A组人轮状病毒VP6蛋白表达及其免疫学分析

发布时间：2018-07-09 17:31

本文选题：A组人轮状病毒 + VP6　；参考：《中国协和医科大学》2008年硕士论文

【摘要】： 轮状病毒(Rotavirus,RV)属呼肠病毒科(Reoviridae)轮状病毒属,是引起人类、哺乳动物类和鸟类腹泻的主要病原体。完整的RV病毒分为三层结构,VP6蛋白位于病毒的内壳中间层,为第6基因片段编码,由397个氨基酸组成,分子量约为44.8KDa。VP6蛋白是轮状病毒中含量仍然最丰富的蛋白,约占病毒总蛋白量的51%,轮状病毒内壳由VP6蛋白构成的260个致密的三聚体所形成。VP6在病毒结构中具有物理受体的作用,在病毒基因转录中也具有重要作用。本研究中的VP6基因克隆自临床样品中分离的A组轮状病毒TB-Chen株。研究中,以VP6全基因为模板,经PCR扩增,基因重组,将VP6蛋白编码基因插入到质粒pET中构建了原核表达质粒pET-VP6,并在大肠杆菌BL21(DE3)中高效表达重组VP6蛋白(rVP6)。rVP6占菌体总蛋白的27.4%。rVP6经纯化后免疫兔,获得动物免疫血清抗体。Western blot检测结果显示,表达的rVP6能被抗-rVP6兔血清抗体特异性识别,能被抗-SA11和抗-Wa的兔免疫血清抗体特异性识别。免疫荧光结果显示MA104细胞中的RVVP6抗原在RV感染3h后即可观察到,在12h左右荧光强度达到最高。病毒的VP6蛋白大部份散布于细胞质间,主要以颗粒状的形式分布。中和实验结果显示,抗-rVP6的兔血清抗体具有中和RV SA11和Wa株病毒的中和病毒感染活性。这些结果为明确A组轮状病毒VP6的生物学和免疫学特性及其在RV亚单位疫苗应用开发中的价值提供了理论实验基础。
[Abstract]:Rotavirus (Rotavirus RV) belongs to the family Reoviridae, which is the main pathogen causing diarrhea in humans, mammals and birds. The complete RV virus is divided into three layers, the VP6 protein is located in the middle layer of the virus's inner shell, which encodes the 6th gene fragment and consists of 397 amino acids. The molecular weight of the protein is about 44.8 KDa.VP6, which is still the most abundant protein in rotavirus. About 51% of the total protein of the virus, rotavirus inner shell is formed by 260 dense trimer composed of VP6 protein. VP6 has the function of physical receptor in the virus structure, and also plays an important role in viral gene transcription. The VP6 gene in this study was cloned from a group A rotavirus TB-Chen strain isolated from clinical samples. In this study, VP6 gene was amplified by PCR and the gene was recombined with VP6 as template. The prokaryotic expression plasmid pET-VP6 was constructed by inserting the VP6 protein coding gene into the plasmid pet. The recombinant VP6 protein (rVP6). RVP6 accounted for 27.4% of the total bacterial protein in E. coli BL21 (DE3). The results showed that the expressed rVP6 could be specifically recognized by anti-rVP6 rabbit serum antibody, and by anti-SA11 and anti-Wa rabbit immune serum antibody. The results of immunofluorescence showed that the RVVP6 antigen in MA104 cells could be observed 3 hours after RV infection, and the highest fluorescence intensity was observed at about 12 h. Most of the VP6 proteins were distributed in the cytoplasm and distributed in granular form. The neutralization results showed that the anti-rVP6 rabbit serum antibody had neutralizing virus infection activity of RV SA11 and WA strains. These results provide a theoretical and experimental basis for clarifying the biological and immunological characteristics of group A rotavirus VP6 and its value in the application and development of RV subunit vaccine.
【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】：2008
【分类号】：R392;R373

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