B组轮状病毒WH-2株vp7基因的原核表达及抗体的制备

发布时间：2018-07-27 17:13

【摘要】： 轮状病毒(Rotavirus，RV)为呼肠孤病毒科，轮状病毒属，已被确认为是引起全世界婴幼儿急性胃肠炎最常见的病因。在成人中，轮状病毒同样也会引起急性腹泻，其中成人腹泻轮状病毒(ADRV)是我国洪涛院士于1983年首先发现的一种人B组轮状病毒，它在我国许多地区曾引起大规模以成人为主的霍乱样腹泻的暴发流行。人B组轮状病毒感染性、传播强度、引起的临床症状等都很严重，远远超出A组轮状病毒，因而引起国内外学者的广泛重视和研究。本文研究体外扩增到人B组轮状病毒WH-2株vp7基因的ORF区，所构建的重组体pGEX-KG-VP7经IPTG诱导，SDS-PAGE分析，表达了pGEX-KG-VP7融合蛋白并制备了多克隆抗血清，并对初步纯化的蛋白做了Western-blot鉴定，结果在预测的相应分子量处得到一条特异的反应带，从而证实该融合蛋白具有良好的免疫学活性。因此所表达的蛋白和制备的抗体不但为研究结构与功能提供了物质基础也可用于建立人GBRV的ELISA方法，为GBRV所引起的疾病预防、诊断和治疗等流行病学研究和临床诊断奠定了基础，具有重要实际应用价值。具体研究内容如下：第一章全面概述了B组轮状病毒的形态结构、理化性质、基因组结构及编码的蛋白质、体外培养、流行病学、实验室检测及临床诊断与治疗等方面的研究进展。第二章将VP7结构蛋白的基因克隆到pGEX-KG载体上并进行了诱导表达纯化与多克隆抗体制备。即从含有人B组轮状病毒WH-2株全长vp7基因的pUCmT-vp7重组质粒上扩增出人B组轮状病毒WH-2株vp7基因ORF区，将扩增产物插入融合表达载体pGEX-KG中构建重组pGEX-KG-VP7质粒。将重组质粒转化入大肠杆菌BL21用IPTG诱导，SDS-PAGE分析显示表达了相对分子量为53.4kDa的pGEX-KG-VP7融合蛋白。将pGEX-KG-VP7融合蛋白作为抗原，免疫新西兰兔，，制备了多克隆抗体。制备的抗血清经同样诱导表达的表达载体pGEX-KG表达产物吸收后1∶500倍稀释后用Western Blot分析，与53.4kDa的pGEX-KG-VP7融合蛋白获得特异性显色信号，从而证实该融合蛋白具有良好的免疫学活性。
[Abstract]:Rotavirus (Rotavirus RV), a family of rotavirus, has been identified as the most common cause of acute gastroenteritis in infants and children all over the world. In adults, rotavirus can also cause acute diarrhea. Adult diarrhea rotavirus (ADRV) is the first human group B rotavirus discovered by Hong Tao academicians in China in 1983. It has caused large-scale outbreaks of cholera-like diarrhea in many areas of China. Human group B rotavirus infection, transmission intensity, clinical symptoms and so on are very serious, far more than group A rotavirus, so domestic and foreign scholars pay attention to and research widely. In this paper, the ORF region of vp7 gene of human group B rotavirus WH-2 strain was amplified in vitro. The constructed recombinant pGEX-KG-VP7 was analyzed by IPTG induced SDS-PAGE, the fusion protein of pGEX-KG-VP7 was expressed and polyclonal antiserum was prepared, and the purified protein was identified by Western-blot. Results A specific reaction band was obtained at the predicted molecular weight, which confirmed that the fusion protein had good immunological activity. Therefore, the expressed protein and the prepared antibody not only provide the material basis for the study of structure and function, but also can be used to establish the ELISA method of human GBRV, which can be used to prevent the disease caused by GBRV. Epidemiological research and clinical diagnosis, such as diagnosis and treatment, have laid the foundation and have important practical application value. The main contents are as follows: in Chapter 1, the morphological structure, physicochemical properties, genomic structure and encoded proteins of group B rotavirus were summarized. Research progress in epidemiology, laboratory detection, clinical diagnosis and treatment. In chapter 2, the gene of VP7 structural protein was cloned into pGEX-KG vector and purified by induction, and the polyclonal antibody was prepared. The ORF region of vp7 gene of group B rotavirus WH-2 strain was amplified from the pUCmT-vp7 recombinant plasmid containing the full-length vp7 gene of human group B rotavirus WH-2 strain. The amplified product was inserted into the fusion expression vector pGEX-KG to construct the recombinant pGEX-KG-VP7 plasmid. The recombinant plasmid was transformed into Escherichia coli BL21 and expressed pGEX-KG-VP7 fusion protein with relative molecular weight of 53.4kDa by IPTG induced SDS-PAGE analysis. Polyclonal antibodies were prepared by immunizing New Zealand rabbits with pGEX-KG-VP7 fusion protein as antigen. The prepared antiserum was absorbed by 1: 500 times of the expression product of pGEX-KG expression vector, and then diluted by Western Blot. The specific color signal was obtained with the pGEX-KG-VP7 fusion protein of 53.4kDa. It was proved that the fusion protein had good immunological activity.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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