned and expressed in E. coli. The expressed protein was purified and its antigenic activity was preliminarily studied. Methods: according to the S gene sequence of HCoV-229E strain provided by Genbank, DQ243973). Synthesis of HCoV-229E S1 protein specific gene fragment of coronavirus. The recombinant plasmid pET-21a-S1 was cloned into the prokaryotic expression vector of pET-21a and identified by PCR. The recombinant vector was transformed into the host strain BL21DE3). The recombinant protein was induced by IPTG and purified by metal chelating affinity chromatography. The recombinant protein was purified by SDS-PAGE electrophoresis and Western. The expressed recombinant protein was identified by blot. Results: the target protein which mainly existed as inclusion body was identified as S1 gene fragment protein by Western blot. Conclusion: the expression vector of HCoV-229E S1 protein was successfully constructed and highly expressed in BL21DDE3. It lays a foundation for the further immunogenicity of the expressed protein and the antiviral protective test of the vaccine.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373;R392

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