CN117986341A - 一种抗菌肽Nigroain-K1-E4KD18K及其制备方法和应用 - Google Patents
一种抗菌肽Nigroain-K1-E4KD18K及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体公开了一种抗菌肽Nigroain‑K1‑E4KD18K及其制备方法和应用，其氨基酸序列如SEQ ID NO.2所示；其是通过对天然来源的Nigroain‑K1肽进行氨基酸替换，将第4位的谷氨酸、第18位的天冬氨酸分别用带有正电荷的赖氨酸替换，同时将替换后的多肽序列N末端和C末端分别为乙酰化和酰胺化修饰，得到抗菌肽Nigroain‑K1‑E4KD18K。与现有技术相比，本发明抗菌肽Nigroain‑K1‑E4KD18K对革兰阴性菌和/或革兰阳性菌有显著的抗菌效果。
Description
技术领域
本发明涉及生物技术领域，特别是一种抗菌肽Nigroain-K1-E4KD18K及其制备方法和应用。
背景技术
日益严重的对常规抗生素的耐药性问题刺激了开发新的抗菌产品的需求。抗菌肽可以有效的抵抗有害微生物。由于抗菌肽的抗菌机制与常规抗生素有明显区别，抗菌肽不易产生耐药性，因此抗菌肽的发展引起了相当大的关注。传统抗生素多是通过抑制细菌特定代谢途径起到杀菌作用，大多数抗菌肽对细菌的作用靶点是细菌膜，通过破坏细菌细胞膜，导致细胞内容物的泄漏，最终导致细菌死亡。由于细菌的细胞膜组成基本不发生变化，因此抗菌肽不易产生耐药性。
抗菌肽多为带有净正电荷的两亲性多肽分子，其结构多为α-螺旋结构；抗菌肽广泛存在于动植物体中，是宿主先天免疫系统的重要组成部分。但天然来源的抗菌肽往往存在结构复杂、序列较长、抗菌活性低等缺点。
中国专利申请号CN201610984016.X公开的一种能抑制和杀灭革兰氏阳性菌和革兰氏阴性菌的抗菌肽，其氨基酸序列如SEQ ID NO.1-3所示。该抗菌肽用于杀灭或抑制以耐甲氧西林金黄色葡萄球菌、李斯特菌为代表的革兰氏阳性菌或以大肠杆菌为代表的革兰氏阴性菌有一定的效果，但是效果不是太显著。
发明内容
为了解决上述技术问题，本发明提供了一种抗菌肽Nigroain-K1-E4KD18K及其制备方法和应用。
为达到上述目的，本发明是按照以下技术方案实施的：
本发明的目的之一是提供一种抗菌肽Nigroain-K1-E4KD18K，其氨基酸序列如SEQID NO.2所示。
本发明的目的之二是提供一种抗菌肽Nigroain-K1-E4KD18K的制备方法，通过对天然来源的Nigroain-K1肽进行氨基酸替换，将第4位的谷氨酸、第18位的天冬氨酸分别用带有正电荷的赖氨酸替换，同时将替换后的多肽序列N末端和C末端分别为乙酰化和酰胺化修饰，得到抗菌肽Nigroain-K1-E4KD18K；具体，称取0.25mM Rink amide MBHAresin树脂活化并脱保护后，以Fmoc-氨基酸为原料，从多肽序列的C端到N端顺序合成；合成过程中，Fmoc-氨基酸加入量为1mM-2mM；20-30℃下100-180转/分振荡孵育合成；待最后一个氨基酸偶联完成后，加入0.5-3Ml乙酸酐对N端进行乙酰化；乙酰化完毕后，将粗肽从树脂上切割下来，切割试剂由TFA、水和三异丙基氯硅烷按照质量比98:1:1混合而成；用含0.1％TFA的A相双蒸水和含0.1％TFA的B相乙腈为流动相，采用AB相梯度混合，用C18色谱柱按1mL/min流速进行多肽粗品的纯化，收集Nigroain-K1-E4KD18K肽肽纯品，冻干保存。
本发明的目的之三是提供一种抗菌肽Nigroain-K1-E4KD18K在制备抑制革兰阴性菌和/或革兰阳性菌产品中的应用。
与现有技术相比，本发明抗菌肽Nigroain-K1-E4KD18K对革兰阴性菌和/或革兰阳性菌有显著的抗菌效果。
附图说明
图1为本发明的制备的抗菌肽Nigroain-K1-E4KD18K的质谱图。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明，并不用于限定发明。
下述实施例中所用原料、试剂、菌株除特殊说明外均为市购所得。
实施例1
抗菌肽Nigroain-K1-E4KD18K的制备：
通过对天然来源的Nigroain-K1肽(其氨基酸序列为
SLWETIKNAGKGFIQNILDKIR，如SEQ ID NO.1所示)进行氨基酸替换，将第4位的谷氨酸、第18位的天冬氨酸分别用带有正电荷的赖氨酸替换，同时将替换后的多肽序列N末端和C末端分别为乙酰化和酰胺化修饰，得到抗菌肽Nigroain-K1-E4KD18K。具体地，首先称取0.25mM Rink amide MBHA resin树脂活化并脱保护后，以Fmoc-氨基酸为原料，从多肽序列的C端到N端顺序合成；合成过程中，Fmoc-氨基酸加入量为1mM-2mM；20-30℃下100-180转/分振荡孵育合成，采用茚三酮染色法检测控制合成过程；待最后一个氨基酸偶联完成后，加入0.5-3Ml乙酸酐对N端进行乙酰化；乙酰化完毕后，将粗肽从树脂上切割下来，切割试剂由TFA、水和三异丙基氯硅烷按照质量比98:1:1混合而成；用含0.1％TFA的双蒸水(A相)和含0.1％TFA的乙腈(B相)为流动相，采用AB相梯度混合，用C18色谱柱按1mL/min流速进行多肽粗品的纯化，收集Nigroain-K1-E4KD18K肽肽纯品，用质谱测定Nigroain-K1-E4KD18K肽分子量，多肽纯品冻干保存。
抗菌肽Nigroain-K1-E4KD18K，其氨基酸序列为
Ac-SLWKTIKNAGKGFIQNLKKIR-NH2，如SEQ ID NO.2所示。将制备的经质谱鉴定，其结果如图1所示，由图1可知，抗菌肽Nigroain-K1-E4KD18K合成无误。
实施例2
抗菌肽Nigroain-K1-E4KD18K最低抑菌浓度测定
采用微量肉汤稀释法测定Nigroain-K1-E4KD18K肽的最低抑菌浓度。所采用的菌株为8种革兰阴性菌菌株与8种革兰阳性菌菌株。革兰阴性菌菌株为大肠埃希氏菌CICC23657、大肠埃希氏菌UB 1005、铜绿假单胞菌CICC 10351、产气肠杆菌CICC 10293、福氏志贺氏菌CICC21534、肠炎沙门氏菌CICC 21513、阪崎肠杆菌CICC 21544、奇异变形杆菌CICC21516。革兰阳性菌菌株为：金黄色葡萄球菌CICC 23656、金黄色葡萄球菌CICC 21600、金黄色葡萄球菌CICC 10306、表皮葡萄球菌CICC 10294、蜡样芽孢杆菌CICC 23828、产气荚膜梭菌CICC 22949、酿脓链球菌CICC 10373、粪链球菌CICC 23658。
挑取单克隆菌落于MH液体培养基中，37℃恒温振荡培养过夜；将菌液用MH培养基稀释到1×106CFU/mL，每孔100μL稀释菌液加入96孔板中；将多肽溶解按照终浓度500μM、250μM、125μM、64μM、32μM、16μM、8μM、4μM、2μM、1μM加入到含菌液的96孔板中，阴性对照为100μL稀释菌液与肽同体积无菌生理盐水；37℃恒温振荡培养24h。MIC定义为抑制细菌生长的最小浓度。
作为对比，将Nigroain-K1按同样方式测定最低抑菌浓度。
抗菌肽Nigroain-K1-E4KD18K和Nigroain-K1对革兰阴性菌的抗菌活性比较结果如表1所示，抗菌肽Nigroain-K1-E4KD18K和Nigroain-K1对革兰阳性菌的抗菌活性比较结果如表2所示。
表1
表2
由表1、表2可知，相对于Nigroain-K1而言，抗菌肽Nigroain-K1-E4KD18K对革兰阴性菌和革兰阳性菌的抗菌活性分别是Nigroain-K1的5.25和3.8倍。这表明抗菌肽Nigroain-K1-E4KD18K的抗菌活性明显提高。
本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。
Claims (4)
1.一种抗菌肽Nigroain-K1-E4KD18K，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
2.一种如权利要求1所述的抗菌肽Nigroain-K1-E4KD18K的制备方法，其特征在于，通过对天然来源的Nigroain-K1肽进行氨基酸替换，将第4位的谷氨酸、第18位的天冬氨酸分别用带有正电荷的赖氨酸替换，同时将替换后的多肽序列N末端和C末端分别为乙酰化和酰胺化修饰，得到抗菌肽Nigroain-K1-E4KD18K。
3.一种如权利要求2所述的抗菌肽Nigroain-K1-E4KD18K的制备方法，其特征在于，具体包括：
称取0.25mM Rink amide MBHAresin树脂活化并脱保护后，以Fmoc-氨基酸为原料，从多肽序列的C端到N端顺序合成；合成过程中，Fmoc-氨基酸加入量为1mM-2mM；20-30℃下100-180转/分振荡孵育合成；待最后一个氨基酸偶联完成后，加入0.5-3Ml乙酸酐对N端进行乙酰化；乙酰化完毕后，将粗肽从树脂上切割下来，切割试剂由TFA、水和三异丙基氯硅烷按照质量比98:1:1混合而成；用含0.1％TFA的A相双蒸水和含0.1％TFA的B相乙腈为流动相，采用AB相梯度混合，用C18色谱柱按1mL/min流速进行多肽粗品的纯化，收集Nigroain-K1-E4KD18K肽肽纯品，冻干保存。
4.一种如权利要求1所述的抗菌肽Nigroain-K1-E4KD18K在制备抑制革兰阴性菌和/或革兰阳性菌产品中的应用。
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