严重急性呼吸综合征（SARS）病毒的形态及其形态发生机制

将SARS患者的咽拭子感染VeroE6细胞 ,用电子显微技术等对SARS病毒进行了研究。结果表明 ,新分离到的病毒粒子没带囊膜时直径大多约 5 0nm ,带有囊膜的直径约 10 0nm。通过RT PCR等证明 ,该病毒是新的冠状病毒。这些病毒可与SARS康复患者的血清呈强烈的阳性反应 ,表明此新的冠状病毒是引起SARS的主要病原。文中还对病毒的发生机制和细胞中的分布进行了探讨。     

血中检测SARS冠状病毒N蛋白在SARS实验室早期诊断中的作用

为明确严重急性呼吸综合症(SARS)冠状病毒N蛋白在SARS实验室早期诊断中的作用,通过微量中和试验及酶联免疫方法、间接免疫荧光法检测疑似病人恢复期血清(大于28天)中SARS-IgG抗体,确诊SARS患者。同时收集发病不同时期SARS及普通发热病人血清,利用酶联免疫方法检测SARS-CoVN蛋白,并与荧光定量PCR早期诊断方法相比较。共检测：广州地区2003年12月～2004．年1月新发4例确诊SARS患者不同时期的血液和咽漱液标本;恢复期血清SARS-CoV中和抗体阳性病人不同时期的血清46份;广州地区2003年1月～4月临床确诊SARS患者159例的血清和56例疑似患者血清;非SARS普通发热病人血清97份;正常人体检血清100份。结果：4例新发SARS患者的不同时期标本中,3例患者急性期血均检出N蛋白,优于常用的荧光定量PCR检测方法。46份SARS-CoV中和抗体阳性的血清标本,N蛋白检出率为100％。159例临床确诊病例中,发病早期5天以内SARS-CoVN蛋白的检出率为92．3％,随后呈现逐步下降的趋势,在发病第18天仍可检出。56例临床疑似患者发病早期也有23．2％检出率。而97例普通发热病人及100份正常人血清中均未能检测出SARS-CoVN蛋白。表明在血清中检测SARS冠状病毒N蛋白的方法敏感性和特异性都好,对SARS实验室早期诊断具有重要作用。     

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 .     

冠状病毒是引起广州地区严重急性呼吸综合征(SARS)的主要病因

为查找引起广州地区流行的严重急性呼吸综合征(SARS)的病原体,采集患者漱口液及尸解标本,用组织培养法接种人胚肺细胞、MDCK细胞、Hep-2细胞和鸡胚分离病毒,用间接免疫荧光法检测患者恢复期血清lgG抗体,确定分离的病原是SARS的主要病因,再用套式RT—PCR、免疫电镜法鉴定病原。结果用人胚肺、Hep-2细胞在75份漱口液和3例尸解组织中分离出13株病原体,经套式RT—PCR扩增出110bp的特异产物,经测序证实为冠状病毒。制备冠状病毒的抗原,检测30份SARS病人恢复期血,其中26份血清lgG抗体阳性。同时检测30份普通发热病人血清作对照,IgG抗体全部阴性。由此证明,经组织培养分离到的病原体是引起SARS的致病因子,用分子生物学方法测序后证实为冠状病毒。     

Proteomic analysis of up-regulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus 3C-like protease

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS CoV) is an important issue for treatment and prevention of SARS. Previously, SARS CoV 3C-like protease (3CLpro) has been demonstrated to induce apoptosis via the activation of caspase-3 and caspase-9 (Lin, C. W., Lin, K. H., Hsieh, T. H., Shiu, S. Y. et al., FEMS Immunol. Med. Microbiol. 2006, 46, 375-380). In this study, proteome analysis of the human promonocyte HL-CZ cells expressing SARS CoV 3CLpro was performed using 2-DE and nanoscale capillary LC/ESI quadrupole-TOF MS. Functional classification of identified up-regulated proteins indicated that protein metabolism and modification, particularly in the ubiquitin proteasome pathway, was the main biological process occurring in SARS CoV 3CLpro-expressing cells. Thirty-six percent of identified up-regulated proteins were located in the mitochondria, including apoptosis-inducing factor, ATP synthase beta chain and cytochrome c oxidase. Interestingly, heat shock cognate 71-kDa protein (HSP70), which antagonizes apoptosis-inducing factor was shown to down-regulate and had a 5.29-fold decrease. In addition, confocal image analysis has shown release of mitochondrial apoptogenic apoptosis-inducing factor and cytochrome c into the cytosol. Our results revealed that SARS CoV 3CLpro could be considered to induce mitochondrial-mediated apoptosis. The study provides system-level insights into the interaction of SARS CoV 3CLpro with host cells, which will be helpful in elucidating the molecular basis of SARS CoV pathogenesis.     

猪繁殖和呼吸综合征病毒膜蛋白和核衣壳蛋白基因在大肠杆菌中的表达

以 Ｐ Ｒ Ｒ Ｓ Ｖ 弱毒株膜蛋白（ Ｍ） 和核衣壳（ Ｎ） 蛋白基因为模板,设计的一对含有 Ｅｃｏ Ｒ Ｉ 和 Ｂａｍ Ｈ Ｉ酶切位点的引物,通过 Ｒ Ｔ Ｐ Ｃ Ｒ 扩增出一约９００ ｂｐ 的 Ｍ Ｎ 基因片段,将此基因片段成功克隆于高效表达载体ｐ Ｂ Ｖ２２０ ,构建成重组质粒ｐ Ｂ Ｖ Ｍ Ｎ,导入大肠杆菌 Ｄ Ｈ５α,经温敏诱导,成功地表达了 Ｍ Ｎ 基因。表达产物经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 电泳和 Ｗｅｓｔｅｒｎ ｂｌｏｔ 印迹分析,其分子量约３４ ｋ Ｄ,与兔抗 Ｐ Ｒ Ｒ Ｓ Ｖ 高免血清发生反应,经光密度扫描分析,表达产物量占菌体总蛋白的１２ ％ 。该研究为 Ｐ Ｒ Ｒ Ｓ 基因诊断抗原的研制奠定了基础     

猪繁殖与呼吸综合征病毒(PRRSV)SD2株ORF5/ORF6双基因真核表达载体在哺乳动物细胞中的表达

为了实现猪繁殖与呼吸综合征病毒(PRRSV)的ORF5和ORF6基因在同一质粒中分别表达各自编码的蛋白,发挥E蛋白的病毒中和优势和M蛋白的细胞免疫优势,将构建成功的pIRES-ORF5/ORF6转移载体用脂质体法转入稳定表达的细胞CHO,经G418加压筛选获得具稳定表达的细胞株。以RT-PCR、SDS-PAGE、Western blot和间接免疫荧光检测目的蛋白的表达情况。结果表明:RT-PCR检测到两种目的基因的转录;SDS-PAGE和West-ern blot检测到同时表达的两种目的蛋白;间接免疫荧光检测到目的蛋白得到表达。     

猪繁殖与呼吸综合征病毒ORF5基因在昆虫细胞中的高效融合表达

对杆状病毒BactoBac表达系统的转座质粒pFastbac1进行改造,即在其多角体蛋白启动子下游插入谷胱苷肽S转移酶（glutathioneStransferase, GST）基因,构建GST融合表达转座质粒pFGST。通过转座和转染Sf9细胞,证实该系统能高水平表达GST。采用PCR方法从pMTgp51质粒中扩增截去N端信号肽序列的猪繁殖与呼吸综合征病毒（PRRSV）YA株ORF5基因,并将截短的ORF5基因片段克隆到pFGST中,使之与GST融合,构建的重组转座质粒pFGST53转染DH10Bac,提取大分子Bacmid DNA,转染Sf9细胞,获得能表达融合蛋白的高滴度重组病毒rvGST53。rvGST53感染Sf9细胞,SDSPAGE和Western印迹分析表明：与GST融合的ORF5基因在Sf9细胞中获得高效表达,表达产物分子量为45kD,能与抗PRRSV E蛋白单克隆抗体发生特异性反应。将表达产物免疫小白鼠,经间接免疫荧光检测,免疫血清能使PRRSV YA株感染的MARC145细胞呈较强的荧光着色,证实表达的融合蛋白具有良好的免疫原性。     

持续性血液滤过联合高流量吸氧治疗重症急性呼吸综合征的疗效及对血清炎症因子水平的影响

目的:探讨持续性血液滤过联合高流量吸氧治疗重症急性呼吸综合征的疗效及对血清炎症因子水平的影响。方法:选择2013年2月至2016年2月我院接诊的60例重症急性呼吸综合征患者,通过随机数表法将其分为观察组(n=30)和对照组(n=30)。观察组采用持续性血液滤过联合高流量吸氧进行治疗,对照组采用持续性血液滤过进行治疗。比较两组临床疗效、治疗前后动脉血氧分压(PaO_2)、动脉血二氧化碳分压(PaCO_2)、氧合指数、氢离子浓度指数(pH)值、呼吸频率(RR)、心率(HR)、血清C反应蛋白(CRP)、白介素6(IL-6)、白介素8(IL-8)、肿瘤坏死因子α(TNF-α)水平的变化及不良反应的发生情况。结果:治疗后,观察组有效率为76.67%,显著高于对照组(50.00%,P0.05)。两组治疗后PaO_2、PaCO_2、氧合指数、pH值、RR、HR均较治疗前明显改善,观察组患者PaO_2、氧合指数明显高于对照组,PaCO_2、pH值、RR、HR、血清CRP、IL-6、IL-8及TNF-α水平均显著低于对照组(P0.05)。治疗期间,观察组患者不良反应总发生率(10.00%)显著低于对照组(36.67%,P0.05);观察组死亡1例(3.33%),对照组死亡6例(20.00%),观察组病死率显著低于对照组(P0.05)。结论:持续性血液滤过联合高流量吸氧治疗重症急性呼吸综合征患者的临床疗效及安全性明显优于单用持续性血液滤过治疗,可能与其更有效减轻炎症反应有关。     

Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus     

Immunogenicity of a recombinant VSV-Vectored SARS-CoV vaccine induced robust immunity in rhesus monkeys after single-dose immunization

Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31st, 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVΔG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVΔG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVΔG-SARS is safe and effective in mice at a dose of 1×10⁶ TCID₅₀. More importantly, only a single-dose immunization of 2×10⁷ TCID₅₀ can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVΔG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.     

猪繁殖与呼吸综合征病毒GP5a多克隆抗血清的制备

构建XX2012株猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)GP5a蛋白基因的真核表达载体,并制备出GP5a蛋白的多克隆抗血清。以XX2012株PRRSV GP5a蛋白的基因序列为扩增模板,设计并合成一对特异性扩增引物,通过RTPCR扩增得到该病毒株的GP5a蛋白全长基因片段,将其定向克隆到真核表达载体p CAGG中,构建含有GP5a蛋白基因的重组表达载体p CAGGS-GP5a,并将获得的p CAGGS-GP5a载体采用基因免疫的方式免疫BALB/c小鼠制备GP5a蛋白的多克隆抗体。结果显示,成功克隆出了XX2012株PRRSV GP5a蛋白全长基因,片段大小为156 bp;构建的p CAGGS-GP5a载体经PCR、双酶切、测序鉴定均无误;通过三次基因免疫小鼠成功制备出了GP5a蛋白的多克隆抗体,间接免疫荧光试验证实制备出的多克隆抗体能特异性识别病毒感染后的细胞。由此获得了PRRSV GP5a蛋白基因的真核表达载体,制备出了GP5a蛋白的特异性多克隆抗血清,为今后开展GP5a蛋白的亚细胞定位及相关功能的研究奠定了基础。     

黄磷及其化合物急性吸入致大鼠急性肺损伤或急性呼吸窘迫综合征模型的制作

目的建立黄磷及其化合物急性吸入致大鼠急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）的模型。方法健康SD大鼠48只随机分为对照组以及实验组（0、4、12、24、48 h时间点处死）。采用自制染毒装置,间歇染毒形成ALI/ARDS模型。观察ALI/ARDS大鼠动脉血气分析以及肺系数和肺组织病理变化。结果肺损伤后大鼠动脉血气分析以及肺组织病理改变明显恶化,肺系数较对照组明显增大。结论成功地建立了黄磷及其化合物急性吸入致大鼠ALI/ARDS的模型,为黄磷及其化合物吸入中毒的防治研究提供良好实验基础,同时也适用于其他气体吸入致ARDS的实验研究。     

Ameliorative Effects of Curcumin on Fibrinogen‐Like Protein‐2 Gene Expression,Some Oxido‐Inflammatory and Apoptotic Markers in a Rat Model of l‐Arginine‐Induced Acute Pancreatitis

The aim of the study was to investigate the ameliorative effects of curcumin on fibrinogen like protein‐2 (fgl‐2), some oxido‐inflammatory and apoptotic markers in rat‐induced acute pancreatitis (AP). Seventy‐five albino rats were divided into control group, l ‐arginine (l ‐Arg)‐induced AP group, curcumin pre‐treated group before AP induction, curcumin post‐treated group after AP induction, and curcumin injected group only. AP group showed severe necrotizing pancreatitis confirmed by histopathological changes and elevations in serum amylase and lipase activities, levels of epithelial neutrophil‐activating peptide 78, tissue content of protein carbonyls, levels of tumor necrosis factor α, and caspase‐3 as well as myeloperoxidase activity. Significant elevation in pancreatic fgl‐2 mRNA expression was detected in AP group. Improvement of all parameters was detected with increase of caspase‐3 in both curcumin‐treated groups that confirmed curcumin ameliorative effects against AP through induction of apoptosis and inhibition of micro‐thrombosis, inflammation, and oxidative stress.     

早产儿急性呼吸窘迫综合征血清1,25-(OH)2D3、PGRN、SIRT1、CTRP3水平与炎症反应和预后的关系研究

摘要 目的：探讨不同病情严重程度早产儿急性呼吸窘迫综合征（ARDS）血清1,25-二羟维生素D₃（1,25-(OH) ₂D₃）、颗粒体蛋白前体（PGRN）、沉默信息调节因子2相关酶1（SIRT1）、C1q/肿瘤坏死因子相关蛋白3（CTRP3）的变化，分析其与炎症反应和预后的关系。方法：选择2018年10月至2019年12月安徽省妇幼保健院收治的ARDS早产儿100例作为ARDS组，另选取同期在我院出生的健康新生儿60例作为对照组。根据《"新生儿急性呼吸窘迫综合征"蒙特勒标准（2017年版）》的病情判定标准将ARDS早产儿分为轻度组（n=40）、中度组（n=32）、重度组（n=28），比较不同病情严重程度ARDS早产儿血清1,25-(OH) ₂D₃、PGRN、SIRT1、CTRP3、炎症反应指标肿瘤坏死子因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）水平变化，采用Pearson法分析ARDS早产儿血清1,25-(OH) ₂D₃、PGRN、SIRT1、CTRP3与炎症反应指标的相关性。另根据ARDS组患儿预后情况分为预后良好组（n=65）和预后不良组（n=35），采用单因素和多因素Logistic回归分析影响ARDS早产儿预后不良的危险因素。结果：ARDS组血清1,25-(OH) ₂D₃、SIRT1、CTRP3、PGRN水平明显低于对照组（P<0.05），ARDS组血清TNF-α、IL-6、IL-1β水平明显高于对照组（P<0.05）。不同病情严重程度ARDS早产儿血清1,25-(OH) ₂D₃、PGRN、SIRT1、CTRP3、炎症反应指标比较差异有统计学意义（P<0.05）。ARDS早产儿血清1,25-(OH) ₂D₃、PGRN、SIRT1、CTRP3水平与TNF-α、IL-6、IL-1β水平呈负相关（P<0.05）。多因素Logistic回归分析结果显示，低出生体重、肺表面活性物质（PS）使用次数≥3次、出现低白蛋白血症是影响ARDS早产儿预后不良的危险因素（P<0.05），血清1,25-(OH)2D3（较高）、PGRN（较高）、SIRT1（较高）、CTRP3（较高）是ARDS早产儿预后不良的保护因素（P<0.05）。结论：血清1,25-(OH) ₂D₃、PGRN、SIRT1、CTRP3可能参与ARDS早产儿的炎症反应过程，与ARDS早产儿的病情进展及预后密切相关，检测血清1,25-(OH) ₂D₃、PGRN、SIRT1、CTRP3有助于评估ARDS早产儿的预后。     

Alanine-scanning mutagenesis of WH2 domains of VopF reveals residues important for conferring lethality in a Saccharomyces cerevisiae model

VopF, the type III effector molecule, has been implicated in the pathogenesis of non-O1, non-O139 strains of Vibrio cholerae. It is a protein of 530 amino acids, comprises of one formin homology 1-like (FH1-like) domain and three WASP homology 2 (WH2) domains. Previous works have demonstrated that WH2 domains are crucial for VopF function as a modulator of cellular actin homeostasis. Furthermore, domain deletion analysis also suggests that VopF variant constituted with only WH2 domain 3 is more efficient in restricting the growth of budding yeast than its congeners containing either only domain 1 or domain 2. Interestingly, a good degree of sequence diversity is present within each WH2 domain of VopF. In order to ascertain the importance of different amino acids in each WH2 domain, a systemic alanine scanning mutagenesis was employed. Using a yeast model system, the alanine derivatives of each amino acid of WH2 domain 1 and 3 of VopF were examined for growth restricting activity. Taken together, our mutagenesis results reveal the identification of critical residues of WH2 domain 1 and 3 of VopF.     

Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites

Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K_d = 0.15 μM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1″ phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.