水稻齿叶矮缩病毒P8蛋白的抗体制备及其在水稻原生质体内的表达动态

水稻齿叶矮缩病毒(rice ragged stunt virus,RRSV)属于呼肠孤病毒科(Reoviridae)水稻病毒属(Oryzavirus)成员。该病毒引起的水稻齿叶矮缩病在我国南方发病严重,成为我国水稻生产上的重要病害。通过建立水稻原生质体培养体系,对RRSV主要外壳蛋白P8在水稻原生质体内的复制与表达情况进行研究。通过原核表达对P8蛋白进行抗体制备,抗血清经Western印迹检测具有特异性,间接ELISA测得其效价为2 500倍左右。以制备的抗血清为探针,通过Western印迹检测到P8蛋白在病毒侵染原生质体后24h开始表达,48h左右达到最大值,60h后开始下降,但仍保持在较高水平;同时,利用荧光定量PCR技术,对病毒侵染水稻原生质体后的P8RNA含量进行检测,结果表明,P8RNA在8h时开始积累,32h左右达到最大值,48h后表达量开始下降到一个平台期。     

单病毒示踪技术在病毒侵染机制及水生动物病毒研究中的应用进展

病毒虽是一类营细胞内寄生的极为简单的微小生物,却是对人类及其他动物生存威胁最大的病原之一,严重威胁人类健康、生物多样性、经济发展和社会稳定。病毒感染宿主的过程复杂且处于不断的动态变化过程中,采用传统的分子生物学技术和电子显微镜超薄切片观察等手段无法对其进行实时动态分析研究。单病毒示踪(Single-virus tracking, SVT)技术的出现,使得研究人员能够实时追踪单个或多个病毒粒子在活细胞中的生命过程,从而全面精细地阐述病毒侵染的时空动态过程及机制。本文综述了单病毒示踪技术方法体系的建立及其在动物病毒生命周期研究特别是水生动物病毒研究中的应用进展,针对该技术研究应用中存在的问题,提出未来应进一步完善病毒、蛋白、核酸和宿主细胞等的标记,发展多维成像和活体成像技术,优化图像处理技术,使动物病毒感染机制研究向着更加精准化和系统化方向发展,以期为有效防控水生动物病毒性疾病暴发与流行提供理论指导和技术支撑。     

siRNA抑制乙肝病毒的研究进展

乙型肝炎作为一种发病率高、死亡率高的传染性疾病,已严重威胁人类健康,乙肝病毒(hepatitis B virus,HBV)是诱发乙型肝炎的重要病因。目前,最主要的治疗方法是运用抗病毒药物控制病情,但这些药物都不能完全治愈乙型肝炎且复发率高。近年来,RNA干扰技术(RNA interference,RNAi)逐渐成为有效、快速治疗乙型肝炎的新疗法。利用RNA干扰技术体外合成针对HBV基因的siRNA,选择适当的载体将其运送至靶细胞,使HBV基因沉默,从而抑制病毒复制,可有效达到治疗乙肝的效果。本文围绕siRNA沉默HBV基因的设计原理、递送载体、靶向策略、以及治疗效果与应用前景等方面进行了系统综述,为今后siRNA治疗乙肝的临床应用提供参考。     

在结肠癌细胞中RNA干扰TGFBR2慢病毒载体的构建及其功能的初步研究

目的:构建TGFBR2基因的RNA干扰慢病毒载体,为研究该基因在结肠癌细胞中的作用奠定基础。方法:合成TGFBR2基因特异性RNAi靶序列,与pcDNATM6.2-GW/EmGFP-miR载体连接,构建干扰载体并鉴定。与TGFBR2质粒共转染HEK293细胞,qRT-PCR和Western blot方法筛选最佳干扰序列,感染SW480细胞。10ng/ml TGF-β1和20ng/ml TNF-a共作用TGFBR2干扰后的SW480细胞和未干扰组细胞72h后,通过细胞侵袭试验计算细胞侵袭数目。结果:成功构建TGFBR2的RNA干扰载体,第4组序列效果最佳,感染SW480后,TGFBR2基因的mRNA表达抑制率为78.45%。TNF-a和TGF-β1处理干扰前后的SW480细胞,通过细胞侵袭试验显示各组细胞迁移的数目:干扰组为89.3±7.9,未干扰组为15.1±8.2,干扰组细胞明显多于对照组(P<0.01)。结论:成功构建人TGFBR2基因特异性的慢病毒干扰载体,并建立稳定的细胞株,为预防和干预结肠癌的早期转移奠定基础。初步提示在TGF-β1和TNF-a因子存在的炎症微环境中,TGFBR2基因突变的结肠癌细胞更具有侵袭性。     

水稻病毒病综合防治方法

水稻病毒病被称为"水稻癌症",是当今国内外难以攻克的"绝症"之一,是水稻上一类重要的病害,严重影响水稻的产量。矮缩病是由RDV病毒引起,其寄主范围广泛,有水稻、燕麦、大麦、小麦、野生稻等30多种禾本科植物,该病毒可由黑尾叶蝉、二点叶蝉和电光叶蝉传播。获毒越冬的二点叶蝉的若虫,翌年春天羽化成成虫从越冬场所开始迁飞到稻田传毒并繁殖,引起水稻发病,无毒的二点叶蝉可通过吸食带病毒植物的汁液而获毒传病,使病害不断扩大、蔓延。条纹叶枯病系由RSV病毒引起,寄主范围较广,有水稻、小麦、玉米、谷子等30多种禾本科植物,这类病毒只能通过昆虫传播,已知灰飞虱为主要媒介,白背飞虱也能传染,灰飞虱体内带毒越冬,成为主要侵染源,在麦田等场所越冬,后迁飞到秧田或本田传毒并繁殖,1年可传播多次。     

PRMT2慢病毒表达载体的构建及鉴定

目的构建精氨酸甲基转移酶2(PRMT2)慢病毒表达载体。方法通过PCR扩增PRMT2 c DNA,将PRMT2 c DNA连接于GV308载体,经测序确认后,将GV308/PRMT2与p Helper 1.0和p Helper 2.0共转染至293T细胞中,收获病毒,通过Real time PCR测定滴度;将PRMT2慢病毒表达载体侵染293T细胞,通过四环素诱导和Western blot检测PRMT2慢病毒表达载体的表达能力。结果在感染PRMT2慢病毒载体293T细胞中能检测到PRMT2-3Flag融合蛋白的表达。结论成功构建PRMT2的慢病毒表达载体。     

FTO过表达对小鼠胰岛β细胞功能的影响以及全基因组表达谱变化研究

为了研究肥胖基因FTO过表达对小鼠胰岛β细胞功能的影响以及基因表达谱的变化,构建小鼠FTO基因过表达慢病毒载体,包装慢病毒颗粒并感染小鼠胰岛MIN6细胞。利用QPCR和Western Blot技术检测FTO基因在MIN6细胞中过表达情况,并检测葡萄糖刺激检测胰岛素的释放情况,进一步利用小鼠全基因芯片检测FTO过表达对小鼠胰岛MIN6细胞表达谱的影响。结果表明:慢病毒载体成功介导了FTO在MIN6细胞中过表达,FTO过表达可以显著抑制MIN6细胞的胰岛素释放。表达芯片的结果显示FTO改变MIN6细胞表达谱,发现多达922个的差异基因。FTO过表达改变了小鼠胰岛细胞的表达谱,差异基因通过一些重要信号通路影响小鼠胰岛β细胞的生物学功能。     

RNA干扰Livin基因对甲状腺乳头状癌的增殖及侵袭抑制作用及其分子机制

目的:探讨Livin对甲状腺乳头状癌TPC-1细胞增殖、凋亡及侵袭的影响以及可能的调控分子机制。方法:构建靶向Livin基因的慢病毒sh RNA载体,转染甲状腺乳头状癌细胞株TPC-1,实验分为干扰组、阴性对照组和空白组。应用RT-PCR和Western印迹检测转染Livin-sh RNA后细胞Livin m RNA和蛋白表达水平的变化。应用MTT、流式细胞仪、Western印迹与Transwell侵袭实验检测Livin对甲状腺乳头状癌细胞增殖、凋亡及侵袭的影响。并接种于裸鼠模型中,进一步确定Livin在体内对甲状腺乳头状癌细胞的影响。结果:慢病毒Livin-sh RNA载体成功抑制TPC-1细胞中Livin m RNA和蛋白的表达;转染Livin-sh RNA后TPC-1细胞的增殖能力受到明显抑制(P<0.01),转染Livin-sh RNA组、阴性对照组和空白组的凋亡率分别为(15.72±0.21)%,(4.54±0.13)%和(4.87±0.22)%,三组比较差异均有统计学意义(P<0.01)。干扰组穿膜细胞数明显少于阴性对照组和空白组(P<0.05)。与空白组和阴性对照组相比,干扰组细胞AKT、p-AKT及PDK1蛋白表达量均下调(P<0.05),而PTEN蛋白明显上调(P<0.05)。甲状腺乳头状癌裸鼠模型结果表明干扰组肿瘤的生长速度、肿瘤质量[(1.11±0.08)g]明显小于空白组[(1.90±0.15)g]和阴性对照组[(2.10±0.12)g]。结论:沉默Livin基因能抑制甲状腺乳头状癌细胞的增殖、侵袭及体内生长,促使甲状腺乳头状癌细胞凋亡的发生,可能是通过抑制PI3K-AKT信号转导通路来发挥作用。     

Pokemon表达慢病毒载体的构建及鉴定

目的构建Pokemon表达慢病毒载体。方法通过PCR扩增Pokemon cDNA,将Pokemon cDNA连接于GV165载体,经测序确认后,将GV165/Pokemon与pHelper 1.0和pHelper 2.0共转染至293T细胞中,收获病毒,通过Real time PCR测定滴度;将Pokemon表达慢病毒载体侵染293T细胞通过免疫荧光和Western blot检测Pokemon表达慢病毒载体的转染效率和Pokemon的表达能力。结果在感染Pokemon慢病毒载体293T细胞中能检查到Pokemon-GFP融合蛋白的表达。结论成功构建表达Pokemon的慢病毒载体。     

人类巨细胞病毒感染对血管平滑肌细胞脂代谢相关基因表达的影响

目的研究人类巨细胞病毒感染对体外培养血管平滑肌细胞脂代谢相关基因表达的影响,探讨该病毒致动脉粥样硬化(As)的机制。方法以1 MOI人类巨细胞病毒感染体外培养的血管平滑肌细胞,利用基因表达谱芯片技术检测细胞脂代谢相关基因的表达变化。结果人类巨细胞病毒感染血管平滑肌细胞后,细胞内与脂代谢有关的基因表达出现明显的异常,其中表达上调的基因主要有低密度脂蛋白受体相关蛋白10、11、12、极低密度脂蛋白受体、B型清道夫受体、HMG-CoA合成酶、HMG-CoA还原酶、酰基辅酶A∶胆固醇酰基转移酶及脂肪酶合成酶等基因;表达下调的主要有载脂蛋白A1、载脂蛋白M和载脂蛋白H等基因。结论人类巨细胞病毒感染可能通过改变血管平滑肌细胞脂代谢相关基因的表达而参与As的发病过程。     

埃博拉——致命的病毒

正埃博拉,一种高致死率的病毒,首先于1976年在非洲被确认。体内出血导致人高热发烧的埃博拉出血热,由某一种埃博拉病毒导致发病,而这种病毒只是五种埃博拉病毒中的一种。五种埃博拉病毒中的四种能引起人和动物发生严重的疾病。第五种,称之为Reston的病毒,只感染某些动物,而不涉及人类。     

Concentration of hepatitis A virus

Cell culture adapted hepatitis A virus (HAV) in 10 and 50 l. of seeded distilled water was respectively concentrated approx. 3300 and 17,000 fold by adsorption-elution on cellulose membrane. HAV (strain CF 53) was adsorbed at pH 4.0 with 0.15 M NaCl to the cellulose filters and eluted by 3% beef-extract at pH 8.5. Eluted virus was further concentrated by precipitation by polyethylene glycol 6000. Hepatitis A antigen (HAAg) was detected by radioimmunoassay and infectious virus was quantified by cell culture titration. The average of HAAg recovery was 76% for 101. and 85% for 501., whereas the average recovery for infectious virus was respectively 89 and 97%. The sensitivity of the method was tested by contaminating 101. with 16 TCID₅₀ only; 63% of initial infectious virus were recovered.     

EB病毒的免疫逃逸

EB病毒(EBV)是一种普遍存在的人类疱疹病毒,人群中90%以上的个体都曾感染过EBV。其中一部分人群为终身潜伏感染,因潜伏感染时病毒复制受抑制、病毒拷贝数低且存在逃避机制,所以不易被人体免疫系统识别。EBV以各种方式逃避免疫系统的监控并导致免疫失调,引发相关疾病乃至肿瘤的发生。此外,在机体抗病毒免疫监控下,EBV具备的多种免疫逃逸机制也可以促进肿瘤的恶性生长。所以研究EBV的免疫逃逸机制是防治与EBV相关疾病的重要部分。本文以EBV对宿主免疫系统的逃逸为切入点,探讨EBV的感染与逃逸机制、免疫失调所造成的疾病以及相关免疫治疗的进展。     

Electrophoretic mobilities of virus adsorbing filter materials

Surface charge characteristics of virus adsorbing filters were studied by electrophoretic measurements of particulate suspensions under conditions commonly encountered in the adsorption technique for virus concentration. A mechanism for virus interaction with filter surfaces, involving cross-complexation by multivalent cations, is discussed.     

Vulnerability of unconfined aquifers to virus contamination

An empirical formula was developed for determining the vulnerability of unconfined sandy aquifers to virus contamination, expressed as a dimensionless setback distance . The formula can be used to calculate the setback distance required for the protection of drinking water production wells against virus contamination. This empirical formula takes into account the intrinsic properties of the virus and the unconfined sandy aquifer. Virus removal is described by a rate coefficient that accounts for virus inactivation and attachment to sand grains. The formula also includes pumping rate, saturated thickness of the aquifer, depth of the screen of the pumping well, and anisotropy of the aquifer. This means that it accounts also for dilution effects as well as horizontal and vertical virus transport. Because the empirical model includes virus source concentration it can be used as an integral part of a quantitative viral risk assessment.     

揭开埃博拉病毒的神秘面纱

<正>埃博拉病毒(Ebola virus)埃博拉病毒是丝状病毒的一种,可导致埃博拉病毒出血热,罹患此病可致人于死,包含数种不同程度的症状,如恶心、呕吐、腹泻、肤色改变、全身酸痛、体内出血、体外出血、发烧等。感染者症状与同为纤维病毒科的马尔堡病毒极为相似,具有50%至90%的致死率。致死原因主要为中风、心肌梗塞、低血容量性休克或器官衰竭。埃博拉病毒,生物安全等级为4级,是对人类危害级别最高     

The effect of sludge digestion on virus infectivity

The rate of inactivation of coxsackievirus B3 during anaerobic sludge digestion was found to be 2 log units in 24 h at pH 7.0 and 35°C. The rate indicates that virus inactivating factor(s) were produced in the digestion. The virus inactivation was enhanced if the digester sludge was pasteurized at 60°C for 1 h. The reason why digesters at sewage treatment plants do not completely inactivate virus is discussed in terms of the continuous nature of digester operation.     

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water

A reliable indicator is needed to predict and reduce the risk of infection associated with fecal contamination of surface water. Since Pepper mild mottle virus (PMMoV), human picobirnaviruses (hPBV) and Torque teno virus (TTV) have been detected at substantial levels in human feces, we explored whether detection of nucleic acids of these viruses is a suitable indicator of fecal contamination in river water. From September 2008 to December 2009, water samples (n = 111) were collected from the Ruhr and Rhine rivers and from the influents and effluents of a wastewater plant (n = 12). Quantitative real time (RT-) PCR was used to determine the abundance of PMMoV, hPBV, and TTV in comparison to human adenoviruses (HAdV) and human polyomaviruses (HPyV) that are frequently detected in surface water and were previously proposed as indicators. While PMMoV was detected in all river water samples, the other viruses were detected less frequently. The concentration of the studied viruses in positive river water ranged from 5 × 10¹ to 1.07 × 10⁶ genome equivalents per liter (gen.equ./l). All wastewater samples were positive for PMMoV, HAdV and HPyV, while TTV and hPBV were detected in 6/12 and 3/12 of samples, respectively. To determine if PMMoV is specific to human-derived fecal waste, fecal samples from human (n = 20) and animal (n = 53) were also tested. In contrast to the ubiquity of PMMoV in human feces (19/20) the virus was only detected at low concentration in a minority of the animal fecal samples tested (7/15 from chicken, 1/10 from Geese and 1/6 from cows). Therefore, in this setting TTV and hPBV do not seem to be suitable indicators of fecal contamination in water. Whereas, the high excretion level and dissemination of PMMoV in human sewage and river water suggest that PMMoV could be a promising indicator of fecal pollution in surface water.     

数据挖掘在计算机网络病毒防御中的应用

互联网信息时代已经来临,网络病毒也在肆虐,计算机网络系统容易受到攻击和破坏,网络病毒威胁着网络用户的使用安全,对用户的数据、文件资料、个人信息进行窃取或破坏。面对这些问题,文章通过数据挖掘在计算机病毒防御中的应用进行探讨和研究,将网络病毒扼杀在摇篮之中,有效地打击网络病毒。这些方法都需要进行探索,以便增强网络使用安全,既减少病毒的威胁,又能够为用户提供一个良好的网络环境。     

Processes controlling virus inactivation in coastal waters

The mechanisms controlling virus inactivation in coastal waters are discussed. Chemical and biological processes appear to be important but a purely thermal mechanism is probably not significant. Viral association with colloidal and particulate materials prolong survival and may enhance the transport of viruses to marine sediments. Sewage management practices are evaluated for efficiency in the control of viral contamination of bathing and shellfishing waters.