CN116143883A - 一种广谱抗微生物肽Scymicrosin7-26及其应用 - Google Patents
一种广谱抗微生物肽Scymicrosin7-26及其应用 Download PDFInfo
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Abstract
本发明公开了一种广谱抗微生物肽Scymicrosin7‑26及其应用,其分子式为C97H178N34O22,其氨基酸序列如SEQ ID NO.01所示,其中,第20位的赖氨酸经‑NH2修饰。本发明由20个氨基酸组成,C端进行酰胺化修饰,分子量为2172.68道尔顿,其中含4个精氨酸和2个甘氨酸。HeliQuest预测该本发明的电荷数为+4,疏水性为0.526,是一条水溶性好、抗菌活性高、杀菌迅速、抗菌谱广且安全、具有广阔应用前景的阳离子短肽;仅需较低微摩尔浓度即可发挥对临床耐药性细菌和新型隐球菌的抑制或杀灭作用,对HEK‑293T细胞无细胞毒性。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种广谱抗微生物肽Scymicrosin7-26及其应用。
背景技术
自1928年,弗莱明发现青霉素以来,多种抗生素陆续被发现,抗生素的出现、应用和普及挽救了无数的生命。然而,在持续性使用抗生素的过程中,由于抗生素的不当或过度使用,导致临床上出现了大量的多重耐药(MDR)、广泛耐药(XDR)和泛耐药(PDR)细菌,使得临床治疗难度加剧、治疗成本增加、病人住院时间延长,甚至出现了无药可用的境地。多粘菌素曾被认为是抗生素的最后一道防线,在细菌中亦出现了对多粘菌素耐药的mcr-1基因。细菌耐药性问题日益严峻,每年有70万以上的人死于耐药性细菌感染,如不采取有效措施,这一数字在2050年将达到1000万。其中,由ESKAPE病原引起的医院内感染最具威胁且治疗困难,给临床治疗带来巨大挑战。ESKAPE病原包括屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌科细菌,ESKAPE病原对多数或所有抗生素均呈现多重耐药问题。
近年来,真菌对抗真菌药物唑类、多烯类、棘白菌素类和氟胞嘧啶的耐药问题层出不穷,最为典型的唑类耐药问题源于唑类药物广泛地在农业中应用。由侵袭性真菌感染的致死率远高于20%,随着免疫功能缺陷人群的增多,侵袭性真菌感染呈上升趋势,每年由白色念珠菌、新型隐球菌、烟曲霉和荚膜组织胞浆菌等引起的侵袭性真菌感染导致的死亡人数高达150万人,严重威胁人类的健康。
细菌和真菌耐药在全球蔓延,耐药率逐年攀升,新药研发相对滞后,新型抗微生物药物开发迫在眉睫。抗菌肽(Antimicrobial Peptide,AMP)又称为宿主防御肽,作为天然免疫的效应分子,通常为两亲性的阳离子多肽,广泛分布于各生物体中,具有广谱抗微生物活性、杀菌迅速、抗菌机制多样、不易产生耐药性,除了直接杀菌作用抗菌肽还具有促进伤口愈合、抗炎及免疫调控等功能,被认为是良好的天然抗生素替代品。已有多种抗菌肽进入临床试验,用于真菌或细菌感染的治疗。鉴于其潜在的临床应用价值,开发和挖掘新型抗菌肽,以期减少和改善临床耐药性问题。
发明内容
本发明目的在于提供一种广谱抗微生物肽Scymicrosin7-26。
本发明的另一目的在于提供上述广谱抗微生物肽Scymicrosin7-26的应用。
本发明的技术方案如下:
一种广谱抗微生物肽Scymicrosin7-26,其分子式为C97H178N34O22,其氨基酸序列如SEQ ID NO.01所示,其中,第20位的赖氨酸经-NH2修饰。
上述广谱抗微生物肽Scymicrosin7-26在制备抗细菌组合物中的应用。
在本发明的一个有选手实施方案中,所述抗细菌组合物对金黄色葡萄球菌、蜡状芽孢杆菌、单核细胞增生李斯特氏菌、屎肠球菌、粪肠球菌、表皮葡萄球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌和阴沟肠杆菌具有抑制和杀灭作用。
一种抗细菌组合物,其有效成分包括上述广谱抗微生物肽Scymicrosin7-26。
在本发明的一个有选手实施方案中,其有效成分为上述广谱抗微生物肽Scymicrosin7-26。
在本发明的一个有选手实施方案中,其对金黄色葡萄球菌、蜡状芽孢杆菌、单核细胞增生李斯特氏菌、屎肠球菌、粪肠球菌、表皮葡萄球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌和阴沟肠杆菌具有抑制和杀灭作用。
上述广谱抗微生物肽Scymicrosin7-26在制备抗新型隐球菌组合物中的应用。
一种抗新型隐球菌组合物,其特征在于:其有效成分包括上述广谱抗微生物肽Scymicrosin7-26。
在本发明的一个有选手实施方案中,其有效成分为上述广谱抗微生物肽Scymicrosin7-26。
本发明的有益效果是:
1、本发明的广谱抗微生物肽Scymicrosin7-26由20个氨基酸组成,C端进行酰胺化修饰,分子量为2172.68道尔顿,其中含4个精氨酸和2个甘氨酸。HeliQuest预测该广谱抗微生物肽Scymicrosin7-26电荷数为+4,疏水性为0.526,是一条水溶性好、抗菌活性高、杀菌迅速、抗菌谱广且安全、具有广阔应用前景的阳离子短肽。
2、本发明的广谱抗微生物肽Scymicrosin7-26仅需较低微摩尔浓度即可发挥对临床耐药性细菌和新型隐球菌的抑制或杀灭作用,对HEK-293T细胞无细胞毒性。
附图说明
图1为本发明实施例3中广谱抗微生物肽Scymicrosin7-26对金黄色葡萄球菌QZ19131、鲍曼不动杆菌CGMCC 1.6769的杀菌动力学曲线图。
图2为本发明实施例4中广谱抗微生物肽Scymicrosin7-26处理金黄色葡萄球菌CGMCC1.2465和铜绿假单胞菌QZ19122引起的形态结构变化图。其中,A为金黄色葡萄球菌,B为金黄色葡萄球菌+3μM Scymicrosin7-26,C为铜绿假单胞菌,D为铜绿假单胞菌+3μMScymicrosin7-26。
图3为本发明实施例5中MTS法检测广谱抗微生物肽Scymicrosin7-26对HEK-293T细胞的细胞毒性实验结果图,其中,横坐标为Scymicrosin7-26蛋白浓度(μM),纵坐标为细胞存活率(%)。
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。
实施例1广谱抗微生物肽Scymicrosin7-26的制备
本实施例中的广谱抗微生物肽Scymicrosin7-26的氨基酸序列为SEQ ID NO:01:GARQLVRRIVPVVLGALSRL-NH2,C端进行酰胺化修饰(-NH2),即第20位的赖氨酸经-NH2修饰。
本实施例委托南京金斯瑞有限公司以固相合成方法合成获得纯度达95%以上的广谱抗微生物肽Scymicrosin7-26,并提供多肽分子量、HPLC等检测信息,用HeliQuest预测其电荷和疏水性,其他理化参数均用ProtParam预测,抗菌肽Scymicrosin7-26理化参数如表1所示。
表1抗菌肽Scymicrosin7-26理化参数
实施例2 Scymicrosin7-26最小杀菌浓度(MBC:Minimum BactericidalConcentration)的测定
涉及到的菌株有:新型隐球菌、白色念珠菌、金黄色葡萄球菌、蜡状芽孢杆菌、单核细胞增生李斯特氏菌、屎肠球菌、粪肠球菌、表皮葡萄球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、嗜水气单胞菌和铜绿假单胞菌购自中国科学院微生物研究所菌种保藏中心;临床耐药菌QZ19131、QZ18080、QZ19138、QZ20141、QZ18109、QZ19122、QZ18106、QZ18103来源于福建医科大学附属第二医院检验科。
测定方法如下:
(1)活化菌种,待菌落生长至合适大小,细菌挑克隆至营养肉汤培养至对数期,真菌则用YPD培养至对数生长期。
(2)离心收集细菌或真菌,细菌用MH液体稀释,使得细菌终浓度约为5×105 cfu/mL;酵母真菌用RPMI1640+0.165M MOPS,真菌终浓度约为5×104cfu/mL。分别加入等体积不同浓度的Scymicrosin7-26。
(3)培养1-2天后,吹打混匀,吸取适量同抗菌肽共孵的菌液至固体培养基表面,观察并记录MBC结果。
Scymicrosin7-26最小杀菌浓度(MBC)观察结果如表2所示,Scymicrosin7-26抗菌活性强,具有广谱抗菌活性。
表2抗菌肽Scymicrosin7-26的抗菌活性
实施例3Scymicrosin7-26杀菌动力学曲线
本实施例涉及到的菌株有:金黄色葡萄球菌QZ19131、鲍曼不动杆菌CGMCC1.6769。
实验方法同实施例2中的方法,抗菌肽和细菌共孵一定时间后,在不同时间点取适量混悬液稀释后涂布于营养肉汤平板,37℃培养箱静置培养过夜,进行菌落计数。广谱抗微生物肽Scymicrosin7-26对金黄色葡萄球菌QZ19131、鲍曼不动杆菌CGMCC 1.6769杀菌动力学曲线如图1所示,Scymicrosin7-26杀菌作用快,低浓度的抗菌肽(1.5μM)在5min内即可杀灭所被测细菌。
实施例4扫描电镜观察Scymicrosin7-26处理后细菌的形态结构变化
本实施例涉及到的菌株有:金黄色葡萄球菌CGMCC1.2465和铜绿假单胞菌QZ19122。
扫描电镜样品的制备按以下步骤进行:
(1)培养细菌至对数生长期,取5×107cfu/mL的菌液+等体积抗菌肽,铜绿假单胞菌37℃共孵30min,金黄色葡萄球菌共孵1h后,离心去上清,用PBS清洗一次后收集菌体。
(2)2.5%戊二醛4℃固定步骤(1)菌体2h。固定后用PBS清洗菌体三次,制成高浓度悬液,滴至玻片上,待菌体黏附后进行乙醇梯度脱水。
(3)临界点干燥、喷金,扫描电镜观察并拍片记录。
广谱抗微生物肽Scymicrosin7-26对金黄色葡萄球菌CGMCC1.2465和铜绿假单胞菌QZ19122形态结构变化如图2所示,经抗菌肽Scymicrosin7-26处理后,金黄色葡萄球菌菌体破裂、胞内物质外流,铜绿假单胞菌菌体表面则出现泡状突起、菌体间的丝状结构减少、胞内物质外流。
实施例5 MTS法检测和评价Scymicrosin7-26的细胞毒性
本实施例选取人肾上皮细胞(HEK-293T)细胞,对广谱抗微生物肽Scymicrosin7-26细胞毒性进行测定,如图3所示,横坐标为Scymicrosin7-26蛋白浓度(μM),纵坐标为细胞存活率(%)。
测定方法如下:
将生长状态良好的细胞接种至96孔细胞培养板,次日,加入含有不同浓度Scymicrosin7-26的培养基,共孵24h后,加入MTS-PMS混合溶液,孵育4h后,酶标仪OD492nm读板,计算细胞存活率。
结果如图3,在所测浓度范围内,Scymicrosin7-26对HEK-293T细胞无细胞毒性。
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 (9)
1.一种广谱抗微生物肽Scymicrosin7-26,其特征在于:其分子式为C97H178N34O22,其氨基酸序列如SEQ ID NO.01所示,其中,第20位的赖氨酸经-NH2修饰。
2.权利要求1所述的广谱抗微生物肽Scymicrosin7-26在制备抗细菌组合物中的应用。
3.如权利要求2所述的应用,其特征在于:所述抗细菌组合物对金黄色葡萄球菌、蜡状芽孢杆菌、单核细胞增生李斯特氏菌、屎肠球菌、粪肠球菌、表皮葡萄球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌和阴沟肠杆菌具有抑制和杀灭作用。
4.一种抗细菌组合物,其特征在于:其有效成分包括如权利要求1所述的广谱抗微生物肽Scymicrosin7-26。
5.如权利要求4所述的一种抗细菌组合物,其特征在于:其有效成分为如权利要求1所述的广谱抗微生物肽Scymicrosin7-26。
6.如权利要求4或5所述的一种抗细菌组合物,其特征在于:其对金黄色葡萄球菌、蜡状芽孢杆菌、单核细胞增生李斯特氏菌、屎肠球菌、粪肠球菌、表皮葡萄球菌、福氏志贺氏菌、鲍曼不动杆菌、大肠埃希菌、铜绿假单胞菌、肺炎克雷伯菌和阴沟肠杆菌具有抑制和杀灭作用。
7.权利要求1所述的广谱抗微生物肽Scymicrosin7-26在制备抗新型隐球菌组合物中的应用。
8.一种抗新型隐球菌组合物,其特征在于:其有效成分包括如权利要求1所述的广谱抗微生物肽Scymicrosin7-26。
9.如权利要求8所述的一种抗新型隐球菌组合物,其特征在于:其有效成分为如权利要求1所述的广谱抗微生物肽Scymicrosin7-26。
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