美洲商陆抗病毒蛋白研究进展

美洲商陆抗病毒蛋白（pokeweed antiviral proteins,PAP)是一类具多种生物功能和活性的蛋白,本在阐述了该类蛋白的生化等特性的同时,综述了该类蛋白在抗植物病毒,抗动物病毒以及用作免疫毒素等方面的研究进展。     

美洲商陆核基因组抗病毒蛋白基因的克隆及序列分析

通过ＰＣＲ扩增,从美洲商陆（Ｐｈｙｔｏｌａｃａａｍｅｒｉｃａｎａ）核基因组中克隆了商陆抗病毒蛋白基因,序列分析表明,该基因含８８５个核苷酸,与已报道的序列比较,核苷酸同源性为９９．３％。     

美洲商陆核基因组抗病毒蛋白基因的克隆及序列分析

通过ＰＣＲ扩增,从美洲商陆（Ｐｈｙｔｏｌａｃａａｍｅｒｉｃａｎａ）核基因组中克隆了商陆抗病毒蛋白基因,序列分析表明,该基因含８８５个核苷酸,与已报道的序列比较,核苷酸同源性为９９３％。     

商陆种子抗病毒蛋白的0.25nm分辨率晶体结构

在高蛋白浓度 ( 1 0 0mg/mL)和高温 ( 33℃ )条件下得到了美洲商陆种子抗病毒蛋白的单晶 ,具有较高的衍射分辨率 .用分子置换法在 0 .2 5nm分辨率确定了该蛋白晶体结构的初始模型 ,经分子动力学修正技术精化 ,晶体学R因子为 1 8.1 5 % ,键长键角对理想值的均方差分别为 0 .0 0 1 6nm和 2 .0 4° .通过与另外两种商陆抗病毒蛋白结构的比较 ,发现连接第 5条 β链段和第 2个α螺旋的环套区位于活性中心附近 ,并且为序列和结构的多变区 ,其上分布着可能的活性残基 ,因而可能是与活性差异有关的区域     

美洲商陆抗真菌蛋白转化烟草的研究和抗病性检测

本研究为美洲商陆抗病毒蛋白(PaAFP)基因首次对植物遗传转化的研究,转入烟草中研究此蛋白对烟草立枯病的抗性。从美洲商陆叶片中获得美洲商陆抗真菌蛋白前体蛋白基因cDNA序列,构建植物表达载体pCAMBIA1300-PaAFP,通过三亲杂交法将其导入根癌农杆菌LBA4404受体菌,转染烟草获得了大量再生转基因植株。PCR、Southern杂交、RT-PCR以及Tris-Tricine-SDS-PAGE检测结果表明目的基因已经整合到烟草基因组中,并且已经得到转译。转基因植株苗期抗立枯病试验表明,转基因烟草植株对立枯丝核菌表现出了抗性。     

英国Agricultural Genetics公司进行商陆植物抗病毒基因研究

英国Agricultural Genetics公司研究人员一直在致力于美洲商陆植物抗病毒蛋白基因的研究.他们计划利用此基因转化作物,使其产生对病毒的抗性.由于Oncogen公司(Seattle,WA)利用此毒素蛋白与单克隆抗体的结合物破坏了艾滋病病毒,因而英国公     

美洲商陆抗病毒蛋白-Ⅱ基因的克隆和表达

根据报道的cDNA序列,用RT-PCR的方法从美洲商陆夏季的叶片中克隆美洲商陆抗病毒蛋白-Ⅱ(pokeweedantiviralproteinⅡ,PAP-Ⅱ)基因。将PAP-Ⅱ基因克隆至表达载体pET-28a( )并在大肠杆菌中表达,SDS-PAGE电泳分析结果表明,PAP-Ⅱ蛋白在BL21(DE3)菌中获得表达,表达产物以不溶性包涵体形式存在,经过溶解包涵体、复性和BBSTNTA树脂柱亲和层析纯化,获得高纯度的PAP-Ⅱ蛋白。用非放射性基于ELISA方法检测经过复性纯化后PAP-Ⅱ蛋白和蓖麻毒素A链(RTA)在体外对HIV-1整合酶有较强的抑制活性,其IC50分别约为303μg/mL,220μg/mL。用MTT法分析PAP-Ⅱ蛋白的生物学活性,复性纯化后蛋白对HEP-G2和Hela细胞有细胞毒作用,IC50分别为93μg/mL,102μg/mL,说明了PAP-Ⅱ蛋白能抑制肿瘤细胞的生长。构建的PAP-Ⅱ表达系统所表达的蛋白经复性后具有生物学活性,为进一步研究PAP-Ⅱ的抗HIV-1机制和抗肿瘤作用奠定了基础。     

商陆抗病毒蛋白重组子筛选及发酵条件初探

目的：筛选体外高表达商陆抗病毒蛋白的毕赤酵母重组菌株,并摸索其适合的发酵条件。方法：通过电击转化将含有商陆抗病毒蛋白（pokeweed antiviral proteins,PAP）基因的分泌型表达载体PIC9K-P导入到毕赤酵母（Pachia pastoris）GS115菌株中。利用免疫印迹法筛选表达量较高的转化子。在摇瓶发酵水平对重组菌株产PAP条件进行初步研究。结果：高表达PAP的重组菌株在发酵时间为96h,10g/L的甲醇浓度,培养基pH值为6.0～6.4时PAP的表达量较高。结论：免疫印迹法适合用于毕赤酵母高表达重组菌株的筛选,重组毕赤酵母的PAP表达量可高达30mg/L。     

商陆抗病毒蛋白基因导入百合愈伤组织初报

利用美洲商陆抗病毒蛋白(Pokeweed antiviral protein,PAP)具有广谱的抗病毒特性,通过冻融法将含有PAP基因的重组表达载体PBll21转入土壤农杆菌LBA4404中,利用叶盘法在农杆菌的介导下转比麝香百合愈伤组织,通过抗性筛选和PCR输测获得了转PAP基因的百合植株。     

美洲商陆毛状根诱导及其离体培养的影响因素

为了探讨利用美洲商陆毛状根生产其药用成分的可能性,研究了美洲商陆毛状根诱导及其离体培养的影响因素。结果表明,美洲商陆叶片外植体被发根农杆菌ATCC 15834感染约18 d后,从其叶片外植体形态学下端叶脉切口处产生毛状根,其中以预培养1 d,农杆菌感染20 min,共培养4 d时的毛状根诱导率最高,达到70%。PCR扩增和硅胶薄层层析结果显示,发根农杆菌Ri质粒的rol C基因以及冠瘿碱合成酶基因已在美洲商陆毛状根基因组中整合和表达。所获得的美洲商陆毛状根系都能在无外源激素的MS固体培养基上快速自主生长;其中以毛状根根系2的生长速度最快、分生侧根能力最强和根表面的根毛密度最高;毛状根根表面呈紫红色或呈白色。在供试的MS、1/2MS、B5和6,7-V液体培养基中,以无外源激素的MS培养基最适合美洲商陆毛状根根系生长。与无外源激素的MS培养基相比,6,7-V培养基更有利于毛状根中商陆皂苷甲的合成与积累。本文所建立的美洲商陆毛状根诱导及其离体培养的适宜条件为今后利用其毛状根株系的规模培养来生产其药用有效成分商陆皂苷甲奠定了实验和技术基础。     

唾液酸糖肽的研究进展

唾液酸糖肽(SGP)是一种含有唾液酸寡糖链的N-糖肽,是蛋黄中的主要组成部分。由于其分支型糖链的结构和唾液酸化糖链的存在,SGP及其相关物质有较好的抗细菌和抗病毒作用。以SGP为基础合成SGP的衍生物,有望成为新的预防性抗细菌和抗病毒药物。我们简要综述了SGP的抗菌、抗病毒研究,SGP相关衍生物的合成及其抗菌、抗病毒的机制。     

海洋药物的抗病毒研究

海洋由于其特殊的生态环境,包含着极其丰富的生物来源的天然产物。自本世纪七十年代以来,已经从海藻类、海绵类、海鞘类、海星类、腹足动物、棘皮动物、腔肠动物、软体动物、珊瑚及海洋微生物等海洋生物中分离出一系列有抗病毒作用的天然化合物,其中有些结构类型已成为抗病毒药物研究的导向化合物。基于现代分离和分析技术的发展、新的实验模型的建立和在病毒学方面分子生物学研究的进展,从海洋生物中寻找新的抗病毒药物已步入一个新的时代。     

Design,Synthesis, Biological Activity Evaluation and Action Mechanism of Myricetin Derivatives Containing Thiazolebisamide

The plant diseases caused by a variety of pathogens such as viruses, bacteria and fungi pose a great threat to global food production and food safety. Therefore, the search for green, efficient and pollution-free pesticides has become an important task. In this article, 23 myricetin derivatives containing thiazolebisamides active groups have been designed and synthesized. Their activities were evaluated by performing in vitro antibacterial and in vivo antiviral assays, microscale thermophoresis (MST) and molecular docking assays. The results of in vivo antiviral assays showed that compounds A4 and A23 exhibited good antiviral activity with EC₅₀ values of 79.0 and 54.1 μg/mL for therapeutic activity and 103.3 and 91.2 μg/mL for protective activity, respectively. The dissociation constants (Kd) values of compounds A4 and A23 against TMV-CP were 0.021 and 0.018 μM, respectively, determined by microscale thermophoresis (MST), which were much smaller than those of the commercial drug ningnanmycin (NNM), which were 2.84 μM. The interaction of compounds A4 , A23 with TMV-CP was further verified at the molecular level. In addition, in vitro antifungal assays of this series of compounds showed that they exhibited some inhibitory activity against a variety of fungi, especially against the phytophthora capsici. Among them, A13 and A20 showed similar inhibitory activity to the control drug azoxystrobin at 100 μg/mL against the phytophthora capsici.     

核苷类药物酶法合成研究进展

由于核苷类似物具有很高的抗病毒活性,因为而已成为医药工作者研究的重点。运用酶法合成核苷类似物．已经显示了巨大的优势。本文综述了酶法合成核苷类似物的产生菌种和酶系,以及它们的催化机理,并罗列了已经用于生产或较有使用价值的菌种。     

应用抑制性消减杂交技术筛选流感病毒感染宿主应答基因

从宿主系统寻找病毒感染特异性相关的生物大分子是研究病毒药物靶标和诊断标志物的新方向 .为了筛选宿主细胞中流感病毒感染特异性基因 ,采用抑制性消减杂交技术 (SSH) ,以流感病毒A 鲁防 93 9(H3N2 )感染MDCK细胞及正常MDCK细胞为材料 ,构建病毒感染特异性差减cDNA文库 ,PCR法扩增鉴定其中插入片段大小 .从差减文库中随机挑取 10 0个克隆进行测序 ,用生物信息学方法对其同源性和基因功能进行分析和预测 .结果显示 ,成功构建了流感病毒感染特异性差减cDNA文库 ,文库中cDNA片段长度在 2 5 0～ 10 0 0bp之间 .从文库中随机选取 10 0个克隆测序 ,获得了 95个有效序列 ,经blast同源性分析发现 ,大部分基因为参与宿主细胞能量代谢和蛋白质生物合成过程中的基因 ;其中 19个为无任何功能线索的新基因片段 .流感病毒感染特异性差减cDNA文库的建立和筛选出病毒感染应答候选新基因cDNA片段 ,为发现新型流感病毒药靶和诊断标志物以及病毒感染机制研究打下基础     

Focus on antivirally active sulfated polysaccharides: from structure-activity analysis to clinical evaluation

In recent years, many compounds having potent antiviral activityin cell culture have been detected and some of these compoundsare currently undergoing either preclinical or clinical evaluation.Among these antiviral substances, naturally occurring sulfatedpolysaccharides and those from synthetic origin are noteworthy.Recently, several controversies over the molecular structuresof sulfated polysaccharides, viral glycoproteins, and cell-surfacereceptors have been resolved, and many aspects of their antiviralactivity have been elucidated. It has become clear that theantiviral properties of sulfated polysaccharides are not onlya simple function of their charge density and chain length butalso their detailed structural features. The in vivo efficacyof these compounds mostly corresponds to their ability to inhibitthe attachment of the virion to the host cell surface althoughin some cases virucidal activity plays an additional role. Thisreview summarizes experimental evidence indicating that sulfatedpolysaccharides might become increasingly important in drugdevelopment for the prevention of sexually transmitted diseasesin the near future.     

广谱抗病毒抑制剂研究进展

研制广谱抗病毒制剂是病毒学前瞻性方向,也是国际医药界倍受瞩目的热点之一.近年来,广谱抗病毒制剂相关研究获得长足进展,突破了抗病毒制剂单一宿主的瓶颈与限制,以及广谱类制剂活性相对较弱的原有思维,部分成果已面向临床应用.本文基于病毒侵染以及宿主细胞防御两个层面,全面综述新型广谱抗病毒抑制剂的研究进展与应用潜力,讨论现存的挑战,并展望了未来研究趋势.     

Type-1 ribosome-inactivating protein from iris bulbs: a useful agronomic tool to engineer virus resistance?

To study the in planta antiviral activity of a type-1 ribosome-inactivating protein from iris bulbs, called IRIP,Nicotiana tabacumcv. Samsun NN was transformed with the IRIP sequence expressed under the control of the 35S cauliflower mosaic virus promoter. Molecular analysis of the transgenic plants and characterization of the purified protein revealed that the recombinant IRIP from tobacco leaves has the same molecular structure and RNA N-glycosidase activity as the native protein from iris bulbs. The tobacco transformants show no apparent phenotypic side effects indicating that ectopically expressed IRIP is not cytotoxic for tobacco cells. No induction of PR-1 could be demonstrated in the transgenic plants expressing IRIP. The in planta antiviral activity of rIRIP was assessed using a bioassay with tobacco mosaic virus. All transformed lines showed a statistically significant lower number of lesions compared to the control plants. The fortunate combination of in planta antiviral activity and lack of cytotoxicity of the ectopically expressed IRIP in transgenic tobacco renders the iris RIP an interesting and useful model for the study and exploitation of the antiviral activity of type-1 RIPs.     

Molnupiravir,favipiravir and other antiviral drugs with proposed potentials for management of COVID-19: a concern on antioxidant aspect

COVID-19 is an important global public health problem that causes millions of infections worldwide. The specific antiviral drug for this new infection is still under research. Some new antiviral drugs, including molnupiravir and favipiravir, are proposed for usefulness in management of COVID-19. Additionally, some classic antiviral drugs used for other viral infections are also reproposed for the potentials for management of COVID-19. In the management of COVID-19, there are several pharmacological actions. An important consideration in antiviral therapy is the management of oxidative stress, which plays important roles in viral infections including to COVID-19. The analysis of antioxidative properties of alternative drugs for management of COVID-19 is interesting and can give basic data for further new antiviral drug researching. Here, the authors perform a molecular analysis on molnupiravir, favipiravir and other antiviral drugs with proposed potentials for management of COVID-19 to determine their antioxidative properties. Data from electron acceptor and donor calculation for each drug is used for further estimating overall antioxidative characteristic. Based on the present study, all studied drugs have overall antioxidative properties. Hence, the advantage of molnupiravir, favipiravir and other antiviral drugs with proposed potentials for the management of COVID-19 is their direct action on viral molecule via binding-blocking process as well as antixodiative process. For management of COVID-19 antioxidative stress, other non-antiviral drugs that are proposed for clinical advantage might also be useful.