SARS冠状病毒结构蛋白在血清学诊断中潜在应用价值的比较

为了确定特异的SARS抗体检测抗原,比较了SARS冠状病毒(SARS-CoV)主要结构蛋白与SARS患者血清的反应性。从SARS死亡患者的肺组织提取的总RNA为模板,用RT-PCR技术分别扩增S、N、M和E4种结构蛋白基因,对3种S截短突变体和N、M、E的重组蛋白在大肠杆菌中进行表达。以表达的蛋白为抗原,应用Western blot跟踪检测11例SARS患者血清54份。结果显示：SARS—CoV的重组N蛋白和s蛋白有很强的抗原性,s蛋白的3个区段的抗原性强弱存在差异,S3抗原性强于S1和s2;在患病第1周、2周、3周及3周以上,N蛋白和s3蛋白抗体检出率分别为40％、65。2％、100％、100％和40％、61％、76.2％、100％;提示SARS-CoV重组N蛋白和S3蛋白在SARS的血清学诊断中有一定的应用价值。     

SARS病毒S蛋白三维结构预测

蛋白质结构类型识别方法可以在没有序列同源性的蛋白质之间检测有没有结构相似性。利用蛋白质结构类型识别方法预测了SARS病毒S蛋白N端区域的结构。模建的SARS病毒S蛋白N端区域是一个全折叠的结构。     

SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究

研究鉴定激活hfgl2凝血酶原酶基因的SARS冠状病毒结构蛋白。从SARS尸检肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、S2和M全长基因序列,再分别克隆到真核表达载体pcDNA3.1( )上。应用免疫组织化学分析鉴定pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2的表达。构建人纤维介素(hfgl2)启动子荧光素酶报告基因质粒,并将SARS冠状病毒结构蛋白表达质粒分别与其共转染以明确激活hfgl2基因转录的SARS冠状病毒结构蛋白。将目的片段克隆至pcDNA3.1( ),经酶切鉴定和测序鉴定无误;免疫组织化学染色可见明显的CHO细胞胞浆棕染。与hfgl2启动子共转染实验阐明SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高4.6倍。SARS冠状病毒N蛋白可增强hfgl2基因的转录活性。     

SARS冠状病毒NS融合蛋白的重组表达纯化与免疫学性质分析(英文)

刺突蛋白(S)和核心蛋白(N)是SARS冠状病毒的主要结构蛋白.在病毒细胞受体结合和病毒包装过程起重要作用.重组融合表达这2种蛋白具有较高的诊断学价值.对SARS病毒N蛋白和S蛋白氨基酸序列进行计算机分析,选择含有优势抗原表位的N蛋白1～227位氨基酸片段和S蛋白450～650位氨基酸片段,采用序列重叠延伸策略(sequenceoverlappingextension,SOE)构建编码N1227LinkerS450650新型融合蛋白的基因片段,导入原核表达载体,实现融合蛋白在大肠杆菌的高效表达.利用组氨酸标签亲和层析的方法纯化,获得高纯度的融合蛋白.对该融合蛋白的结构特征模拟分析的结果显示,其免疫化学性质均无显著改变.采用ELISA和Western印迹方法对其识别SARS冠状病毒特异性抗体的能力进行初步鉴定,显示该融合蛋白具有较好的抗原性和特异性,可有效特异性地检测恢复期SARS病人血清中抗SARS冠状病毒结构蛋白的抗体,可以作为SARS冠状病毒感染的辅助诊断手段.     

SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究

研究鉴定激活hfgl2凝血酶原酶基因的SARS冠状病毒结构蛋白.从SARS尸检肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、S2和M全长基因序列,再分别克隆到真核表达载体pcDNA3.1(+)上.应用免疫组织化学分析鉴定pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2的表达.构建人纤维介素(hfgl2)启动子荧光素酶报告基因质粒,并将SARS冠状病毒结构蛋白表达质粒分别与其共转染以明确激活hfgl2基因转录的SARS冠状病毒结构蛋白.将目的片段克隆至pcDNA3.1(+),经酶切鉴定和测序鉴定无误;免疫组织化学染色可见明显的CHO细胞胞浆棕染.与hfgl2启动子共转染实验阐明SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高4.6倍.SARS冠状病毒N蛋白可增强hfgl2基因的转录活性.     

新冠病毒刺突糖蛋白结构与功能的生物信息学分析及原核表达

SARS-CoV-2是一种高致病性且传播迅速的病原体,通过刺突糖蛋白(Spike glycoprotein,S蛋白)识别宿主细胞表面的受体来实现入侵和感染。对S蛋白进行系统的生物信息学分析和原核表达,有助于深入理解S蛋白的功能和阐明该蛋白介导病毒感染的分子机制。本文采用Protparam、Pfam、TMHMM、ExPASy-ProtScale、PSORTⅡ、SignalP、UniProt、NetPhos 3.1、NetNGlyc 1.0、NetOGlyc 4.0和BLAST等生物信息学软件和数据库对S蛋白的理化性质、亚细胞定位、翻译后修饰及相互作用网络等生物学特性进行了系统分析。利用Clustal X2和MEGA7.0软件对该蛋白进行了基于氨基酸序列的同源性分析和系统进化分析。最后,通过分子克隆技术构建重组表达载体pET-22b-S并进行原核表达。结果显示,S蛋白由1273个氨基酸组成,分子量141.2 kD,等电点6.24,有两个卷曲螺旋结构,一个跨膜螺旋区,疏水性较强。S蛋白包含刺突受体结合结构域和S2糖蛋白结构域,主要分布于宿主细胞的内质网膜和细胞膜,含有136个潜在的磷酸化位点和20个可能的糖基化位点。与SARS-CoV-2 S蛋白序列一致性最高的是SARS冠状病毒、SARS冠状病毒WH20和蝙蝠冠状病毒HKU3,均为76%。SARS-CoV-2与SARS冠状病毒和蝙蝠冠状病毒聚为一大支,提示它们可能具有共同的祖先。S蛋白主要在细菌裂解液离心之后的沉淀中表达,这为后续的结构分析和疫苗研发奠定了基础。S蛋白在SARS冠状病毒和蝙蝠冠状病毒之间保守性较高,提示其在病毒入侵过程中具有重要功能。SARS-CoV-2与SARS冠状病毒和蝙蝠冠状病毒可能具有共同的祖先。本研究为SARS-CoV-2 S蛋白的表达纯化、结构与功能研究提供了重要的数据基础,有助于全面揭示S蛋白的生物学功能,同时为设计和筛选靶向S蛋白的新型抗病毒药物提供了科学依据。     

科技动态

两种SARS候选疫苗攻毒效果评价SARS疾病流行已经过去3年多了,但是对SARS防治的研究还在继续。加拿大的科学家在病毒遗传学杂志上报道了他们新开发的两种抗SARS病毒疫苗,并利用129S6/SvEv小鼠模型比较了它们的有效性。这两种疫苗,一个为灭活疫苗(用β-丙内酯,beta-propiolactone灭活),另一个为两种腺病毒载体重组的疫苗,分别表达SARS-CoV的N蛋白和S蛋白(命名为Ad S/N)。评价方式包括血清中和抗体滴度、细胞免疫反应和对SARS-CoV在肺部复制的抑制作用。灭活疫苗经皮下注射129S6/SvEv小鼠,而Ad S/N疫苗分别采取鼻腔吸入和肌肉…     

SARS病毒S和N蛋白抗原表位的基因合成、融合表达及纯化鉴定

通过计算机分析SARS病毒N蛋白和S蛋白的氨基酸序列 ,初步确定含强抗原表位的N蛋白片段和S蛋白片段 ,共 5 6 0个氨基酸。选择真核和原核生物均偏爱的密码子 ,化学合成全新的SARS病毒N蛋白片段和S蛋白片段的基因序列 ,利用基因工程技术将两个基因片段串联 ,克隆至质粒Pet2 8a(+)内的NcoⅠ/EcoRⅠ位点 ,表达S蛋白片段和N蛋白片段的融合蛋白。将重组质粒转化大肠杆菌BL21(DE3) ,筛选获得了高效表达SARS病毒S蛋白片段和N蛋白片段融合蛋白的工程菌 ,表达的SARS病毒的融合蛋白约占菌体蛋白总量的 30 %左右 ,部分以可溶性形式存在。经离子交换柱和反相高压液相纯化获得了表达的融合蛋白 ,经初步鉴定 ,显示该融合蛋白有较好的抗原性和特异性.     

SARS冠状病毒PLpro蛋白酶的结构与功能

SARS冠状病毒基因组编码2种病毒蛋白酶,即木瓜样蛋白酶（PLpro）和3C样蛋白酶(3CLpro).其中,PLpro蛋白酶结构与功能研究是近年来冠状病毒分子生物学研究的热点之一. PLpro蛋白酶参与SARS冠状病毒1a(1ab)复制酶多聚蛋白N端部分的切割加工,是SARS冠状病毒复制酶复合体(RC)形成的重要调节蛋白分子;最新研究表明,SARS冠状病毒PLpro蛋白酶是一种病毒编码的去泛素化酶（DUB）,对细胞蛋白具有明显去泛素化作用;而且对泛素(Ub)和泛素样分子ISG15均具有活性. PLpro蛋白酶对宿主抗病毒天然免疫反应具有负调节作用,是SARS冠状病毒的一种重要干扰素拮抗分子.PLpro蛋白酶是一种多功能病毒蛋白酶.本文结合作者课题组研究工作,对SARS冠状病毒PLpro蛋白酶结构和功能研究最新进展进行综述.     

SARS 冠状病毒 S 蛋白受体结合结构域的表达及其表位作图

严重急性呼吸综合征 (SARS) 是一种新出现的人类传染病,该病的病原是 SARS 冠状病毒 (SARS-CoV). S 蛋白是 SARS 冠状病毒的一种主要结构蛋白,它在病毒与宿主细胞受体结合以及诱导机体产生中和抗体中起重要作用 . 研究表明 S 蛋白与受体结合的核心区域为第 318 ~ 510 氨基酸残基的片段 . 首先克隆并用 pGEX-6p-1 载体融合表达了该受体结合结构域,并且通过蛋白质印迹分析表明,该受体结合结构域融合蛋白能被 SARS 康复患者血清和 S 蛋白特异的单克隆抗体所识别 . 为了对这一区域进行抗原表位作图,进一步设计了一套 23 个覆盖受体结合结构域的长 16 个氨基酸残基的部分重叠短肽,并进行了 GST 融合表达 . 用免疫动物血清和单克隆抗体 D3D1 对 23 个融合蛋白进行蛋白质印迹和 ELISA 免疫反应性分析,结果鉴定出两个抗原表位 SRBD3(F334PSVYAWERKKISNCV349) 和表位 D3D1 (K447LRPFERDI455). 其结果对进一步分析 S 蛋白结构与功能以及诊断试剂和基因工程疫苗的研究有一定意义 .     

SARS相关冠状病毒及其基因组

正在全球部分地区流行的严重急性呼吸综合征(SARS),由于其传染性强、危害性大而引起了广泛关注。各国实验室密切协作,在数月时间内分离出了SARS冠状病毒(SARSCoV),测定了病毒基因组序列,并在猴体内初步再现出SARSCoV所致肺部疾病与人相似,这些工作为遏制SARS的蔓延发挥了重要作用。现就SARSCoV的鉴定、基因组及其产物的结构与功能做一综述。     

Preparedness for SARS in the UK in 2003

Severe acute respiratory syndrome (SARS) has been described as the first major emerging infectious disease of the twenty-first century. Having initially emerged, almost unnoticed, in southern China, it rapidly spread across the globe. It severely tested national public health and health systems. However, it also resulted in rapid, intensive international collaboration, led by the World Health Organization, to elucidate its characteristics and cause and to contain its spread. The UK mounted a vigorous public health response. Some particular issues concerned: the practicalities of implementing exit screening had this been required; the likely efficacy of this and other control measures; the legal base for public health action; and the surge capacity in all systems should the disease have taken hold in the UK. We have used this experience of 2003 to inform our preparation of a framework for an integrated, escalating response to a future re-emergence of SARS according to the levels of disease activity worldwide. Recent cases confirm that SARS has not "gone away". We cannot be complacent about our contingency planning.     

Understanding SARS with Wolfram approach

Stepping acquired immunodeficiency syndrome (AIDS), severe acute respiratory syndrome (SARS) as another type of disease has been threatening mankind since late last year. Many scientists worldwide are making great efforts to study the etiology of this disease with different approaches. 13 species of SARS virus have been sequenced. However, most people still largely rely on the traditional methods with some disadvantages. In this work, we used Wolfram approach to study the relationship among SARS viruses and between SARS viruses and other types of viruses, the effect of variations on the whole genome and the advantages in the analysis of SARS based on this novel approach. As a result, the similarities between SARS viruses and other coronavirusxes are not really higher than those between SARS viruses and non-coronaviruses.     

Immunopathogenesis of coronavirus infections: implications for SARS

At the end of 2002, the first cases of severe acute respiratory syndrome (SARS) were reported, and in the following year, SARS resulted in considerable mortality and morbidity worldwide. SARS is caused by a novel species of coronavirus (SARS-CoV) and is the most severe coronavirus-mediated human disease that has been described so far. On the basis of similarities with other coronavirus infections, SARS might, in part, be immune mediated. As discussed in this Review, studies of animals that are infected with other coronaviruses indicate that excessive and sometimes dysregulated responses by macrophages and other pro-inflammatory cells might be particularly important in the pathogenesis of disease that is caused by infection with these viruses. It is hoped that lessons from such studies will help us to understand more about the pathogenesis of SARS in humans and to prevent or control outbreaks of SARS in the future.     

Scientific research progress of COVID‐19/SARS‐CoV‐2 in the first five months

A cluster of pneumonia (COVID‐19) cases have been found in Wuhan China in late December, 2019, and subsequently, a novel coronavirus with a positive stranded RNA was identified to be the aetiological virus (severe acute respiratory syndrome coronavirus 2, SARS‐CoV‐2), which has a phylogenetic similarity to severe acute respiratory syndrome coronavirus (SARS‐CoV). SARS‐CoV‐2 transmits mainly through droplets and close contact and the elder or people with chronic diseases are high‐risk population. People affected by SARS‐CoV‐2 can be asymptomatic, which brings about more difficulties to control the transmission. COVID‐19 has become pandemic rapidly after onset, and so far the infected people have been above 2 000 000 and more than 130 000 died worldwide according to COVID‐19 situation dashboard of World Health Organization ( https://covid19.who.int ). Here, we summarized the current known knowledge regarding epidemiological, pathogenesis, pathology, clinical features, comorbidities and treatment of COVID‐19/ SARS‐CoV‐2 as reference for the prevention and control COVID‐19.     

De Novo synthesis of PCR templates for the development of SARS diagnostic assay

A novel coronavirus was identified as the cause for severe acute respiratory syndrome (SARS). The complete sequence of SARS genome has provided an opportunity for the development of molecular diagnostic assays. To restrain further outbreak of SARS, the World Health Organization has posted several pairs of polymerase chain reaction (PCR) primers for early diagnosis and urged more research to be done on PCR protocols. Here we report a strategy for the de novo synthesis of PCR templates complimentary to the SARS virus genome, which has the advantage of working on PCR templates without concern about viral infection and also has the advantage that it can be used by those who do not have access to the SARS virus. This highly efficient and safe strategy for obtaining SARS gene fragments is useful for the development of PCR assays, as well as for the preparation of reliable positive controls for PCR testing kits.     

临床应用重组SARS病毒双抗原夹心诊断试剂盒的检测效果评价

严重急性呼吸综合征(severe acute respiralory syndrome,SARS)的临床表现为非典型性肺炎.继加拿大首次完成SARS病毒株Tor2的全基因组测序后[1],世界卫生组织(WTO)宣布一种新型的冠状病毒(coronavirus)是引发SARS的病原体[2,3].由于SARS具有极强的传染性和较高的病死率(5%～15%),且早期疾病体征较难与某些非SARS病毒引起的非典型肺炎相区分[4],由此导致大量疑似病例无法确诊,以及因误诊引起的交叉感染给人们造成巨大的心理压力和社会恐慌.所以,建立快速、准确的早期诊断方法显得尤为重要.目前的实验室诊断方法中,主要有基于病毒抗体检测的免疫荧光法和酶联免疫吸附试验(ELISA),以及基于基因检测的多聚酶链式反应(PCR)和基因芯片法.其中ELISA主要是使用病毒裂解抗原,检测病毒IgG及IgM抗体的间接ELISA.由于病毒裂解抗原的复杂性,以及间接ELISA中二抗带来的假阳性结果,间接ELISA试剂在正常人群中有1.5%～2%的阳性结果.     

Current topics on SARS coronavirus

The serious respiratory disease, SARS (Severe Acute Respiratory Syndrome), outbreaking in winter of 2003 to 2004 remained in a sporadic patient's generating at this winter. However, there is also a possibility that wild animals as the source of infection may not be specified and that it may be much in fashion again. The paper regarding SARS and SARS-CoV is published at one per day now which has passed since fashion of SARS in one or so year. There are many papers which the researchers of other viruses enter into the research field of SARS-CoV using their own technology in addition to the researchers of coronavirus. Topics of the research on the present SARS-research field are development of vaccine, inspecting of medicine and establishment of diagnostic method. Here, the newest information is offered about these researches.     

Aging and diabetes drive the COVID-19 forwards; unveiling nature and existing therapies for the treatment

Molecular and Cellular Biochemistry - Human SARS Coronavirus-2 (SARS-CoV-2) has infected more than 170 million people worldwide and resulted in more than 3.5 million deaths so far. The infection...     

SARS及其病原体研究进展

严重急性呼吸道综合症(SARS)的病原体己被认定为一种新的冠状病毒-SARS冠状病毒。关于SARS冠状病毒的基因组和蛋白组研究也已取得重要进展,这为探索SARS病毒的来源与进化、研制SARS诊断试剂、开发SARS疫苗和治疗药物奠定了坚实基础。本文对严重急性呼吸道综合症病原学研究取得的一些进展进行了综述。