CN115414366A - 11-酮基-beta-乳香酸在制备治疗猫杯状病毒病的药物中的应用 - Google Patents
11-酮基-beta-乳香酸在制备治疗猫杯状病毒病的药物中的应用 Download PDFInfo
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Abstract
本发明公开了11‑酮基‑beta‑乳香酸在制备治疗猫杯状病毒病的药物中的应用，涉及生物医药技术领域。本发明将11‑酮基‑beta‑乳香酸引入到抑制猫杯状病毒的应用中，通过间接免疫荧光、WesternBlot检测猫杯状病毒变化量，TCID50检测猫杯状病毒量滴度变化，表明11‑酮基‑beta‑乳香酸具有明显的抗猫杯状病毒的作用，随药物浓度增加，对猫杯状病毒抑制作用增强，最低有效浓度为6.848μM。
Description
技术领域
本发明涉及生物医药技术领域，特别是涉及11-酮基-beta-乳香酸在制备治疗猫杯状病毒病的药物中的应用。
背景技术
猫杯状病毒(FCV)是一种高度传染性的病原体，在猫科动物种群中广泛分布。FCV属于杯状病毒科Vesivirus属。杯状病毒包括人类的重要病原体(例如诺如病毒，这是人类感染性胃肠炎的最常见原因之一)和其他动物物种，包括欧洲棕兔综合征病毒和兔出血性疾病病毒。杯状病毒颗粒呈六角形或星形，在电镜下呈杯状凹陷；该病毒有一个小的(约7.5kb)单链RNA基因组，具有正(信使)极性，这使其能够快速进化。它是一种无包膜病毒，缺乏脂质包膜，有助于其在环境中持久存在，这也解释了为什么该病毒不与宿主细胞的细胞磷脂膜融合，而是使用替代途径进入它们。FCV的基因组RNA(gRNA)是非分段的，包含三个功能性开放阅读框(ORF1-3)。ORF1编码一种多蛋白，该多蛋白在翻译后被切割成非结构蛋白(蛋白酶、聚合酶等)，而ORF2编码一种多蛋白，该多蛋白随后被加工以释放一种小蛋白，即衣壳的前导蛋白，以及主要衣壳蛋白(VP1)，ORF3编码次要衣壳蛋白(VP2)。
11-酮基-beta-乳香酸(11-Keto-β-boswellicacid)是一种五环三萜酸，来自俗称印度乳香(IndianFrankincense)的乳香树(Boswelliaserrate)树皮的油树脂。11-Keto-beta-boswellicacid具有抗炎活性，主要是由于抑制5-脂氧合酶(5-lipoxygenase；5-LOX)，白三烯(leukotriene)，NF-κB的激活和肿瘤坏死因子α的产生。
查阅相关资料可知，目前仍未见11-酮基-beta-乳香酸在猫杯状病毒方面的相关报道。因此，将11-酮基-beta-乳香酸引入抑制猫杯状病毒中，具有重大现实意义。
发明内容
本发明的目的是提供11-酮基-beta-乳香酸在制备治疗猫杯状病毒病的药物中的应用，以解决上述现有技术存在的问题，本发明将11-酮基-beta-乳香酸引入到抑制猫杯状病毒的应用中，采用间接免疫荧光、TCID50、Western Blot等方法验证了其可以有效抑制猫杯状病毒。
为实现上述目的，本发明提供了如下方案：
本发明提供11-酮基-beta-乳香酸在制备治疗猫杯状病毒病的药物中的应用。
本发明还提供11-酮基-beta-乳香酸在制备抑制猫杯状病毒的药物中的应用。
本发明还提供一种治疗猫杯状病毒病的药物，有效成分包括11-酮基-beta-乳香酸。
本发明还提供一种抑制猫杯状病毒的药物，有效成分包括11-酮基-beta-乳香酸。
本发明公开了以下技术效果：
本发明通过将11-酮基-beta-乳香酸引入到抑制猫杯状病毒的应用中，通过间接免疫荧光、Western Blot检测猫杯状病毒变化量，TCID50检测猫杯状病毒量滴度变化，表明11-酮基-beta-乳香酸具有明显的抗猫杯状病毒的作用，随药物浓度增加，对猫杯状病毒抑制作用增强，最低有效浓度为6.848μM。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为11-酮基-beta-乳香酸在细胞水平对病毒的抑制效果；其中，A为细胞对照，B为猫杯状感染CCL-94 24h后病变图片，C为将11-酮基-beta-乳香酸加入感染细胞后细胞感染图片；
图2为11-酮基-beta-乳香酸对细胞毒性作用的CC50的测定；
图3为11-酮基-beta-乳香酸抗病毒活性TCID50的测定；其中ns代表无显著差异，**代表有显著统计学差异，***代表有极其显著统计学差异；
图4为11-酮基-beta-乳香酸抗病毒活性TCID50的测定；其中，Postive组为猫杯状病毒感染猫肾细胞，negative组为猫肾细胞对照；
图5为11-酮基-beta-乳香酸抗病毒活性EC50的测定；
图6为11-酮基-beta-乳香酸对病毒活性Western Blot的测定。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值，以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
以下实施例中的病毒FCVWZ-1株(Gene bank:KX371573.1)；猫肾细胞(CCL-94)购买于ATCC；11-酮基-beta-乳香酸购自MCE。
实施例1
11-酮基-beta-乳香酸对细胞活性的检测
1)取生长状态良好的CCL-94细胞进行消化传代，用细胞生长液调整细胞密度为1×105/mL，接种于96孔板，每孔100μL，置于37℃、5％CO2培养箱培养24h；
2)14h后，取出96孔板，弃去孔中培养基，用无菌PBS清洗三次；
3)将11-酮基-beta-乳香酸用细胞维持液稀释成25μM、12.5μM、6.26μM、3.125μM、1.5625μM和0.78125μM，然后依次加入到CCL-94细胞中，并用一纵列加入100μL细胞维持液做为对照。
4)48h后，用MTS试剂进行细胞活力检测。
如图1所示，镜下可以观察到正常的CCL-94细胞生长状态良好，形态完整，界限清晰。而用病毒接种感染的CCL-94细胞，3天后细胞出现皱缩，变圆，葡萄串珠样病变，可见细胞核聚集，病变非常明显。用11-酮基-beta-乳香酸和病毒同时处理细胞后，细胞病变明显减少，细胞生长较好。
测定细胞存活率能反应11-酮基-beta-乳香酸对细胞的毒性作用，根据公式计算出细胞存活率(％)＝经过11-酮基-beta-乳香酸处理的细胞发光值平均值/细胞对照组发光值平均值。11-酮基-beta-乳香酸对CCL-94细胞的毒性作用较小，其CC50大于50μM(图2)。
半最大效应浓度(EC50)能反应11-酮基-beta-乳香酸对病毒的抑制效果，即能达到50％最大生物效应(抑制病毒)对应的药物浓度。从图5的EC50曲线图可知11-酮基-beta-乳香酸对猫杯状病毒的治疗效果很好，EC50的具体数值为6.848μM。
实施例2
11-酮基-beta-乳香酸对病毒活性TCID50的测定
1)将CCL-94细胞进行消化传代，用细胞生长液调整细胞密度为1×105/mL，接种12孔板1mL/孔，37℃、5％CO2培养箱培养24h；
2)24h后，取出12孔板，做好标记，分别为20μM、10μM、5μM、2.5μM、1.25μM和0.625μM。准备EP管，加入细胞维持液与11-酮基-beta-乳香酸，使其终浓度为上述8个浓度，振荡器上混匀至少5s；
3)弃去12孔板孔中培养基，用无菌PBS清洗三次，甩干后加入已经稀释好的11-酮基-beta-乳香酸。置于37℃、5％CO2培养箱孵育1h；
4)1h后，取出12孔板，每孔接种0.01MOI的病毒，轻晃12孔板混匀，每块12孔板设置病毒对照与细胞对照。于37℃、5％CO2培养箱培养；
5)约48h后，病毒对照出现70％病变，将12孔板转移至-80℃超低温冰箱冻融一次；
6)收集每孔液体至EP管中，4000rpm离心10min后收集上清测毒价。
7)准备生长状态良好的细胞，待长到80％左右后用胰酶消化，铺到96孔板中。
8)待96孔板中的细胞长到80％左右时，将病毒在灭菌的2mL EP管中进行10倍倍比稀释(首次用10-1到10-12)，每孔病毒悬液量为100μL，每个稀释度作8孔，并做好对照孔。
9)操作需在冰上进行，保证毒价的稳定，并且要震荡均匀，防止病毒聚团，导致测出的毒价偏差较大。
10)平稳地将96板移至含有5％CO2，37℃细胞培养箱。
11)48h后观察结果，取出96孔板在显微镜上看细胞病变，并记录结果。
12)按Reed和Muench两氏法计算病毒TCID50。
TCID50检测(图3)11-酮基-beta-乳香酸在20μM、10μM、5μM、2.5μM、1.25μM和0μM的TCID50分别为10-5.28/mL、10-5.39/mL、10-5.66/mL、10-6.22/mL、10-7.26/mL和10-7.53/mL。如图3所示，在**μM时可以下降**个滴度，大于**μM时病毒的毒价已经检测不出。
实施例3
11-酮基-beta-乳香酸对病毒活性的间接免疫荧光检测
1)取生长状态良好的CCL-94细胞进行消化传代，用细胞生长液调整细胞密度为1×105/mL，接种于96孔板100μL/孔，置于37℃、5％CO2培养箱培养12h；
2)12h后细胞长满单层，取出96孔板用无血清DMEM培养基清洗两遍，换成细胞维持液，加入11-酮基-beta-乳香酸进行2倍倍比稀释，同时加入0.01mol病毒液，使11-酮基-beta-乳香酸终浓度为20μM、10μM、5μM、2.5μM、1.25μM、和0.625μM，同时设置病毒对照组和细胞对照组；
3)待细胞出现60％病变，弃掉孔中培养基，用PBS清洗2遍后加入4％多聚甲醛室温作用10min；
4)弃掉废液，用PBS洗2次后加入含1％BSA的PBST的封闭液，在37℃静置作用1h；
5)PBS洗2次后，加入稀释了1000倍的一抗(FIPVN蛋白兔多克隆抗体)，37℃静置作用1h；
6)PBST洗4次后，加入稀释了2000倍的羊抗兔荧光二抗，37℃避光静置作用1h；
7)弃掉二抗，用PBS洗4次，置于荧光显微镜下观察拍照。
间接免疫荧光检测(图4)可以看出11-酮基-beta-乳香酸在10μM时，检测到猫杯状病毒明显减少。随着药物浓度的下降，检测到的猫杯状病毒不断增加，在1.25μM时，可以在细胞中检测到大量的猫杯状病毒。
实施例4
11-酮基-beta-乳香酸对病毒活性Western Blot的测定
1)将不同浓度药物与病毒共同加入细胞中，待细胞病变约70％，先弃掉细胞培养液，用预冷的PBS洗一次细胞，吸干PBS后，再用1mL(6孔板)的PBS重悬细胞，装入2mL的EP管中；
2)4℃4000r/min离心5min，弃掉上清，加入120μL(6孔板)的细胞裂解液，重悬，在4℃旋转作用25min；
3)4℃12000r/min离心20min，收集上清，加入5×loading buffer，沸水中煮10min后冰上10min，4℃保存；
4)制备12％分离胶，混匀后加入玻璃板中，并在分离胶上加入一层去离子水，置于通风橱中室温放置30min左右；
5)待分离胶完全聚合后，将配置好的5％的浓缩胶加入分离胶之上，插入梳子，于通风橱中室温放置30min左右；
6)将配制好的聚丙烯酰胺凝胶置于垂直电泳槽中，往中间槽及外槽加入适量的1×SDS-PAGE电泳缓冲液，于点样孔中加入适量体积的处理好的蛋白样品和蛋白Marker；
7)接上电源80V30mim，再120V 1h 20min进行电泳；
8)转膜：SDS-PAGE电泳结束后，根据目的蛋白的分子量，以蛋白Marker为对照进行切胶并裁剪合适大小的滤纸和PVDF膜。将切好的滤纸放入电转缓冲液中，将切好的PVDF膜放入甲醇中浸泡，然后分别放入纯水和电转缓冲液中浸泡，放入转膜槽中以120V电压转膜，根据蛋白大小，转膜时间60min；
9)封闭：轻轻用镊子将膜转入一个干净的平皿内，加入适量的封闭液(5％脱脂牛奶)，置于摇床温育2h；
10)一抗孵育：将膜用TBST涮洗一下，放入含事先稀释好一抗(1：1000)的平皿中，于室温摇床上孵育2.5h；
11)二抗孵育：将膜用TBST洗3次，每次15min，然后将膜放入含事先稀释好二抗(1：1000)的平皿中，于室温摇床上孵育2h，孵育完后将膜用TBST洗六遍，每次5min；
12)显色：将A液和B液等体积混匀，于显色仪器中显色；
13)显色拍照后用TBST将膜清洗六遍，每次5min。同样方法孵育GAPDH一抗与羊抗兔二抗，显色后拍照保存。
Western Blot的实验结果：
Western blot检测(图6)可以看出随着药物浓度增加，细胞中检测到的病毒蛋白量不断减少，有明显剂量依耐性。在10μM药物浓度下，基本检测不到病毒蛋白。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (4)
1.11-酮基-beta-乳香酸在制备治疗猫杯状病毒病的药物中的应用。
2.11-酮基-beta-乳香酸在制备抑制猫杯状病毒的药物中的应用。
3.一种治疗猫杯状病毒病的药物，其特征在于，有效成分包括11-酮基-beta-乳香酸。
4.一种抑制猫杯状病毒的药物，其特征在于，有效成分包括11-酮基-beta-乳香酸。
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