CN117126260A - 一种抗菌肽cath2及其制备的产品和应用 - Google Patents
一种抗菌肽cath2及其制备的产品和应用 Download PDFInfo
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- CN117126260A CN117126260A CN202311245010.7A CN202311245010A CN117126260A CN 117126260 A CN117126260 A CN 117126260A CN 202311245010 A CN202311245010 A CN 202311245010A CN 117126260 A CN117126260 A CN 117126260A
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Abstract
本发明提供了一种抗菌肽CATH2及其制备的产品和应用,属于抗菌肽制备技术领域。一种抗菌肽CATH2,氨基酸序列如SEQ ID NO:1所示。所述抗菌肽CATH2具有广谱抗菌活性,同时与现有抗菌肽相比,具有溶血活性低、无细胞毒性和稳定性高的特点,优势显著,应用前景广阔。同时本发明还提供了所述抗菌肽CATH2在制备抑制微生物的产品中的应用,制备得到的产品可以抑制多种微生物,增加了抗菌肽CATH2的用途。
Description
技术领域
本发明属于抗菌肽技术领域,具体涉及一种抗菌肽CATH2及其制备的产品和应用。
背景技术
抗生素的滥用,会导致越来越多的耐药病原微生物的出现,这不仅给人类健康带来巨大的威胁,也会给环境安全造成严重挑战。目前临床上应对耐药抗病菌的主要措施是使用新的或者替代性的抗生素或者几种抗生素联合用药,因此持续开发新型抗病原菌药物的问题迫在眉睫。
抗菌肽是一种天然小分子多肽,是生物体免疫系统的不可缺少的一份子,对细菌、真菌、病毒甚至原虫均具有直接的杀灭作用。抗菌肽具有分子量小、结构简单、抗菌活性强、毒性低和不易引起耐药性等优点,因此抗菌肽就被认为是具有极大开发和应用前景的新一代抗生素。到目前为止,已从不同生物体中发现超过三千多种不同的抗菌肽。然而不同种类的抗菌肽在抗菌活性和抗菌谱方面存在较大差异,此外,抗病原菌药物还需满足较低的溶血活性和较低的细胞毒性的要求,这无形中限制了部分多肽的应用。
发明内容
有鉴于此,本发明的目的在于提供一种抗菌肽CATH2,在具备高抗菌活性的同时还具有较低的溶血活性和无细胞毒性,适用于抗菌产品的制备。
本发明提供了一种抗菌肽CATH2,氨基酸序列如SEQ ID NO:1所示。
本发明提供了一种抗菌产品,包括所述抗菌肽CATH2和辅料。
优选的,所述辅料包括以下至少一种:填充剂、润湿剂、粘合剂、崩解剂、润滑剂和表面活性剂。
优选的,所述抗菌产品还包括具有抗菌性能的抗生素、蛋白、多肽、植物提取物和细胞因子中的一种或几种。
优选的,所述抗菌产品包括药品和/或护肤品。
本发明提供了所述抗菌肽CATH2在制备杀灭病原菌或抑制病原菌的产品中的应用。
优选的,所述病原菌包括细菌。
优选的,所述细菌包括革兰氏阳性菌和/或革兰氏阴性菌。
优选的,所述革兰氏阴性菌包括以下至少一种:大肠杆菌(Escherichia coli)、铜绿假单胞菌(Pseudomonas aeruginosa)、志贺杆菌(Shigella castellani)和鲍曼不动杆菌(Acinetobacter baumnnii);
所述革兰氏阳性菌包括金黄色葡萄球菌(Staphylococcus aureus)和/或产气荚膜杆菌(Clostridiumperfringens)。
优选的,所述抗菌肽CATH2的浓度为37.5μg/mL以上。
本发明提供了一种抗菌肽CATH2,氨基酸序列如SEQ ID NO:1所示。本发明以抗菌肽CATH2为活性成分分别测定对大肠杆菌、铜绿假单胞菌、鲍曼不动杆菌、金黄色葡萄球菌和产气荚膜梭菌的最小抑菌浓度,依次为37.5μg/mL、75μg/mL、37.5μg/mL、18.75μg/mL和37.5μg/mL。同时,本发明抑菌动力学实验表明,所述抗菌肽CATH2具有杀灭细胞快速的特点,多肽CATH2在60min内即可杀死以大肠杆菌为代表的革兰氏阳性细菌,而阳性对照氨苄青霉素(ampicillin)需要120min才能杀死大肠杆菌;多肽CATH2可在180min内杀死以金黄色葡萄球菌为代表的革兰氏阴性细菌,而阳性对照氨苄青霉素在180min时仍无法杀死金黄色葡萄球菌。这表明本发明保护的抗菌肽CATH2杀菌作用高效,杀菌速度快于氨苄青霉素。此外,经溶血活性和细胞毒性实验表明,抗菌肽CATH2在不同浓度下的溶血活性均较低,并且也没有细胞毒性,具有良好的细胞安全性,可以运用于抗细菌感染药物或者护肤品尤其是医美产品的制备。
附图说明
图1为抗菌肽CATH2的杀菌速度测定结果;
图2为抗菌肽CATH2的溶血活性测定结果;
图3为抗菌肽CATH2的细胞毒性测定结果。
具体实施方式
本发明提供了一种抗菌肽CATH2,氨基酸序列如SEQ ID NO:1(RFRLPFRRPPIRIHPPPFYPPFRRFLGRR)所示。
在本发明中,所述抗菌肽CATH2分子量为3,955.63Da;通过等电聚焦电泳测定抗菌肽CATH2的等电点为12.54。所述抗菌肽CATH2的C末端修饰有氨基(-NH2),以便维持抗菌肽CATH2的结构稳定性。
在本发明中,所述抗菌肽CATH2的来源为山羊颌下腺,具体以1月龄、6月龄和12月龄母山羊的颌下腺组织为材料,利用转录组学分析差异基因得到。
本发明对所述抗菌肽CATH2的制备方法并没有特殊限定,优选包括直接合成、原核表达和真核表达。在本发明实施例中,采用固相合成法制备所述抗菌肽CATH2。本发明对所述固相合成法的具体步骤没有特殊限定,依据具体步骤得到所述抗菌肽CATH2即可。本发明优选采用高效液相色谱纯化所述抗菌肽,本发明对所述高效液相色谱的纯化方法没有特殊限定,采用本领域常规步骤即可。在本发明的实施例中具体给出了制备抗菌肽CATH2的一种固相合成和纯化步骤,但是不能仅仅将其认定为本发明的全部保护范围。
本发明提供了一种抗菌产品,包括所述抗菌肽CATH2和辅料。
在本发明中,所述抗菌肽CATH2的浓度优选为0.01%~5%,更优选为0.2%。
在本发明中,所述辅料优选包括以下至少一种:填充剂、润湿剂、粘合剂、崩解剂、润滑剂和表面活性剂。所述填充剂优选包括淀粉、糖粉、糊精、乳糖、可压性淀粉、微晶纤维素、硫酸钙、磷酸氢钙和甘露醇中的一种或多种;所述湿润剂和粘合剂优选包括蒸馏水、乙醇、淀粉浆、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素和乙基纤维素、羟丙基甲基纤维素、明胶溶液、蔗糖溶液、聚乙烯吡咯烷酮(pVp)的水溶液或醇溶液;所述崩解剂优选包括干淀粉、甲基淀粉钠、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮和交联羧甲基纤维素钠中的一种或多种;所述润滑剂优选包括硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇类和月桂醇硫酸镁中的一种或多种;所述表面活性剂优选包括十二烷基硫酸钠。
在本发明中,所述抗菌产品优选还包括具有抗菌性能的抗生素、蛋白、多肽、植物提取物和细胞因子中的一种或几种。所述抗菌产品优选包括药品和/或护肤品。所述护肤品优选为医美产品和常规护肤品,更优选为医美产品。本发明对所述护肤品的类型没有特殊限定,本发明所述抗菌肽CATH2可用于制备本领域任意类型的护肤品。
本发明提供了所述抗菌肽CATH2在制备杀灭病原菌或抑制病原菌的产品中的应用。
在本发明中,所述病原菌优选包括细菌。所述细菌包括革兰氏阳性菌和/或革兰氏阴性菌。所述革兰氏阴性菌包括以下至少一种:大肠杆菌(Escherichia coli)、铜绿假单胞菌(Pseudomonas aeruginosa)、志贺杆菌(Shigella castellani)和鲍曼不动杆菌(Acinetobacter baumnnii);所述革兰氏阳性菌包括金黄色葡萄球菌(Staphylococcusaureus)和/或产气荚膜杆菌(Clostridium perfringens)。
在本发明中,所述抗菌肽CATH2的浓度优选为37.5μg/mL以上。
在本发明实施例中,所述抗菌肽CATH2对大肠杆菌、铜绿假单胞菌、鲍曼不动杆菌、金黄色葡萄球菌和产气荚膜梭菌的最小抑菌浓度依次为37.5μg/mL、75μg/mL、37.5μg/mL、18.75μg/mL和37.5μg/mL,除对铜绿假单胞菌、鲍曼不动杆菌的最小抑菌浓度低于青霉素外,对其他三种菌的最小抑菌浓度均不同程度高于青霉素的MIC值。然而根据抑菌动力学实验表明,所述抗菌肽CATH2具有良好的高效杀灭细菌的作用,具体分别以大肠杆菌作为革兰氏阳性菌的代表,以金黄色葡萄球菌为革兰氏阴性菌的代表,抗菌肽CATH2杀菌迅速,CATH2在60min内即可杀死大肠杆菌,而阳性对照氨苄青霉素(ampicillin)需要120min才能杀死大肠杆菌。与之类似,CATH2可在180min内杀死金黄色葡萄球菌,而阳性对照氨苄青霉素在180min时仍无法杀死金黄色葡萄球菌。
下面结合实施例对本发明提供的一种抗菌肽CATH2及其制备的产品和应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
抗菌肽CATH2的制备方法,由以下步骤组成:
1、根据设计的氨基酸序列:
Arg-Phe-Arg-Leu-Pro-Phe-Arg-Arg-Pro-Pro-Ile-Arg-Ile-His-Pro-Pro-Pro-Ph e-Tyr-Pro-Pro-Phe-Arg-Arg-Phe-Leu-Gly-Arg-Arg-NH2用固相合成法合成得到粗多肽;
2、将粗多肽通过HPLC反相柱层析脱盐纯化,鉴定其纯度,具体方法为:
将0.1mg步骤1中制备得到的粗多肽溶于1mL含有0.1%三氟醋酸的超纯水中,若有不溶解的杂质,用0.45μm滤膜过滤,流动相A为0.1%三氟醋酸-水,流动相B为0.1%三氟醋酸-乙腈,待基线平稳后开始上样,上样量为50μL;色谱柱为硅胶烷基键合相C18柱(4.6mm×300mm,胶粒大小5μm,孔径大小为100A),采用二元流动相梯度洗脱系统,进行梯度洗脱,即在30min内,流动相B在洗脱剂中的含量从0%-80%按线性关系增长,流速1mL/min,检测波长215nm,25℃下测定,直到多肽的纯度不低于95%,得到纯化的多肽,即抗菌肽CATH2;
3、通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF)测定步骤2中得到的抗菌肽CATH2,具体方法为:
将步骤2中得到的抗菌肽CATH2溶于去离子水中,配置成1μmol/mL的溶液,取10μL与等体积的饱和基质溶液(将α-氰基-4-羟基肉桂酸溶于含0.1%三氟醋酸的50%乙腈溶液中,制成饱和溶液,离心,取上清液)混合后测定,得到抗菌肽CATH2的分子量为3,955.63Da;
4、通过等电聚焦电泳测定抗菌肽CATH2的等电点为12.54,并采用自动氨基酸测序仪测定纯化后多肽的氨基酸序列结构,确定为Arg-Phe-Arg-Leu-Pro-Phe-Arg-Arg-Pro-Pro-Ile-Arg-Ile-His-Pro-Pro-Pro-Phe-Ty r-Pro-Pro-Phe-Arg-Arg-Phe-Leu-Gly-Arg-Arg-NH2。
实施例2
抗菌肽CATH2的抗菌活性
最小抑菌浓度的定义:能够抑制细菌生长、繁殖的最低药物浓度。
1.本实施例采用二倍梯度稀释的方法进行定量抑菌分析,具体操作如下:
用灭菌超纯水配制2mg/mL CATH2样品备用。分别准备好新鲜的金黄色葡萄球菌菌液、大肠杆菌菌液和鲍曼不动杆菌菌液,使用紫外分光光度计检测菌液的OD600,按照1OD600为1×109CFU/mL的浓度,用新鲜LB液体培养基将上述菌液浓度分别稀释调整至2×105CFU/mL。
随后预先在无菌的96孔板中加入90μL生理盐水,在第一孔内加入配制好的CATH2样品(以氨苄青霉素为阳性对照),依次对待测样品进行二倍梯度稀释,再在每孔内加入100μL浓度为2×105CFU/mL的菌液(金黄色葡萄球菌菌液、大肠杆菌菌液和鲍曼不动杆菌菌液均采用此检测方法独立进行,不再赘述),用移液枪将其吹打混匀,混匀后置于37℃恒温培养箱中过夜培养,最后用酶标仪检测菌液在600nm处的光吸收值,以检测不到细菌生长的孔和相邻孔的样品浓度的平均值作为最小抑菌浓度,即MIC值,抗菌肽CATH2的抑菌活性见表1。
表1抗菌肽CATH2的最小抑菌浓度
由表1可以得出,抗菌肽CATH2对Escherichia coli CMCC44102(大肠杆菌)、Escherichia coli ATCC25922(大肠杆菌)、Pseudomonas aeruginosa CMCC10104(铜绿假单胞菌)、Acinetobacter baumnnii ATCC19606(鲍曼不动杆菌)、ShigellacastellaniATCC12022(志贺杆菌)、Staphylococcus aureus CMCC26003(金黄色葡萄球菌)和Clostridiumperfringens ATCC13124(产气荚膜梭菌),37.5μg/mL、37.5μg/mL、75μg/mL、37.5μg/mL、75μg/mL、18.75μg/mL和37.5μg/mL。
2.2将Escherichia coli CMCC44102接种到LB固体培养基平板上,于37℃培养箱倒置培养至菌落长出。用接种环挑取单菌落接种到LB液体培养基中,37℃振荡培养箱中培养到对数生长期。用新鲜的LB液体培养基将菌液稀释至1×106CFU/mL,将样品加入稀释好的菌液中,使其终浓度为1×、5×和10×MIC(阳性对照加入相应体积5×MIC氨苄青霉素,阴性对照加入相应体积的灭菌超纯水)。加入样品的菌液迅速放入37℃振荡培养箱中,150rpm振荡培养,分别于0min、0.1min、1min、10min、30min、60min、180min、360min取10μL菌液用灭菌的生理盐水稀释1×103倍,取50μL涂布LB固体培养基平板,然后放入37℃培养箱中倒置培养16h,菌落计数。每组做3个平行,结果如图1所示:
由图1可以看出:抗菌肽CATH2杀菌迅速,CATH2在60min内即可杀死大肠杆菌,而阳性对照氨苄青霉素(ampicillin)需要120min才能杀死大肠杆菌。与之类似,CATH2可在180min内杀死金黄色葡萄球菌,而阳性对照氨苄青霉素在180min时仍无法杀死金黄色葡萄球菌。CATH2杀菌作用高效,杀菌速度快于氨苄青霉素。
实施例3
抗菌肽CATH2的溶血活性和细胞毒性测定,具体步骤为:
1.用生理盐水把洗涤好的红细胞的稀释调整为107~108个/mL,然后将红细胞悬浮液与不同浓度(50~200μg/ml)的CATH2多肽样品混合,37℃孵育30min。1000rpm离心5min,上清液转移至96孔板中于540nm检测紫外吸收值。阴性对照为生理盐水,阳性对照为TritonX-100。作为阳性对照,溶血活性则与540nm处的光吸收值成正比,溶血活性见图2;
由图2可以看出:本发明抗菌肽CATH2在540nm处的光吸收值均显著低于阳性对照TritonX-100,而与阴性对照非常接近,由此可以得出,抗菌肽CATH2在不同浓度下的溶血活性均较低。
2.将活化好的hacat、HepG2和4T1细胞铺于培养皿中,待细胞生长铺满培养皿底面积80%左右时,用0.25%的胰蛋白酶消化,细胞刮轻轻刮下,轻轻吹打至单细胞后转移到灭菌离心管中,用离心机1000rpm离心5min,弃上清,用细胞培养液RPMI1640重悬,吹打混匀后,用血球计数板计数。计数完毕后稀释至试验所需细胞浓度,即为种板细胞。
取无菌96孔板,向各孔中加入10,000个种板细胞,RPMI1640调至每孔总体积100μL。培养至细胞贴壁后,加入不同浓度(50~200μg/ml)的CATH2多肽样品,并设孔板对照与阳性对照组,37℃培养24h后加入20μLMTT溶液(5mg/mL,现配现用,避光保存),继续培养4h,然后弃去各孔细胞培养液,并加入150μL的DMSO(二甲基亚砜),轻微震荡使结晶物溶解,用全波长酶标仪检测490nm处的吸光值,结果如如图3所示。
由图3可以看出,与对照组NC相比,不同浓度下的抗菌肽CATH2均没有细胞毒性,本发明所述抗菌肽CATH2的安全性较高。
由以上实施例可以得出,本发明提供的抗菌肽CATH2可直接杀灭病原菌,功能显著,作用快速,且CATH2溶血活性低、无细胞毒性,稳定性较高,可以运用于抗细菌感染药物或者护肤品尤其是医美产品的制备。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种抗菌肽CATH2,其特征在于,氨基酸序列如SEQ ID NO:1所示。
2.一种抗菌产品,其特征在于,包括权利要求1所述抗菌肽CATH2和辅料。
3.根据权利要求2所述抗菌产品,其特征在于,所述辅料包括以下至少一种:填充剂、润湿剂、粘合剂、崩解剂、润滑剂和表面活性剂。
4.根据权利要求2所述抗菌产品,其特征在于,所述抗菌产品还包括具有抗菌性能的抗生素、蛋白、多肽、植物提取物和细胞因子中的一种或几种。
5.根据权利要求2~4中任意一项所述抗菌产品,其特征在于,所述抗菌产品包括药品和/或护肤品。
6.权利要求1所述抗菌肽CATH2在制备杀灭病原菌或抑制病原菌的产品中的应用。
7.根据权利要求6所述应用,其特征在于,所述病原菌包括细菌。
8.根据权利要求7所述应用,其特征在于,所述细菌包括革兰氏阳性菌和/或革兰氏阴性菌。
9.根据权利要求8所述应用,其特征在于,所述革兰氏阴性菌包括以下至少一种:大肠杆菌(Escherichia coli)、铜绿假单胞菌(Pseudomonas aeruginosa)、志贺杆菌(Shigellacastellani)和鲍曼不动杆菌(Acinetobacter baumnnii);
所述革兰氏阳性菌包括金黄色葡萄球菌(Staphylococcus aureus)和/或产气荚膜杆菌(Clostridiumperfringens)。
10.根据权利要求6~9中任意一项所述应用,其特征在于,所述抗菌肽CATH2的浓度为37.5μg/mL以上。
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