反向遗传构建A组轮状病毒重配毒株的研究
发布时间：2018-12-11 07:25
【摘要】： 轮状病毒(Rotavirus,RV)属于呼肠孤病毒科,轮状病毒属;已被确认为引起全世界婴幼儿急性胃肠炎最常见的病原。轮状病毒感染每年导致全球约1.25亿婴幼儿发生腹泻,轮状病毒腹泻每年造成全球90万5岁以下婴幼儿死亡,严重影响了婴幼儿的健康并给全球带来巨大的疾病负担。轮状病毒引起的腹泻至今尚无特效药物进行治疗,疫苗是目前证实唯一最有效的预防方法。本论文研究内容包括A组轮状病毒(GARV)武汉地区G9型流行毒株CC-1的分离及vp7基因的克隆与分析;A组轮状病毒vp7基因转录载体的构建与检测;A组轮状病毒重配株的构建。具体内容如下: 第一章全面概述了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、以及A组轮状病毒的流行病学、轮状病毒疫苗研究现状,重点论述了G9型轮状病毒的流行特征及轮状病毒的反向遗传学研究。 第二章武汉地区G9型轮状病毒的分离和vp7基因的克隆与序列分析采用MA104细胞从粪便样本中分离CC-1株,RT-PCR获得vp7基因,并克隆至pGEMT-easy载体,获得质粒pGEMvp7-G9。对质粒插入片断测序并进行序列分析。 第三章A组轮状病毒全长vp7基因转录载体的构建与检测。采用Overlap PCR方法构建5'端上游有T7-RNA聚合酶启动子、下游有HDV核酶序列与T7-RNA聚合酶终止子的vp7基因,构建vp7转录载体pGEMvp7-Rz。该质粒转染预先感染vTF7-3的哺乳动物细胞后,在哺乳动物细胞中获得具有正确的正确5'与3'末端的vp7+sRNA。 第四章反向遗传学方法构建A组轮状病毒重配株。采用补骨质素-紫外线A使重组痘病毒vTF7-3失去感染性,但仍保持表达T7-RNA聚合酶的活性。将重组痘病毒vTF7-3感染Vero细胞,采用G1型A组轮状病毒vp7的全长cNDA转录载体转染感染了vTF7-3的Vero细胞,在细胞内获得具有正确5'与3'端的轮状病毒vp7基因+sRNA。然后将另一株G10型A组轮状病毒感染轮状病毒敏感的vp7细胞,在病毒的包装过程中vp7发生重配。然后使用单克隆抗体对重配病毒进行筛选。
[Abstract]:Rotavirus (Rotavirus,RV) belongs to the family Reoviridae and has been identified as the most common cause of acute gastroenteritis in infants and children in the world. Rotavirus infection causes diarrhea in about 125 million infants and young children in the world every year. Rotavirus diarrhea causes deaths of children under 5 years of age in the world every year, which seriously affects the health of infants and children and brings a huge burden of disease to the whole world. There is no effective drug to treat rotavirus-induced diarrhea, and vaccine is the only effective method to prevent diarrhea caused by rotavirus. This thesis includes the isolation of CC-1 and the cloning and analysis of vp7 gene of group A rotavirus (GARV) strain G9 in Wuhan region, the construction and detection of vp7 gene transcription vector of group A rotavirus. Construction of Group A rotavirus Recombinant. The main contents are as follows: in the first chapter, the morphology, physicochemical properties, genome structure and encoded protein of rotavirus, culture in vitro, epidemiology of group A rotavirus and the status of rotavirus vaccine were summarized. The epidemic characteristics of G 9 rotavirus and the reverse genetics of rotavirus were discussed. Chapter 2 isolation of G9 rotavirus and cloning and sequence Analysis of vp7 Gene in Wuhan region CC-1 strain was isolated from fecal samples by MA104 cells. Vp7 gene was obtained by RT-PCR and cloned into pGEMT-easy vector to obtain plasmid pGEMvp7-G9.. The plasmid insert fragment was sequenced and sequenced. Chapter 3: construction and detection of full-length vp7 transcription vector of group A rotavirus. The vp7 gene with HDV ribozyme sequence and T7-RNA polymerase Terminator was constructed by Overlap PCR method. The vp7 transcription vector pGEMvp7-Rz. was constructed. After transfection of the plasmid into mammalian cells preinfected with vTF7-3, the correct 5 'and 3' terminal vp7 sRNA. were obtained in mammalian cells. Chapter 4 reverse genetics method was used to construct group A rotavirus recombinant strain. The recombinant poxvirus (vTF7-3) was infected by UVA, but the activity of T7-RNA polymerase was maintained. The recombinant poxvirus vTF7-3 was infected with Vero cells. The Vero cells infected with vTF7-3 were transfected with the full-length cNDA transcriptional vector of group G1 rotavirus vp7. The 5 'and 3'end rotavirus vp7 gene sRNA. was obtained in the cells. Then another G10 group A rotavirus strain was infected with rotavirus sensitive vp7 cells and vp7 reassorted during the packaging of the virus. Then monoclonal antibodies were used to screen the reassorted virus.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
本文编号：2372140
[Abstract]:Rotavirus (Rotavirus,RV) belongs to the family Reoviridae and has been identified as the most common cause of acute gastroenteritis in infants and children in the world. Rotavirus infection causes diarrhea in about 125 million infants and young children in the world every year. Rotavirus diarrhea causes deaths of children under 5 years of age in the world every year, which seriously affects the health of infants and children and brings a huge burden of disease to the whole world. There is no effective drug to treat rotavirus-induced diarrhea, and vaccine is the only effective method to prevent diarrhea caused by rotavirus. This thesis includes the isolation of CC-1 and the cloning and analysis of vp7 gene of group A rotavirus (GARV) strain G9 in Wuhan region, the construction and detection of vp7 gene transcription vector of group A rotavirus. Construction of Group A rotavirus Recombinant. The main contents are as follows: in the first chapter, the morphology, physicochemical properties, genome structure and encoded protein of rotavirus, culture in vitro, epidemiology of group A rotavirus and the status of rotavirus vaccine were summarized. The epidemic characteristics of G 9 rotavirus and the reverse genetics of rotavirus were discussed. Chapter 2 isolation of G9 rotavirus and cloning and sequence Analysis of vp7 Gene in Wuhan region CC-1 strain was isolated from fecal samples by MA104 cells. Vp7 gene was obtained by RT-PCR and cloned into pGEMT-easy vector to obtain plasmid pGEMvp7-G9.. The plasmid insert fragment was sequenced and sequenced. Chapter 3: construction and detection of full-length vp7 transcription vector of group A rotavirus. The vp7 gene with HDV ribozyme sequence and T7-RNA polymerase Terminator was constructed by Overlap PCR method. The vp7 transcription vector pGEMvp7-Rz. was constructed. After transfection of the plasmid into mammalian cells preinfected with vTF7-3, the correct 5 'and 3' terminal vp7 sRNA. were obtained in mammalian cells. Chapter 4 reverse genetics method was used to construct group A rotavirus recombinant strain. The recombinant poxvirus (vTF7-3) was infected by UVA, but the activity of T7-RNA polymerase was maintained. The recombinant poxvirus vTF7-3 was infected with Vero cells. The Vero cells infected with vTF7-3 were transfected with the full-length cNDA transcriptional vector of group G1 rotavirus vp7. The 5 'and 3'end rotavirus vp7 gene sRNA. was obtained in the cells. Then another G10 group A rotavirus strain was infected with rotavirus sensitive vp7 cells and vp7 reassorted during the packaging of the virus. Then monoclonal antibodies were used to screen the reassorted virus.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
【参考文献】
相关期刊论文 前5条
1 钱渊，袁丽娟，，熊朝晖，张又，刘军，关德华，王之梁;在我国腹泻患儿中发现G9型轮状病毒感染[J];病毒学报;1994年03期
2 童志礼;婴幼儿轮状病毒流行的新趋势——G9型轮状病毒全球流行综述[J];国外医学.病毒学分册;2002年04期
3 温乐英;轮状病毒蛋白的研究进展[J];国外医学.病毒学分册;1996年03期
4 汪洋;白萱;余晖;孙红;杨继红;;2006年武汉地区A组轮状病毒VP7基因型初步研究[J];黄冈师范学院学报;2006年06期
5 石长信;王长安;王湛;周伟;洪涛;;A组轮状病毒外壳蛋白VP4的原核表达[J];中华实验和临床病毒学杂志;1996年02期
本文编号：2372140
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2372140.html
最近更新
教材专著
