A组人轮状病毒NSP2基因克

发布时间：2018-08-13 20:31

【摘要】： 轮状病毒(Rotavirus)属于呼肠病毒科(Reoviridae)轮状病毒属(Rotavirus),是一个直径约为70 nm、无包膜、具有三层衣壳的RNA病毒。1973年,RV被发现为引起婴幼儿非细菌性肠胃炎的主要病原体。目前每年全球约有600,000儿童死于RV感染所导致的重症腹泻中。RV基因组包含11个分节段的双链RNA,编码6个结构蛋白和6个非结构蛋白。根据结构蛋白VP6的抗原性差异,可将轮状病毒分为A-G 7个组,其中,A组为引起婴幼儿腹泻的主要病原。 A组RV非结构蛋白NSP2一般由317个氨基酸组成,是一个大小约为35 KDa的碱性蛋白质,N末端和C末端由一个带正电的25 A深的裂缝分开,里面含有能结合和水解NTPs的残基。NSP2参与RV复制,它能与RV其它蛋白,如NSP5相互作用形成毒质体。在毒质体内,病毒基因组双链RNA被复制并包装进入前病毒核心颗粒中。重组表达的NSP2也具有Mg~(2+)依赖性NTPase活性,能够水解NTPs上的γ磷酸基团,起到NTP酶的作用。为了深入了解NSP2的重组表达、亚细胞分布及其免疫学性质,本研究克隆了A组人RV TB-Chen株NSP2全基因,构建了重组表达质粒pET-NSP2,并在大肠杆菌BL21(DE3)中高效表达了重组蛋白NSP2(rNSP2)。研究结果表明,rNSP2表达量占菌体总蛋白量的44.54%。利用纯化的rNSP2免疫豚鼠,能够诱发豚鼠产生抗NSP2特异性血清抗体。Western blot和免疫荧光检测表明所得抗体不仅能与该rNSP2蛋白发生特异性反应,而且可以与SA11株或Wa株RV感染的MA104细胞中表达的病毒NSP2发生反应。研究结果还表明,在菌体细胞中表达的rNSP2以包涵体形式存在,充满了整个菌体;SA11株或Wa株RV感染的MA104细胞中表达的病毒NSP2在病毒感染3h时就能检测到,并且主要以毒质体形式存在于细胞质内,在细胞核周围有密集分布。以上研究结果为进一步研究该NSP2蛋白的结构、功能、免疫学性质及其开发应用奠定了很好的基础。
[Abstract]:Rotavirus (Rotavirus) belongs to the (Reoviridae) rotavirus genus (Rotavirus), of the family Reoviridae, which is a RNA virus with a diameter of about 70 nm, no capsule and three capsids. In 1973, RV was found to be the main pathogen causing non-bacterial gastroenteritis in infants and young children. At present, every year about 600000 children die of severe diarrhea caused by RV infection. The genome of .RV contains 11 segments of double-stranded RNAs, encoding 6 structural proteins and 6 non-structural proteins. According to the antigenicity difference of structural protein VP6, rotavirus can be divided into 7 groups. Group A is the main pathogen of infantile diarrhea. Group A RV non-structural protein NSP2 is composed of 317 amino acids. The N and C ends of a basic protein about 35 KDa in size are separated by a positively charged 25 A deep fissure, containing residues capable of binding and hydrolyzing NTPs. NSP2 is involved in RV replication, and it can interact with other RV proteins. For example, NSP5 interacts to form toxic plastids. In vivo, the viral genome double-stranded RNA is copied and packaged into the core particles of the virus. The recombinant NSP2 also has Mg2-dependent NTPase activity, which can hydrolyze 纬 -phosphate group on NTPs and play the role of NTP enzyme. In order to understand the recombinant expression, subcellular distribution and immunological properties of NSP2, the whole NSP2 gene of group A human RV TB-Chen strain was cloned, the recombinant expression plasmid pET-NSP2 was constructed, and the recombinant protein NSP2 (rNSP2) was highly expressed in Escherichia coli BL21 (DE3). The results showed that rNSP2 expression accounted for 44.54% of the total bacterial protein. Using purified rNSP2 to immunize guinea pigs, guinea pigs were induced to produce anti NSP2 specific serum antibody. Western blot and immunofluorescence assay showed that the obtained antibody could not only react specifically with the rNSP2 protein. Moreover, it can react with viral NSP2 expressed in MA104 cells infected with SA11 or WA strain RV. The results also showed that the rNSP2 expressed in bacterial cells existed in the form of inclusion bodies, and the viral NSP2 expressed in MA104 cells infected with SA11 or WA strain RV could be detected at 3 h after infection. And it mainly exists in cytoplasm in the form of toxic plastids, and has dense distribution around the nucleus. These results provide a good basis for the further study of the structure, function, immunological properties and development and application of the NSP2 protein.
【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】：2009
【分类号】：R373

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