【摘要】： 輪狀病毒(Rotavirus，RV)為呼腸孤病毒科，輪狀病毒屬，已被確認(rèn)為是引起全世界嬰幼兒急性胃腸炎最常見的病因。在成人中，輪狀病毒同樣也會引起急性腹瀉，其中成人腹瀉輪狀病毒(ADRV)是我國洪濤院士于1983年首先發(fā)現(xiàn)的一種人B組輪狀病毒，它在我國許多地區(qū)曾引起大規(guī)模以成人為主的霍亂樣腹瀉的暴發(fā)流行。人B組輪狀病毒感染性、傳播強度、引起的臨床癥狀等都很嚴(yán)重，遠(yuǎn)遠(yuǎn)超出A組輪狀病毒，因而引起國內(nèi)外學(xué)者的廣泛重視和研究。本文研究體外擴(kuò)增到人B組輪狀病毒W(wǎng)H-2株vp7基因的ORF區(qū)，所構(gòu)建的重組體pGEX-KG-VP7經(jīng)IPTG誘導(dǎo)，SDS-PAGE分析，表達(dá)了pGEX-KG-VP7融合蛋白并制備了多克隆抗血清，并對初步純化的蛋白做了Western-blot鑒定，結(jié)果在預(yù)測的相應(yīng)分子量處得到一條特異的反應(yīng)帶，從而證實該融合蛋白具有良好的免疫學(xué)活性。因此所表達(dá)的蛋白和制備的抗體不但為研究結(jié)構(gòu)與功能提供了物質(zhì)基礎(chǔ)也可用于建立人GBRV的ELISA方法，為GBRV所引起的疾病預(yù)防、診斷和治療等流行病學(xué)研究和臨床診斷奠定了基礎(chǔ)，具有重要實際應(yīng)用價值。具體研究內(nèi)容如下： 第一章全面概述了B組輪狀病毒的形態(tài)結(jié)構(gòu)、理化性質(zhì)、基因組結(jié)構(gòu)及編碼的蛋白質(zhì)、體外培養(yǎng)、流行病學(xué)、實驗室檢測及臨床診斷與治療等方面的研究進(jìn)展。 第二章將VP7結(jié)構(gòu)蛋白的基因克隆到pGEX-KG載體上并進(jìn)行了誘導(dǎo)表達(dá)純化與多克隆抗體制備。即從含有人B組輪狀病毒W(wǎng)H-2株全長vp7基因的pUCmT-vp7重組質(zhì)粒上擴(kuò)增出人B組輪狀病毒W(wǎng)H-2株vp7基因ORF區(qū)，將擴(kuò)增產(chǎn)物插入融合表達(dá)載體pGEX-KG中構(gòu)建重組pGEX-KG-VP7質(zhì)粒。將重組質(zhì)粒轉(zhuǎn)化入大腸桿菌BL21用IPTG誘導(dǎo)，SDS-PAGE分析顯示表達(dá)了相對分子量為53.4kDa的pGEX-KG-VP7融合蛋白。將pGEX-KG-VP7融合蛋白作為抗原，免疫新西蘭兔，，制備了多克隆抗體。制備的抗血清經(jīng)同樣誘導(dǎo)表達(dá)的表達(dá)載體pGEX-KG表達(dá)產(chǎn)物吸收后1∶500倍稀釋后用Western Blot分析，與53.4kDa的pGEX-KG-VP7融合蛋白獲得特異性顯色信號，從而證實該融合蛋白具有良好的免疫學(xué)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.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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