CN116370449B - 一种小分子抑制剂px478在鱼类抗病毒中的应用 - Google Patents
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Abstract
本发明公开了小分子抑制剂PX478在制备预防或治疗鱼类鲤春病毒血症病毒感染的药物或饲料添加剂中的用途，通过斑马鱼活体实验发现，在PX478浓度为10μM时，PX478可有效增强斑马鱼抗病毒相关基因（ifn1，mxc）的表达，同时显著抑制病毒SVCV的复制能力，从而提高SVCV感染后斑马鱼的存活率。因此，PX478可用于制备预防或治疗鱼类抗病毒（尤其是鲤春病毒血症病毒）的药物或饲料添加剂。
Description
技术领域
本发明属于水产养殖技术领域，具体涉及小分子抑制剂PX478在鱼类抗病毒中的应用。
背景技术
鱼类病原性疾病包括细菌性、真菌性、病毒性和寄生虫性疾病等类型，其中病毒性疾病潜伏期长，不易发现，一旦发病，速度较快，不易控制。鲤春病毒血症(Spring viraemiaof carp，SVC)是一种严重危害鲤科鱼类的水生动物病毒性疾病，具有致死率高、传染性强、发病急等特点。鲤春病毒血症已被世界动物卫生组织(World Organization for AnimalHealth；OIE)列入到必须通报的动物传染疾病名录，也被中国农业相关部门列入到一类动物疫病。鲤春病毒血症的直接病原是鲤春病毒血症病毒(Spring viraemia of carpvirus，SVCV)，SVCV属于弹状病毒科病毒，能感染青鱼、草鱼等我国淡水养殖核心品种，常导致鱼类大量死亡，造成巨大的经济损失。目前针对鲤春病毒血症尚无商品化的疫苗和特效药物，因此该疾病的防治难度大。
PX478是低氧诱导因子HIF1α的特异抑制剂，其分子量为394.12，分子式为C13H20Cl4N2O3，结构式见图1。已有研究表明，PX478可穿透血脑屏障，并具有抗癌活性。PX478也能够增强前列腺癌PC3细胞的放射敏感性。在高脂饮食小鼠体内，PX478减少它们脂肪组织中的纤维化病变和炎症浸润。但是PX478在抗病毒先天免疫中的研究目前没有相关报道。
发明内容
本发明的目的在于提供小分子抑制剂PX478在制备预防或治疗鱼类鲤春病毒血症病毒感染的药物或饲料添加剂中的用途。
为了实现上述目的，本发明采用以下技术方案：
本发明通过斑马鱼活体实验发现，在PX478的浓度为10μM和20μM时，作用48h后对斑马鱼幼鱼均无明显毒性；在PX478浓度为10μM时，PX478可有效增强斑马鱼抗病毒相关基因(ifn1，mxc)的表达，同时显著抑制病毒SVCV的复制能力，从而提高SVCV感染后斑马鱼的存活率。因此，PX478可用于制备预防或治疗鱼类抗病毒(尤其是鲤春病毒血症病毒)的药物或饲料添加剂。
与现有技术相比，本发明的有益效果是：本发明首次发现并证明PX478是一种具有抗鲤春病毒血症病毒SVCV病毒活性的小分子抑制剂，通过对斑马鱼幼鱼进行SVCV病毒的攻毒实验发现，PX478具有抗鱼类病毒的特点，可以用于制备抗病毒药物或饲料添加剂。
附图说明
图1为小分子抑制剂PX478的结构式。
图2为不同浓度PX478对斑马鱼毒性的影响。
图3为PX478增强斑马鱼抗病毒的能力。
图4为PX478对SVCV感染后斑马鱼存活率的影响。
图5为PX478增强SVCV感染后斑马鱼抗病毒相关基因(ifn1，mxc)的表达。
图6为PX478抑制SVCV的复制。
具体实施方式
下列实施例涉及到的材料和试剂，实验条件和方法如下：
一、材料和试剂
材料：PX478(购于Selleck，#S7612)，溶剂DMSO(购于Selleck)；
病毒：鲤春病毒血症病毒SVCV；
斑马鱼：AB品系斑马鱼来自国家斑马鱼资源中心。
二、方法及步骤
PX478储液配制：将购得的100mg PX478粉末溶于2.54ml DMSO中，配得100mM的PX478储液。
实施例1PX478对斑马鱼的毒性实验
利用AB品系斑马鱼成鱼进行雌雄交配，获得足够数量的三天龄的斑马鱼幼鱼，将三天龄的斑马鱼幼鱼按照30条为一组随机分为3组，分别使用60mm培养皿培养。分别向每组鱼中加入4ml培养水，之后向空白对照组(PX478 0μM)中加入1ml含有1μl DMSO的培养水，向PX478工作浓度为10μM组中加入1ml含有0.5μl PX478储液的培养水，向PX478工作浓度为20μM组中加入1ml含有1μl PX478储液的培养水。将斑马鱼幼鱼置于28℃恒温培养箱中，48h后显微镜下观察斑马鱼状态并拍照记录，如图2所示。
根据显微镜拍照结果表明，小分子抑制剂PX478在浓度分别为10μM、20μM时，均对斑马鱼幼鱼的生命活动及生存状态没有影响，说明PX478对斑马鱼幼鱼无毒性。
实施例2PX478增强斑马鱼抗病毒能力
利用AB品系斑马鱼成鱼进行雌雄交配，获得足够数量的三天龄的斑马鱼幼鱼，将三天龄的斑马鱼幼鱼按照30条为一组随机分为2组，分别使用60mm培养皿培养。分别向每组鱼中加入2ml培养水，之后向空白对照组(DMSO组)中加入1ml含有1μl DMSO的培养水，向实验组PX478组中加入1ml含有1μlPX478储液的培养水，此时PX478的工作浓度是10μM，之后向每组中加入2ml SVCV病毒(～1.0×108TCID50/ml)。将斑马鱼幼鱼置于28℃恒温培养箱中，每隔4h观察斑马鱼幼鱼状态并统计死亡数量，病毒感染36h后显微镜拍照记录，如图3所示，红色箭头表示死亡的斑马鱼幼鱼。利用graphpad prism软件统计斑马鱼存活情况，结果如图4所示。
根据显微镜拍照结果表明，小分子抑制剂PX478在工作浓度为10μM时，在SVCV病毒感染36h后，对照组DMSO组斑马鱼死亡数量明显多于实验组PX478组，这说明PX478可以增强斑马鱼抗SVCV病毒的能力。同时，图4的死亡曲线表明，对照组斑马鱼在SVCV病毒感染52h后全部死亡，而PX478处理后的斑马鱼此时存活率仍在70％以上，这说明PX478可以显著提高斑马鱼受SVCV病毒感染后的存活率。
实施例3PX478增强斑马鱼抗病毒相关基因的表达
利用AB品系斑马鱼成鱼进行交配，获得足够数量的三天龄的斑马鱼幼鱼，将三天龄的斑马鱼幼鱼按照30条为一组随机分为4组，分别使用60mm培养皿培养。向其中两组中分别加入4ml培养水，之后一组中加入1ml含有1μl DMSO的培养水，另一组中加入1ml含有1μlPX478储液的培养水，此时PX478的终浓度为10μM，这两组均为无刺激组(即-SVCV)；向另外两组分别加入2ml培养水，之后向对照组(DMSO组)中加入1ml含有1μl DMSO的培养水，向实验组PX478组中加入1ml含有1μl PX478储液的培养水，此时PX478终浓度为10μM，之后分别向这两组中加入2ml SVCV病毒(～1.0×108TCID50/ml)，因此这两组为刺激组(即+SVCV)。将斑马鱼幼鱼置于28℃恒温培养箱中培养24h后将培养水去掉，将斑马鱼收集于1.5ml离心管中，利用Trizol法对斑马鱼总RNA进行提取，之后使用反转录试剂盒获得cDNA，根据SYBRGreen qPCR mix说明书要求，进行实时荧光定量PCR，检测斑马鱼抗病毒相关基因(ifn1，mxc)的表达情况，荧光定量PCR引物序列如表1所示。实验结果如图5所示。
实验结果表明，病毒SVCV可明显激活斑马鱼抗病毒相关基因ifn1、mxc的表达，同时，PX478可显著增强抗病毒基因ifn1、mxc的表达水平。
表1
实施例4PX478抑制SVCV病毒的复制能力
利用AB品系斑马鱼成鱼进行交配，获得足够数量的三天龄的斑马鱼幼鱼，将三天龄的斑马鱼幼鱼按照30条为一组随机分为4组，分别使用60mm培养皿培养。向其中两组中分别加入4ml培养水，之后一组中加入1ml含有1μl DMSO的培养水，另一组中加入1ml含有1μlPX478储液的培养水，此时PX478的终浓度为10μM，这两组均为无刺激组(即-SVCV)；向另外两组分别加入2ml培养水，之后向对照组(DMSO组)中加入1ml含有1μl DMSO的培养水，向实验组PX478组中加入1ml含有1μl PX478储液的培养水，此时PX478终浓度为10μM，之后分别向这两组中加入2ml SVCV病毒(～1.0×108TCID50/ml)，因此这两组为刺激组(即+SVCV)。将斑马鱼幼鱼置于28℃恒温培养箱中培养24h后将培养水去掉，将斑马鱼收集于1.5ml离心管中，利用Trizol法对斑马鱼总RNA进行提取，之后使用反转录试剂盒获得cDNA，根据SYBRGreen qPCR mix说明书要求，进行实时荧光定量PCR，检测SVCV病毒五个基因(SVCV-P，SVCV-G，SVCV-N，SVCV-M，SVCV-L)的表达情况，荧光定量PCR引物序列如表1所示。实验结果如图6所示。
实验结果表明，PX478可显著抑制病毒SVCV各组分基因的表达情况，进而说明PX478显著抑制SVCV的复制能力。
Claims (2)
1.PX478在制备用于预防或者治疗鱼类鲤春病毒血症病毒感染的药物或饲料添加剂中的应用，其特征在于，所述PX478的结构式为
2.根据权利要求1所述的应用，其特征在于，PX478的使用浓度为10μM。
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| CN101168063A (zh) * | 2007-11-07 | 2008-04-30 | 中山大学 | 一种鲤春病毒血症病毒dna疫苗及其制备方法 |
| WO2011057220A2 (en) * | 2009-11-09 | 2011-05-12 | Northwestern University | Compositions and methods for treating lymphoma |
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| Title |
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| 鲤春病毒血症病毒研究进展;尚信池等;中国兽医杂志;第56卷(第6期);61-64 * |
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