CN117050148A - 一种抗菌肽map34-b、抑菌剂及其应用 - Google Patents
一种抗菌肽map34-b、抑菌剂及其应用 Download PDFInfo
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- CN117050148A CN117050148A CN202311245007.5A CN202311245007A CN117050148A CN 117050148 A CN117050148 A CN 117050148A CN 202311245007 A CN202311245007 A CN 202311245007A CN 117050148 A CN117050148 A CN 117050148A
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- map34
- antibacterial peptide
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Classifications
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Abstract
本发明提供了一种抗菌肽MAP34‑B、抑菌剂及其应用，属于抗菌肽制备技术领域；所述抗菌肽具有广谱抗菌活性，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性，优势显著，应用前景广阔。本发明还提供了所述抗菌肽MAP34‑B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MAP34‑B的用途。
Description
技术领域
本发明属于抗菌肽制备技术领域，具体涉及一种抗菌肽MAP34-B、抑菌剂及其应用。
背景技术
抗菌肽具有强大、快速和广谱的抗菌活性。动物抗微生物肽是先天免疫系统必不可少的组成部分，被称为具有低耐药性发展速度的宿主防御肽，是多细胞生物免疫系统的一部分，广泛分布于自然界各种生物体内。这些多肽在大小、组成、作用机制和抗菌特性的范围上各不相同。它们在许多组织、中性粒细胞、巨噬细胞和粘膜上皮细胞中都有表达。它们也可以作为模型来测试抗菌肽的疗效、伤口愈合效果和免疫学特性。
抗菌肽通过多种作用机制如抑制膜蛋白和DNA的合成、单链DNA的断裂、与DNA的相互作用、过氧化氢的产生、诱导真核细胞的细菌自溶和凋亡等对某些病原体具有非特异性的活性。随着耐药菌株数量的增加，新型抗菌药物的研发成为亟待解决的问题。利用抗菌肽的先天免疫特点及不易产生耐药性的特点，是未来药物设计和疾病治疗药物研发的重要方向。抗菌肽具有杀菌速度快、稳定性好且不易产生耐药性的特点，在感染类药物开发方面有着广阔的应用前景。但是，现有抗菌肽具有生物学活性好，溶血、细胞毒性等副作用和体内稳定性差等缺陷，对临床应用造成困难。
发明内容
本发明的目的在于提供一种抗菌肽MAP34-B、抑菌剂及其应用，本发明的抗菌肽MAP34-B在具备较高抗菌活性的基础上，同时具有溶血活性低、无细胞毒性和稳定性高的特性。
本发明提供了一种抗菌肽MAP34-B，氨基酸序列如SEQ ID NO.1所示。
优选的，所述抗菌肽MAP34-B的C末端还带有酰胺基修饰。
本发明还提供了上述方案所述的抗菌肽MAP34-B在制备抑制微生物的产品中的应用。
优选的，所述微生物包括细菌；所述细菌包括革兰氏阳性菌和/或革兰氏阴性菌。
优选的，所述革兰氏阳性菌包括金黄色葡萄球菌和/或产气荚膜梭菌；所述革兰氏阴性菌包括大肠杆菌、绿脓杆菌、痢疾杆菌和鲍曼不动杆菌中的一种或几种。
优选的，所述抑制微生物包括微生物消杀和/或抑制微生物繁殖。
本发明还提供了一种抑菌剂，包括上述方案所述的抗菌肽MAP34-B。
优选的，所述抑菌剂还包括辅料和/或其他有效成分。
优选的，所述辅料包括填充剂、润湿剂、粘合剂、崩解剂、润滑剂和表面活性剂中的一种或多种；
所述其他有效成分包括抗生素、蛋白、多肽、植物提取物和细胞因子中的一种或多种。
优选的，所述抑菌剂包括药品、护肤品、消毒剂、食品防腐剂或保鲜剂。
本发明提供了一种抗菌肽MAP34-B，氨基酸序列如SEQ ID NO.1所示；所述抗菌肽MAP34-B具备抗菌谱广、抑菌活性强，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性，优势显著，应用前景广阔。
本发明还提供了抗菌肽MAP34-B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MAP34-B的用途。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为实施例2中抗菌肽MAP34-B对大肠杆菌和金黄色葡萄球菌的杀菌动力学结果；
图2为实施例3中抗菌肽MAP34-B的溶血活性测定结果；
图3为实施例3中抗菌肽MAP34-B的细胞毒性测定结果；
图4为实施例4中抗菌肽MAP34-B的稳定性测定结果。
具体实施方式
本发明提供了一种抗菌肽MAP34-B，氨基酸序列如SEQ ID NO.1所示，具体为：N端-GLFGRLRDSLRRGGQKILEKVERIGDRIKDIFRG-C端。
在本发明中，所述抗菌肽MAP34-B的C末端优选的还带有酰胺基修饰，具体为：GLFGRLRDSLRRGGQKILEKVERIGDRIKDIFRG-NH2；所述酰胺基修饰用以保证多肽的稳定性，且不会影响多肽的活性。
在本发明中，所述抗菌肽MAP34-B的分子量为3,955.63Da，等电点为11.29。
本发明对所述抗菌多肽MAP34-B的制备方法并没有特殊限定，优选包括直接合成、原核表达或真核表达，更优选采用固相合成法制备所述抗菌肽MAP34-B；本发明对所述固相合成法的具体步骤没有特殊限定，依据常规步骤得到所述抗菌肽MAP34-B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MAP34-B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。
本发明还提供了上述方案所述的抗菌肽MAP34-B在制备抑制微生物的产品中的应用。
在本发明中，所述抑制微生物优选的包括微生物消杀和/或抑制微生物繁殖。
在本发明中，所述微生物优选的包括细菌；所述细菌优选的包括革兰氏阳性菌和/或革兰氏阴性菌，进一步优选包括革兰氏阳性菌和革兰氏阴性菌；所述革兰氏阳性菌优选的包括金黄色葡萄球菌和/或产气荚膜梭菌，进一步优选为金黄色葡萄球菌；所述革兰氏阴性菌包括大肠杆菌、绿脓杆菌、痢疾杆菌和鲍曼不动杆菌中的一种或几种，进一步优选为大肠杆菌和/或绿脓杆菌。
在本发明中，所述大肠杆菌包括Escherichia coli CMCC44102，最小抑菌浓度MIC为18.75μg/mL；所述绿脓杆菌包括Pseudomonas aeruginosa CMCC10104，最小抑菌浓度MIC为75μg/mL；所述痢疾杆菌包括Shigella castellaniATCC12022，最小抑菌浓度MIC为37.5μg/mL；所述鲍曼不动杆菌包括Acinetobacter baumnniiATCC19606，最小抑菌浓度MIC为18.75μg/mL；所述金黄色葡萄球菌包括Staphylococcus aureus CMCC26003，最小抑菌浓度MIC为18.75μg/mL；所述产气荚膜梭菌包括Clostridiumperfringens ATCC13124，最小抑菌浓度MIC为9.38μg/mL。
本发明所述抗菌肽MAP34-B具备较高的抑菌活性，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性，可用于制备抑制微生物的产品。
本发明还提供了一种抑菌剂，包括上述方案所述的抗菌肽MAP34-B。
在本发明中，所述抗菌肽MAP34-B优选为所述抑菌剂的唯一活性成分。
在本发明中，所述抑菌剂优选的还包括辅料和/或其他有效成分。
在本发明中，所述辅料优选的包括填充剂、润湿剂、粘合剂、崩解剂、润滑剂和表面活性剂中的一种或多种；所述填充剂优选的包括淀粉、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分别优选的包括蒸馏水、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；所述崩解剂优选的包括干淀粉、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；所述润滑剂优选的包括硬脂酸镁、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的包括十二烷基硫酸钠；所述其他有效成分优选的包括抗生素、蛋白、多肽、植物提取物和细胞因子中的一种或多种。
在本发明中，所述抑菌剂包括药品、护肤品、消毒剂、食品防腐剂或保鲜剂；所述护肤品优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MAP34-B可用于制备本领域任意类型的护肤品。在本发明中，所述药品的剂型包括注射剂或者外用制剂；所述外用制剂包括喷剂。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的一种抗菌肽MAP34-B、抑菌剂及其应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
抗菌肽MAP34-B的制备方法，由以下步骤组成：
1、根据设计的氨基酸序列：
Gly-Leu-Phe-Gly-Arg-Leu-Arg-Asp-Ser-Leu-Arg-Arg-Gly-Gly-Gln-Lys-IleLeu-Glu-Lys-Val-Glu-Arg-Ile-Gly-Asp-Arg-Ile-Lys-Asp-Ile-Phe-Arg-Gly-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含有体积浓度为0.1％的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为体积浓度0.1％的三氟醋酸-水，流动相B为体积浓度0.1％的三氟醋酸-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MAP34-B；
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MAP34-B，具体方法为：
将步骤2中得到的抗菌肽MAP34-B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MAP34-B的分子量为3,955.63Da；
4、通过等电聚焦电泳测定抗菌肽MAP34-B的等电点为11.29，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Gly-Leu-Phe-Gly-Arg-Leu-Arg-Asp-Ser-Leu-Arg-Arg-Gly-Gly-Gln-Lys-IleLeu-Glu-Lys-Val-Glu-Arg-Ile-Gly-Asp-Arg-Ile-Lys-Asp-Ile-Phe-Arg-Gly-NH2。
实施例2
抗菌肽MAP34-B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/mLMAP34-B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＝1×109CFU/mL，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MAP34-B样品(以氨苄青霉素和美罗培南为阳性对照)，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MAP34-B的抑菌活性见表1。
表1抗菌肽MAP34-B的最小抑菌浓度
由表1可以得出，抗菌肽MAP34-B对Escherichia coli CMCC44102(大肠杆菌)、Pseudomonas aeruginosa CMCC10104(绿脓杆菌)、Shigella castellani ATCC12022(痢疾杆菌)、AcinetobacterbaumnniiATCC19606(鲍曼不动杆菌)、Staphylococcus aureusCMCC26003(金黄色葡萄球菌)和Clostridiumperfringens ATCC13124(产气荚膜梭菌)的最小抑菌浓度分别为：18.75μg/mL、75μg/mL、37.5μg/mL、18.75μg/mL、18.75μg/mL和9.38μg/mL。
2.将Escherichia coli CMCC44102和Staphylococcus aureus CMCC26003分别接种到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MAP34-B杀菌迅速，MAP34-B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，MAP34-B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。MAP34-B杀菌作用高效，杀菌速度快于氨苄青霉素。
实施例3
抗菌肽MAP34-B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的多肽样品混合，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Triton X-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MAP34-B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MAP34-B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，加入各浓度梯度的多肽样品，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MAP34-B均没有细胞毒性，本发明所述抗菌肽MAP34-B的安全性较高。
实施例4
抗菌肽MAP34-B的稳定性
用无菌注射器获取10mL人体血液储存于抗凝管中，在4℃、3500rpm的条件下离心30min，小心吸取黄色上清，即为所需血浆。用无菌生理盐水将上述血浆稀释一倍，加入抗菌肽MAP34-B，控制MAP34-B的终浓度为10mg/mL。随后，把溶有抗菌肽MAP34-B的血浆放入37℃的恒温培养箱中进行孵育，分别在0、1、2、4、6、8和10h这7个时间点各取10μL，用抑菌圈法检测抗菌肽MAP34-B与血浆共孵育后对鲍曼不动杆菌的抗菌活性，每个时间点设置两个重复，结果如图4所示。
由图4可以看出：在将抗菌肽MAP34-B与血清孵育12h，仍然可以检测到抗菌活性，由此可以证明所述抗菌肽MAP34-B的稳定性较高。
由以上实施例可以得出，本发明提供的抗菌肽MAP34-B可直接杀灭病原菌，功能显著，作用快速，且MAP34-B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。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (10)
1.一种抗菌肽MAP34-B，其特征在于，氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的抗菌肽MAP34-B，其特征在于，所述抗菌肽MAP34-B的C末端还带有酰胺基修饰。
3.权利要求1或2所述的抗菌肽MAP34-B在制备抑制微生物的产品中的应用。
4.根据权利要求3所述的应用，其特征在于，所述微生物包括细菌；所述细菌包括革兰氏阳性菌和/或革兰氏阴性菌。
5.根据权利要求4所述的应用，其特征在于，所述革兰氏阳性菌包括金黄色葡萄球菌和/或产气荚膜梭菌；所述革兰氏阴性菌包括大肠杆菌、绿脓杆菌、痢疾杆菌和鲍曼不动杆菌中的一种或几种。
6.根据权利要求3所述的应用，其特征在于，所述抑制微生物包括微生物消杀和/或抑制微生物繁殖。
7.一种抑菌剂，其特征在于，包括权利要求1或2所述的抗菌肽MAP34-B。
8.根据权利要求7所述的抑菌剂，其特征在于，所述抑菌剂还包括辅料和/或其他有效成分。
9.根据权利要求8所述的抑菌剂，其特征在于，所述辅料包括填充剂、润湿剂、粘合剂、崩解剂、润滑剂和表面活性剂中的一种或多种；
所述其他有效成分包括抗生素、蛋白、多肽、植物提取物和细胞因子中的一种或多种。
10.根据权利要求7～9任意一项所述的抑菌剂，其特征在于，所述抑菌剂包括药品、护肤品、消毒剂、食品防腐剂或保鲜剂。
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