Structure studies on hepatitis E virus capsid protein and its underlying neutralization epitope

戊型肝炎是由戊型肝炎病毒(Hepatitis E virus,HEV)引起的病毒性肝炎,被证实为人兽共患病,该病症对免疫力低下人群和已有基础性肝病的患者的危害更为显著。戊肝病毒主要通过粪口途径传播,猪是其主要的天然宿主。近年来,关于戊肝病毒的衣壳蛋白及其表位结构的研究取得了重要的进展,对于戊肝病毒的病毒学研究和戊肝疫苗的机制探讨起着重要的指导意义。本文将综述HEV病毒衣壳蛋白的结构研究和中和表位的结构基础,着重说明戊肝病毒中和表位存在型特异性和型交叉两种特征,提示型交叉表位与人兽共患现象的关系。Hepatitis E virus(HEV)is a causative pathogen of hepatitis E,one of the major acute infectious diseases,which has been recently proved as a zoonosis with main virus reservoir in domestic pigs.The virus is mainly spread by fecal-oral route and it's more likely to infect the immunocompromised population and those who have suffered from liver disease.To date,there are numbers progress on the structure of hepatitis E virus capsid proteins and their immune complex with neutralizing monoclonal antibodies.These knowledge advances our understanding on the virology and immunology of hepatitis E virus,and subsequently benefits for the research and development of hepatitis E vaccine.Here,we review the key points in regard to the structure determination and immunologically functional basis of hepatitis E virus capsid protein.The summarized advances might shed lights on the theoretical basis for genotype specificity,cross-genotype feature and their correlation to zoonotic hepatitis E disease

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基于2009-2013年第8次全国森林资源连续清查数据,利用生物量扩展因子法,采用改良的计算参数,从不同龄组、林型等方面进行考虑,对西南5省市区森林资源的生物量、碳储量及碳密度进行了估算。结果表明:我国第8次森林资源清查中,西南5省市区森林植被总生物量为5 308.18×10~6t,碳储量总量为2 752.05×10~6tC,林分碳储量为2 546.74×10~6tC;西藏藏族自治区森林植被碳储量在西南5省市区碳储量中占最大份额,为980.46×10~6tC,占西南5省森林植被碳储量的35.63%;重庆市其森林植被碳储量只占西南5省市区森林植被碳储量的2.55%。在碳密度方面,西藏藏族自治区林..

Forest Carbon Storage in Yunnan during 2008-2013

根据第七、第八次全国森林资源连续清查数据,采用生物量扩展因子法,从不同龄组、林型、起源对云南省森林植被碳储量和碳密度进行估算。结果表明,2008年云南省森林植被总碳储量798.31×10~6 t,2013年增至831.81×10~6 t;2013年的调查结果显示各林型(除灌木林)碳储量普遍增加,平均碳密度却有所降低,由第七次调查时的49.98 t∕hm~2,降至2013年的39.78 t∕hm~2;人工林各龄组间碳储量变化明显,平均变化率达55.28%;天然林碳储量却只增加了23.02×10~6 t,增长率为3.26%。适当扩大人工林面积可增加碳储量,但这种方式已经不适合云南碳汇项目的发展

Antigenicity and Immunogenicity of Influenza M2 e Protein Fused with Intramolecular Adjuvant CRM197

流感病毒严重威胁人类健康且流行株不断变化,传统疫苗对流感预防具有局限性。流感病毒M2蛋白胞外区(M2; e)氨基酸序列高度保守,可诱导产生特异性抗体,是通用流感疫苗研究的主要靶标之一。白喉毒素突变体(CRM197)是一种理想的蛋白载体,可增强小分子; 抗原的免疫原性,商业化的肺炎球菌疫苗采用CRM197结合形式提高多糖分子的免疫原性。本实验通过重组蛋白融合的方式,将M2 e与CRM197(aa; 1~535)、CRM197-N190 (aa 1~190)、CRM197-N389(aa; 1~389)的C端或N端进行融合表达,并考察融合蛋白的反应活性以及免疫原性。融合蛋白中的M2; e和CRM197均具有反应活性;超速离心分析和四聚体特异构象单抗反应均表明融合蛋白(CRM197-N190)-M2 e及M2; e-(CRM197-N190)主要以类似M2 e天然构象的四聚体形式存在;融合蛋白可与多株流感M2; e特异性单抗以及CRM197特异性单抗反应,M2; e融合至CRM197N末端形成的重组蛋白的反应活性高于C端融合蛋白;相比融合GST,与CRM197-N190的融合显著增强了M2; e蛋白的免疫原性,为流感通用疫苗研究提供了新思路。New strategies are needed urgently to develop broad-spectrum vaccines; against the influenza virus and to reduce the risk of pandemics.The; conserved M2 eprotein is a potential candidate for development of a; universal vaccine against influenza viruses.In this work,M2 ewas fused; to the N-or C-terminus of CRM197(aa1~535),CRM197-N389(aa1~389)and; CRM197-N190(aa1~190)by gene engineering.Data from SDS-PAGE and western; blotting showed that these fusion proteins formed multimers,and that the; monomer and dimer bands were the most prominent.Moreover,AUC data; demonstrated that the tetramers of some fusion proteins were present in; solutions that might reflect the native conformation of the M2 e; protein.The fusion protein of M2 efused to the N-terminus of; CRM197,CRM197-N389,and CRM197- N190 showed excellent reactivity to the; characterized monoclonal antibodies O19 and L18 as compared with M2; efused to the C-terminus of CRM197,CRM197-N389,and; CRM197-N190.Furthermore,the immunogenicity of these fusion proteins was; evaluated in mice.Results demonstrated that the immunogenicity of M2; ewas enhanced significantly when being fused to CRM197-N190 than to; GST.These findings provide some clues for the development of a M2; e-based,broad-spectrum influenza vaccine.国家自然科学基

Expression,Purification,Structure Determination and Immunogenicity Assay of Hepatitis E Virus Capsid Protein p495 Derived from Baculovirus-based Insect Cell

本研究目的是利用杆状病毒-昆虫细胞表达系统建立戊型肝炎病毒(HEV)p495蛋白的高效稳定表达和可放大纯化的方法,研究其理化性质和解析三维结构,与上市戊肝疫苗益可宁(Hecolin)进行免疫原性的比较。本研究选择基因1型HEV毒株的开放读码框2(ORF2)的aa112-606片段构建杆状病毒,感染昆虫细胞进行重组蛋白的表达,经过PEG沉淀和阴离子交换层析的纯化,获得纯度为95%以上的p495蛋白,每升细胞培养液最终获得15mg的目的蛋白。经ELISA、分析超离、分子排阻色谱和电镜负染等分析显示p495蛋白具有良好的抗原性且呈现为均一的病毒样颗粒(VLP),沉降系数为51.3S,颗粒直径约20nm。冷冻电镜三维结构解析获得分辨率为3.8的三维结构,揭示p495形成了T=1的等二十面体结构,与已报道的晶体结构相一致。小鼠免疫实验表明p495蛋白与益可宁中p239抗原的免疫原性相当。本研究为进一步开展HEV的受体鉴定、表位结构研究以及疫苗改进等奠定良好的基础。Our objective was to establish a robust method for the expression and purification of hepatitis E virus(HEV)p495protein using a baculovirus-based insect cell expression system;to determine the properties and cryo-EM structure of the resulting virus-like particles(VLPs);and to compare their immunogenicity with p239 particles in the commercial hepatitis E vaccine(Hecolin).The sequence spanning HEV ORF2 amino acids 112-606 in the genotype I HEV isolate was cloned into baculovirus to express recombinant p495 protein.ELISA,analytical ultracentrifugation,size-exclusion chromatography and negativestaining transmission electron microscopy(TEM)were carried out to characterize the physicochemical properties of p495.Recombinant p495 VLPs were obtained successfully from the insect cell expression system with purity of>95%and yield of 15mg/L.The recombinant HEV p495 protein was homogeneous in solutions.The 3Dstructure of p495 VLPs was determined by cryo-EM;it was icosahedral with T=1arrangement,and showed good congruency with the crystal structure in the literature(PDB ID:2ZZQ).In mouse vaccination experiments,p495 conferred comparable immunogenicity with that of p239 antigen in Hecolin.Thus,a robust and scalable approach to obtain homogeneous,immunogenic HEV p495 VLPs has been established.This study may assist investigations of HEV receptors,epitope mapping,vaccine improvement and so on.国家自然科学基金(项目号:81571996

JUNO Sensitivity on Proton Decay p→νˉK+p\to \bar\nu K^+ Searches

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this paper, the potential on searching for proton decay in $p\to \bar\nu K^+$ mode with JUNO is investigated.The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits to suppress the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar\nu K^+$ is 36.9% with a background level of 0.2 events after 10 years of data taking. The estimated sensitivity based on 200 kton-years exposure is $9.6 \times 10^{33}$ years, competitive with the current best limits on the proton lifetime in this channel

JUNO sensitivity on proton decay p → ν K + searches*

The Jiangmen Underground Neutrino Observatory (JUNO) is a large liquid scintillator detector designed to explore many topics in fundamental physics. In this study, the potential of searching for proton decay in the $p\to \bar{\nu} K^+$ mode with JUNO is investigated. The kaon and its decay particles feature a clear three-fold coincidence signature that results in a high efficiency for identification. Moreover, the excellent energy resolution of JUNO permits suppression of the sizable background caused by other delayed signals. Based on these advantages, the detection efficiency for the proton decay via $p\to \bar{\nu} K^+$ is 36.9% ± 4.9% with a background level of $0.2\pm 0.05({\rm syst})\pm$$0.2({\rm stat})$ events after 10 years of data collection. The estimated sensitivity based on 200 kton-years of exposure is $9.6 \times 10^{33}$ years, which is competitive with the current best limits on the proton lifetime in this channel and complements the use of different detection technologies