肠道病毒71型外壳蛋白VP2在Pichia.pastoris酵母中的表达

利用逆转录聚合酶链式反应(RT-PCR)的方法,克隆肠道病毒71型(Enterovirus 71,EV71)SHZH03株外壳蛋白VP2基因,构建酵母分泌型表达质粒pPIC9K/VP2,经序列测定证实α-factor信号肽和VP2的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导筛选到高效表达VP2蛋白的工程菌株.用饱和硫酸铵法对重组蛋白VP2进行了较好的纯化.Western Blot分析表明,重组蛋白VP2具有较好的反应原性,从而为发展EV71快速、高效、廉价诊断试剂奠定了基础.     

交河故城古车师人的线粒体DNA分析

从距今2000～2500年左右的交河故城古代人骨中提取古DNA,用4对重叠引物对线粒体基因组的调控区(363bp)进行了扩增及测序.线粒体基因组编码区的扩增片段用于限制性片段多样性分析.结果显示4个个体中具有3个DNA序列,其中来自不同墓穴的两个个体的序列相同,说明这两者间有密切的母系遗传关系.系统发育分析表明古车师的这4个个体分散分布在现代新疆维吾尔人的序列之中.从这些结果可以初步得出结论,即古车师人群并不是一个同源群体,在早期铁器时代,欧亚人群的混合就已经存在了.     

交河矿城古车师人的线粒体DNA分析

从距今2000－2500年左右的交河故城古代中提取古DNA，用4对重叠引物对线粒体基因组的调控区(363bp)进行了扩增及测序，线粒体基因组编码区的扩增片段用于限制性片段多样性分析。结果显示4个个体中具有3个DNA序列，其中来自不同墓穴的两个个体的序列相同，说明这两者间有密切的母系遗传关系。系统发育分析表明古车师的4个个体分散分布在现代新疆维吾尔人的序列之中。从这些结果可以初步得出结论，即古车师人群并不是一个同源群体，在早期铁器时代，欧亚人群的混合就已经存在了。     

禽流感病毒H5N1病毒颗粒中鸡胚宿主蛋白的质谱分析

在A型流感病毒等许多包膜病毒的病毒颗粒中,除包含病毒基因组编码的结构蛋白外,还包含来源于宿主细胞的多种蛋白。然而,在鸡胚内增殖的病毒颗粒所包含的宿主蛋白的种类尚不清楚。本研究采用20%～60%(w/w)蔗糖密度梯度离心法分离纯化了繁殖于鸡胚的流感病毒,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)结合质谱法对纯化的流感病毒颗粒进行了全面的蛋白质组学分析。结果表明,在病毒颗粒中,除包含9种病毒编码蛋白外,还发现了12种来源于鸡胚的蛋白,如膜联蛋白A2、肽酰脯氨酰顺反异构酶B、过氧化物酶1、磷酸甘油酸激酶、丙酮酸激酶肌组织异构酶等,以及2种细胞骨架蛋白(微管蛋白b-3和肌动蛋白)。     

中国人促红细胞生成素基因组的克隆及其在COS-7细胞中的表达

本文利用PCR技术,从正常人胎肝染色体DNA库中分离克隆了长度分别为506bp,465bp的中国人促红细胞生成素(EPO)基因组片段。506bp基因片段包括外显子2(信号肽),内含子2和外显子3;465bp片段包括外显子4,内含子4和外显子5。通过在引物中设置的酶切位点将两个克隆片段进行了正确的拼接,从而得到了约1.0kb的EPO次全基因组片段,它包括除信号肽中第2,3,4位氨基酸外的所有编码区及两个内含子序列。 所克隆的次全EPO基因组插入表达载体pSV2-dhfr中的不同克隆位点,构建了3种不同的转移载体质粒,分别转染导入COS-7细胞后,3种转移载体质粒转染的细胞上清液都有明显的EPO活性。本工作证明仅含有内含子2,4序列,去除负调控区和氧敏感区序列的人次全EPO基因组可以在COS-7细胞中获得表达。     

马铃薯Y病毒基因组3′-端区域的克隆和序列分析

以马铃薯Y病毒(PVY,中国分离株)基因组RNA的cDNA为模板,我们用聚合酶连锁反应(PCR)合成了完整的外壳蛋白(CP)基因及部分3′末端非编码区序列。在5′-引物Y5中引入了限制性酶NcoI位点及起始密码AUG,3′-引物Y3中引入了Sall位点。PCR产物经NcoI及Sall双酶切后克隆入pGEM衍生质粒,并测定了其中一个克隆pPCY6的CP基因的序列,以此克隆的CP基因片段作探针,从cDNA库中钓出相关的几个克隆,并测定了序列,由此我们获得了包含部分NIb基因,全部的CP基因及3′-非编码区共1317个碱基。序列分析表明,该株系CP基因核苷酸序列与0株系同源性(94.2％)较与N株系(89.6％)同源性稍高,但氨基酸序列的同源性差别不大(分别为93.3％及91.8％)。目前我们已将该基因克隆入双元载体pBI121.1,并通过脓杆菌转化马铃薯,以期得到抗PVY的马铃薯工程株。     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

丝氨酸蛋白酶抑制剂超家族的一个新成员——痘苗病毒(天坛株)基因组HindⅢK片段编码的K1蛋白

本文测定了位于痘苗病毒(天坛株)基因组的Hind Ⅲ K片段左端,由1893个碱基所组成的开放读码框架K1(ORF K1)的核苷酸排列顺序,并利用微机对ORFK1所编码K1蛋白的序列进行了研究。结果发现,K1蛋白与丝氨酸蛋白酶抑制剂超家族的成员在蛋白质一级结构上有着显著的同源性,通过点阵分析和序列同源排列,证明K1蛋白是丝氨酸蛋白酶抑制剂超家族的一个新成员,它可能具有与丝氨酸蛋白酶抑制剂超家族其他成员相似的活性中心及其他特征序列。这一发现对于研究痘苗病毒的进化地位及所编码蛋白的功能均有一定意义。     

乙型肝炎病毒相关性肝癌患者的比较蛋白质组学研究

筛选并鉴定了乙型肝炎病毒(HBV)相关性肝癌的血清差异表达蛋白. 采用蛋白芯片及表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术对正常人与乙型肝炎病毒相关性肝癌患者术前血清分别进行检测, 共发现了44个差异蛋白, 其中21个上调, 23个下调. 通过高效液相色谱技术分离纯化其中表达差异最明显的蛋白, 并进行质谱鉴定. 通过蛋白功能结果分析表明, 这些蛋白的差异表达可能与乙型肝炎病毒相关性肝癌的发生机制密切相关.     

兔早期胚泡特异肽的cDNA克隆

从家兔受精后第6天(着床前)的早期胚泡构建cDNA文库,经阶段特异分子杂交筛选,克隆了仅在此时期表达的9种胚泡特异肽类的cDNA对其中一种丰度较高的cDNA克隆进行了研究,测出其编码区含有285碱基对,5'和3'侧翼区各有132和170碱基对.经DNA序列分析,推导出一种分泌性多肽的一级结构,由75个氨基酸组成.计算机分析显示它与迄今报道的所有基因和蛋白在结构上无任何同源性.     

Photoinactivation of Virus Infectivity by Hypocrellin A

Abstract— We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration-dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or bT-carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.     

Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane‐Associated Protein IFITM3 and Modified Variants

Interferon‐induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133‐residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi‐milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C‐terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.     

Self-assembly of anisotropic tobacco mosaic virus nanoparticles on gold substrate

A facile approach to assembled virus film with tunable structure is presented.Rod-like tobacco mosaic virus (TMV) was selected as the prototype in this study for its anisotropic structural feature.TMV can either lie down or stand up on gold substrate by tuning the solution pH.A quartz crystal microbalance with dissipation monitoring was used to monitor the pH-dependent self-assembly behavior of TMV nanoparticles,and atomic force microscopy and single molecule force spectroscopy further confirmed the differe...     

Amphipathic and membrane-destabilizing properties of the cationic acrylate polymer Eudragit E100

The cationic acrylate polymer Eudragit E100 (E100) produces a biphasic effect on the stability of casein micelles disrupting their internal structure. These results suggested that this polymer could have some amphipathic character. Therefore, in this study the polymer was characterized with respect to its interaction with different amphipathic systems (bile-acid micelles, lipoproteins and liposomes), cell membranes (red blood cells) and virus membranes (Herpes simplex type 2 virus). As with caseins, a biphasic effect was observed with bile acids with a precipitation phase at low polymer/bile acid ratio and a solubilization phase when the polymer concentration was increased. Upon interaction with human plasma, an important reduction in cholesterol and triglycerides was observed upon remotion of E100 by a rise in pH to 8.5 and centrifugation. In agreement with this finding, an important reduction in plasma lipoproteins was observed upon its treatment with E100 and further remotion by pH rise and centrifugation. However, the amount of the major protein components of human plasma and the activity of several enzymes and antibodies were not affected by their treatment with E100. The membrane-destabilizing properties of E100 were confirmed by its lytic activity on liposomes and red blood cells and by an important antiviral effect of E100 on Herpes simplex virus type 2. Altogether, these results show that, despite its water solubility and cationic character, E100 displays a significative amphipathic and membrane-destabilizing character with potential biotechnological applications. [diagram in text].     

Topology-Matching Design of an Influenza-Neutralizing Spiky Nanoparticle-Based Inhibitor with a Dual Mode of Action

In this study, we demonstrate the concept of “topology-matching design” for virus inhibitors. With the current knowledge of influenza A virus (IAV), we designed a nanoparticle-based inhibitor (nano-inhibitor) that has a matched nanotopology to IAV virions and shows heteromultivalent inhibitory effects on hemagglutinin and neuraminidase. The synthesized nano-inhibitor can neutralize the viral particle extracellularly and block its attachment and entry to the host cells. The virus replication was significantly reduced by 6 orders of magnitude in the presence of the reverse designed nano-inhibitors. Even when used 24 hours after the infection, more than 99.999 % inhibition is still achieved, which indicates such a nano-inhibitor might be a potent antiviral for the treatment of influenza infection.     

HYPOCRELLIN, FROM HYPOCRELLA BAMBUASE, IS PHOTOTOXIC TO HUMAN IMMUNODEFICIENCY VIRUS

Abstract Hypocrellin, a photodynamic perylene quinonoid isolated from the Chinese medicinal fungus Hypocrella bambuase , was evaluated for antiviral activity against the human immunodeficiency virus (HIV-1). Hypocrellin was phototoxic to HIV-1, almost as good as the structurally similar plant pigment hypericin, and like hypericin its activity required visible light. In contrast peroxyhypocrellin had little or no effect on the virus.     

流感病毒糖蛋白糖链的作用和功能研究

流感病毒是近几年的研究热点之一.糖链在流感病毒生活周期中发挥重要作用,例如宿主细胞表面的唾液酸化糖链是病毒侵染细胞时的特异性受体,宿主决定的病毒糖蛋白糖链结构影响病毒的宿主范围和毒力.本文从糖组学角度综述糖链在甲型流感病毒生活周期中的重要作用,着重阐述病毒血凝素糖基化的影响因素及其对病毒宿主范围、毒力的影响和在病毒演化...     

Synthesis of nontrivial quinopimaric acid derivatives by oxidation with dimethyldioxirane

Oxidation of dihydroquinopimaric acid methyl ester and its derivatives with dimethyldioxirane results in the formation of a nontrivial derivative and hemiacetals in high yields with excellent regioselectivity, as is demonstrated for an unsaturated diol. The structures of the products were confirmed by X-ray crystallographic analysis. Dihydroquinopimaric acid and its nontrivial derivative are found to be moderately active against influenza virus type A and the papilloma virus.     

Precise Photoremovable Perturbation of a Virus–Host Interaction

Viruses utilize distinct binding interactions with a variety of host factors to gain entry into host cells. A chemical strategy is described to precisely perturb a specific molecular interaction between adeno‐associated virus and its host cell, which can be rapidly reversed by light. This strategy enables pausing the virus entry process at a specific stage and then restart it rapidly with a non‐invasive stimulus. The ability to synchronize the invading virus population at a discrete step in its entry pathway will be highly valuable for enabling facile experimental characterization of the molecular processes underlying this process. Additionally, adeno‐associated virus has demonstrated outstanding potential for human gene therapy. This work further provides a potential approach to create therapeutic vectors that can be photoactivated in vivo with high spatial and temporal control.     

Topology‐Matching Design of an Influenza‐Neutralizing Spiky Nanoparticle‐Based Inhibitor with a Dual Mode of Action

In this study, we demonstrate the concept of “topology‐matching design” for virus inhibitors. With the current knowledge of influenza A virus (IAV), we designed a nanoparticle‐based inhibitor (nano‐inhibitor) that has a matched nanotopology to IAV virions and shows heteromultivalent inhibitory effects on hemagglutinin and neuraminidase. The synthesized nano‐inhibitor can neutralize the viral particle extracellularly and block its attachment and entry to the host cells. The virus replication was significantly reduced by 6 orders of magnitude in the presence of the reverse designed nano‐inhibitors. Even when used 24 hours after the infection, more than 99.999 % inhibition is still achieved, which indicates such a nano‐inhibitor might be a potent antiviral for the treatment of influenza infection.