A组轮状病毒NSP1基因的克隆表达及免疫学性质研究

发布时间：2018-01-02 13:23

本文关键词：A组轮状病毒NSP1基因的克隆表达及免疫学性质研究　出处：《中国协和医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 轮状病毒(rotavirus, RV)属于呼肠病毒科(Reoviridae)轮状病毒属(Rotavirus),是一个直径约为70nm,无包膜,具有三层衣壳蛋白组成的正二十面体双链RNA病毒。RV是引起哺乳类和鸟类及人类腹泻的主要病原体,每年全球因RV感染所导致的重症腹泻而死亡的儿童约600000。RV基因组包含11个分节段的双链RNA,编码6个结构蛋白(VP1-VP4, VP6, VP7)和6个非结构蛋白(NSP1-NSP6)。根据结构蛋白VP6的抗原性差异,可将已发现的轮状病毒分为A-G7个组,其中,A组为引起婴幼儿腹泻的主要病原。 RV NSP1在病毒与宿主的相互作用中发挥着重要的功能。本研究运用基因克隆和重组表达技术在大肠杆菌中表达了RV TB-Chen株NSP1蛋白,进行了NSP1的免疫学性质和RV感染细胞中NSP1蛋白的合成与分布以及NSP1的系统进化和基因分型研究。结果表明,大肠杆菌BL21(DE3)能高效表达重组NSP1蛋白(rNSP1), rNSP1表达量约占菌体总蛋白的34.4%。rNSP1能诱导免疫豚鼠产生特异性血清抗体。Western blot及免疫荧光检测结果表明,抗rNSP1血清抗体能特异性识别自身蛋白,对SA11, Wa株的NSP1蛋白有交叉反应性;免疫荧光结果还表明,SA11感染的MA 104细胞中合成的NSP1蛋白在细胞质中区域化聚集形成辐射状排列的颗粒状结构,而Wa株的NSP1不能形成此样结构。分子系统进化研究结果表明,至今发现的A组RV至少可以分为16个不同的NSP1基因型,TB-Chen株NSP1为A2型。不同NSP1基因型病毒具有独特的敏感宿主范围,同一NSP1基因型可能感染不同种动物,同一种动物也可能感染不同基因型。本研究结果为进一步研究NSP1蛋白质的结构功能及其应用开发奠定了很好的基础。
[Abstract]:Rotavirus (RV) belongs to the family Reoviridae.Rotavirus belongs to Rotavirus.RV is about 70 nm in diameter. The 20 hedron double-stranded RNA virus. RV, which is composed of three layers of capsid protein, is the main pathogen causing diarrhea in mammals, birds and humans. About 600000.RV genome contains 11 segments of double-stranded RNAs that encode 6 structural proteins, VP1-VP4, in children who die of severe diarrhea caused by RV infection every year. VP6 (VP7) and 6 nonstructural proteins NSP1-NSP6. According to the antigenicity difference of structural protein VP6, the rotavirus can be divided into A-G7 groups. Group A was the main cause of infantile diarrhea. RV NSP1 plays an important role in the interaction between virus and host. In this study, RV was expressed in Escherichia coli by gene cloning and recombinant expression techniques. NSP1 protein of TB-Chen strain. The immunological properties of NSP1, the synthesis and distribution of NSP1 protein in RV infected cells, and the phylogeny and genotyping of NSP1 were studied. Escherichia coli BL21 DE3) can efficiently express recombinant NSP1 protein rNSP1). The expression of rNSP1 was about 34.4% of the total bacterial protein. RNSP1 could induce the production of specific serum antibody. Western blot and immunofluorescence assay showed that rNSP1 could induce specific antibody production in guinea pigs. The anti rNSP1 serum antibody can specifically recognize the autoprotein and cross react to the NSP1 protein of SA11 and WA strains. The results of immunofluorescence also showed that the NSP1 protein synthesized in MA104 cells infected with SA11 was localized and aggregated in the cytoplasm to form a radial granular structure. But the NSP1 of WA strain could not form this kind of structure. The results of molecular phylogenetic studies showed that the group A RV found so far could be divided into at least 16 different NSP1 genotypes. NSP1 of TB-Chen strain is A2. Different NSP1 genotypes have unique sensitive host range, and the same NSP1 genotype may infect different species of animals. Different genotypes may also be infected in the same animal. The results of this study provide a good basis for further study on the structure and function of NSP1 protein and its application and development.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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