轮状病毒结构蛋白在大肠杆菌中的表达及其类病毒颗粒的体外组装
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摘要
A组轮状病毒是引起婴幼儿严重腹泻的主要病原体,几乎所有儿童在5岁前至少感染过一次轮状病毒,每年因感染轮状病毒而导致的婴幼儿腹泻高达2亿,住院人数超过200万,死亡高达100万人,仅在美国,每年因轮状病毒而产生的医疗费用就高达1亿美元。
自1973年轮状病毒在人体发现以来,各国科研人员对其进行了大量的研究,对其结构和感染机制的研究日益深入,但到目前为止,尚无针对轮状病毒的特效药物,因此,快速、准确的诊断和安全、有效的疫苗对控制轮状病毒的感染极其重要。病毒样颗粒(Virus-Like Particles,VLPs)作为轮状病毒的候选疫苗具有广阔的发展前景,对进一步研究轮状病毒的感染机制和组装机制也具有重要的意义。
我们在大肠杆菌中表达了轮状病毒的结构蛋白VP2,首次在体外成功的组装了轮状病毒核心样颗粒(Core-like Particle,CLP)并初步研究了环境因素对其稳定性的影响,为进一步研究其免疫原性和免疫保护性奠定了基础;通过缺失突变研究了VP6的自身相互作用的区域,发现243-334aa为VP6形成三聚体的最小区域,通过定点突变研究了半胱氨酸在VP6聚体形成中的作用并通过免疫印迹研究了缺失突变和定点突变对VP6免疫反应性的影响,通过同源建模构建了VP6的结构并分析了定点突变对VP6结构的影响;构建了VP4和VP7的表达质粒;此外,还制备了轮状病毒的单克隆抗体,初步实验证实7H11、2A9和5C2具有较好的广谱性和特异性。该研究为轮状病毒VLP的体外组装及VLP疫苗的研究奠定了基础,对进一步研究轮状病毒的感染机制和组装机制也具有重要的意义。
Rotavirus is the most common cause of severe gastroenteritis among children worldwide,Almost every child has Infected rotavirus once or more before 5 years old, resulting in approximately 200 million diarrhea cases,more than 2 000 000 hospitalization and 1 million death every year worldwide,the healthcare cost of rotavirus is over 1 billion dollars every year in the United States.
A lot of research on rotavirus has been under way since it was discovered in human in 1973,we understand more deeply in the structure and infection mechanism of the virus,but there is no specific medicine to rotavirus diarrhea,thus quick, accurate diagnostics and safe,effective vaccines are important in rotavirus prevention and control.VLPs has great Opportunity as a rotavirus candidate vaccine and is important in the research of both infection and assembly mechanism.
First,we expressed the structure protein VP2 in E.coli and assembled it into CLPs in-vitro for the first time,we also studied the role of buffers and additives on its stability,and it laid the foundation for the study of VP2 immunogenicity and protectivity;Second,We expressed the inner layer protein VP6 and its deletion mutants,we found that 243-334aa is important for VP6 trimerization,at the same time, we expressed single point mutants of VP6 and found Cys197 is important for VP6 polymerization,the WB results show the influence of deletion and point mutation on VP6 immuno-reactivity;third,we constructed VP4 and VP7 expression plasmids; furthermore,we prepared mAb to rotavirus,mainly to VP6 and primary results show that 7H11,2A9 and 5C2 are excellent in both sensitivity and specificity.This study laid the foundation for rotavirus VLP in-vitro assembly and vaccine development,it is important for the study of rotavirus infection and assembly mechanism.
自1973年轮状病毒在人体发现以来,各国科研人员对其进行了大量的研究,对其结构和感染机制的研究日益深入,但到目前为止,尚无针对轮状病毒的特效药物,因此,快速、准确的诊断和安全、有效的疫苗对控制轮状病毒的感染极其重要。病毒样颗粒(Virus-Like Particles,VLPs)作为轮状病毒的候选疫苗具有广阔的发展前景,对进一步研究轮状病毒的感染机制和组装机制也具有重要的意义。
我们在大肠杆菌中表达了轮状病毒的结构蛋白VP2,首次在体外成功的组装了轮状病毒核心样颗粒(Core-like Particle,CLP)并初步研究了环境因素对其稳定性的影响,为进一步研究其免疫原性和免疫保护性奠定了基础;通过缺失突变研究了VP6的自身相互作用的区域,发现243-334aa为VP6形成三聚体的最小区域,通过定点突变研究了半胱氨酸在VP6聚体形成中的作用并通过免疫印迹研究了缺失突变和定点突变对VP6免疫反应性的影响,通过同源建模构建了VP6的结构并分析了定点突变对VP6结构的影响;构建了VP4和VP7的表达质粒;此外,还制备了轮状病毒的单克隆抗体,初步实验证实7H11、2A9和5C2具有较好的广谱性和特异性。该研究为轮状病毒VLP的体外组装及VLP疫苗的研究奠定了基础,对进一步研究轮状病毒的感染机制和组装机制也具有重要的意义。
Rotavirus is the most common cause of severe gastroenteritis among children worldwide,Almost every child has Infected rotavirus once or more before 5 years old, resulting in approximately 200 million diarrhea cases,more than 2 000 000 hospitalization and 1 million death every year worldwide,the healthcare cost of rotavirus is over 1 billion dollars every year in the United States.
A lot of research on rotavirus has been under way since it was discovered in human in 1973,we understand more deeply in the structure and infection mechanism of the virus,but there is no specific medicine to rotavirus diarrhea,thus quick, accurate diagnostics and safe,effective vaccines are important in rotavirus prevention and control.VLPs has great Opportunity as a rotavirus candidate vaccine and is important in the research of both infection and assembly mechanism.
First,we expressed the structure protein VP2 in E.coli and assembled it into CLPs in-vitro for the first time,we also studied the role of buffers and additives on its stability,and it laid the foundation for the study of VP2 immunogenicity and protectivity;Second,We expressed the inner layer protein VP6 and its deletion mutants,we found that 243-334aa is important for VP6 trimerization,at the same time, we expressed single point mutants of VP6 and found Cys197 is important for VP6 polymerization,the WB results show the influence of deletion and point mutation on VP6 immuno-reactivity;third,we constructed VP4 and VP7 expression plasmids; furthermore,we prepared mAb to rotavirus,mainly to VP6 and primary results show that 7H11,2A9 and 5C2 are excellent in both sensitivity and specificity.This study laid the foundation for rotavirus VLP in-vitro assembly and vaccine development,it is important for the study of rotavirus infection and assembly mechanism.
引文
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