CN116585448A - 黑鲷抗菌肽AS-hepc3(48-56)在制备抗新生隐球菌组合物中的应用 - Google Patents
黑鲷抗菌肽AS-hepc3(48-56)在制备抗新生隐球菌组合物中的应用 Download PDFInfo
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Abstract
本发明公开了黑鲷抗菌肽AS‑hepc3(48‑56)在制备抗新生隐球菌组合物中的应用。本发明通过检测黑鲷抗菌肽AS‑hepc3(48‑56)对新生隐球菌CGMCC2.1563和新生隐球菌H99的抗菌活性,分析新生隐球菌H99对AS‑hepc3(48‑56)的耐药性,确定黑鲷抗菌肽AS‑hepc3(48‑56)具有抗新生隐球菌活性,同时为研发天然肽类抗隐球菌药物奠定基础。
Description
技术领域
本发明属于海洋分子生物学技术领域,具体涉及黑鲷抗菌肽AS-hepc3(48-56)在制备抗新生隐球菌组合物中的应用。
背景技术
真菌病是由真菌感染引起的疾病,根据感染人体的部位可以分为浅表真菌病、皮肤真菌病、皮下真菌病和系统性真菌病四种类型。尽管系统性真菌病的发病率相对较低,但与这些感染相关疾病的死亡率却非常高,因此更值得被关注。系统性真菌病主要发生于免疫功能低下的患者,据统计全球每年有近200万人死于系统性真菌病。在所有与系统性真菌病相关的死亡病例中,90%以上是由隐球菌属、念珠菌属、曲霉菌属和肺孢子菌属感染引起的。新生隐球菌(Cryptococcus neoformans)是一类环境来源的病原真菌,能够以干酵母或孢子形式,通过人类宿主的呼吸道进入肺部定殖。当宿主免疫系统受损时,潜伏的新生隐球菌会被激活,穿越肺泡毛细血管屏障进入血液,跟随血液循环扩散至肾脏和骨髓等身体各个部位,并能突破血脑屏障进入中枢神经系统。据统计,新生隐球菌每年导致的全球新发感染病例数超过100万例,其中约62.5万人死亡,死亡率超过60%,严重威胁公众生命健康。
发明内容
本发明目的在于克服现有技术缺陷,提供黑鲷抗菌肽AS-hepc3(48-56)在制备抗新生隐球菌组合物中的应用。
本发明的技术方案如下:
黑鲷抗菌肽AS-hepc3(48-56)在制备抗新生隐球菌组合物中的应用。
在本发明的一个优选实施方案中,所述抗新生隐球菌组合物对新生隐球菌CGMCC2.1563和新生隐球菌H99有抑制和杀灭作用。
一种抗新生隐球菌组合物,其特征在于:其有效成分包括黑鲷抗菌肽AS-hepc3(48-56)。
在本发明的一个优选实施方案中,其有效成分为黑鲷抗菌肽AS-hepc3(48-56)。
在本发明的一个优选实施方案中,其对新生隐球菌CGMCC2.1563和新生隐球菌H99有抑制和杀灭作用。
本发明的有益效果是:在所有与隐球菌有关的感染中,大约95%的感染是由新生隐球菌格鲁比变种菌株(新生隐球菌H99)引起,其余4%至5%的感染是由新生隐球菌新生变种或格特隐球菌引起。本发明通过检测黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌CGMCC2.1563和新生隐球菌H99的抗菌活性,分析新生隐球菌H99对AS-hepc3(48-56)的耐药性,确定黑鲷抗菌肽AS-hepc3(48-56)具有抗新生隐球菌活性,同时为研发天然肽类抗隐球菌药物奠定基础。
附图说明
图1为本发明实施例3中黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌CGMCC2.1563和新生隐球菌H99的杀菌动力学图,其中横坐标为时间(min),纵坐标为真菌数量[log(CFU/mL)]。
图2为本发明实施例4中黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌CGMCC2.1563和新生隐球菌H99的生物膜抑制活性图,其中横坐标为AS-hepc3(48-56)蛋白浓度(μM),纵坐标为细胞活性(%)。
图3为本发明实施例5中新生隐球菌H99对黑鲷抗菌肽AS-hepc3(48-56)和抗生素氟康唑耐药性的分析;其中横坐标为时间(day),纵坐标为最小抑菌浓度(minimuminhibitory concentration,MIC)与初始MIC的比值。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
以下实施例中所涉及的新生隐球菌CGMCC2.1563购买自中国科学院微生物研究所菌种保藏中心(China General Microbiological Culture Collection Center,CGMCC);所涉及的新生隐球菌H99由中国科学院微生物研究所王琳淇研究员馈赠,以上菌株均由申请人保种贮藏。
实施例1黑鲷抗菌肽AS-hepc3(48-56)的制备
本实施例黑鲷抗菌肽AS-hepc3(48-56)的氨基酸序列为:
Arg-Arg-Arg-Arg-Cys-Arg-Phe-Cys-Cys(SEQ ID NO.01)
采用现有的固相化学合成的方法即可得到纯度达95%以上黑鲷抗菌肽AS-hepc3(48-56)。本实施例中的黑鲷抗菌肽AS-hepc3(48-56)委托吉尔生化(上海)有限公司以固相合成方法合成获得,并提供高效液相色谱和质谱检测信息。
实施例2黑鲷抗菌肽AS-hepc3(48-56)抗新生隐球菌活性测定
(1)将实验室保种的新生隐球菌接种至YPD平板,于28℃培养箱倒置培养48h。待菌落生长起来后,挑取单克隆菌落接种至YPD液体培养基中,28℃,230rpm,培养18h;
(2)5000g,离心5min,弃上清,用RPMI-MOPS重悬菌体并调整菌浓度至2×104CFU/mL;
(3)用无菌的Milli-Q水将AS-hepc3(48-56)粉末溶解并使用0.22μm滤膜进行过滤,置于冰上备用;
(4)在无菌96孔细胞培养板中进行AS-hepc3(48-56)抗新生隐球菌活性测定,分别设置空白对照组、阴性对照组和待测实验组,每组设置三个平行:
空白对照组:50μL待测样品和50μL培养基
阴性对照组:50μL无菌Milli-Q水和50μL菌悬液
待测实验组:50μL待测样品和50μL菌悬液
(5)将96孔细胞培养板置于28℃培养箱中,培养72h,观察MIC结果;随后将各组的澄清溶液吹打混匀后,吸取5μL滴于YPD平板上,于28℃培养箱中倒置培养48h,观察最小杀菌浓度(minimum fungicidal concentration,MFC)结果。
结果如表1所示,黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌具有明显的抗菌活性,其中对新生隐球菌CGMCC2.1563的MIC和MFC均为3-6μM,对新生隐球菌H99的MIC和MFC均为6-12μM。
表1黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌的抗菌活性
注:最小抑菌浓度(MIC)用a-b表示。a:肉眼可见菌体生长的最高蛋白浓度;b:肉眼未见菌体生长的最低蛋白浓度。最小杀菌浓度(MFC)用a-b表示。a:平板可见菌落生长的最高蛋白浓度;b:平板未见菌落生长的最低蛋白浓度。
实施例3黑鲷抗菌肽AS-hepc3(48-56)杀菌动力学曲线
(1)将实验室保种的新生隐球菌接种至YPD平板,于28℃培养箱倒置培养48h。待菌落生长起来后,挑取单克隆菌落接种至YPD液体培养基中,28℃,230rpm,培养18h;
(2)5000g,离心5min,弃上清,用RPMI-MOPS重悬菌体并调整菌终浓度至1×107CFU/mL;
(3)调整AS-hepc3(48-56)浓度至24μM和48μM,置于冰上备用;
(4)将新生隐球菌悬液与AS-hepc3(48-56)在EP管中混匀,置于28℃培养箱;
(5)在孵育后的15、30、60、120和240min,取共孵的混合液梯度稀释后涂布至YPD平板上,28℃倒置培养48h,记录新生隐球菌单克隆数量。
黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌的杀菌动力学曲线结果如图1所示,新生隐球菌菌量随着与AS-hepc3(48-56)共孵时间的延长而不断降低,并且与AS-hepc3(48-56)的浓度呈正相关。当48μM的AS-hepc3(48-56)与新生隐球菌共孵1h时,能杀死90%以上的新生隐球菌CGMCC2.1563或新生隐球菌H99;共孵4h时,能杀死所有的新生隐球菌CGMCC2.1563或新生隐球菌H99。
实施例4黑鲷抗菌肽AS-hepc3(48-56)对新生隐球菌生物膜抑制活性
(1)将实验室保种的新生隐球菌接种至YPD平板,于28℃培养箱倒置培养48h。待菌落生长起来后,挑取单克隆菌落接种至YPD液体培养基中,28℃,230rpm,培养18h;
(2)5000g,离心5min,收集上述培养的菌体,弃上清,随后用PBS清洗菌体2次;
(3)用生物膜基本培养基(20mg/mL硫胺素,30mM葡萄糖,26mM甘氨酸,20mMMgSO4·7H2O,58.8mM KH2PO4)重悬菌体并调整菌浓度至1×107CFU/mL;
(4)将100μL上述菌液加入96孔板中,置于37℃培养箱静置培养48h以形成生物膜;
(5)生物膜形成后,吸去上清,用PBS清洗3次以去除未粘附的隐球菌;
(6)将100μL RPMI-MOPS和100μL AS-hepc3(48-56)溶液加入96孔板中,置于37℃培养箱孵育24h;
(7)加入20μL MTS-PMS溶液,37℃避光孵育2h,使用酶标仪测得A492值。
结果如图2所示,AS-hepc3(48-56)在3-6μM时即可抑制新生隐球菌生物膜的活性,当抗菌肽浓度为48μM时,AS-hepc3(48-56)对新生隐球菌CGMCC2.1563和新生隐球菌H99生物膜的抑制率分别为93.50%和70.76%。
实施例5新生隐球菌H99对黑鲷抗菌肽AS-hepc3(48-56)耐药性分析
(1)将实验室保种的新生隐球菌H99接种至YPD平板,于28℃培养箱倒置培养48h。待菌落生长起来后,随机挑取6个单克隆菌落(Colony 1-Colony 6)接种至YPD液体培养基中,28℃,230rpm,培养18h;
(2)5000g,离心5min,弃上清,用RPMI-MOPS重悬菌体并调整菌浓度至2×104CFU/mL;
(3)在无菌96孔细胞培养板中检测AS-hepc3(48-56)对新生隐球菌H99的抗菌活性,临床常用抗生素氟康唑作为对照;
(4)将96孔细胞培养板置于28℃培养箱中培养72h,观察并记录MIC结果;
(5)从各组中取10μL在抗菌肽或抗生素条件下生长的菌液,加入990μL新鲜的RPMI-MOPS混匀;
(6)按照上述(3)、(4)、(5)步骤,重复30次。
结果如图3所示,新生隐球菌H99在临床常用抗生素氟康唑连续处理90天后,氟康唑对其MIC是初始MIC的32-64倍,证明本实施例可以诱导新生隐球菌H99产生耐药性;而在AS-hepc3(48-56)连续处理90天后,AS-hepc3(48-56)对其MIC仍与初始MIC一致,说明新生隐球菌H99未对AS-hepc3(48-56)产生明显耐药性。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (5)
1.黑鲷抗菌肽AS-hepc3(48-56)在制备抗新生隐球菌组合物中的应用。
2.如权利要求1所述的应用,其特征在于:所述抗新生隐球菌组合物对新生隐球菌CGMCC2.1563和新生隐球菌H99有抑制和杀灭作用。
3.一种抗新生隐球菌组合物,其特征在于:其有效成分包括黑鲷抗菌肽AS-hepc3(48-56)。
4.如权利要求3所述的一种抗新生隐球菌组合物,其特征在于:其有效成分为黑鲷抗菌肽AS-hepc3(48-56)。
5.如权利要求3或4所述的一种抗新生隐球菌组合物,其特征在于:其对新生隐球菌CGMCC2.1563和新生隐球菌H99有抑制和杀灭作用。
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