CN110317247B - 一种抗口腔致病菌多肽及应用 - Google Patents
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Abstract
本项发明属于医药应用领域，具体涉及一种抗口腔致病菌多肽及应用。该多肽的氨基酸序列为：Gly‑Phe‑Gly‑Arg‑Ile‑Gly‑Arg‑Ile‑Val‑Leu‑Arg‑Asn‑Ala‑Leu‑Gln‑His，如SEQ ID NO:1所示。本发明所述的抗牙龈卟啉单胞菌及具核酸杆菌多肽适宜制成牙龈卟啉单胞菌及具核酸杆菌感染性疾病药物、漱口水及牙膏添加剂。该多肽对牙龈卟啉单胞菌及具核酸杆菌具有较强的杀灭作用，且对人血红细胞毒性极小。
Description
技术领域
本项发明属于医药应用领域，具体涉及一种抗口腔致病菌多肽及应用。
背景技术
口腔致病菌牙龈卟啉单胞菌及具核酸杆菌在牙周炎、侵袭性牙周炎、牙周脓肿、以及牙髓感染等口腔疾病的发生发展过程中发挥着重要作用，同时还是引起口臭的主要病原菌。目前在牙周疾病的治疗过程中主要运用抗生素来抑制杀灭牙龈卟啉单胞菌及具核酸杆菌等病原菌，这些抗生素的长期使用，不仅会诱导细菌产生耐药性，同时还会杀灭部分口腔及胃肠道中的正常菌群，进而导致菌群失调，并引起相关疾病。抗菌肽作为新型抗菌药物研发中的明星分子，不易诱导细菌产生耐药性，而特异性作用于病原菌的抗菌多肽具有很好的开发应用价值。
发明内容
本发明旨在提供一种抗口腔致病菌多肽，该抗菌肽能够治疗牙龈卟啉单胞菌及具核酸杆菌相关感染，且无毒副作用。
本发明的第一个目的是提供一种抗口腔致病菌多肽，所述多肽的氨基酸序列为：Gly-Phe-Gly-Arg-Ile-Gly-Arg-Ile-Val-Leu-Arg-Asn-Ala-Leu-Gln-His，氨基酸序列如SEQ ID NO:1所示。
本发明的第二个目的是提供上述多肽在制备用于治疗牙龈卟啉单胞菌及具核酸杆菌感染性疾病药物中的应用。
本发明的第三个目的是提供上述多肽在制备漱口水中的应用。
本发明的第四个目的是提供上述多肽在制备牙膏添加剂中的应用。
与现有技术相比，本发明具有如下有效果：
本发明所述的抗口腔致病菌多肽可杀灭牙龈卟啉单胞菌及具核酸杆菌，且对人血红细胞毒性极小，具有开发成抗牙龈卟啉单胞菌及具核酸杆菌药物、牙膏添加剂和漱口水的潜力，可用于防治牙龈卟啉单胞菌及具核酸杆菌引起的牙周疾病。
附图说明
图1为实施例3中抗口腔致病菌多肽抑菌效果图(图中，A：牙龈卟啉单胞菌不加多肽处理；B：牙龈卟啉单胞菌加多肽处理；C：具核酸杆菌加多肽处理；D：具核酸杆菌不加多肽处理)。
图2为实施例3中培养时间与OD值的关系图。
图3为实施例4中抗口腔致病菌多肽抑菌多肽的溶血活性图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
抗口腔致病菌多肽的筛选及获得
本发明提出的抗菌多肽来源于我们所构建的中药蛴螬多肽序列数据库，其氨基酸序列如下：Gly-Phe-Gly-Arg-Ile-Gly-Arg-Ile-Val-Leu-Arg-Asn-Ala-Leu-Gln-His，如SEQ ID NO:1所示。
根据所示的氨基酸序列，经吉尔生化(上海)有限公司合成得到对应的抗菌多肽，其纯度>95％。
实施例2
抗口腔致病菌多肽的抑菌活性
将多肽配置成为一定浓度储存备用，以生理盐为稀释液，通过二倍稀释法配置系列梯度的多肽溶液。将对数生长期的待测菌液用新鲜的培养基调整为约105-106CFU/mL用于接种，其中牙龈卟啉单胞菌及具核酸杆菌所用培养基为ATCC Medium 2722，正常菌株所用培养基为LB。分别取上述多肽溶液和菌液100μL加入无菌的96孔细胞培养版中，每个多肽浓度3个平行。37℃恒温培养，牙龈卟啉单胞菌及具核酸杆菌厌氧培养48h，常规菌正常培养18-24h,以肉眼无可见细菌生长的最低多肽浓度为该多肽对检测菌的最小抑菌浓度。
抗口腔致病菌多肽的抗菌活性如表1所示，由表1可以看出，该多肽主要对牙龈卟啉单胞菌和具核酸杆菌具有很好的抗菌活性，其最小抑菌浓度均为12.5μg/mL。而在100μg/mL的浓度下对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌和白色念珠菌没有抗菌活性。说明该多肽具有很好的细菌选择性。
表1
实施例3
抗口腔致病菌多肽抑菌效果的有效性
将对数生长期的待测菌液用新鲜的培养基ATCC Medium 2722调整为约105-106CFU/mL，加入抗菌多肽，抗菌多肽终浓度为100μg/mL，不加抗菌多肽为对照。37℃恒温培养，牙龈卟啉单胞菌及具核酸杆菌厌氧培养120h，观测细菌生长情况并检测培养体系OD630吸光值。
抗菌多肽的抗菌有效性如图1所示，图中A：牙龈卟啉单胞菌不加多肽处理；B：牙龈卟啉单胞菌加多肽处理；C：具核酸杆菌加多肽处理；D：具核酸杆菌不加多肽处理。
由图1可以看出，经多肽处理的牙龈卟啉单胞菌(试管B)和具核酸杆菌(试管C)培养了120h后，培养体系依然为透明的；而未经多肽处理的牙龈卟啉单胞菌(试管A)和具核酸杆菌(试管D)培养了120h后，培养体系为浑浊的。如图2所示，经多肽处理后的培养体系其OD630吸光值在120h依然为基础吸光值。由此可以说明该多肽对牙龈卟啉单胞菌和具核酸杆菌具有很好的抗菌有效性，有效性最少可维持120h。说明该多肽具有持久或者高效的抑菌作用。
实施例4
抗口腔致病菌多肽的溶血活性
采集健康人新鲜血液抗凝血2mL，1000g离心5min，弃血浆收集红细胞。将收集到的红细胞用生理盐洗涤三遍，按2％(V/V)的比例重悬血细胞。在无菌的96孔细胞培养板中加入100μL上述红细胞重悬液，再分别加入不同浓度的多肽溶液，每个多肽浓度设置3个平行。以0.1％Tritonx-100作为完全溶血的阳性对照，以生理盐组作为不溶血的阴性对照。37℃孵育1h，1000g离心10min。离心后将上清转移到新的无菌96孔细胞培养板中，并利用酶标仪检测570nm处的光吸收值。计算多肽的溶血活性。
抗牙龈卟啉单胞菌及具核酸杆菌多肽溶血活性如图3所示，该多肽在800μg/mL的浓度下仅表现出不足2％的溶血活性，说明其毒副作用非常低。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 河南科技大学
<120> 一种抗口腔致病菌多肽及应用
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Gly Phe Gly Arg Ile Gly Arg Ile Val Leu Arg Asn Ala Leu Gln His
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Claims (4)
1.一种抗口腔致病菌多肽，其特征在于，所述多肽的氨基酸序列为：Gly-Phe-Gly-Arg-Ile-Gly-Arg-Ile-Val-Leu-Arg-Asn-Ala-Leu-Gln-His，如SEQ ID NO:1所示。
2.如权利要求1所述抗口腔致病菌多肽在制备用于治疗牙龈卟啉单胞菌及具核酸杆菌感染性疾病药物中的应用。
3.如权利要求1所述抗口腔致病菌多肽在制备漱口水中的应用。
4.如权利要求1所述抗口腔致病菌多肽在制备牙膏添加剂中的应用。
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| US20050183150A1 (en) * | 2004-02-13 | 2005-08-18 | Rebecca Torisky | Method for regenerating and transforming St. Augustinegrass from embryogenic callus |
| CN106432520B (zh) * | 2016-09-22 | 2019-03-12 | 曹蔚 | 一种蛴螬多糖组分及其制备方法和用途 |
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