Multimerization and protective efficacy of rotavirus VP4 truncated proteins

轮状病毒是全球范围内引起5岁以下婴幼儿腹泻的主要病原体，在全球每年由于轮状病毒感染导致的死亡病例高达40-60万。疫苗是预防轮状病毒感染及发病最有效的途径，目前已经有两种轮状病毒疫苗（Rotateq和Rotarix）在全球范围内推广使用，70多个国家已经将轮状病毒疫苗纳入免疫规划。随着轮状病毒疫苗的推广，轮状病毒导致的年死亡病例由40-60万下降到20万左右。但是目前的轮状病毒疫苗存在地区有效性差异大、会诱发小儿肠套叠等问题，因此更加安全、有效的轮状病毒亚单位疫苗的研究对于进一步降低轮状病毒导致的发病率和死亡率具有重要意义。VP4作为刺突蛋白，介导了轮状病毒的吸附和入胞过程，其抗体可以阻断轮状...Rotavirus is a major pathogen that causes diarrhea in infants and young children under 5 years of age worldwide, with 40 to 60 million deaths worldwide due to rotavirus infection. Vaccine is the most effective way to prevent rotavirus infections. Two rotavirus vaccines (Rotateq and Rotarix) are now available worldwide, and more than 70 countries have included rotavirus vaccines in immunization pro...学位：医学硕士院系专业：公共卫生学院_转化医学学号：3262014115057

Research progress in rotavirus VP4 subunit vaccine

轮状病毒是全球范围内导致5岁以下婴幼儿严重腹泻的主要病原体,造成了巨大的经济负担和社会负担。疫苗预防接种是控制轮状病毒感染最为有效的手段,但在轮; 状病毒导致的死亡率较高的非洲和亚洲部分低收入国家,目前已经上市的轮状病毒疫苗的有效性较低,且会增加肠套叠的风险。更加安全、有效的轮状病毒疫苗对于; 降低轮状病毒感染导致的发病率和死亡率具有重要意义。目前,各国科研人员试图从多个方面提高轮状病毒疫苗的有效性,非复制型基因工程亚单位疫苗是目前轮状; 病毒疫苗研究的主要方向。文中就目前轮状病毒亚单位疫苗,特别是基于VP4蛋白的亚单位疫苗的研究进展进行了综述,以期对轮状病毒疫苗的发展提供借鉴意义; 。Rotaviruses are leading causes of worldwide acute diarrhea in children; younger than 5 years old, with severe consequence of social and economic; burden. Vaccination is the most effective way to control rotavirus; infection, however, the licensed rotavirus vaccines are ineffective in; some low-income countries of Africa and Asia, where the mortality caused; by rotavirus is higher than other areas. In addition, there are also; safety concerns such as increased risk of intussusception. Therefore, it; is urgent to improve the efficiency and safety of rotavirus vaccine to; reduce the morbidity and mortality caused by rotavirus. Till now, many; efforts are made to improve the effectiveness of rotavirus vaccines, and; the inactive vaccine becomes the main rend in the research of rotavirus; vaccine. The developments in recombinant rotavirus vaccines, especially; in VP4 subunit vaccines are summarized in this review, and it could be; helpful to develop effective recombinant rotavirus vaccines in further; studies.国家自然科学基

轮状病毒VP4~*高聚体的制备及其免疫保护性评价

在前期工作中发现,截短的轮状病毒VP4~*蛋白(aa26–476)在大肠杆菌中能够以可溶形式表达,且在小鼠模型中具有较高的免疫原性和免疫保护性。本研究通过颗粒化进一步提高VP4~*蛋白的免疫保护性。通过37℃水浴加热处理24h使VP4~*蛋白多聚化,通过高效液相色谱、透射电镜、分析超离等分析VP4~*蛋白颗粒化程度,通过酶联免疫吸附试验分析颗粒化对VP4~*蛋白与中和抗体反应性的影响;通过差示量热法分析VP4~*高聚体的热稳定性;最后,通过小鼠母传抗体模型研究颗粒化对VP4~*免疫原性和免疫保护性的影响。结果表明,VP4~*蛋白高聚体结构均一,并且相比三聚体,具有更高热稳定性和中和抗体结合活性;在内毒素<20 EU/mg的条件下,与铝佐剂混合,刺激小鼠产生更高滴度的中和抗体;对轮状病毒导致的腹泻具有更高的免疫保护性。综上所述,VP4~*高聚体的研究为轮状病毒基因工程亚单位疫苗的研制提供了更广阔的思路。国家自然科学基金(No.81501741);;福建省科学技术创新平台(No.2014Y2004)资助~

Construction of a Real-time Fluorescent Quantitative RT-PCR for Detection of Group A Rotavirus

建立一种通用的检测不同物种来源A组轮状病毒的逆转录实时荧光定量PCR方法(RT-qPCR)。根据轮状病毒保守的NSP5基因的保守序列设计引物和探; 针,建立并优化RT-qPCR检测体系;同时通过9份轮状病毒毒株和96份临床标本的检测对该方法和目前常用的基于NSP3基因的2种RT-qPCR方法; 进行灵敏度和检出率的比较。新建立的RT-qPCR方法特异性高,重复性好;与基于NSP3基因的方法相比:(1)在临床标本检测中,检出率无差异;(2; )9株培养病毒检测中,NSP5体系可全部检出,两个NSP3对照体系NSP3-1和NSP3-2的检出率分别为78.78%(7/9)和88.89%(; 8/9);(3)NSP5体系与对照NSP3-1体系灵敏度相当,检测下限比NSP3-2体系低100倍。本研究建立的基于NSP5的荧光定量PCR体系; 灵敏度高,可检测毒株范围广,为检测不同物种来源的A组轮状病毒提供一种新的选择。We wished to establish a more universal real-time fluorescent; quantitative reverse transcriptionpolymerase chain; reaction(RT-PCR)method for detection of group A rotavirus.Primers and; probes were designed based on the conserved genome sequence of; non-structural protein 5(NSP5),and a TaqManRTPCR was constructed.Nine; strains of rotavirus and 72clinical samples were detected using; NSP5-based real- time RT-PCR and the commonly used non-structural; rotavirus protein 3 (NSP3)- based real-time RTPCR in parallel.The; RT-PCR system based on NSP5had good specificity and efficiency.Compared; with NSP3-based real-time RT-PCR:(i)there was no significant difference; between different real-time RT-PCR assays in the detection of clinical; samples;(ii)NSP5-based real-time RT-PCR constructed in the present study; could be used to detect more strains of cultured rotavirus;(iii)the; sensitivity of NSP5-based RTPCR was equivalent to that for; NSP3-1,whereas it was 100-fold higher than that for NSP3-2.The universal; real-time RT-PCR that we constructed could be a new way to detect; rotavirus from different species.国家自然科学基