CN113999297B - 一种抗菌肽hrNCM及其制备方法与应用 - Google Patents
一种抗菌肽hrNCM及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种抗菌肽hrNCM及其制备方法与应用。本发明根据绿海龟抗菌肽的氨基酸序列，利用分子改造方法设计改造体hrNCM，该改造体具有广谱高效的抗菌活性和很强的抗炎活性，此外具有分子量小、结构简单、溶血活性低、制备方法简单、稳定性高等有益特点。
Description
技术领域
本发明涉及一种抗菌肽hrNCM及其制备方法与应用，属于生物医学技术领域。
背景技术
近年来传统抗生素的大规模开发和滥用导致越来越严重的病原微生物耐药问题，给人类健康带来巨大的威胁。临床上应对耐药微生物感染的措施是使用对耐药微生物尚未使用过的新的或者替代性的抗生素，因此这就需要持续开发新的抗微生物药物。
抗菌肽是生物体基因编码的一种天然小分子多肽，是生物体免疫系统的一种重要分子，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、杀菌机制独特、毒性低和不易引起耐药性等优点，因此自发现之日起就被认为是具有极大开发潜力的新一代抗生素。到目前为止，已从不同生物体中发现超过2600多种不同的抗菌肽，而且其数目还在增加。但是，有些天然抗菌肽还存在抗菌活性低、细胞毒性高、稳定性差等问题。
发明内容
为解决上述技术问题，本发明提供一种绿海龟(Chelonia mydas)抗菌肽Cm-CATH2的改造体抗菌肽hrNCM及其制备方法和应用。
本发明的第一个目的是提供一种抗菌肽hrNCM，所述抗菌肽hrNCM是对氨基酸序列如SEQ ID NO.1所示的绿海龟抗菌肽Cm-CATH2进行改造获得。
进一步地，所述的抗菌肽hrNCM的氨基酸序列为Phe1 Har2 Har3 Val4 Har5 Har6Gln7 Leu8Gly9 Har10 Val11 Leu12 Har13 His14 Ser15 Har16 Ile17 Thr18 Val19 Gln20 Gln21Har22 Met23 Har24 Phe25。
进一步地，所述的抗菌肽hrNCM为直链多肽。
进一步地，所述的抗菌肽hrNCM的N端为α螺旋，C端为无规结构。
进一步地，所述的抗菌肽hrNCM的分子量为3237.12Da。
本发明的第二个目的是提供所述的抗菌肽hrNCM的制备方法，所述方法包括采用多肽固相合成法合成抗菌肽hrNCM的全序列，以及采用HPLC反相柱层析脱盐。
本发明的第三个目的是提供所述的抗菌肽hrNCM在制备抗菌药物或组合物、抑制细菌生长药物或组合物、抗炎药物或组合物、防腐剂、动物饲料添加剂或化妆品添加剂中的应用。
本发明的有益效果是：
本发明根据绿海龟抗菌肽的氨基酸序列，利用分子改造方法设计改造体hrNCM，该改造体具有广谱高效的抗菌活性和很强的抗炎活性，此外具有分子量小、结构简单、溶血活性低、制备方法简单、稳定性高等有益特点。
附图说明
图1为hrNCM的抗炎活性；
图2为hrNCM和NCM4的稳定性；
图3为hrNCM与NCM4的峰面积变化。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：
绿海龟改造体抗菌肽hrNCM的化学合成
绿海龟抗菌肽Cm-CATH2是基因编码的一种多肽，含有33个氨基酸残基，分子量4089.9Da，等电点12.96。绿海龟抗菌肽Cm-CATH2全序列为：Arg1 Arg2 Ser3 Arg4 Phe5Gly6Arg7 Phe8 Phe9 Lys10 Lys11Val12 Arg13 Lys14 Gln15 Leu16 Gly17 Arg18 Val19 Lys20Arg21 His22 Ser23Arg24 Ile25 Thr26 Val27 Gly28 Gly29 Arg30 Met31 Arg32 Phe33(SEQ IDNO.1)。根据绿海龟抗菌肽 Cm-CATH2的氨基酸序列，利用分子改造方法设计获得一系列肽链缩短肽，经过抗菌、抗炎活性研究，以及细胞毒性溶血活性研究筛选出改造体N-CM4，然后为了提高抗菌肽的稳定性，进一步对N-CM4中的阳离子氨基酸替换成高精氨酸，得到抗菌肽hrNCM，并利用多肽固相合成的方法对其进行了化学合成，
具体制备方法如下：
Ⅰ、hrNCM的制备方法：根据上述hrNCM的氨基酸序列，用自动多肽合成仪(433A,Applied Biosystems)合成其全序列，利用HPLC反相柱层析脱盐。
Ⅱ、分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)。
Ⅲ、纯化的hrNCM用高效液相色谱HPLC方法鉴定其纯度，分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)，用自动氨基酸测序仪测定氨基酸序列结构。
测定结果为：
hrNCM是绿海龟抗菌肽Cm-CATH2的一种改造体。hrNCM是一种直链多肽，其是N 端为α螺旋，C端为无规结构的，含有25个氨基酸残基的抗菌肽，分子量3237.12Da。hrNCM 全序列为：Phe1 Har2 Har3 Val4 Har5 Har6 Gln7 Leu8 Gly9 Har10 Val11 Leu12 Har13 His14Ser15 Har16Ile17 Thr18 Val19 Gln20 Gln21 Har22 Met23 Har24 Phe25。
实施例2：
hrNCM药理实验：
1.hrNCM抗菌活性测定：
(1)分别挑取保存于斜面上的试验菌株均匀涂布于MH固体培养基(北京索莱宝科技有限公司)平板上，将经过灭菌的0.5cm直径的滤纸片置于培养基表面，滴加溶解于灭菌去离子水的2mg/ml的抗菌肽hrNCM样品溶液10μl，于37℃倒置培养18－20小时，观察抑菌圈形成与否。若样品具有抗菌活性，则会在滤纸片周围形成清晰透明的抑菌圈，抑菌圈越大表明样品抗菌活性越强。
(2)抗菌肽hrNCM最小抑菌浓度(Minimum Inhibitory Concentration)测定(2倍稀释法)：
选择上步实验中具有抑菌圈的菌株进行MIC测定实验。试验菌株接种到MH液体培养基(北京索莱宝科技有限公司)中，37℃振荡培养到对数生长期，而后用新鲜MH液体培养基将培养至对数生长期的培养液稀释到2×105cfu/ml待用。
在无菌96孔板各孔中预先加入100μl MH液体培养基，然后在第一孔中加入100μl用 MH液体培养基稀释到一定浓度的经0.22μm孔滤膜过滤的的抗菌肽hrNCM样品溶液，混匀后取100μl加入第2孔，依次倍比稀释(参见表1)，自第9孔吸出100μl弃去，第10孔系对照管。
表1.稀释方法
将上述各管混匀后放置37℃缓慢振荡培养18小时，于600nm波长处测定光吸收。最小抑菌浓度为看不见细菌生长的最低样品浓度。结果如表2所示。
由表2可见，抗菌肽hrNCM对革兰氏阳性细菌、革兰氏阴性细菌和真菌均表现出很强的抗菌活性，其中包括部分临床分离致病菌，MIC值处于4.69-18.75μg/ml的范围。
表2.抗菌肽hrNCM抗菌活性
MIC：最小抑菌浓度，以上结果为三次独立重复实验平均值。
2.hrNCM溶血活性测定：
将采集的兔血与阿氏液混合抗凝，生理盐水洗涤2次并重悬成107-108cell/ml的悬浮液。上述稀释好的红细胞悬液与溶解于生理盐水的hrNCM样品混合，37℃保温30min，再于1000 rpm离心5min，上清液于540nm测吸收值。阴性对照使用生理盐水，阳性对照使用Triton X-100，溶血百分比按以下公式计算：溶血百分比H％＝A样品-A阴性对照/A阳性对照×100％。结果表明样品浓度为100μg/ml，hrNCM的溶血百分比为1.26％。说明hrNCM具有较低的溶血活性，不易引起哺乳动物红细胞破裂溶解。尤其抗菌活性范围内，安全性高。
3.hrNCM抗炎活性测定：
提取6-8周龄C57小鼠腹腔巨噬细胞，用含10％血清的1640培养基培养过夜，次日换成含2％血清的1640培养基，然后用终浓度为100ng/mL的大肠杆菌LPS(Sigma,美国)刺激细胞，同时给多肽hrNCM，终浓度为20μg/mL，设不给多肽和LPS的空白对照组与仅给 LPS的阳性对照组，共孵育16h，取上清，用ELISA试剂盒(R&D,美国)检测上清液中促炎因子IL-6和TNF-α的含量。每个做三个平行。
结果如图1所示，hrNCM能够显著抑制小鼠腹腔巨噬细胞中LPS诱导的促炎因子IL-6 和TNF-α表达，表明hrNCM具有极强的抗炎活性。
4.hrNCM酶稳定性实验研究：
将细胞消化用的0.25％胰酶与多肽样品，按照摩尔比1：200的比例混合，37℃下孵育，分别在0、6、12、24h时取样50μL，然后用多肽溶剂将所取样品稀释1倍，用0.22μm的滤膜过滤，取20μL用反向高效液相色谱测定多肽样品的残留量。其中A相用含0.1％三氟乙酸(TFA)的纯水，B相用含0.1％TFA的乙腈，进行梯度洗脱，得到多肽样品hrNCM与胰酶混合后不同时间点的洗脱峰和积分面积，然后用软件Origin2018作图。结果如图2所示，抗菌肽hrNCM在6h开始被降解，但是24h以内仍然有未被降解的本体，与NCM4相比较，稳定性相当。如图3所示是hrNCM和NCM4与酶作用后，在不同时间点的峰面积差别图，可见hrNCM在与酶作用后，峰面积变小，但是比NCM4降解的速度慢，说明hrNCM的稳定性比NCM4强。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 苏州市第九人民医院
<120> 一种抗菌肽hrNCM及其制备方法与应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> PRT
<213> （人工序列）
<400> 1
Arg Arg Ser Arg Phe Gly Arg Phe Phe Lys Lys Val Arg Lys Gln Leu
1 5 10 15
Gly Arg Val Lys Arg His Ser Arg Ile Thr Val Gly Gly Arg Met Arg
20 25 30
Phe
Claims (6)
1.一种抗菌肽hrNCM，其特征在于，所述的抗菌肽hrNCM的氨基酸序列为Phe1 Har2 Har3 Val4 Har5 Har6 Gln7 Leu8 Gly9 Har10 Val11 Leu12 Har13 His14 Ser15 Har16 Ile17 Thr18Val19 Gln20 Gln21 Har22 Met23 Har24 Phe25。
2.根据权利要求1所述的抗菌肽hrNCM，其特征在于，所述的抗菌肽hrNCM为直链多肽。
3.根据权利要求2所述的抗菌肽hrNCM，其特征在于，所述的抗菌肽hrNCM的N端为α螺旋，C端为无规结构。
4.根据权利要求1所述的抗菌肽hrNCM，其特征在于，所述的抗菌肽hrNCM的分子量为3237.12 Da。
5.一种权利要求1~4任一项所述的抗菌肽hrNCM的制备方法，其特征在于，所述方法包括采用多肽固相合成法合成抗菌肽hrNCM的全序列，以及采用HPLC反相柱层析脱盐。
6.权利要求1~4任一项所述的抗菌肽hrNCM在制备抗菌药物或组合物、抑制细菌生长药物或组合物、抗炎药物或组合物、防腐剂或动物饲料添加剂中的应用。
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