CN112641929B - 美洲大蠊宿主防御肽的抗病毒应用 - Google Patents
美洲大蠊宿主防御肽的抗病毒应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，特别涉及美洲大蠊宿主防御肽的抗病毒应用，通过对美洲大蠊宿主防御肽进行研究，发现美洲大蠊宿主防御肽PaDefensin5是具有较好稳定性的阳离子肽，将其在果蝇感染病毒模型中进行验证，结果发现美洲大蠊宿主防御肽PaDefensin5对果蝇C病毒具有很好的抗性，因此，该多肽有望成为治疗由病毒感染的疾病治疗药物，具有良好的药用价值前景，可作为新型抗病药物开发。
Description
技术领域
本发明涉及生物技术领域，特别涉及美洲大蠊宿主防御肽的抗病毒应用。
背景技术
美洲大蠊(Periplaneta americana)属昆虫纲，蜚蠊目，蜚蠊科，俗称蟑螂、偷油郎等。早期研究发现，蟑螂大约在3.5亿年前的志留纪时期(Mya)进化而来，与螳螂一起从一种同样会产生白蚁的祖先谱系中分化出来，是世界上生命力最强、最古老、至今繁衍最成功的的昆虫类群之一。蟑螂可携带多种致病的病毒、细菌、真菌原虫、以及寄生虫蠕虫的卵。至少携带16个属的32种细菌，包括枯草芽孢杆菌，大肠杆菌和9株沙门氏菌等。此外蟑螂还被发现藏有7种寄生虫的卵，至少17种真菌，3种原生动物和2种脊髓灰质炎病毒，但至今仍未发现或分离出任何一种能够感染美洲大蠊的病毒。
宿主防御肽(host defense peptides,HDPs)是由动物机体免疫和上皮等组织细胞表达与分泌的一类具有抗菌、抗癌、修复损伤、免疫调节等作用的多功能肽，是先天免疫系统的重要成员，是机体抵抗病原入侵的第一道屏障。宿主防御肽已被证明在昆虫抗病毒的免疫反应中发挥重要作用。比如研究表明，在受到Alphavirus病毒侵染后，果蝇中抗病毒多肽Diptericin B和Attacin C的RNA表达量显著上升。而敲除抗病毒多肽基因dptB和attC后，Alphaviruses的病毒滴度显著上升，果蝇发育受到显著影响，证明了抗病毒多肽与果蝇抗Alphavirus有密切联系。第一个研究抗病毒多肽的药物作用的报道发表于1986年，揭示了防御素在体外可以有效抑制单纯性疱疹病毒和巨细胞病毒的复制。随后更多的抗病毒多肽被应用于抗病毒性疾病的治疗上。比如柞蚕素治疗乙型肝炎具有良好效果。已有研究发现从美洲大蠊中提取分离得到的抗菌肽成分对大肠杆菌有杀灭作用。美洲大蠊提取物对体外培养的牙龈卟啉单胞菌的生长有抑制作用。美洲大蠊醇提取物具有抗乙肝病毒的功效，以其为主要原料的“肝龙胶囊”已被证实可使慢性乙型肝炎患者HbeAg和HBV-DNA感染指标转阴率大大提高，临床上具有抗乙型肝炎病毒活性和免疫活性。然而，关于美洲大蠊宿主防御肽对其它病毒是否有防治作用，目前并不清楚。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明提供了美洲大蠊宿主防御肽的抗病毒应用，具体用于抗果蝇C病毒。
本发明的技术方案如下文所示。
本发明一方面提供了美洲大蠊宿主防御肽在制备抗病毒药物中的应用，所述病毒为小RNA病毒科病毒。
在本发明的一些实施方式中，所述小RNA病毒科病毒为果蝇C病毒。
在本发明的一些实施方式中，所述美洲大蠊宿主防御肽为PaDefensin5。
在本发明的一些实施方式中，所述PaDefensin5的氨基酸序列如SEQ ID NO：1所示或如SEQ ID NO:1所示的氨基酸序列经缺失、插入或替换所获得的具有相同功能的氨基酸序列。
在本发明的一些实施方式中，采用固相合成的方法获得所述PaDefensin5。
在本发明的一些实施方式中，所述药物还包括其它抗病毒药物。
本发明人运用生物信息学从美洲大蠊基因组信息库进行宿主防御肽的预测和筛选，包括一级结构分析(带点性质、稳定性等)，二级结构预测(盘曲折叠方式，亲疏水性等)，三维结构的预测以及抗病毒能力的预测等。对预测所得的部分宿主防御肽通过Fmoc法进行多肽的固相合成，并在果蝇感染病毒模型中进行验证，结果发现美洲大蠊宿主防御肽PaDefensin5对果蝇C病毒(DCV)具有很好的抗性，因此，该多肽有望成为治疗由病毒感染的疾病治疗药物，具有良好的药用价值前景，可作为新型抗病药物开发。
附图说明
图1为PaDefensin5序列中信号肽与成熟肽的分布图；
图2为PaDefensin5的质谱图；
图3为不同浓度PaDefensin5对细胞的毒害作用图；
图4为不同处理组在不同时间的细胞形态图；
图5为不同处理组中细胞内病毒拷贝数图；
图6为不同处理组的细胞流式图和细胞凋亡情况图；
图7为不同处理组中细胞活力线粒体差异图；
图8为不同处理组的凋亡相关蛋白表达情况图。
具体实施方式
以下结合具体的实施例对本发明的技术方案和技术效果做进一步说明和阐释，但本发明并不限于这些具体实施方式。实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均可从商业途径得到的试剂和材料。所有操作处理、试剂保存等，如无特殊说明，均在室温下进行操作和保存。
实施例1美洲大蠊宿主防御肽的生物信息学分析
从美洲大蠊基因组库中筛选出5种美洲大蠊宿主防御肽，为Defensin家族多肽，其序列如表1所示。
表1.美洲大蠊5种宿主防御肽的氨基酸序列
对PaDefensin1、PaDefensin2、PaDefensin3、PaDefensin4、PaDefensin5这5种多肽进行带电荷的计算、稳定性预测、亲疏水性预测、二级结构预测以及是否为抗病毒多肽的预测、氨基酸组成成分分析等生物信息学分析，其结果如表2所示。根据表2的结果发现，PaDefensin5为具有较好的稳定性的阳离子多肽。对PaDefensin5进一步分析，发现其为分泌型多肽，其信号肽裂解位点位于第18-19个氨基酸之间，信号肽与成熟肽的分布如图1所示。
表2.5种美洲大蠊宿主防御肽的理化性质
实施例2美洲大蠊宿主防御肽PaDefensin 5的合成
采用Fmoc/PyBOP固相合成法，在431A自动合成仪上合成PaDefensin5。Fmoc保护基团的脱除用30％六氢吡啶的DMF溶液；肽链从树脂上的切落用切肽试剂(三氟乙酸/结晶苯酚/水/乙二硫醇/甲乙硫醚/＝81.5/5/5/5/2.5/1)。使用Waters600E色谱C18反相柱子纯化并收集目标肽，然后做质谱鉴定。
经质谱鉴定(如图2所示)为PaDefensin5，纯度为95％。
实施例3美洲大蠊宿主防御肽PaDefensin 5细胞毒性的验证
材料：
A、果蝇KC细胞的培养。
果蝇KC细胞培养在加入5％的胎牛血清(HyClone)和1％的青链霉素混合液的施耐德果蝇培养基(Schneider's Drosophila Medium,Lonza)中，27℃恒温培养箱培养，每隔2-3天后以1:4的比例稀释培养基作细胞传代培养。
B、多肽药物：PaDefensin5(实施例2制备得到)
溶于灭菌的超纯水中。
试验步骤：
KC细胞接种于96孔板，加入KC细胞培养基，使得细胞单层贴壁生长(约24h)，把细胞分成5组，分别为1、2、3、4、5组，第1组加入10μL浓度为0mg/mL的PaDefensin5、第2组加入10μL浓度为0.0001mg/mL的PaDefensin5、第3组加入10μL浓度为0.0010mg/mL的PaDefensin5、第4组加入10μL浓度为0.0100mg/mL的PaDefensin5、第5组加入10μL浓度为0.1000mg/mL的PaDefensin5。共同培养48h后通过MTT法检测细胞存活率，结果如图3所示,发现从高到低浓度的PaDefensin5对KC细胞均无毒害作用。
实施例4美洲大蠊宿主防御肽PaDefensin 5抗病毒药效验证
材料：
A、果蝇KC细胞
果蝇KC细胞培养在加入5％的胎牛血清(HyClone)和1％的青链霉素混合液的施耐德果蝇培养基(Schneider's Drosophila Medium,Lonza)中，27℃恒温培养箱培养，每隔2-3天后以1:4的比例稀释培养基作细胞传代培养。
B、病毒株
本研究使用的果蝇C病毒(Drosophila Cvirus)来源于中科院武汉病毒所。本实验所用DCV病毒滴度为TCID50＝10-9.3/mL。
C、多肽药物：PaDefensin5
溶于灭菌的超纯水中。
1、PaDefensin5处理对感染病毒细胞的病毒拷贝数的影响
KC细胞接种于24孔板，加入KC细胞培养基，使得细胞单层贴壁生长(约24h)，把细胞分成3组，编号为A组、B组、C组，A组为空白对照组，B组为DCV感染组，C组为DCV感染+PaDefensin5处理组(PaDefensin5在DCV感染细胞前提前混入培养基中，使得PaDefensin5浓度为0.1000mg/mL；其在DCV感染结束后也持续存在)。
分别在DCV感染细胞后24h，48h进行显微镜观察，观察结果如图4所示。结果显示病毒感染24h后，B组和C组未有明显的差异变化，48h后B组细胞病变状况比D组严重。
根据这一现象，分别取DCV感染48h后的A组、B组、C组细胞进行实时荧光定量PCR检测DCV的拷贝数的变化。结果如图5所示，得到的荧光定量PCR结果与显微结果一致，B组DCV拷贝数明显高于C组。
2、PaDefensin5药物处理对感染病毒细胞的凋亡影响
1)流式细胞仪检测
KC细胞接种于24孔板，加入KC细胞培养基，使得细胞单层贴壁生长(约24h)，把细胞分成4组，编号为A组、B组、C组，D组。A组为空白对照组，B组为DCV感染组，C组为PaDefensin5单独处理组，D组为DCV感染+PaDefensin5处理组(PaDefensin5在DCV感染细胞前提前混入培养基中，使得PaDefensin5浓度为0.1000mg/mL；其在DCV感染结束后也持续存在)。
在DCV感染细胞后24h收集细胞用Annexin-VFICT试剂盒对细胞膜进行染色再通过流式细胞仪进行检测。其结果如图6所示，结果显示，B组凋亡细胞比率明显高于A组与C组，而D组与之相比，凋亡细胞比率有所下降。
2)细胞活力线粒体检测
KC细胞接种于24孔板，加入KC细胞培养基，使得细胞单层贴壁生长(约24h)，把细胞分成4组，编号为A组、B组、C组，D组。A组为空白对照组，B组为DCV感染组，C组为PaDefensin5单独处理组，D组为DCV感染+PaDefensin5处理组(PaDefensin5在DCV感染细胞前提前混入培养基中，并在DCV感染结束后也持续存在)。
在DCV感染细胞后24h收集细胞用JC-1荧光试剂对细胞线粒体膜进行染色，结果如图7所示。结果显示A组、C组、D组活性线粒体的比率显著高于B组的活性线粒体的比率。活性线粒体能够反映出细胞的凋亡情况，线粒体膜电位通过JC-1染色后，会从红色荧光到绿色荧光的转变，通过此法可以很容易地检测到细胞膜电位的下降与否，线粒体膜电位的升降降能反映线粒体的活性情况，可作为细胞凋亡早期的一个检测指标。
3)蛋白免疫印迹检测
KC细胞接种于6孔板，加入KC细胞培养基，使得细胞单层贴壁生长(约24h)，把细胞分成4组，编号为A组、B组、C组，D组，A组为空白对照组，B组为DCV感染组，C组为PaDefensin5单独处理组，D组为DCV感染+PaDefensin5处理组(PaDefensin5在DCV感染细胞前提前混入培养基中，并在DCV感染结束后也持续存在)。
在DCV感染细胞后24h收集细胞并提取蛋白进行蛋白免疫印迹检测，通过对凋亡蛋白drICE的切割情况来判断细胞的凋亡情况，其结果如图8所示。在果蝇细胞里，完整的drICE蛋白条带位于38kDa的位置，而当凋亡发生时drICE蛋白会被剪切，而剪切后的蛋白cleaveddrICE会出现在21kDa中出现。从图中看出，B组和D组的38kDa的drICE蛋白较A组和C组有所下降；A组与C组在21kDa位置的cleaveddrICE没有出现条带，而B组与D组在21kDa位置的出现了明显的条带，并且B组的条带亮度要高于D组的条带。
三种不同的方法都验证了PaDefensin5处理能抑制DCV感染细胞的凋亡。说明PaDefensin5具有抗果蝇C病毒的作用，且无副作用，对细胞无明显毒害作用，有望治疗病毒感染引起的疾病，具有良好的药用价值前景。
尽管已参照具体实施方式公开了本发明，但是显而易见的是，在不背离本发明的真正精神和范围的情况下，本领域的其他技术人员可以设计本发明的其它实施方式和变化，所附权利要求书目的在于被解释为包括所有这样的实施方式和等价的变化。此外，本文引用的所有参考文献的内容据此引入本文以供参考。
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Claims (3)
1.美洲大蠊宿主防御肽在制备抗病毒药物中的应用，其特征在于，所述病毒为小RNA病毒科病毒；
所述小RNA病毒科病毒为果蝇C病毒；
所述美洲大蠊宿主防御肽为PaDefensin5；
所述PaDefensin5的氨基酸序列如SEQ ID NO：1所示。
2.根据权利要求1所述的应用，其特征在于，采用固相合成的方法获得所述PaDefensin5。
3.根据权利要求1所述的应用，其特征在于，所述药物还包括其它抗病毒药物。
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| CN107759677B (zh) * | 2016-12-22 | 2021-04-20 | 四川好医生攀西药业有限责任公司 | 一种美洲大蠊过敏原蛋白ba2及其表达方法 |
| CN110590930B (zh) * | 2018-06-13 | 2023-03-31 | 四川好医生攀西药业有限责任公司 | 美洲大蠊生长因子pdgf在制备损伤修复药物中的应用 |
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| CN101289501A (zh) * | 2008-03-17 | 2008-10-22 | 深圳市圣西马生物技术有限公司 | 美洲商陆抗菌肽、其前体肽及多核苷酸与应用 |
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