CN116444678B - 一种具有真菌抗菌抑菌性能的新型多肽 - Google Patents
一种具有真菌抗菌抑菌性能的新型多肽 Download PDFInfo
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- CN116444678B CN116444678B CN202211275185.8A CN202211275185A CN116444678B CN 116444678 B CN116444678 B CN 116444678B CN 202211275185 A CN202211275185 A CN 202211275185A CN 116444678 B CN116444678 B CN 116444678B
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- malassezia
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Abstract
本发明涉及新型抗真菌肽及其用途。本发明利用现有数据库中的不同功能模块，通过半理性设计，将细胞膜渗透肽（CPP）与二价离子结合肽模块的融合，获得对真菌具有强效抗菌抑菌作用的新型抗真菌肽，开发强效、绿色、安全替代产品；同时可用于解决抗生素滥用及耐药问题，可在洗护及化妆品中替代化学品的使用，具有绿色、温和无刺激、安全的优点，以期能产生巨大的社会经济效益。
Description
技术领域
本发明属于生物技术领域，具体涉及一种具有真菌抗菌抑菌性能的新型多肽。
背景技术
一个多世纪以来，人类应对病菌的主要工具是抗生素。然而，近几十年来，新抗生素的发现严重不足，更多是现有药物基础上变体。不可忽视的是，由于长期以来抗生素滥用，导致已有大量病菌产生抗生素耐药，给人类社会及经济带来巨大的安全威胁，因此寻找抗生素替代品迫在眉睫。
真菌在皮肤微生态中普遍存在。在皮肤健康领域，包括马拉色菌（Malassezia）在内的真菌被认为与多种皮肤炎症相关，如头皮头屑、头/颈部皮炎，溢脂性皮炎，毛囊炎，以及各类皮癣等（Ditte M. L. Saunte, George Gaitanis and Roderick James Hay,Malassezia-Associated Skin Diseases, the Use of Diagnostics and Treatment.Front. Cell. Infect. Microbiol., 20 March 2020）。
洗护及去屑用品领域，目前主要的活性成分为化学品，如ZPT，OCT，CLM，生产方式不可持续，产品本身具有刺激性。其中ZPT因其生殖毒性（Reproductive toxicity），欧盟将会在2022全面禁止其在洗护产品中使用。
OCT通过抑菌、抗氧化作用和分解氧化物等阻断头屑产生的外部因素，但因在去屑香波、护发素产品中使用时不容易沉积在头皮上去屑的效果有限。CLM主要通过非竞争性阻断 P450 酶中羊毛甾醇 14α-去甲基化酶(也称为真菌 CYP51)的合成，从而抑制等离子体真菌细胞膜的重要组成部分麦角固醇的生物合成，使真菌细胞死亡。ZPT的应用能够造成真菌细胞内二价金属离子（如锌离子、铜离子和亚铁离子）不平衡，导致铁硫蛋白活性降低或者丢失，从而抑制真菌的增殖（Nancy L Reeder, Jerry Kaplan, and Jun Xu, et al.Zinc pyrithione inhibits yeast growth through copper influx and inactivationof iron-sulfur proteins. Antimicrob Agents Chemother. 2011 Dec;55(12):5753-60）。
抗真菌肽。随着抗生素耐药性问题的发展，免疫缺陷和/或免疫抑制相关疾病数量的增加，加之以有限的可用治疗手段，正在引发对新替代品的寻找。抗菌肽以对宿主的毒性小，具有针对性或更广的抗菌谱和低抗药性的可能性等优势，备受人们关注。基于此，具有抗真菌特性的抗菌肽，由于其功效和高选择性而成为强有力的候选者（Miguel Fernándezde Ullivarri, Sara Arbulu, Enriqueta Garcia-Gutierrez and Paul D. Cotter.Antifungal Peptides as Therapeutic Agents. Front. Cell. Infect. Microbiol.,17 March 2020 Sec. Clinical Microbiology）。
近20-30年来市场上所使用的抗生素都是上个世纪80年代发现的传统抗生素药物分子结构上的变体。据世界卫生组织统计，近年来正投入于临床开发阶段的近40种抗生素依然未能发现能够充分解决威胁人类生命安全的细菌耐药性问题。总而言之，临床研发进展和最新批准的抗生素药物都不足以应对我们所面对的挑战。
化学抑制剂本身生物兼容性差，安全性不足，甚至可能致癌；可降解性低，对环境造成二次污染，生产方式不可持续。其中某些产品面临逐渐被禁用及淘汰。而抗菌肽的开发主要依赖于天然肽的发现、半合成及全合成，开发难度大，功效不理想。
发明内容
本发明利用现有数据库中的不同功能模块，通过半理性设计，将细胞膜渗透肽（CPP）与二价离子结合肽模块的融合，获得对真菌具有强效抗菌抑菌作用的新型抗真菌肽，开发强效、绿色、安全替代产品；同时可用于解决抗生素滥用及耐药问题，可在洗护及化妆品中替代化学品的使用，具有绿色、温和无刺激、安全的优点，以期能产生巨大的社会经济效益。
本发明中的新型抗菌肽设计意图是模拟ZPT的作用方式，通过破坏真菌细胞内二价离子浓度的不平衡，从而由抑制真菌的增殖。该新型多肽由两个模块组成，其中一个组成模块（模块一）是来自CPPsite 2.0（https://webs.iiitd.edu.in/raghava/cppsite/）中的任何一种细胞膜渗透肽；另一组成部分是一种具有结合锌离子的短肽模块（zinc-bindingmotif sequence, ZBMS），HEX1X2…XnH（模块二）（Vazeux, 1996, THE JOURNAL OFBIOLOGICAL CHEMISTRY , Identification of Glutamate Residues Essential forCatalytic Activity and Zinc Coordination in Aminopeptidase A；Evans MENACH,Yasuhiko HASHIDA, Kiyoshi YASUKAWA, and Kuniyo INOUYE. Effects of Conversionof the Zinc-Binding Motif Sequence of Thermolysin, HEXXH, to That ofDipeptidyl Peptidase III, HEXXXH, on the Activity and Stability ofThermolysin. Biosci. Biotechnol. Biochem., 77 (9), 1901–1906, 2013），其中X可以是20种天然必需氨基酸中的任何一个，n代表X的个数（n≥2）。在组合方式上，ZBMS模块可以直接连接在CPP模块的N端，也可以连接在CPP模块的C端。
本发明所使用的锌离子结合肽ZBMS本身具有结合锌离子的能力，但由于不具备膜结合能力，因此无法与细胞膜结合，因此不能实现通过破坏真菌细胞内二价离子浓度的不平衡，从而由抑制真菌的生长的目的。
本发明所使用的细胞膜渗透肽CPP（来自CPPsite 2.0），可能属于三种情况：1）本身不具有抗菌换抑菌的功效；2）属于已知抗菌肽但未发现具有抗真菌或者特定的真菌，如马拉色菌（见实施例）作用；3）属于已知抗菌肽且具有抗真菌或者特定的真菌，如马拉色菌（见实施例）作用。通过上述两种模块的融合而生成的新肽，对真菌（或者特定的真菌，见实施例）起到高效的杀菌作用。
因此，本发明提供一种抗菌肽，其由细胞膜渗透肽和具有结合锌离子的短肽串联连接而成，优选地，具有结合锌离子的短肽具有HEX1X2…XnH的结构，其中X可以是20种天然必需氨基酸中的任何一个，n代表X的个数（n≥2）；更优选地，具有结合锌离子的短肽直接连接在细胞膜渗透肽的N端或C端。
优选地，具有结合锌离子的短肽是HELVH；所述细胞膜渗透肽选自下述之一：
CPP-1 LLIILRRRIRKQAHAHSK
CPP-2 YGRKKRRQRRR
CPP-3 RQIKIWFQNRRMKWKK
CPP-4 RRWRRWNRFNRRRCR
CPP-5 LGTYTQDFNKFHTFPQTAIGVGAP
CPP-6 RWRWKSCKK
CPP-7 WLRLLORWLOLWROLLRLW
CPP-8 KLALKLALKALKAALKLAGC
CPP-9 NYRWRCKNQN
CPP-10 RKKNPNCRRH
CPP-11 LCLRPVG
CPP-12 CGRKKRRQRRRPPQ
CPP-13 GRCTKSIPPICFPD
CPP-14 ACSSSPSKHCG。
具体地，其是下述任意之一：
FAMP-1：HELVHLLIILRRRIRKQAHAHSK；
FAMP-2：LLIILRRRIRKQAHAHSKHELVH；
FAMP-3：YGRKKRRQRRRHELVH；
FAMP-4：HELVHYGRKKRRQRRR；
FAMP-5：RQIKIWFQNRRMKWKKHELVH；
FAMP-6：HELVHRQIKIWFQNRRMKWKK；
FAMP-7：RRWRRWNRFNRRRCRHELVH；
FAMP-8：HELVHRRWRRWNRFNRRRCR；
FAMP-9：LGTYTQDFNKFHTFPQTAIGVGAPHELVH；
FAMP-10：HELVHLGTYTQDFNKFHTFPQTAIGVGAP；
FAMP-11：RWRWKSCKKHELVH；
FAMP-12：HELVHRWRWKSCKK；
FAMP-13：WLRLLORWLOLWROLLRLWHELVH；
FAMP-14：HELVHWLRLLORWLOLWROLLRLW；
FAMP-15：KLALKLALKALKAALKLAGCHELVH；
FAMP-16：HELVHKLALKLALKALKAALKLAGC；
FAMP-17：NYRWRCKNQNHELVH；
FAMP-18：HELVHNYRWRCKNQN；
FAMP-19：RKKNPNCRRHHELVH；
FAMP-20：HELVHRKKNPNCRRH；
FAMP-21：LCLRPVGHELVH；
FAMP-22：HELVHLCLRPVG；
FAMP-23：CGRKKRRQRRRPPQHELVH；
FAMP-24：HELVHCGRKKRRQRRRPPQ；
FAMP-25：GRCTKSIPPICFPDHELVH；
FAMP-26：HELVHGRCTKSIPPICFPD；
FAMP-27：HELVHACSSSPSKHCG；
FAMP-28：ACSSSPSKHCGHELVH。
本发明也提供编码所述的抗菌肽的核酸，以及含有所述的核酸的表达载体和重组细胞。
本发明尤其提供所述的抗菌肽或其编码核酸在制备抗菌产品中的应用。具体地，所述抗菌是指抗马拉色菌、隐球菌、以及红酵母。
本发明可广泛应用于包括但不限于医药、化妆品、洗护用品、食品、食品包装、抗菌涂层、农药等领域及相关产品。故本发明提供所述的抗菌肽或其编码核酸在制备抗菌产品中的应用。
本发明的优点在于，本发明多肽既可通过化学合成，也可通过微生物进行生物合成，生产方式安全、环保，产品生物兼容性优异，无毒无害；而且通过锌离子结合模块与细胞膜渗透肽模块的结合，赋予新型多肽抗真菌能力，其可以针对导致皮肤病的致病真菌，包括但不限于马拉色菌（Malassezia）、隐球菌（Cryptococcus）、以及红酵母（Rhodotorula）等。
具体实施方式
结合实施实例，进一步阐述本项发明。除非特别说明，以下实施例中使用的试剂和仪器均为市售可得产品。
本发明实施例中的实验材料及试剂：
实验所用菌株：糠秕马拉色菌（Malassezia furfur,M.fufur）。
主要试剂与仪器：蛋白胨、葡萄糖、吐温80、琼脂等营养成分、Na2HPO4·2H2O、KH2PO4等无机盐试剂、96孔板购自生工生物工程（上海）有限公司。
实验所用培养基及常规试剂配制
MF 培养基：称取10 g蛋白胨、40 g 葡萄糖、2 g 吐温80，加入约500 ml 蒸馏水将其充分溶解，然后用量筒定容至1 L即为液体MF培养基。固体MF培养基为每200 ml 液体培养基添加4 g 琼脂。最终都要使用灭菌锅进行115 ℃高温灭菌20 min，密封保存。
2*MF 液体培养基：称取20 g 蛋白胨、80 g 葡萄糖、4 g 吐温80，加入约500 ml蒸馏水将其充分溶解，然后用量筒定容至1 L 即为2*MF液体培养基，115 ℃高温灭菌20min，密封保存。
25mM PBS溶液(pH=7.17)：称取2.488g Na2HPO4、1.02 g KH2PO4溶于100 ml 蒸馏水，用容量瓶定容至1 L，再调节pH至7.17，121 ℃高温灭菌15 min。
25mM PBS溶液(pH=5.29)：称取0.22 g Na2HPO4、3.23 g KH2PO4溶于100 ml 蒸馏水，用容量瓶定容至1 L，再调节pH至5.29，121 ℃高温灭菌15 min。
10% Zn2+的ZnSO4溶液：称取76.9 g ZnSO4溶于100 ml 蒸馏水，121 ℃高温灭菌15 min。
本发明实施例中的马拉色菌抑菌定性和定量分析实验方法：
（1）配制多肽母液：将目标多肽溶于ddH2O中，浓度为2mg/ml备用；
（2）用MF培养基于30℃培养糠秕马拉色菌备用；
（3）取多肽母液，用缓冲液稀释至不同浓度梯度（定量实验用）或一个确浓度（定性实验），并添加Zn2+离子至最终浓度为0.1%；
（4）在微孔板中添加150μl培养体系，包括75μl多肽溶液（1 mg/ml）+15μl PBS+60μl 菌液；其中150μl体系中马拉色菌最终OD值为0.05，每一个多肽设置至少3个平行组；
（5）微孔板于培养箱中在30℃，900 rpm条件下进行孵育；
（6）取上述孵育后的液体3μl进行倒MF平板，然后置于培养箱中30℃倒置培养48h。
（7）培养结束后评估平板中菌的生长情况。与阴性对照进行比较，对比培养平板上菌落生长情况。如果是定性评估则根据抑菌情况标明相关符号（-表示无作用，+表示有弱抑菌作用，++表示有中等抑菌作用，+++表示有强抑菌作用）；
如果定量测定，则计数菌落数，根据以下公式计算出抑菌率：
抑菌率（%）=（对照组菌落数-实验组菌落数）/对照组菌落数*100。
实施例1、CPP对马拉色菌的抗菌性验证
对选取的14个CPP肽，在抗菌肽库（AMPdatabase）及文献中进行检索，除CPP-1/2/3/4三个多肽外，均未发现有报道具有任何抗菌抑菌功效（表1）。
表1 本发明中部分CPP及相关信息汇总
说明：从CPP数据库中选择不同类型的CPP肽，如蛋白质来源、人工合成的、线性的、及环状的。
经检索，除了CPP-1/3外，均未发现对马拉色菌有抗菌抑菌功效。经本发明进行抗菌抑菌功效检测，验证了CPP1和CPP3对马拉色菌有抗菌功效。同时新发现CPP4对马拉色菌有抗菌功效，此前并无文献报道。经验证，除CPP1、CPP3和CPP4外，其它CPP肽对马拉色菌均无效（见表2）。
表2 CPP对马拉色菌的抗菌定性结果
说明：-表示无作用，+表示有弱抑菌作用，++表示有中等抑菌作用，+++表示有强抑菌作用。
实施例2、融合抗菌肽定性检测结果
本发明基于半理性设计，将CPP元件与ZMBS元件进行组合，具体见表3的组合方式得到相应的融合抗菌肽。
同时进行了抗菌抑菌性验证，结果见表3所示。
表3 融合抗菌肽对马拉色菌的抗菌的定性检测结果
| 融合肽编号 | 序列 | 融合组成 | 定性结果 |
| FAMP-1 | HELVHLLIILRRRIRKQAHAHSK | ZMBS+CPP-1 | ++ |
| FAMP-2 | LLIILRRRIRKQAHAHSKHELVH | CPP-1+ZMBS | ++ |
| FAMP-3 | YGRKKRRQRRRHELVH | CPP-2+ZMBS | +++ |
| FAMP-4 | HELVHYGRKKRRQRRR | ZMBS+CPP-2 | +++ |
| FAMP-5 | RQIKIWFQNRRMKWKKHELVH | CPP-3+ZMBS | ++ |
| FAMP-6 | HELVHRQIKIWFQNRRMKWKK | ZMBS+CPP-3 | ++ |
| FAMP-7 | RRWRRWNRFNRRRCRHELVH | CPP-4+ZMBS | +++ |
| FAMP-8 | HELVHRRWRRWNRFNRRRCR | ZMBS+CPP-4 | +++ |
| FAMP-9 | LGTYTQDFNKFHTFPQTAIGVGAPHELVH | CPP-5+ZMBS | ++ |
| FAMP-10 | HELVHLGTYTQDFNKFHTFPQTAIGVGAP | ZMBS+CPP-5 | ++ |
| FAMP-11 | RWRWKSCKKHELVH | CPP-6+ZMBS | +++ |
| FAMP-12 | HELVHRWRWKSCKK | ZMBS+CPP-6 | +++ |
| FAMP-13 | WLRLLORWLOLWROLLRLWHELVH | CPP-7+ZMBS | ++ |
| FAMP-14 | HELVHWLRLLORWLOLWROLLRLW | ZMBS+CPP-7 | ++ |
| FAMP-15 | KLALKLALKALKAALKLAGCHELVH | CPP-8+ZMBS | + |
| FAMP-16 | HELVHKLALKLALKALKAALKLAGC | ZMBS+CPP-8 | + |
| FAMP-17 | NYRWRCKNQNHELVH | CPP-9+ZMBS | +++ |
| FAMP-18 | HELVHNYRWRCKNQN | ZMBS+CPP-9 | +++ |
| FAMP-19 | RKKNPNCRRHHELVH | CPP-10+ZMBS | +++ |
| FAMP-20 | HELVHRKKNPNCRRH | ZMBS+CPP-10 | +++ |
| FAMP-21 | LCLRPVGHELVH | CPP-11+ZMBS | ++ |
| FAMP-22 | HELVHLCLRPVG | ZMBS+CPP-11 | ++ |
| FAMP-23 | CGRKKRRQRRRPPQHELVH | CPP-12+ZMBS | +++ |
| FAMP-24 | HELVHCGRKKRRQRRRPPQ | ZMBS+CPP-12 | +++ |
| FAMP-25 | GRCTKSIPPICFPDHELVH | CPP-13+ZMBS | ++ |
| FAMP-26 | HELVHGRCTKSIPPICFPD | ZMBS+CPP-13 | ++ |
| FAMP-27 | HELVHACSSSPSKHCG | ZMBS+CPP-14 | + |
| FAMP-28 | ACSSSPSKHCGHELVH | CPP-14+ZMBS | + |
说明：+表示有弱抑菌作用，++表示有中等抑菌作用，+++表示有强抑菌作用。
上述结果表明，1）锌离子结合模块ZMBS通过N端或都C端融合CPP肽均可以实现新增对马拉色菌同等的抗菌抑菌功能；2）抗菌抑菌效果的强弱因CPP的差异而不同；3）与ZMBS融合对已知CPP的抗菌抑菌功能无明显影响。
实施例3 融合抗菌肽对马拉色菌抗菌功效的定量检测
选取了抗菌肽FAMP-4、抗菌肽FAMP-8、抗菌肽FAMP-11、抗菌肽FAMP-17和抗菌肽FAMP-23测定不同浓度下对马拉色菌的抗菌抑菌效果。结果分别如下：
表4 抗菌肽FAMP-4的定量检测结果
| 多肽浓度(μg/ml) | 64 | 6.4 | 5.1 |
| 抑菌率 | >99% | 95% | 85% |
表5 抗菌肽FAMP-8的定量检测结果
| 多肽浓度(μg/ml) | 61 | 6.1 | 4.9 |
| 抑菌率 | >99% | 98% | 75% |
表6 抗菌肽FAMP-11的定量检测结果
| 浓度(μg/ml) | 31 | 18 |
| 抑菌率 | 98% | 65% |
表7 抗菌肽FAMP-17的定量检测结果
| 浓度(μg/ml) | 90 | 60 |
| 抑菌率 | >99% | 85% |
表8 抗菌肽FAMP-23的定量检测结果
| 浓度(μg/ml) | 100 | 60 |
| 抑菌率 | >99% | 91% |
上述结果表明，融合抗菌肽对马拉色菌具有较显著的抗菌抑菌性能：
所选取的融合抗菌肽在较低的浓度下杀菌率达98%以上，也即：FAMP4，FAMP8，FAMP11，FAMP17和FAMP23的最小抑菌浓度（MIC）分别为：64，61，31，90，100 μg/ml。其中，抗菌肽FAMP-4在5.1μg/ml浓度下抑菌率依然达85%以上；抗菌肽FAMP-8在4.9μg/ml浓度下抑菌率依然达75%以上；抗菌肽FAMP-11在18μg/ml浓度下抑菌率依然达65%以上；抗菌肽FAMP-17在60μg/ml浓度下抑菌率依然达85%以上；抗菌肽FAMP-23在60 μg/ml浓度下抑菌率依然达90%以上。
Claims (6)
1.一种抗菌肽，其特征在于，由细胞膜渗透肽和具有结合锌离子的短肽串联连接而成，其是下述任意之一的多肽：
FAMP-3：YGRKKRRQRRRHELVH；
FAMP-4：HELVHYGRKKRRQRRR；
FAMP-7：RRWRRWNRFNRRRCRHELVH；
FAMP-8：HELVHRRWRRWNRFNRRRCR；
FAMP-9：LGTYTQDFNKFHTFPQTAIGVGAPHELVH；
FAMP-10：HELVHLGTYTQDFNKFHTFPQTAIGVGAP；
FAMP-11：RWRWKSCKKHELVH；
FAMP-12：HELVHRWRWKSCKK；
FAMP-13：WLRLLORWLOLWROLLRLWHELVH；
FAMP-14：HELVHWLRLLORWLOLWROLLRLW；
FAMP-15：KLALKLALKALKAALKLAGCHELVH；
FAMP-16：HELVHKLALKLALKALKAALKLAGC；
FAMP-17：NYRWRCKNQNHELVH；
FAMP-18：HELVHNYRWRCKNQN；
FAMP-19：RKKNPNCRRHHELVH；
FAMP-20：HELVHRKKNPNCRRH；
FAMP-21：LCLRPVGHELVH；
FAMP-22：HELVHLCLRPVG；
FAMP-23：CGRKKRRQRRRPPQHELVH；
FAMP-24：HELVHCGRKKRRQRRRPPQ；
FAMP-25：GRCTKSIPPICFPDHELVH；
FAMP-26：HELVHGRCTKSIPPICFPD；
FAMP-27：HELVHACSSSPSKHCG；
FAMP-28：ACSSSPSKHCGHELVH。
2.编码如权利要求1所述的抗菌肽的核酸。
3.含如权利要求2所述的核酸的表达载体。
4.含如权利要求2所述的核酸或如权利要求3所述的表达载体的重组细胞。
5.如权利要求1所述的抗菌肽或如权利要求2所述的核酸在制备抗菌产品中的应用；所述抗菌是指抗马拉色菌；所述产品是指药品、化妆品、洗护用品、食品包装、抗菌涂层或农药。
6.一种抗菌肽在制备抗菌产品中的应用；所述抗菌是指抗马拉色菌；所述产品是指药品、化妆品、洗护用品、食品包装、抗菌涂层或农药；
所述的抗菌肽选自任意之一的多肽：
FAMP-1：HELVHLLIILRRRIRKQAHAHSK；
FAMP-2：LLIILRRRIRKQAHAHSKHELVH；
FAMP-5：RQIKIWFQNRRMKWKKHELVH；
FAMP-6：HELVHRQIKIWFQNRRMKWKK。
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