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2008年23卷5期

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天然植物产物抗单纯疱疹病毒1型研究进展

向阳飞, 裴赢, 王一飞

2008, 23(5): 305 doi: 10.1007/s12250-008-2962-7

目前使用核苷类似物是治疗疱疹病毒1型(HSV-1)感染的主要手段,但随着耐药株的出现,有必要开发新的抗HSV-1药物。天然产物为多数新药提供了结构前体,也是抗HSV-1药物的重要来源。本文对近年来国内外发现的抗HSV-1天然植物产物作一综述。研究表明,多种多糖、多酚及萜类化合物具有抗HSV-1活性,具有潜在开发前景。

Abstract: Nucleoside analogues have been the mainstay of clinical treatment of herpes simplex virus 1 (HSV-1) infections since their development. However, the emergence of drug resistant strains has underlined the urgency of the discovery of novel anti-HSV-1 drugs. Natural products, which provided many novel drug leads, are known to be an important source of anti-HSV-1 agents. Herein, we present an overview of natural products with anti-HSV-1 activities isolated from a variety of plants reported in recent years. Several different compounds, mainly belonging to the three groups of polysaccharides, polyphenols and terpenes, showed antiviral effects against HSV-1, indicating their potential to be promising anti-HSV-1 agents

抗病毒适体在肝炎中的应用

Hui FENG, hong HU

2008, 23(5): 315 doi: 10.1007/s12250-008-2979-y

适体是高亲和性的核酸配子,在建立随机的分子文库后通过重复地进行多轮分离和扩增从而得到。和传统的抗体比较,适体除了具备高度的亲和性和特异性以外,还有一些独特的优点;因此,在筛选抗乙肝病毒、抗丙肝病毒以及抗其他的病原物的药物中受到广泛关注。本篇综述总结了近年来抗病毒适体在肝炎中的应用,发展适体对诊断和发现新药均具备重要意义。

Abstract: Aptamers are short nucleic acids or peptides that strongly bind to a protein of interest and functionally inhibit a given target protein at the intracellular level. Besides high affinity and specificity, aptamers have several advantages over traditional antibodies. Hence, they have been broadly selected to develop antiviral agents for therapeutic applications against hepatitis B and C viruses (HBV, HCV). This review provides a summary of in vitro selection and characterization of aptamers against viral hepatitis, which is of practical significance in drug discovery.

棉铃虫核多角体病毒ORF113编码有活性的成纤维细胞因子

李祥, 梁昌镛, 宋建华, 陈新文

2008, 23(5): 321 doi: 10.1007/s12250-008-2969-0

FGF是一个非常重要的调节因子,它能影响许多细胞的生长、发育和迁移。在鳞翅目杆状病毒中发现了一个FGF同源家族(vFGF)。已经报道AcMNPV和BmNPV的vFGF具有趋化能力,可能在口服感染中起一定作用。本文主要研究棉铃虫核多角体病毒(HearNPV)vFGF的基本特性及其功能。RT-PCR结果发现HearNPV vfgf 从感染后的18 h开始转录,转录水平随时间延长而持续增加,在感染96 h后还能检测到。对病毒感染不同时间的细胞进行Western blotting检测发现,在感染后24 h到96 h都能检测到一条36 kDa特异带;同时在病毒感染细胞的上清中也检测到了vHaFGF的存在,表明vHaFGF能分泌到细胞外,暗示了它作为胞外的配体能跟肝素结合在信号传导过程中起作用。趋化实验结果显示,HearNPV vFGF只能特异地趋化Hz-AM1细胞,而对其它的Sf9 、Se-UCR、293和HepG2细胞没有趋化能力,这与AcMNPV和BmNPV的vFGF不同,它们能趋化多种不同来源的昆虫细胞。另外,我们还发现vHaFGF作为BV的囊膜蛋白而存在,而ODV上没有,这与BV的趋化实验的结果一致,同时也意味着vHaFGF在BV的感染中起作用

Abstract: Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group II NPV. The HearNPV vfgf transcripts were detected from 18 to 96 h post-infection (hpi) of Hz-AM1 cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.

ELISpot质控品的初步探讨研究

张春涛, 吴瑜, 赵晨燕, 洪坤学, 刘春雨, 王盈, 钟平, 聂建辉, 吴雪伶, 王佑春

2008, 23(5): 330 doi: 10.1007/s12250-008-2933-2

目前ELISPOT方法广泛的应用在HIV-1疫苗或肿瘤疫苗临床试验的细胞免疫应答评价中,但由于缺乏标准操作程序(SOP)以及不同实验室之间的验证,临床试验结果不能客观全面的反映疫苗诱导的细胞免疫应答反应,因此必须建立用于不同实验室质控的ELISPOT标准品。本课题分离健康成人外周血单个核淋巴细胞,冻存于液氮中,在不同的冻存时间复苏细胞后检测细胞存活率和回收率,同时在7家不同的实验室应用ELISPOT方法检测CEF肽库刺激分泌IFN-γ形成的斑点数。实验结果表明不同冻存时间复苏的细胞回收率和存活率无显著性差异,CEF肽库刺激复苏后细胞所产生的IFN-γ的能力是在一定范围内波动的,但是同新鲜分离细胞相比无明显区别。7家不同实验室之间对回收率、存活率和ELISPOT的实验结果存在显著性差异,说明冻存的外周血单个核细胞可用于制备ELISPOT的质控品,但在不同实验室之间必须建立统一的标准操作程序。

Abstract: The ELISpot assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories.

沉默UBP43可增强干扰素抗HBV的作用

樊和斌, 王宝菊, 卢银平, 郝友华, 杨新星, 陆蒙吉, 杨东亮

2008, 23(5): 339 doi: 10.1007/s12250-008-2960-9

以前的研究发现对干扰素无应答的患者肝内干扰素刺激基因(ISG)15的蛋白酶UBP43上升 。因此推测UBP43可能阻碍干扰素抑制HBV复制的作用。本研究在HepG2.2.15细胞中,观察了通过载体介导的shRNA沉默UBP43是否能增加干扰素抑制HBV复制的作用。结果显示HBeAg的表达和HBV DNA的复制显著下降,干扰素的抗病毒活性增强。因此,UBP43影响干扰素的抗病毒活性,是治疗HBV感染的一个可能靶点。

Abstract: Previous studies have shown that expression of the interferon-sensitive gene (ISG)15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by H1(psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, so vector-based siRNA promoted by H1 (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

一种新型细胞培养体系用于病毒的鉴定与分离研究

夏宇尘, 丘志娟, 马仲彬, 王华林, 胡志红, 邓菲

2008, 23(5): 345 doi: 10.1007/s12250-008-2968-1

细胞培养技术在病毒学研究中起了重要的作用。它不仅为病毒的检测、分离提供了平台,而且为病毒的生物化学和分子生物学研究提供了有效的研究手段。在本研究中,我们发展了一种可以支持多种不同的细胞系同时在一个容器中培养的新型细胞培养系统。该系统中,各细胞系生长于同一容器的独立区域,因此我们称此系统为细胞的独立共培养系统。我们利用腺病毒模拟感染实验验证了该系统,结果显示此系统可以有效的用于病毒的分离培养和诊断。摘要:细胞培养技术在病毒学研究中起了重要的作用。它不仅为病毒的检测、分离提供了平台,而且为病毒的生物化学和分子生物学研究提供了有效的研究手段。在本研究中,我们发展了一种可以支持多种不同的细胞系同时在一个容器中培养的新型细胞培养系统。该系统中,各细胞系生长于同一容器的独立区域,因此我们称此系统为细胞的独立共培养系统。我们利用腺病毒模拟感染实验验证了该系统,结果显示此系统可以有效的用于病毒的分离培养和诊断。

Abstract: Cell culture has played an important role in virology. It provides a platform for the detection and isolation of viruses as well as for the biochemistry and molecular biology study of viruses. In the present study, a new system that could support multiple different cell lines to be simultaneous cultured in one dish was developed. In the system, each cell line was cultured in an isolated zone in the same dish or well and the system is therefore called isolated co-cultured system. The usefulness of this novel approach for virus isolation was demonstrated using a model system based on adenovirus.

猪瘟病毒C株E2基因主要抗原区的分泌型表达 及间接ELISA方法的建立

蔺国珍, 邱昌庆*, 郑福英, 周继章, 曹小安

2008, 23(5): 363 doi: 10.1007/s12250-008-2970-7

将猪瘟病毒C株E2基因主要抗原区原核表达载体pGEX-E2转化进大肠杆菌BL21–CodonPlus(DE3)–RIL中,经条件优化,获得可溶性表达的目的蛋白。应用Glutathione Sepharose TM4B介质纯化重组目的蛋白,Western blot试验表明纯化的目的蛋白能被猪瘟病毒阳性血清识别,具有很好的免疫原性。以纯化的目的蛋白作为包被抗原,初步建立了检测猪瘟病毒抗体的iE2-ELISA诊断方法。结果表明,抗原的最佳包被浓度为0.6 g/mL,血清的最佳稀释度为180。阳性标准为待检血清OD 490 / 阴性血清OD 490≥2.1。应用iE2-ELISA和猪瘟间接血凝试验对100份田间血清样品检测,结果表明两种方法的相对敏感性和特异性分别为90.3% 和 94.7%。特异性实验表明,牛病毒性腹泻血清和猪瘟E2蛋白间没有交叉反应

Abstract: The sequence encoding an E2 main antigen glycoprotein of the C strain of classical swine fever virus (CSFV) was highly expressed in the host cell E. coli BL21–CodonPlus(DE3)–RIL using the pGEX-4T-1 expression vector and the soluble recombinant product purified with Glutathione Sepharose TM4B by centrifugation. The soluble recombinant protein showed good immune reactions and was confirmed by Western blot using anti-CSFV-specific antibodies. Then an indirect ELISA with the purified E2 protein as the coating antigen was established to detect antibody against CSFV. The result revealed that the optimal concentration of coated antigen was 0.6 g/mL and the optimal dilution of serum was 180. The positive cut-off value of this ELISA assay was OD tested serum / OD negative serum≥2.1. The E2-ELISA method was evaluated by comparison with the indirect hemagglutination test (IHAT). When a total of 100 field serum samples were tested the sensitivity and specificity were 90.3% and 94.7% respectively. Specificity analysis showed that there were no cross-reactions between BVD serum and the purified E2 protein in the E2-ELISA.

内源性反转录病毒ev/J gp85基因表达及其免疫活性分析

杨玉莹, 秦爱建, 梁雄燕, 童淑梅

2008, 23(5): 369 doi: 10.1007/s12250-008-2971-6

内源性反转录病毒ev/J是新近鉴定的内源性禽反转录病毒序列,其gp85基因与ALV-J禽白血病病毒具有高度的同源性。本研究利用英杰Bac-toBac杆状病毒表达系统表达了源自商品肉鸡的ev/J gp85基因。使用ALV-J特异性单克隆抗体JE9 (ADOL-4817)、抗ev/J gp85基因编码物(SU)阳性鼠血清和ALV-J自然感染鸡阳性血清,通过免疫荧光、Western blot、间接ELISA和阻断ELISA分析了重组内源性ev/J gp85 SU的免疫原性和免疫反应性。结果表明ev/J SU可以特异识别JE9单抗和ALV-J自然感染鸡阳性血清,而且抗ev/J SU鼠血清能够阻断外源性ALV-J gp85 SU与ALV-J自然感染鸡阳性血清之间的免疫反应。结果表明内源性ev/J gp85基因重组表达物与外源性ALV-J囊膜蛋白(ADOL-4817 and IMC10200)之间有紧密的免疫学相关性。

Abstract: The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC10200 strain).

用RT-PCR技术扩增、鉴定自然感染牛精液中口蹄疫病毒

邵军军, 常惠芸*, 林 彤, 丛国正, 独军政, 郭建宏, 包慧芳, 尚佑军, 杨亚明, 刘湘涛, 刘在新, 柳纪省

2008, 23(5): 378 doi: 10.1007/s12250-008-2980-5

为了调查自然感染种牛的精液是否被口蹄疫病毒污染(在生物学上的安全性),本文用RT-PCR和病毒分离方法对收集的15份精液样品(包括5份有口蹄疫典型症状牛的样品)进行病原学鉴定。结果显示,自然感染牛的精液被口蹄疫病毒污染,但从无症状牛的精液样品中没有检测出口蹄疫病毒。这也是我国首次证实自然感染牛的精液被口蹄疫病毒污染。对VP1基因部分序列的分析显示,精液样品分离株与水疱皮分离株的基因序列的同源性达97.9%,属于同一基因亚群。

Abstract: To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group.