二肽基肽酶4为人类中东呼吸道综合征冠状病毒(MERS-CoV)功能性细胞受体

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.

MERS-CoV调控非小细胞肺癌细胞A549补体系统抑制因子C4BPA的机制

目的探讨关于中东呼吸综合征-冠状病毒(middle east respiratory syndrome-coronavirus,MERS-CoV)调控非小细胞肺癌细胞A549补体系统抑制因子补体成分4结合蛋白Alpha(complement component 4 binding protein alpha,C4BPA)的机制。方法以是否MERS-CoV感染非小细胞肺癌细胞A549分为感染组和对照组,通过酶联免疫吸附试验(ELISA)、免疫印迹和实时荧光定量PCR(qPCR)分别检测2组细胞C4BPA的蛋白和mRNA表达情况。TargetScanHuman预测与C4BPA结合的miRNA,实时荧光定量PCR和荧光素酶报告试验确定靶向C4BPA的miRNA。在MERS-CoV感染的情况下,过表达或敲低靶向C4BPA的miRNA,ELISA或qPCR检测C4BPA的表达和分泌情况。同时分析人肺成纤维细胞和小鼠感染MERS-CoV后,C4BPA和miR-451b的表达情况。结果非小细胞肺癌细胞A549感染MERS-CoV后,其分泌的C4BPA增加(P<0.05)。通过TargetScanHuman在线分析和qPCR发现MERS-CoV抑制非小细胞肺癌细胞A549 miR-451b的表达(P<0.05),并且miR-451b靶向C4BPA的3‘UTR(P<0.05)。过表达miR-451b后,C4BPA的表达和分泌受到抑制(P<0.05)。敲低miR-451b后,C4BPA的表达和分泌受到促进(P<0.05)。同时,MERS-CoV感染人肺成纤维细胞和小鼠后,miR-451b的表达受到抑制(P<0.05),C4BPA的表达增强。并且,MERSCoV感染引起的C4BPA增多能够抑制补体经典系统的溶血活性。结论MERS-CoV通过下调miR-451b促进C4BPA的分泌。

Molecular Characteristics,Functions,and Related Pathogenicity of MERS-CoV Proteins

Middle East respiratory syndrome(MERS)is a viral respiratory disease caused by a de novo coronavirus—MERS-CoV—that is associated with high mortality.However,the mechanism by which MERS-CoV infects humans remains unclear.To date,there is no effective vaccine or antibody for human immunity and treatment,other than the safety and tolerability of the fully human polyclonal Immunoglobulin G(IgG)antibody(SAB-301)as a putative therapeutic agent specific for MERS.Although rapid diagnostic and public health measures are currently being implemented,new cases of MERS-CoV infection are still being reported.Therefore,various effective measures should be taken to prevent the serious impact of similar epidemics in the future.Further investigation of the epidemiology and pathogenesis of the virus,as well as the development of effective therapeutic and prophylactic anti-MERS-CoV infections,is necessary.For this purpose,detailed information on MERS-CoV proteins is needed.In this review,we describe the major structural and nonstructural proteins of MERS-CoV and summarize different potential strategies for limiting the outbreak of MERS-CoV.The combination of computational biology and virology can accelerate the advanced design and development of effective peptide therapeutics against MERS-CoV.In summary,this review provides important information about the progress of the elimination of MERS,from prevention to treatment.

Newcastle disease virus-based MERS-CoV candidate vaccine elicits high-level and lasting neutralizing antibodies in Bactrian camels

Middle East respiratory syndrome coronavirus （MERS-CoV）, a member of the Coronavifidae family, is the causative pathogen for MERS that is characterized by high fever, pneumonia, acute respiratory distress syndrome （ARDS）, as well as extrapul- monary manifestations. Currently, there are no approved treatment regimens or vaccines for MERS. Here~ we generated recombinant nonvirulent Newcastle disease virus （NDV） LaSota strain expressing MERS-CoV S protein （designated as rLa- MERS-S）, and evaluated its immunogenicity in mice and Bactrian camels. The results revealed that rLa-MERS-S showed similar growth properties to those of LaSota in embryonated chicken eggs, while animal immunization studies showed that rLa-MERS-S induced MERS-CoV neutralizing antibodies in mice and camels. Our findings suggest that recombinant rLa- MERS-S may be a potential MERS-CoV veterinary vaccine candidate for camels and other animals affected by MERS.

SARS-CoV、MERS-CoV、SARS-CoV-2的基因结构特点与致病机制

高致病性冠状病毒具有高发病率、高病死率以及全球大流行等特点,严重威胁人类健康,给社会经济带来巨大挑战。目前关于高致病冠状病毒的致病机制,主要从直接细胞病变效应和间接免疫病理损伤两方面进行讨论,但具体作用基因的研究尚需进一步深入。本文从高致病性冠状病毒的基因结构特点与致病机制的相关性进行综述,为预防和治疗高致病性冠状病毒感染提供参考。

Diabetes and coronavirus infections (SARS-CoV, MERS-CoV, and SARS-CoV-2)

Diabetes,as the major cause of hyperglycemia,is a chronic metabolic disease.Immune system disorders caused by diabetes can increase the risk of respiratory diseases.Thus,diabetes is considered to be a major risk factor for viral respiratory infections such as coronavirus infections.Coronaviruses are members of the Coronaviridae,which has caused three outbreaks from 2003 to 2020.Patients with coronavirus infection in the lower and upper respiratory tract could show mild to severe symptoms.In this review,we focus on the relationship between diabetes and three coronaviruses:SARS-CoV,MERS-CoV,and SARS-CoV-2.

Coronaviruses in pregnant women in Saudi Arabia:A systematic comparative review of MERS-CoV and SARS-CoV-2

Objective:To compare the effects of coronaviruses,such as Middle East respiratory syndrome coronavirus(MERS-CoV)or coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus-2(SARS-CoV-2),on pregnant women in Saudi Arabia,and to understand the disease dynamics of these coronaviruses so as to konw how to provide care and management of infected pregnant women and infants.Methods:We conducted a systematic review of all published papers on MERS-CoV and SARS-CoV-2 infections in pregnant women in Saudi Arabia to identify knowledge gaps.Inclusion and exclusion criteria were based on the PRISMA system.The search included all papers which were published between September 1,2012 and November 29,2020 on pregnant women with MERS-CoV and/or COVID-19 in Saudi Arabia.Of 26 papers screened,five were included in the analysis.Results:A total of 11 pregnant women with MERS-CoV and four with COVID-19 were reported in Saudi Arabia in the medical literature during the review period.The mean ages of the women were 33 and 31 years old for MERS-CoV and COVID-19,respectively.Maternal and fetal mortality in cases of MERSCoV were 35%and 30%,respectively,while no maternal or fetal mortalities were reported in COVID-19 cases.Conclusions:Very limited data has emerged from Saudi Arabia on pregnant women MERS-CoV and COVID-19.With such high mortality observed with MERS-CoV compared to COVID-19,there is a need for greater reporting of cases to truly grasp the extent of these infections in pregnant women in a country where both coronaviruses are circulating.

Convalescent plasma as a therapeutic agent for SARS-CoV, MERS-CoV and SARS-CoV-2: A scoping review

Severe acute respiratory syndrome coronavirus(SARS-CoV),Middle East respiratory syndrome coronavirus(MERS-CoV),and SARS-CoV-2 are three kinds of coronaviruses that are exceptionally pathogenic to humans via zoonotic infections.The outbreaks of SARS-CoV and MERS-CoV,and SARS-CoV-2,to some extent,posed a severe threat to human health,daily activities as well as the economic status of many countries.When faced with these emerging viruses and no accessible vaccines and drugs,convalescent plasma(CP)is required as passive immunotherapy,since CP has the potential to neutralize and eliminate the virus from blood circulation.The sources of CP are individuals who have recovered from the viruses.Currently,CP is administered as emergency use and investigational treatment.Some studies have shown that CP is effective to treat infected individuals with viral pandemics such as influenza A,Ebola virus,SARS-CoV,and MERS-CoV.Moreover,following the deadly outbreak of SARS-CoV-2 in 2019,plenty of non-randomized clinical studies have been done on the effectiveness of CP for the treatment of Coronavirus Disease 2019(COVID-19),and most of these studies have indicated that CP therapy is promising and saved many critically-ill patients.Therefore,CP is a helpful immune therapeutic agent for the immediate response of such pandemics because of its clinical efficacy,immediate availability,cost-effectiveness,ease of production,delivery,and storage.This review aims to summarize the effectiveness of CP in the treatment of these three coronaviruses,i.e.SARS-CoV,MERS-CoV,and SARS-CoV-2.

Spanish Flu, SARS, MERS-CoV by CO₂Emission and Maximal Sunspot Number

The 1918 Spanish flu, 2002 severe acute respiratory syndrome (SARS), and 2012 Middle East respiratory syndrome coronavirus (MERS-CoV) were investigated to reveal their causes and routes of transmission. They have the common features such as 1) the induction by viruses (Spanish flu;avian influenza virus (AIV), SARS and MERS-CoV;coronavirus), 2) the outbreak during the maximal sunspot number, 3) the aeolian desert dust region (Spanish flu;Saharan, SARS;Asian, MERS-CoV;Arabian), and 4) similar incubation period (AIV;5, SARS;2 - 7, MERS-CoV;5 days), 5) different transmission reservoir (Spanish flu;aquatic bird/swine, SARS;bat, MERS-CoV;bat/dromedary camel). When carbon dioxide (CO2) combustion emissions were simultaneously high at the maximal sunspot number, UV radiation in the Poles was so extensive to mutate the aquatic virus through the food web to be the fundamental reason for these pandemic. Guangdong Province and Hong Kong in China are the source of 2002-2003 SARS. The stranded dead whales and dolphins along the coast of the Persian Gulf might be fed on by coastal animals in the Arabian Desert to transmit MERS-CoV. Mutations in the hot temperature in the Arabian Desert and Persian Gulf, and the maximal sunspot numbers were observed in Saudi Arabia. Saudi crude burn in power plants since 2009 with coincidental outbreaks of MERS-CoV in Saudi Arabia since 2012 peaking in 2014. The reduction of CO2 emissions by nuclear power plants is a unique solution to decrease MERS-CoV outbreaks.

A Paucity of Detection of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Nucleic Acids in the Vaginal Secretions and the Urine of Some Naturally Infected Dromedary Camels

The dromedary camels play some critical roles in the evolution and transmission cycle of the MERS-CoV. There are no current available vaccines or antiviral medications to do prevention or to treat humans against the virus, respectively. Few studies reported the course of the MERS-CoV infection in dromedary camels under both the experiment and natural infection approaches. The curve of the viral particle, as well as its nucleic acids in nasal secretions of the dromedary camels, was reported in more details. However, MERS-CoV shedding in some other secretions such as vaginal secretions still understudied. The main goal of the current study was to check the possibility of MERS-CoV shedding of the MERS-CoV through the vaginal mucous membranes of some female camels. To achieve these goals, we conducted molecular surveillance on one dromedary camel herd in eastern Saudi Arabia. We collected the following swabs per each animal (nasal, rectal, and vaginal) as well as urine samples during the period from Jan 2020-April 2020. Testing of MERS-CoV nucleic acid (NA) was conducted in these swabs by the real-time PCR kits. Our results are showing that (47%, 21%, 0%, and 0%) of the (nasal, rectal, and vaginal) swabs, as well as the urine, were positive, respectively. All the tested vaginal swabs were negative. This may suggest the absence of MERS-CoV shedding in the female genital tract and urine samples despite the detection of the viral NA in the nasal swabs. This study is showing for the first time the failure of detection of the MERS-CoV in the vaginal secretions of some naturally infected dromedary camels. This result is enriching our understanding of the MERS-CoV shedding in various body secretions and excretions.

MERS-CoV S1蛋白的表达及鉴定

目的真核表达具有天然构象的中东呼吸综合征冠状病毒(MERS-CoV)S1蛋白,并进行免疫反应性鉴定。方法以pcDNA3.1-S重组质粒为模板,PCR扩增S1基因,连接至pFastBac1质粒,构建重组质粒pFB1-S1。将pFB1-S1转化至DH10Bac感受态中,构建重组杆粒BpFB1-S1,然后转染sf9细胞获得重组杆状病毒rpFB1-S1并连续传代。将第四代rpFB1-S1接种sf9细胞后悬浮培养96h,取上清,用PEG8000浓缩并进行免疫亲和层析纯化,采用SDS-PAGE和Western blot鉴定其分子质量及免疫反应性;另取感染rpFB1-S1 48h的sf9细胞进行IFA鉴定。结果 S1基因PCR扩增产物大小为2 212bp,与预期相符。重组质粒pFB1-S1经酶切和测序分析鉴定构建正确。重组杆粒BpFB1-S1经PCR鉴定构建正确。IFA试验可见感染rpFB1-S1的sf9细胞出现特异性绿色荧光,即有目的蛋白表达。SDSPAGE显示纯化的重组S1为单一100×103(Mr)蛋白条带,Western blot检测该蛋白能被鼠抗S-RBD蛋白单克隆抗体识别。结论成功表达了具有免疫反应性的MERS-CoV S1蛋白,为MERS-CoV快速诊断方法的建立及候选疫苗的构建奠定了基础。

基于受体结合区(RBD)的ELISA在MERS-CoV抗体检测中的应用评价

目的比较中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)不同形式受体结合区(receptor binding domain,RBD)蛋白在抗体检测中的应用特点.方法以293T和ExpiCHO-STM系统表达并纯化制备MERS-CoV RBD,采用非变性凝胶电泳和Western blot对纯化RBD蛋白进行鉴定.然后以等量的上述RBD蛋白或昆虫细胞表达的RBD作为包被抗原,建立间接酶联免疫检测方法(enzyme-linked immune sorbent assay,ELISA),用于MERS-CoV IgG抗体检测.同时以商品化的欧蒙MERS-CoV IgG抗体检测试剂盒(以S1为包被抗原)为对照,利用WHO提供的MERS-CoV抗体参比品对基于不同RBD蛋白抗原的ELISA方法进行应用效果比较分析.结果采用293T和ExpiCHO-STM系统成功制备MERS-CoV RBD单体/聚体抗原;不同来源与形式的RBD抗原均能特异识别MERS-CoV特异抗体,与正常成人血清无交叉反应;基于RBD所建立的ELISA应用于MERS-CoV抗体参比品检测,与欧蒙商品化试剂盒检测结果具有较好的一致性,但RBD-T4f、RBD-GCN4聚体RBD检测阳性标本的读值高且较集中,单体/二聚体RBD(昆虫细胞或哺乳动物细胞)阳性标本的读值低且较分散.结论建立了基于MERS-CoV不同单体与聚体RBD蛋白抗原的间接ELISA检测方法,可替代欧蒙商品化试剂盒用于临床检测MERS-CoV特异IgG抗体.RBD-T4f、RBD-GCN4包被抗原的应用结果优于RBD抗原.

MERS-CoV棘突蛋白主要特性及B/T细胞抗原表位预测分析

目的应用生物信息学方法分析中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)棘突蛋白(Spike,S)的主要特性,预测可能的B/T细胞抗原表位。结果 MERS-CoV的S蛋白氨基酸序列基本保守,具有多个糖基化、酰胺化及磷酸化翻译后修饰位点。综合各种单参数预测MERS-CoV S蛋白可能存在4个B细胞抗原表位、7个CTL细胞表位和5个Th细胞表位。方法以MERS-CoV S蛋白的氨基酸序列一级结构为基础分析其保守性,采用ProtParam预测S蛋白的理化特性,SOPMA预测其二级结构,MotifScan预测翻译后修饰位点,DNAStar分析其序列的亲水性指数、柔韧性指数、可及性参数以及抗原指数,推测B细胞抗原表位的可能位置,采用SYFPEITHI的T细胞表位预测工具预测CTL和Th细胞表位。结论生物信息学方法预测MERS-CoV S蛋白氨基酸保守序列,该蛋白可能含有B/T细胞抗原表位,为MERS-CoV疫苗研制及其他相关研究奠定了基础。

人类高致病性冠状病毒SARS-CoV和MERS-CoV的流行与突变--共性与个性特征的启示

人类高致病性冠状病毒目前主要包括严重急性呼吸道综合征冠状病毒(SARS-Co V)和中东呼吸综合征冠状病毒(MERS-Co V),其广泛的流行、较强的致病性,给全球的经济造成了较大损失,也给人类的健康带来了严重威胁。虽然SARS-Co V和MERS-Co V都属于动物源性病毒,但其流行与突变中都存在各自的特征。现着重就SARS-Co V与MERS-Co V的病原学、流行及基因突变等方面进行对比综述,以分析揭示高致病性冠状病毒在以上方面的共性和个性特征,为未来疫情的监测和防控提供新的思路。

真核表达MERS-CoV刺突蛋白亚单位的信号肽序列优化研究

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-S^(TM)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-S^(TM)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。

The structural and accessory proteins M,ORF 4a,ORF 4b,and ORF 5 of Middle East respiratory syndrome coronavirus(MERS-CoV)are potent interferon antagonists

The newly emerged Middle East respiratory syndrome coronavirus(MERS-CoV)is a highly pathogenic respira-tory virus with pathogenic mechanisms that may be driven by innate immune pathways.The goal of this study is to characterize the expression of the structural(S,E,M,N)and accessory(ORF 3,ORF 4a,ORF 4b,ORF 5)proteins of MERS-CoV and to determine whether any of these pro-teins acts as an interferon antagonist.Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells,and their native expression and subcellular localiza-tion were assessed using Wes tern blotting and indirect immunofl uorescence.While ORF 4b demonstrated majorly nuclear localization,all of the other proteins demonstrated cytoplasmic localization.In addition,for the fi rst time,our experiments revealed that the M,ORF 4a,ORF 4b,and ORF 5 proteins are potent interferon antagonists.Further exami-nation revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production(IFN-βpromoter activity,IRF-3/7 and NF-κB activation)and ISRE promoter element signaling pathways.Together,our re-sults provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.

Molecular aspects of MERS-CoV

Middle East respiratory syndrome coronavirus （MERS-CoV） is a betacoronavirus which can cause acute respiratory distress in humans and is associated with a relatively high mortality rate. Since it was first identified in a patient who died in a Jeddah hospital in 2012, the World Health Organization has been notified of 1735 laboratory-confirmed cases from 27 countries, including 628 deaths. Most cases have occurred in Saudi Arabia. MERS-CoV ancestors may be found in Old World bats of the Vespertilionidae family. After a proposed bat to camel switching event, transmission of MERS-CoV to humans is likely to have been the result of multiple zoonotic transfers from dromedary camels. Human-to-human transmission appears to require close contact with infected persons, with outbreaks mainly occurring in hospital environments. Outbreaks have been associated with inadequate infection prevention and control implementation, resulting in recommendations on basic and more advanced infection prevention and control measures by the World Health Organization, and issuing of government guidelines based on these recommendations in affected countries including Saudi Arabia. Evolutionary changes in the virus, particularly in the viral spike protein which mediates virus-host cell contact may potentially increase transmission of this virus. Efforts are on-going to identify specific evidence-based therapies or vaccines. The broad-spectrum antiviral nitazoxanide has been shown to have in vitro activity against MERS-CoV. Synthetic peptides and candidate vaccines based on regions of the spike protein have shown promise in rodent and non-human primate models. GLS-5300, a prophylactic DNA-plasmid vaccine encoding S protein, is the first MERS-CoV vaccine to be tested in humans, while monoclonal antibody, m336 has given promising results in animal models and has potential for use in outbreak situations.

SARS-COV和MERS-COV以及SARS-COV-2的研究进展

β-冠状病毒(β-coronavirus,β-COV)是一类可导致人类呼吸道感染的相关病毒,在自然界中广泛存在,并可对人或动物造成严重危害。该病毒感染具有传染性高、流行性广、突发性强且无有效预防用药等特点,对世界各国及其公民造成较大的医疗负担。本文主要对严重急性呼吸综合征冠状病毒(severe acute respiratory syndrome coronavirus,SARS-COV)和中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-COV),以及2019年12月以来的新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-COV-2)的流行概况、传播途径、致病性及致病机制、药物与疫苗等最新进展进行综述,旨在为更好地防控高致病性人冠状病毒提供依据。

Genetic Evidence of Middle East Respiratory Syndrome Coronavirus(MERS-Cov) and Widespread Seroprevalence among Camels in Kenya

We describe the first genome isolation of Middle East respiratory syndrome coronavirus(MERS-CoV) in Kenya. This fatal zoonotic pathogen was first described in the Kingdom of Saudi Arabia in 2012. Epidemiological and molecular evidence revealed zoonotic transmission from camels to humans and between humans. Currently, MERS-CoV is classified by the WHO as having high pandemic potential requiring greater surveillance. Previous studies of MERS-CoV in Kenya mainly focused on site-specific and archived camel and human serum samples for antibodies. We conducted active nationwide cross-sectional surveillance of camels and humans in Kenya, targeting both nasal swabs and plasma samples from 1,163 camels and 486 humans collected from January 2016 to June 2018. A total of 792 camel plasma samples were positive by ELISA. Seroprevalence increased with age, and the highest prevalence was observed in adult camels(82.37%, 95%confidence interval(CI) 79.50–84.91). More female camels were significantly seropositive(74.28%, 95% CI 71.14–77.19)than male camels(P \ 0.001)(53.74%, 95% CI 48.48–58.90). Only 11 camel nasal swabs were positive for MERS-CoV by reverse transcription-quantitative PCR. Phylogenetic analysis of whole genome sequences showed that Kenyan MERSCoV clustered within sub-clade C2, which is associated with the African clade, but did not contain signature deletions of orf4 b in African viruses. None of the human plasma screened contained neutralizing antibodies against MERS-CoV. This study confirms the geographically widespread occurrence of MERS-CoV in Kenyan camels. Further one-health surveillance approaches in camels, wildlife, and human populations are needed.

MERS-CoV E蛋白诱导293T细胞自噬的研究

目的本研究旨在探讨中东呼吸综合征冠状病毒包膜蛋白(MERS-CoV E蛋白)诱导293T细胞自噬其及作用机制。方法构建表达载体pEGFP-E、pEGFP-ERT、pEGFP-ORF5-E,分别将其转染293T细胞,采用荧光显微镜和Western blot方法检测目的蛋白的表达,采用Western blot方法检测LC3的表达量,采用Real-time PCR(RT-PCR)和Western blot方法检测自噬相关基因Atg4,Atg5,Atg7,P62,PI3KC3的表达情况。结果成功构建pEGFP-E、pEGFP-ERT、pEGFP-ORF5-E表达载体,在293T细胞中可检测到目的蛋白的表达。转染后24 h,与阴性对照组相比,pEGFP-E实验组LC3 II的表达量显著高于转染pEGFP-N1阴性对照组(P<0.05),但转染pEGFP-E的实验组自噬相关基因(P62、PI3KC3、Atg4、Atg5、Atg7)的mRNA水平以及相应蛋白的表达量与阴性对照组相比,差异无统计学意义(均P>0.05),这表明在293T细胞中过表达MERS-CoV E蛋白能增加LC3 II的表达,但并不引起自噬相关基因转录和相应蛋白表达水平的变化。结论MERS-CoV E蛋白可诱导293T细胞自噬,其机制不依赖于Atg蛋白。