本文關(guān)鍵詞：A組輪狀病毒NSP1基因的克隆表達及免疫學性質(zhì)研究　出處：《中國協(xié)和醫(yī)科大學》2010年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 輪狀病毒 NSP1 重組表達 免疫學性質(zhì) 亞細胞分布 基因分型

【摘要】： 輪狀病毒(rotavirus, RV)屬于呼腸病毒科(Reoviridae)輪狀病毒屬(Rotavirus),是一個直徑約為70nm,無包膜,具有三層衣殼蛋白組成的正二十面體雙鏈RNA病毒。RV是引起哺乳類和鳥類及人類腹瀉的主要病原體,每年全球因RV感染所導致的重癥腹瀉而死亡的兒童約600000。RV基因組包含11個分節(jié)段的雙鏈RNA,編碼6個結(jié)構(gòu)蛋白(VP1-VP4, VP6, VP7)和6個非結(jié)構(gòu)蛋白(NSP1-NSP6)。根據(jù)結(jié)構(gòu)蛋白VP6的抗原性差異,可將已發(fā)現(xiàn)的輪狀病毒分為A-G7個組,其中,A組為引起嬰幼兒腹瀉的主要病原。 RV NSP1在病毒與宿主的相互作用中發(fā)揮著重要的功能。本研究運用基因克隆和重組表達技術(shù)在大腸桿菌中表達了RV TB-Chen株NSP1蛋白,進行了NSP1的免疫學性質(zhì)和RV感染細胞中NSP1蛋白的合成與分布以及NSP1的系統(tǒng)進化和基因分型研究。結(jié)果表明,大腸桿菌BL21(DE3)能高效表達重組NSP1蛋白(rNSP1), rNSP1表達量約占菌體總蛋白的34.4%。rNSP1能誘導免疫豚鼠產(chǎn)生特異性血清抗體。Western blot及免疫熒光檢測結(jié)果表明,抗rNSP1血清抗體能特異性識別自身蛋白,對SA11, Wa株的NSP1蛋白有交叉反應(yīng)性;免疫熒光結(jié)果還表明,SA11感染的MA 104細胞中合成的NSP1蛋白在細胞質(zhì)中區(qū)域化聚集形成輻射狀排列的顆粒狀結(jié)構(gòu),而Wa株的NSP1不能形成此樣結(jié)構(gòu)。分子系統(tǒng)進化研究結(jié)果表明,至今發(fā)現(xiàn)的A組RV至少可以分為16個不同的NSP1基因型,TB-Chen株NSP1為A2型。不同NSP1基因型病毒具有獨特的敏感宿主范圍,同一NSP1基因型可能感染不同種動物,同一種動物也可能感染不同基因型。本研究結(jié)果為進一步研究NSP1蛋白質(zhì)的結(jié)構(gòu)功能及其應(yīng)用開發(fā)奠定了很好的基礎(chǔ)。
[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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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