CN110066321A - 一种抗菌水凝胶及其制备方法和应用 - Google Patents
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Abstract
本发明属于抗菌剂技术领域，具体涉及一种抗菌水凝胶及其制备方法和应用。通过两个甘氨酸(G)将短抗菌肽序列(AMP)与水凝胶序列(RADA)连接，得到抗菌水凝胶(AMP‑RADA)，其中，RADA在一定浓度的NaCl溶液中能形成稳定的水凝胶，AMP具有极好的抗菌效果，两者结合能够形成既具有水凝胶特性，又具有抗菌效果的抗菌水凝胶。本发明结构简单、合成方便、抗菌活性高，可用于金黄色葡萄球菌引起的疾病的预防和治疗。
Description
技术领域
本发明属于抗菌剂技术领域，具体涉及一种抗菌水凝胶及其制备方法和应用。
背景技术
多药耐药性细菌感染已成为人类健康的严重威胁，抗菌肽(AMP)是由少于50个氨基酸组成的阳离子两亲性肽，多种生物体能够产生，因为其优异的杀菌活性，对生物体本身的低细胞毒性，它们已被认为是抗生素的潜在替代品。
RADA是一种多肽，当暴露于生理介质或盐溶液时，其自发组装成稳定的二级结构并进一步形成有序的纳米结构，它在细胞培养，生物工程，纳米医学和其他领域具有潜在的应用价值。RADA水凝胶由大于99％的水组成，其中纳米纤维均匀分散，能够很好地模拟天然细胞外基质，为细胞培养和组织再生提供有利的3D微环境。
虽然抗菌肽具有良好的抗菌效果，但由于其易被酶水解的性质，导致其应用受到限制。因此将抗菌肽与水凝胶结合，形成一种抗菌水凝胶，对于抗菌肽的应用具有重要意义。
发明内容
本发明的目的在于为了解决现有技术中的不足，提供了一种结构简单，抗菌活性高的抗菌水凝胶及其合成方法和应用，本发明选择的抗菌肽序列与水凝胶序列结合能够很好地成胶，且制备的抗菌水凝胶具有较好的抗菌效果，在抑菌、杀菌领域均有广泛的应用前景。为了实现上述目的，本发明采用如下技术方案：
本发明的抗菌水凝胶由水凝胶和抗菌肽组成，抗菌水凝胶的全序列包括但不限于:GKRWWKWWRRGGRADARADARADARADA(AMP-RADA)。
其中，水凝胶的序列为：RADARADARADARADA(RADA)；
抗菌肽的序列为：GKRWWKWWRR(AMP)；
抗菌水凝胶的合成，采用固相化学合成法从C末端向N末端合成，具体制备方法步骤如下：
1、基本材料：
固相Fmoc法选择的树脂为Rink-Amide-ChemMatrix树脂，具有反应条件温和，副反应少，产率高等优点，采用HBTU和HOBt作为连接分子，将多肽分子固定到树脂上。
2、反应步骤
第一步，将第一个氨基酸共价连接到树脂上
加入适当的缩合剂如HBTU和HOBt，使被保护氨基酸羧基端与树脂形成共脂以完成氨基酸的固定；
第二步，去保护
采用碱性溶剂20％哌啶去除氨基上的Fmoc，暴露出氨基。
第三步，激活和交联
采用活化剂HBTU和HOBt活化下一个氨基上的羧基，与树脂上的氨基交联，形成肽键。
第四步，重复第二步和第三步，反复循环添加单体氨基酸，按照序列直到合成完成。
3、合成后处理
(1)洗脱和脱保护：脱保护基后，用切割液三氟乙酸(TFA)将肽链从树脂上切下来。
(2)HPLC分析纯化，冻干。
通过上述方法，合成多肽序列AMP-RADA。
所述抗菌肽通过两个甘氨酸(G)与水凝胶序列RADA连接起来，所述水凝胶序列包括但不限于RADA。
与现有技术相比，本发明具有以下有益效果：
(1)本发明制备得到的抗菌水凝胶对金黄色葡萄球菌具有持续、显著的抑制作用，还可以避免抗菌肽水解，可广泛应用于抑菌、杀菌领域中；
(2)本发明公开的制备工艺简单，结构稳定，反应温和，适合推广使用。
附图说明
图1为抗菌水凝胶的HPLC图。
图2为抗菌水凝胶AMP-RADA对金黄色葡萄球菌抗菌效果的测定曲线图。
图3为抗菌水凝胶AMP-RADA成胶实验图。
图4为抗菌水凝胶AMP-AMP-RADA成胶实验图。
具体实施方式：
以下结合实施例对本发明进行详细阐述，但这些实施例仅为例示说明之用，而不应被解释为对本发明实施的限制。
实施例1
本发明所述的方法采用常规的固相Fmoc法，即固相树脂上被Fmoc保护的单体氨基酸去保护后露出氨基，通过缩合反应与溶液中氨基酸的羧基形成肽键，从而将氨基酸连接到树脂上，使肽链从C端向N端延伸。
1、基本材料：
固相Fmoc法选择的树脂为Rink-Amide-ChemMatrix树脂，具有反应条件温和，副反应少，产率高等优点，采用HBTU和HOBt作为连接分子，将多肽分子固定到树脂上。
2、反应步骤
第一步，将第一个氨基酸共价连接到树脂上
加入适当的缩合剂如HBTU和HOBt，使被保护氨基酸羧基端与树脂形成共脂以完成氨基酸的固定；
第二步，去保护
采用碱性溶剂20％哌啶去除氨基上的Fmoc，暴露出氨基。
第三步，激活和交联
采用活化剂HBTU和HOBt活化下一个氨基上的羧基，与树脂上的氨基交联，形成肽键。
第四步，重复第二步和第三步，反复循环添加单体氨基酸，按照序列直到合成完成。
3、合成后处理
(1)洗脱和脱保护：脱保护基后，用切割液三氟乙酸(TFA)将肽链从树脂上切下来。
(2)HPLC分析纯化，冻干。
通过上述方法，合成多肽序列AMP-RADA。
4、抗菌效果测定
通过测定不同浓度抗菌水凝胶来反映其抗菌效果。首先将固定量被测多肽溶于蒸馏水中，得到一定浓度多肽溶液。实验中需用到金黄色葡萄球菌，首先将细菌放入TSB培养基中培养过夜，然后设置样品浓度进行涂板实验，根据最终细菌生长情况可以看出抗菌水凝胶对于金黄色葡萄球菌具有较好的抑制作用(图3)。
对比实施例1
采用常规的固相Fmoc法合成双链抗菌水凝胶
反复循环添加单体氨基酸，使肽链从C末端向N末端延伸，直到合成完成。其他步骤同实施例1。
该对比实施例得到的多肽不能形成水凝胶，具体见图4。
以上述依据本发明的理想实施例为启示，通过上述的说明内容，相关工作人员完全可以在不偏离本项发明技术思想的范围内，进行多样的变更及修改。本项发明的技术性范围并不局限于说明书上的内容，必须要根据权利要求范围来确定其技术范围。
Claims (5)
1.一种抗菌水凝胶，其特征在于，所述抗菌水凝胶由水凝胶和抗菌肽组成，抗菌水凝胶的氨基酸序列为:GKRWWKWWRRGGRADARADARADARADA(AMP-RADA)。
2.一种根据权利要求1所述的抗菌水凝胶的制备方法，其特征在于，所述抗菌水凝胶由Fmoc固相合成法制得。
3.根据权利要求2所述的抗菌水凝胶的制备方法，其特征在于，所述抗菌水凝胶通过HPLC进行分析、纯化。
4.根据权利要求2所述的抗菌水凝胶的制备方法，其特征在于，所述抗菌水凝胶通过两个甘氨酸(G)将抗菌肽与水凝胶序列连接起来。
5.一种根据权利要求1所述的抗菌水凝胶的应用，其特征在于，所述抗菌水凝胶用作金黄色葡萄球菌的抗菌药物。
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| CN110669147A (zh) * | 2019-10-24 | 2020-01-10 | 常州大学 | 一种用于Saureus菌检测的含明胶酶酶切序列的抗菌剂FAM-AMP |
| CN112321680A (zh) * | 2020-09-24 | 2021-02-05 | 南京斯泰尔医药科技有限公司 | 一种能够特异性识别并靶向S.aureus菌的抗菌肽 |
| CN112868668A (zh) * | 2021-03-19 | 2021-06-01 | 常州英诺升康生物医药科技有限公司 | 一种Fe3O4-DA-AMP纳米复合抗菌材料及其制备方法和应用 |
| CN113018507A (zh) * | 2021-04-02 | 2021-06-25 | 常州大学 | 一种具有光热性能的抗菌水凝胶及其制备方法和应用 |
| CN113384743A (zh) * | 2021-07-09 | 2021-09-14 | 吉林省国大生物工程有限公司 | 一种具有促进组织修复及抗菌的温敏敷料的制备方法 |
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| CN113018507A (zh) * | 2021-04-02 | 2021-06-25 | 常州大学 | 一种具有光热性能的抗菌水凝胶及其制备方法和应用 |
| CN113018507B (zh) * | 2021-04-02 | 2022-05-17 | 常州大学 | 一种具有光热性能的抗菌水凝胶及其制备方法和应用 |
| CN113384743A (zh) * | 2021-07-09 | 2021-09-14 | 吉林省国大生物工程有限公司 | 一种具有促进组织修复及抗菌的温敏敷料的制备方法 |
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