CN115040517A - 石蒜碱在防治svcv感染中的应用 - Google Patents
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Abstract
本发明公开了石蒜碱在防治鲤春病毒血症病毒（Spring viraemia of carp virus，SVCV）感染中的应用，细胞感染模型的建立显示，石蒜碱能够对SVCV感染细胞起到显著的保护作用，结合qPCR实验结果发现石蒜碱处理后SVCV相关基因转录水平被显著抑制，Western Blot实验结果显示石蒜碱处理后SVCV病毒在蛋白水平被清除。因此，石蒜碱能够作为防治SVCV感染的药物或添加剂，并具有使用剂量小，保护效果佳的优点。
Description
技术领域
本发明属于水产养殖技术领域，涉及石蒜碱的新用途，具体涉及石蒜碱用于制备预防或治疗SVCV病毒感染的药物或添加剂。
背景技术
石蒜碱(Lycorine hydrochloride)是一种存在于石蒜科植物石蒜的鳞茎内的环形生物碱，是石蒜提取物中主要活性物质。近期的研究表明，石蒜碱的药理活性包括抗肿瘤作用，抗炎作用，调控脂质合成作用等。在抗肿瘤活性方面，石蒜碱通过抑制肿瘤转移的重要调节因子表达，失活机体自噬信号通路，促进肿瘤细胞凋亡等方式防止肿瘤扩散。在抗炎作用方面，石蒜碱被发现对于细菌脂多糖引起的炎症反应具有良好的抑制效果，主要通过抑制微管相关蛋白1A/1B轻链 3B(LC3Ⅱ)的酯化形式形成并增加螯合体蛋白(p62)的表达，从而抑制脂多糖诱导的机体自噬。在调控脂质合成作用方面，石蒜碱能够促进调控脂质生物合成的重要转录因子固醇调节原件结合蛋白(SREBPs)降解，进而改善高脂饮食引发的肥胖、高血脂和胰岛素抵抗。此外，石蒜碱被发现能够在体内、体外有效改善心脏纤维化，或对心血管疾病治疗具有重要意义。目前，石蒜碱针对水生病毒的作用尚不清楚，尚未有将石蒜碱应用于防治水生动物病毒病的实例。
鲤春病毒血症病毒(Spring viraemia of carp virus，SVCV)属弹状病毒科(Rhabdoviridae)，水疱病毒属(Vesiculovirus)，是一种单股负义链RNA病毒。 SVCV引起的鲤春病毒血症(SVC)是一种具高传染性、高致死性的水产疾病，能够造成鲤科鱼类大量死亡，形成巨大的经济损失。SVCV基因组主要编码5种结构蛋白，分别为核蛋白(Nucleoprotein，N)、磷蛋白(Phosphoprotein，P)、基质蛋白(Matrix protein，M)、糖蛋白(Glycoprotein，G)和RNA聚合酶蛋白 (RNA polymerase，L)。目前，SVCV的结构及遗传多样性已有较多研究和报道。同样地，SVCV的致病机理被发现包括影响细胞凋亡，造成氧化应激损伤，针对免疫系统实现免疫逃逸等。
尽管SVCV的研究较为丰富，药物研究较多，仍然缺乏行之有效的防治手段，尚无商品化的特效药。目前，研究人员尝试使用新手段治疗SVCV的感染，例如利用RNA干扰诱导病毒基因表达的沉默，但是，该方式具有成本较高，干扰RNA保存困难的缺点。另外，SVCV的疫苗开发目前尚处于探索阶段，包括灭活疫苗、弱毒疫苗、核酸疫苗和亚单位疫苗等，但尚未研发出保护率、成本、接种方式三项均满足规模化生产，能够实现市场投放的疫苗。因此，在当前的研究背景下，寻找高效安全的药物作为SVCV的防治手段仍然是首选。
发明内容
本发明的目的在于提供石蒜碱在防治鲤春病毒血症病毒(SVCV)感染中的应用，石蒜碱能够显著抑制鱼类离体培养细胞中SVCV病毒相关基因(包括g， l，m，n和p)的转录，TCID50实验证实石蒜碱对于SVCV感染起到保护作用，石蒜碱能够用于制备防治SVCV感染的药物或添加剂。
为了实现上述目的，本发明采用以下技术方案：
石蒜碱在防治SVCV病毒感染中的应用：在本发明的具体实施例中，感染 SVCV的EPC细胞系在加入20μM石蒜碱处理后，病毒造成的细胞病变效应被显著抑制，SVCV滴度显著降低。进一步检测细胞中病毒相关基因扩增情况显示，石蒜碱处理之后，EPC细胞中病毒相关基因(包括g，l，m，n和p)的表达被显著抑制，细胞中SVCV病毒蛋白N、P的表达水平显著降低，SVCV病毒在蛋白水平被清除，同时细胞系中抗病毒免疫相关基因(包括epcifn，epcisg15和 epcvig1)表达量基本恢复正常水平。
本发明实施例中选取鱼类细胞系源于SVCV病毒重要宿主，因此通过建立细胞感染病毒的疾病模型，以及对病毒相关基因表达的检测，可知石蒜碱适用于 SVCV的病毒防治。
石蒜碱作为预防或治疗SVCV病毒感染的药物或添加剂，药物可以以注射剂或口服剂形式使用，具有给药剂量低，成分单一，没有药物残留风险的优点。
附图说明
图1为石蒜碱抑制SVCV在细胞中增殖。A为病变效应，B为病毒滴度。
图2为石蒜碱处理显著降低SVCV病毒基因转录。
图3为石蒜碱处理显著抑制SVCV病毒的蛋白表达。N指核蛋白，P指磷酸化蛋白，β-actin指肌动蛋白(作为内参)。
图4为石蒜碱处理感染SVCV细胞后宿主抗病毒免疫相关基因表达水平恢复。
具体实施方式
实施例1：石蒜碱对SVCV感染细胞的影响
抗病毒实验用于检测SVCV在细胞中的滴度。在24孔细胞培养板及96孔细胞培养板中进行。EPC细胞用于检测SVCV病毒滴度。取生长状态良好的EPC 细胞传代接入24孔板，28℃培养过夜，接种MOI＝10的SVCV于EPC细胞，同时加入20μM石蒜碱处理，接种后继续培养72h，将贴壁细胞使用4％细胞组织固定液固定1h，1％结晶紫染色过夜。次日对染色完成的细胞进行拍照，观察病变效应。结果表明石蒜碱处理使细胞病变效应明显被抑制(图1A)。固定细胞前收集上清，保存于4℃备测病毒滴度。取生长状态良好的EPC细胞传代接入 96孔板，28℃培养过夜；接种收集的上清后，分别使用TCID50方法计算石蒜碱未处理组和处理组病毒滴度。结果显示，20μM石蒜碱处理后的感染病毒细胞中，病毒滴度显著降低(图1B)。
实施例2：石蒜碱对SVCV病毒基因转录的影响
检测感染SVCV之后的细胞用20μM石蒜碱处理后，EPC细胞中病毒相关基因的转录水平。取生长状态良好的EPC细胞传代接入6孔板，28℃培养过夜；按照实施例1中描述的方法接种病毒及药物处理；继续培养24h后，吸尽细胞培养基，用TRIzol(Invitrogen)裂解细胞沉淀后提取细胞总RNA，由GoScript 逆转录试剂盒(Promega)逆转录得到cDNA。通过qPCR检测病毒相关基因(包括g，l，m，n和p)的转录水平，相关方法参照文献[1]。结果表明：20μM石蒜碱处理后，细胞中SVCV病毒相关基因(包括g，l，m，n和p)的转录水平显著下降(图2)。
实施例3：石蒜碱对SVCV增殖的影响
检测感染SVCV之后的EPC细胞用20μM石蒜碱处理后，细胞中病毒相关蛋白的表达情况。取生长状态良好的EPC细胞传代接入6孔板，28℃培养过夜；按照实施例1中描述的方法接种病毒及药物处理；继续培养24h后，吸尽细胞培养基，PBS润洗一遍后放入-80℃冰箱保存；向冰冻保存的细胞沉淀中加入适量RIPA弱裂解液，置于4℃裂解1小时，使细胞充分裂解；吸取细胞裂解物至新的1.5mL离心管中，样品加入1/4体积的5×SDS Sample Buffer，涡旋混匀， 100℃沸水浴10min，置于冰上备用。使用Western Blot实验[2]检测石蒜碱处理后SVCV蛋白表达水平(核蛋白N、磷酸化蛋白P)。实验结果表明，20μM石蒜碱处理后，细胞中SVCV病毒蛋白N、P的表达水平显著降低，SVCV在蛋白水平被清除(图3)。
实施例4：石蒜碱对宿主抗病毒免疫相关基因表达水平的影响
感染SVCV之后的EPC细胞用20μM石蒜碱处理后，检测细胞中抗病毒免疫相关基因的表达情况。取生长状态良好的EPC细胞传代接入6孔板，28℃培养过夜；按照实施例1中描述的方法接种病毒及药物处理；继续培养24h后，用TRIzol(Invitrogen)裂解贴壁细胞后提取细胞总RNA，由GoScript逆转录试剂盒(Promega)逆转录得到cDNA。使用qPCR检测抗病毒免疫相关因子的转录水平(包括epcifn，epcisg15和epcvig1)，相关方法参照文献[1]。实验结果表明，20μM石蒜碱处理后，经SVCV刺激后表达水平较高的抗病毒免疫相关因子 (包括epcifn,epcisg15和epcvig1)在石蒜碱处理后显著恢复(图4)。
参考文献
[1]LU L F,ZHANG C,LI Z C,et al.Grass Carp Reovirus VP35 Degrades MAVSThrough the Autophagy Pathway to Inhibit Fish Interferon Production[J].FrontImmunol,2021,12:613145.
[2]LU L F,ZHANG C,LI Z C,et al.A novel role of Zebrafish TMEM33 innegative regulation of interferon production by two distinct mechanisms[J].PLoS Pathog, 2021,17(2):e1009317。
Claims (2)
1.石蒜碱在制备防治鲤春病毒血症病毒感染的药物中的应用。
2.石蒜碱在制备防治鲤春病毒血症病毒感染的添加剂中的应用。
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