SARS冠状病毒S蛋白研究进展

SARS冠状病毒是引起传染性非典型肺炎的病原体,它具有4种结构蛋白,其中S蛋白含有主要的中和抗原表位,并有受体结合和膜融合活性,与SARS冠状病毒的组织嗜性、细胞融合性和病毒毒力密切相关,是制备保护性疫苗的重要靶位.     

SARS冠状病毒S蛋白研究进展

随着严重急性呼吸综合征 (severeacuterespiratorysyn drome,SARS)病原体的确定 ,对SARS冠状病毒 (SARScorona virus,SARS CoV)的研究已取得了突飞猛进的发展。许多研究表明SARS CoV的S糖蛋白 (spikeprotein)在病毒的感染过程中起重要作用。它在病毒与宿主细胞表面受体结合及介导膜融合进入细胞的过程中 ,起关键性作用 ,也是冠状病毒的主要抗原。因此本文将就SARS CoVS蛋白的基因、蛋白质结构及其可能的受体和抗原表位等方面的研究进展作一综述。1　编码S蛋白的基因SARS CoV基因组全长 2 975 1nt(TOR2株 ) ,有 11个预测的…     

SARS冠状病毒Mc区克隆及序列分析

目的：构建SARS冠状病毒（SARS-CoV）GD322株M基因膜内区（Mc区）重组表达质粒，并对目的序列进行序列分析。方法：根据GenBank中公布的SARS-CoV Tor2株M基因序列，设计一对引物，采用RT-PCR法从SARS-CoV GD322株基因组中扩增Mc区．克隆至pET-32a（＋）载体中，转化大肠杆菌BL21后测序，利用Clustal X分析所测序列翻译的氨基酸与81株SARS-CoVMc区翻译的氨基酸序列的差异。结果：测序结果表明该区与Tor2株对应核苷酸序列同源性为100％。与已收集的81株SARS-CoVMc区所译氨基酸（Mc蛋白）相比，与77株（占95．1％）Mc蛋白完全同源．与4株（占4．9％）仅有一个氨基酸改变。结论：本试验成功构建SARS-CoV Mc区重组表达质粒：SARS-CoV各毒株间Mc蛋白氨基酸序列差异极小，提示利用任一毒株Mc蛋白制备单抗和疫苗具有普遍意义．     

SARS 冠状病毒主蛋白酶结构的研究进展

急性呼吸道综合征冠状病毒(SARS-CoV)是SARS的病原体,其基因为单股正链RNA,通过比较发现,SARS-CoV主蛋白酶(MPRO)与其他冠状病毒如人冠状病毒株229E(HCoV229E)及一种在猪体内引发传染性胃肠炎的病毒(TGEV)MPRO结构具有高度同源性,SARS-CoVMPRO由于在病毒复制中的重要性,使它成为抗病毒药物设计的重要靶点。本文针对SARS-CoVMPRO结构作一综述,以期研制或筛选出以针对SARS-CoVMPRO的特效药物。     

冠状病毒及SARS病毒概述

目前已确定世界范围内流行的非典型肺炎的病原体为一种新变异的冠状病毒，本文就冠状病毒的结构、分类及致病性以及SARS冠状病毒的特征作一综述。     

SARS-CoV S蛋白全长基因克隆及其表达的初步研究

目的:克隆表达SARS-CoVS蛋白,构建SARS全长基因疫苗。方法:从SARS病毒的总cDNA中以PCR方法扩增编码人SARS-CoVS1和S2蛋白的编码序列,再将二者分别克隆入真核表达载体pVAX1,构建重组表达载体。然后进一步将S1和S2连接到pVAX1中。酶切和测序鉴定阳性克隆。采用脂质体法转染VeroE6细胞,用Western检测S蛋白的表达。结果:PCR方法分别扩增出了1900bp和1800bp左右的基因片段,两片段插入pVAX1构建重组表达载体后,经序列测定证实该插入片段为SARS病毒S蛋白编码序列。将重组子转染VeroE6细胞,收集细胞总蛋白,Western检测获得特异蛋白带。结论:成功克隆并表达了SARS-CoVS蛋白,为其作为SARS基因疫苗研究打下基础。     

SARS冠状病毒核衣壳蛋白在酿酒酵母中的表达和纯化

目的:构建重组表达质粒pYES6-N,诱导SARS冠状病毒核衣壳蛋白(nucleocapsidprotein)在酿酒酵母中的表达和纯化。方法:逆转录获得SARS冠状病毒DNA片段,PCR获得核衣壳蛋白基因互相重叠的两部分片段,酶切连接成全长,在E.coliJM109中构建重组表达克隆pYES6-N,在酿酒酵母中诱导表达并纯化。结果:重组克隆pyes6-N经酶切、测序鉴定确定,插入片段大小、方向、碱基匹配与预期一致,获得一个完整的核衣壳蛋白基因,表达产物纯化后,电泳鉴定,相对分子质量近45000。结论:成功克隆SARS冠状病毒核衣壳蛋白基因全长,并在酿酒酵母中诱导表达成功,有助于抗SARS分子疫苗的研究。     

蛋白S研究进展

蛋白S是一种抗凝因子 ,本文主要介绍了其结构 ,理化作用及其缺乏的原因。在其活性状态 -游离蛋白S缺乏的情况下 ,可以导致多部位血栓形成。且目前已经逐渐认识到 :特别是在年青人中 ,蛋白S的缺陷可能是缺血性卒中的危险因素。     

蛋白S研究进展

蛋白S是一种抗凝因子，本文主要介绍了其结构，理化作用及其缺乏的原因。在其活性状态-游离蛋白S缺之的情况下，可以导致多部位血栓形成。且目前已经逐渐认识到：特别是在年青人中，蛋白S的缺陷可能是缺血性卒中的危险因素。     

SARS病毒M蛋白真核表达载体的构建与表达

目的 构建SARS病毒M蛋白片段真核表达载体,并检测其在Vero细胞中的表达.方法 在PCR引物下游引入Flag序列,以PCR从质粒pGEX-6P-1-SARS-M中扩增M蛋白编码基因片段,将酶切后的PCR产物克隆至pcDNA3.1(+)中,构建并鉴定重组质粒pcDNA3.1(+)-SARS-M.重组质粒经Superfect转染Vero细胞,Western blot检测基因表达情况.结果 重组质粒经酶切鉴定和基因测序显示构建正确;Western blot检测表明重组M蛋白片段在Vero细胞中获得正确表达.结论 成功构建SARS病毒M蛋白片段真核表达载体,并在Vero细胞中获得正确表达,为进一步研究M蛋白的功能奠定了基础.     

Synthetic peptide studies on the severe acute respiratory syndrome (SARS) coronavirus spike glycoprotein: perspective for SARS vaccine development

BACKGROUND: The S (spike) protein of the etiologic coronavirus (CoV) agent of severe acute respiratory syndrome (SARS) plays a central role in mediating viral infection via receptor binding and membrane fusion between the virion and the host cell. We focused on using synthetic peptides for developing antibodies against SARS-CoV, which aimed to block viral invasion by eliciting an immune response specific to the native SARS-CoV S protein. METHODS: Six peptide sequences corresponding to the surface regions of SARS-CoV S protein were designed and investigated by use of combined bioinformatics and structural analysis. These synthetic peptides were used to immunize both rabbits and monkeys. Antisera collected 1 week after the second immunization were analyzed by ELISA and tested for antibody specificity against SARS-CoV by immunofluorescent confocal microscopy. RESULTS: Four of our six synthetic peptides (S2, S3, S5, and S6) elicited SARS-CoV-specific antibodies, of which S5 (residues 788-820) and S6 (residues 1002-1030) exhibited immunogenic responses similar to those found in a parallel investigation using truncated recombinant protein analogs of the SARS-CoV S protein. This suggested that our S5 and S6 peptides may represent two minimum biologically active sequences of the immunogenic regions of the SARS-CoV S protein. CONCLUSIONS: Synthetic peptides can elicit specific antibodies to SARS-CoV. The study provides insights for the future development of SARS vaccine via the synthetic-peptide-based approach.     

SARS coronavirus

SARS is a new type of infectious pneumonia that emerged in China in November 2002. It spread around the world through an outbreak in Hong Kong in March 2003. Patients were reported in 29 countries, and around 800 people died, although a majority of these cases were in Asian countries. Within a month after the virus that causes SARS was isolated, its entire genome sequence became available. The result is the acknowledgement of a novel coronavirus, now called SARS coronavirus (SCoV), that is different from other already known existing human and animal coronaviruses. Since a number of scientists from distinct fields have participated in the research on this emerging virus, a plenty of information has become available, and now SARS-CoV has become one of the best-studied members among coronaviruses. In this review, I would like describe on SCoV virology by comparison with other well-studied coronaviruses. It covers the taxonomy, structure of virion, viral genome and proteins, unique replication strategy, receptors and viral pathogenesis. In the last part, I mention with my personal speculation about the origin and evolution of SCoV.     

Synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of SARS-associated coronavirus

A novel severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. We previously isolated and characterized SARS-CoV and SARS-CoV-like viruses from human and animals, respectively, suggesting that SARS could be transmitted from wild/farmed animals to humans. Comparison of the viral genomes indicated that sequence variation between animal and human isolates existed mainly in the spike (S) gene. We hypothesized that these variations may underlie a change of binding specificity of the S protein to the host cells, permitting viral transmission from animals to humans. Here we report that four 20-mer synthetic peptides (S protein fragments), designed to span these sequence variation hotspots, exhibited significant antiviral activities in a cell line. SARS-CoV infectivity was reduced over 10 000-fold through pre-incubation with two of these peptides, while it was completely inhibited in the presence of three peptides. Molecular modelling of the SARS-CoV peplomer suggests that three of these antiviral peptides map to the interfaces between the three monomers of the trimeric peplomer rather than the heptad repeat region from which short peptides are known to inhibit viral entry. Our results revealed novel regions in the spike protein that can be targeted to inhibit viral infection. The peptides identified in this study could be further developed into antiviral drugs.     

SARS康复者及医护人员冠状病毒的初步调查

目的　采用荧光聚合酶链反应 (F PCR)和基因芯片技术检测严重急性呼吸综合征 (SARS)冠状病毒 ,并探讨其临床应用价值。方法　应用 SARS Co V F PCR诊断试剂盒及基因芯片技术检测了 6 0份确诊 SARS患者血清、发热门诊医护人员血清 2 0份和漱口液样本 2 0份以及 1份 SARS疑似患者血清的 c DNA。结果　中山大学达安基因股份有限公司及上海复星实业股份有限公司的两种 F PCR检测试剂盒及晶宇芯片反应 3种检测方法所测 80份血清和 2 0份痰液样本均为阴性 ;但 1例 SARS疑似患者血清的 c DNA可经荧光定量 PCR反应扩增出病毒特异 RNA片段。结论　 SARS康复者及密切接触医护人员血液和漱口水中均未检测到 SARS病毒特异 RNA片段。     

Potential antivirals and antiviral strategies against SARS coronavirus infections

There are a number of antivirals as well as antiviral strategies that could be envisaged to prevent or treat severe acute respiratory syndrome (SARS) (or similar) coronavirus (CoV) infections. Targets for the prophylactic or therapeutic interventions include interaction of the spike (S) glycoprotein (S1 domain) with the host cell receptor, fusion of the S2 domain with the host cell membrane, processing of the replicase polyproteins by the virus-encoded proteases (3C-like cysteine protease [3CLpro] and papain-like cysteine protease) and other virus-encoded enzymes such as the NTPase/helicase and RNA-dependent RNA polymerase. Human monoclonal antibody blocking S1 may play an important role in the immunoprophylaxis of SARS. Fusion inhibitors reminiscent of enfuvirtide in the case of HIV may also be developed for SARS-CoV. Various peptidomimetic and nonpeptidic inhibitors of 3CLpro have been described, the best ones inhibiting SARS-CoV replication with a selectivity index greater than 1000. Human interferons, in particular alpha- and beta-interferon, as well as short interfering RNAs could further be pursued for the control of SARS. Various other compounds, often with an ill-defined mode of action but selectivity indexes up to 100, have been reported to exhibit in vitro activity against SARS-CoV: valinomycin, glycopeptide antibiotics, plant lectins, hesperetin, glycyrrhizin, aurintricarboxylic acid, chloroquine, niclosamide, nelfinavir and calpain inhibitors.     

Potential antivirals and antiviral strategies against SARS coronavirus infections

There are a number of antivirals as well as antiviral strategies that could be envisaged to prevent or treat severe acute respiratory syndrome (SARS) (or similar) coronavirus (CoV) infections. Targets for the prophylactic or therapeutic interventions include interaction of the spike (S) glycoprotein (S1 domain) with the host cell receptor, fusion of the S2 domain with the host cell membrane, processing of the replicase polyproteins by the virus-encoded proteases (3C-like cysteine protease [3CLpro] and papain-like cysteine protease) and other virus-encoded enzymes such as the NTPase/helicase and RNA-dependent RNA polymerase. Human monoclonal antibody blocking S1 may play an important role in the immunoprophylaxis of SARS. Fusion inhibitors reminiscent of enfuvirtide in the case of HIV may also be developed for SARS-CoV. Various peptidomimetic and nonpeptidic inhibitors of 3CLpro have been described, the best ones inhibiting SARS-CoV replication with a selectivity index greater than 1000. Human interferons, in particular α- and β-interferon, as well as short interfering RNAs could further be pursued for the control of SARS. Various other compounds, often with an ill-defined mode of action but selectivity indexes up to 100, have been reported to exhibit in vitro activity against SARS-CoV: valinomycin, glycopeptide antibiotics, plant lectins, hesperetin, glycyrrhizin, aurintricarboxylic acid, chloroquine, niclosamide, nelfinavir and calpain inhibitors.     

Indomethacin has a potent antiviral activity against SARS coronavirus

Severe acute respiratory syndrome (SARS) is a newly emerging, highly transmissible and fatal disease caused by a previously unknown coronavirus (SARS-CoV). Existing in non-identified animal reservoirs, SARS-CoV continues to represent a threat to humans because there is no effective specific antiviral therapy for coronavirus infections. OBJECTIVES: Starting from the observation that cyclopentenone cyclooxygenase (COX) metabolites are active against several RNA viruses, we investigated the effect of the COX inhibitor indomethacin on coronavirus replication. METHODS: Work involving infectious SARS-CoV was performed in biosafety level 3 facilities. SARS-CoV was grown in monkey VERO cells and human lung epithelial A549 cells, while canine coronavirus (CCoV) was grown in A72 canine cells. Antiviral activity was analysed by determining infective virus titres by TCID50, viral RNA synthesis by Northern blot analysis and real-time RT-PCR, and viral protein synthesis by SDS-PAGE analysis after 35S-methionine-labelling. Antiviral efficacy in vivo was determined by evaluating virus titres in CCoV-infected dogs treated orally with 1 mg/kg body weight indomethacin (INDO). RESULTS: Unexpectedly, we found that INDO has a potent direct antiviral activity against the coronaviruses SARS-CoV and CCoV. INDO does not affect coronavirus binding or entry into host cells, but acts by blocking viral RNA synthesis at cytoprotective doses. This effect is independent of cyclooxygenase inhibition. INDO's potent antiviral activity (>1,000-fold reduction in virus yield) was confirmed in vivo in CCoV-infected dogs. CONCLUSIONS: The results identify INDO as a potent inhibitor of coronavirus replication and suggest that, having both anti-inflammatory and antiviral activity, INDO could be beneficial in SARS therapy.