B組輪狀病毒WH-2株vp7基因的原核表達及抗體的制備
發(fā)布時間：2018-07-27 17:13
【摘要】： 輪狀病毒(Rotavirus，RV)為呼腸孤病毒科，輪狀病毒屬，已被確認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重，遠遠超出A組輪狀病毒，因而引起國內外學者的廣泛重視和研究。本文研究體外擴增到人B組輪狀病毒WH-2株vp7基因的ORF區(qū)，所構建的重組體pGEX-KG-VP7經IPTG誘導，SDS-PAGE分析，表達了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，結果在預測的相應分子量處得到一條特異的反應帶，從而證實該融合蛋白具有良好的免疫學活性。因此所表達的蛋白和制備的抗體不但為研究結構與功能提供了物質基礎也可用于建立人GBRV的ELISA方法，為GBRV所引起的疾病預防、診斷和治療等流行病學研究和臨床診斷奠定了基礎，具有重要實際應用價值。具體研究內容如下： 第一章全面概述了B組輪狀病毒的形態(tài)結構、理化性質、基因組結構及編碼的蛋白質、體外培養(yǎng)、流行病學、實驗室檢測及臨床診斷與治療等方面的研究進展。 第二章將VP7結構蛋白的基因克隆到pGEX-KG載體上并進行了誘導表達純化與多克隆抗體制備。即從含有人B組輪狀病毒WH-2株全長vp7基因的pUCmT-vp7重組質粒上擴增出人B組輪狀病毒WH-2株vp7基因ORF區(qū)，將擴增產物插入融合表達載體pGEX-KG中構建重組pGEX-KG-VP7質粒。將重組質粒轉化入大腸桿菌BL21用IPTG誘導，SDS-PAGE分析顯示表達了相對分子量為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。制備的抗血清經同樣誘導表達的表達載體pGEX-KG表達產物吸收后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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
本文編號：2148561
[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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392
【參考文獻】
相關期刊論文 前10條
1 王長安,胡超文,黃發(fā)利,陳湘,洪濤;在我國患急性腹瀉成人中發(fā)現一種新輪狀病毒[J];病毒學報;1987年04期
2 王正黨,王林卿,單文魯,張彥,郭艷華,支鳳英;從綿羊羔和山羊羔腹瀉糞樣中檢出B組輪狀病毒[J];病毒學報;1989年02期
3 何小源,周廣和;應用聚合酶鏈式反應擴增和光敏生物素標記的cDNA探針檢測馬鈴薯紡錘塊莖類病毒[J];病毒學報;1992年04期
4 韓新兵,王正黨,黃嘉駟;B組輪狀病毒RT-PCR來源的cDNA探針核酸診斷方法的建立[J];病毒學報;1998年02期
5 譚志平,黃亮群,鄭多,房海燕,夏昆,夏家輝;p11融合蛋白表達、純化及抗血清的制備[J];湖南醫(yī)科大學學報;2002年03期
6 張志珍,張忠,劉玉欣,張書香,陳書芬,周明河,侯通,尚全林,李升奎;某大學一起新型成人輪狀病毒感染調查[J];疾病監(jiān)測;1998年01期
7 吳輝緒,席云珍,銀濤,傅建國,李秉龍,陳世望;一起成人輪狀病毒腹瀉暴發(fā)的流行病學調查[J];解放軍預防醫(yī)學雜志;2003年01期
8 李迪克,葉軍,譚詩龍,楊文清;18例病毒性胃腸炎流行病學調查[J];解放軍預防醫(yī)學雜志;1999年01期
9 王正連，劉漢領，徐海棠，邱方，王彥厚;從成人流行腹瀉糞便中檢出B群輪狀病毒[J];臨床檢驗雜志;1995年S1期
10 馮小黎;重組包涵體蛋白質的折疊復性[J];生物化學與生物物理進展;2001年04期
本文編號：2148561
本文鏈接：http://www.sikaile.net/yixuelunwen/binglixuelunwen/2148561.html
最近更新
教材專著
