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目的:为了避免中东呼吸综合征冠状病毒(MERS-CoV)感染与中和试验中操作活病毒带来的生物安全隐患,构建只具有一次感染能力而无复制能力的MERS假病毒,建立MERS假病毒系统,并应用于中和抗体检测。方法:构建含有MERS-CoV S基因的重组质粒pcDNA3.1-MERS-S,与缺失Env基因、含有萤光素酶报告基因的HIV-1骨架质粒pNL4-3.Luc.RE共转染293T细胞,收获含有假病毒的上清;通过Western印迹、细胞感染实验和血清中和试验,确定是否包装出MERS假病毒,及是否能有效应用于细胞感染与中和试验。结果:MERS假病毒pMERS-S培养上清经Western印迹鉴定出相对分子质量为25×103的HIV-1 P24蛋白和相对分子质量为180×103的MERS-CoV S蛋白;与阴性对照假病毒pEnv-相比,pMERS-S能有效感染MERS-CoV敏感细胞系Huh-7,在感染细胞中产生荧光信号,感染细胞的假病毒量与产生的荧光信号呈明显的量效关系;在MERS假病毒中和试验中,pMERS-S能被MERS-CoV中和抗体中和而失去感染力,反映抗体对MERS-CoV的中和活性。结论:建立了不依赖于BSL-3高等级生物安全条件的MERS假病毒系统,并有效应用于中和抗体检测,为MERS-CoV疫苗、药物评价及病毒致病机制研究提供了良好的技术支撑手段。  相似文献   

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Middle East respiratory syndrome coronavirus (MERS‐CoV) is a single‐stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS‐CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS‐CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS‐CoV. Using a recombinant full‐length S protein, an indirect ELISA was developed and found to detect MERS‐CoV‐specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS‐CoV S protein. Neither ELISA system exhibited significant intra‐assay or inter‐assay variation, indicating good reproducibility. Moreover, the inter‐day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS‐CoV.
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中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)的刺突蛋白(Spike,S)亚单位1(S1)是引起宿主免疫反应和产生中和抗体的主要靶抗原,也是疫苗研发和病原检测的重要靶标,选用适宜的真核表达系统高效表达S1蛋白是进行相关研究的基础。为确定MERS-CoV S1在哺乳动物细胞中高效分泌性表达的信号肽序列,构建了含高斯荧光素酶(Gaussia luciferase,GLuc)、人组织纤溶酶原激活剂(Tissue plasminogen activator,tPA)及小鼠免疫球蛋白G的2a亚型(Mouse immunoglobular G subtype 2a,MIgG2a)7个信号肽(原始序列和改造序列)序列的MERS-CoV S1表达质粒,瞬时转染细胞后,通过Western Blot检测并比较细胞培养上清和裂解液中S1的表达水平及分泌表达效率(条带密度灰度扫描比),并对哺乳动物细胞表达的S1蛋白的纯度与抗原特性进行了分析。结果表明7种信号肽在293T、BHK21和ExpiCHO-STM三种细胞系统中介导MERS-CoV S1的高效分泌表达的效率各有不同,其中tPA-1信号肽介导S1抗原在ExpiCHO-STM中具有较高的分泌表达效率与产量,纯化的S1蛋白保持了较好的抗原性。本研究为进一步研发基于MERS-CoV S1的亚单位疫苗及免疫学检测试剂奠定了基础。  相似文献   

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中东呼吸道综合征冠状病毒(Middle East respiratory syndrome coronavirus, MERS-CoV)是继SARS冠状病毒(SARS-CoV)之后新近出现的又一种能够引发严重呼吸道感染的人类新发冠状病毒. MERS-CoV于2012年9月首次在中东一些国家被发现,截至2013年9月7日,MERS-CoV已经引起114例感染病例,其中54人死亡,死亡率约50%. 病毒受体研究为MERS-CoV等人类新发冠状病毒进化和跨种传播机制提供重要依据.最近,Raj等在Nature发表文章,首次报道了二肽基肽酶4(dipeptidyl peptidase 4,DPP4;又名CD26)为MERS-CoV感染细胞的功能性受体.MERS-CoV功能性受体的发现为人类新冠状病毒溯源和跨种进化研究、病毒传染和流行病学特征分析以及抗病毒药物和疫苗研究提供重要基础.  相似文献   

5.
2019新型冠状病毒的暴发持续至今,导致了世界各地数以百万计的感染个例,更夺去了数十万人的生命.世界卫生组织在2020年2月将此病毒引起的疾病定名为2019冠状病毒病(Coronavirus disease 2019,COVID-19),而国际病毒分类委员会也将此病毒命名为SARS-CoV-2. COVID-19的典型...  相似文献   

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Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.  相似文献   

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can trigger a cytokine storm in the pulmonary tissue by releasing various types of mediators, leading to acute respiratory distress syndrome (ARDS). Increased neutrophil-to-lymphocyte ratio, as well as CD4+ T lymphopenia, is reported in cases with novel coronavirus disease (COVID-19), meanwhile, lymphopenia is a significant finding in the majority of COVID-19 cases with a severe phenotype. Moreover, excessive activation of monocyte/macrophage and cytokine storms are associated with the severity of the disease and the related complications in SARS-CoV-2 infection. Understanding the immune response dysregulation in COVID-19 is essential to develop more effective diagnostic, therapeutic, and prophylactic strategies in this pandemic.  相似文献   

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Middle East respiratory syndrome coronavirus (MERS‐CoV) is a human pathogen responsible for a severe respiratory illness that emerged in 2012. Structural information about the proteins that constitute the viral particle is scarce. In order to contribute to a better understanding of the nucleoprotein (N) in charge of RNA genome encapsidation, the structure of the C‐terminal domain of N from MERS‐CoV obtained using single‐crystal X‐ray diffraction is reported here at 1.97 Å resolution. The molecule is present as a dimer in the crystal structure and this oligomerization state is confirmed in solution, as measured by additional methods including small‐angle X‐ray scattering measurements. Comparisons with the structures of the C‐terminal domains of N from other coronaviruses reveals a high degree of structural conservation despite low sequence conservation, and differences in electrostatic potential at the surface of the protein.  相似文献   

10.
严重急性呼吸综合征2019(sever acute respiratory syndrome,SARS)、中东呼吸综合征(Middle East respiratory syndrome,MERS)和2019冠状病毒病(corona virus disease 2019,COVID-19)对全世界人民造成了严重的经济损...  相似文献   

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Ⅰ型膜融合蛋白(class I membrane fusion protein)在Ⅰ型包膜病毒入侵宿主细胞过程中发挥重要作用。基于抑制六螺旋结构形成的多肽类融合抑制剂设计的原理是模拟该蛋白融合区域的自身序列,与病毒融合蛋白结合形成异源六聚体,从而阻断病毒与靶细胞膜的融合。此类融合抑制剂的传统设计主要基于一级与二级结构,但为进一步强化其抗病毒活性,通常需依赖病毒膜融合蛋白三级结构信息,从而限制了对那些尚无病毒蛋白三级结构信息的新发病毒的多肽类融合抑制剂的快速优化和研发。本研究提出了不依赖蛋白三级结构信息,而利用I-Mutant2.0软件来辅助设计和优化多肽类病毒膜融合抑制剂的设想。根据I-Mutant2.0的预测结果,以中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)为模型,分析该病毒HR2区融合抑制剂序列中若干适合与不适合优化的位点,并设计了一系列多肽。结果发现,对适合优化的位点进行调整的多肽,其对HR1的结合能力及对病毒的抑制活性均有所提升;反之,多肽活性明显下降。结果表明,利用I-Mutant2.0辅助设计与优化病毒融合抑制多肽的方法具有一定的可行性,为进一步开发新的融合抑制剂设计方法奠定了基础。  相似文献   

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近二十多年,全球范围内先后爆发了由严重急性呼吸综合征冠状病毒(severe acute respiratory syndrome coronavirus,SARS-CoV)、中东呼吸综合征冠状病毒(middle east respiratory syndrome coronavirus,MERS-CoV)和严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)3种高致病性冠状病毒导致的疫情。这3种高致病性冠状病毒感染通常伴随着免疫系统功能失调,临床表现有淋巴细胞减少症、细胞因子风暴、急性呼吸系统窘迫综合征,甚至多器官衰竭而导致死亡。揭示高致病性冠状病毒在免疫应答中的作用机制,对于预防与控制冠状病毒感染具有重要意义。本文总结了SARS-CoV、MRES-CoV和SARS-CoV-2的进入机制和受体特征、固有免疫应答和适应性免疫应答失调方面的研究进展,强调了高致病性冠状病毒与宿主免疫应答之间的复杂相互作用,以期为防治冠状病毒感染提供参考。  相似文献   

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A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing the S protein from SL-CoV(rAd-Rp3-S)to investigate its immune characterization.Our results showed that immunized mice generated strong humoral immune responses against the SL-CoV S protein.Moreover,a strong cellular immune response demonstrated by elevated IFN-γand IL-6 levels was also observed in these mice.However,the induced antibody from these mice had weaker cross-reaction with the SARS-CoV S protein,and did not neutralize HIV pseudotyped with SARS-CoV S protein.These results demonstrated that the immunogenicity of the SL-CoV S protein is distinct from that of SARS-CoV,which may cause the immunological differences between human SARS-CoV and bat SL-CoV.Furthermore,the recombinant virus could serve as a potential vaccine candidate against bat SL-CoV infection.  相似文献   

15.
A group of SARS-like coronaviruses(SL-CoV)have been identified in horseshoe bats.Despite SL-CoVs and SARS-CoV share identical genome structure and high-level sequence similarity,SL-CoV does not bind to the same cellular receptor as for SARS-CoV and the N-terminus of the S proteins only share 64%amino acid identity,suggesting there are fundamental differences between these two groups of coronaviruses.To gain insight into the basis of this difference,we established a recombinant adenovirus system expressing t...  相似文献   

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董慧君  李彤  庄辉  向宽辉 《微生物学报》2023,63(4):1329-1339
目前新型冠状病毒(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)感染所致的新型冠状病毒肺炎(corona virus disease, COVID-19)已成为威胁人类健康和安全的全球性流行性疾病。随着新突变株的不断出现,寻找有效治疗药物和靶点迫在眉睫。干扰素刺激基因(interferon-stimulated genes, ISGs)是由干扰素(interferons, IFNs)诱导后表达上调的一类基因,在宿主抵抗病毒感染过程中发挥着至关重要的作用。研究表明,ISGs能够靶向许多病毒复制的不同阶段发挥抗病毒作用,然而SARS-CoV-2也进化出各种策略干扰或逃避宿主天然免疫。因此,全面了解SARS-CoV-2与ISGs相互作用,对于设计抗病毒策略至关重要。本文简要综述不同ISGs抵抗SARS-CoV-2的作用机制,为开发新型的抗病毒药物提供思路和理论依据。  相似文献   

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Molecules expressed by the pathogenic ameobaEntamoeba histolytica but weakly expressed or absent from the non-pathogenic ameobaEntamoeba dispar could be used by intestinal epithelial cells to discriminate between the two species and to initiate an appropriate inflammatory response. Among the possible molecules involved in this identification are the Gal/GalNac lectin and the lipophosphoglycan. Once the inflammatory response is initiated,E. histolytica trophozoites have to protect themselves against reactive nitrogen intermediates produced by intestinal epithelial cells, oxygen intermediates, and cytotoxic molecules released by activated neutrophils. By screening theE. histolytica genome, we have identified proteins that may play a role in the defence strategy of the parasite. One of these proteins, a serine proteinase inhibitor, inhibits human neutrophil cathepsin G, a key component of the host defence.  相似文献   

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严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)自被发现以来就引起了人们的广泛关注,开发针对该病毒的安全、有效疫苗成为近期的研究热点。本文以高致病性冠状病毒疫苗(包括灭活疫苗、重组亚单位疫苗、重组病毒载体疫苗和核酸疫苗)的研究展开综述,为研制SARS-CoV-2疫苗提供参考。  相似文献   

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Autophagy-dependent cell death is a prominent mechanism that majorly contributes to homeostasis by maintaining the turnover of organelles under stressful conditions. Several viruses, including coronaviruses (CoVs), take advantage of cellular autophagy to facilitate their own replication. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-coronavirus (β-CoVs) that mediates its replication through a dependent or independent ATG5 pathway using specific double-membrane vesicles that can be considered as similar to autophagosomes. With due attention to several mutations in NSP6, a nonstructural protein with a positive regulatory effect on autophagosome formation, a potential correlation between SARS-CoV-2 pathogenesis mechanisms and autophagy can be expected. Certain medications, albeit limited in number, have been indicated to negatively regulate autophagy flux, potentially in a way similar to the inhibitory effect of β-CoVs on the process of autophagy. However, there is no conclusive evidence to support their direct antagonizing effect on CoVs. Off-target accumulation of a major fraction of FDA-approved autophagy modulating drugs may result in adverse effects. Therefore, medications that have modulatory effects on autophagy could be considered as potential lead compounds for the development of new treatments against this virus. This review discusses the role of autophagy/virophagy in controlling SARS-CoV-2, focusing on the potential therapeutic implications.  相似文献   

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