CN105294838B - 一种抗菌肽及其应用 - Google Patents
一种抗菌肽及其应用 Download PDFInfo
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Abstract
本发明公开了一种抗菌肽，属于分子生物学领域。该抗菌肽具有如SEQ ID NO：2所示的氨基酸序列：Asn Trp Phe Ala Ile Thr Asn Trp Leu Trp Tyr Ile Lys Lys Lys Lys。本发明的抗菌肽不但对大肠杆菌、枯草杆菌和表皮葡萄球菌等革兰氏阴性菌和阳性菌都有明显的抑制作用，而且具有很低的溶血活性和细胞毒性，稳定性好，抗菌活性强，具有高效广谱抑菌作用，可作为抗生素替代品使用。
Description
技术领域
本发明属于分子生物学领域，具体涉及一种抗菌肽及其应用。
背景技术
近年来由于传统抗生素的滥用导致越来越严重的病原微生物的耐药性问题，给人类的健康带来巨大威胁，迫切需要开发新的抗菌药物。为了解决这个问题，抗菌肽受到了越来越多的关注。抗菌肽是生物体在抵御外来病原微生物入侵时产生的一种有活性的多肽物质，是生物体先天性免疫应答的重要组成部分。一般由20-60个氨基酸组成，分子量40kDa左右。至今为止，已经在动物，植物，微生物和人的体内发现了超过1500种抗菌肽，抗菌肽具有分子量低，抗菌谱广，不易产生耐药性的优点。
然而，天然的抗菌肽部分存在免疫原性、抗菌活性低，稳定性差，细胞毒性强，溶血率高等问题，限制了其作为抗菌药物的推广应用。因此，寻找抗菌活性强、安全性高、稳定性好的抗菌肽是实现抗菌肽部分替代抗生素的关键因素。
发明内容
本发明的目的是提供一种抗菌肽，其分子量小，稳定性好，细胞毒性小，溶血活性低，抗菌活性强。
本发明的另一目的是提供上述抗菌肽的应用。
为了实现上述目的，本发明的抗菌肽，该抗菌肽具有SEQ ID NO：2所示的氨基酸序列：Asn Trp Phe Ala Ile Thr Asn Trp Leu Trp Tyr Ile Lys Lys Lys Lys。
所述抗菌肽的分子量为2181.63D，等电点为9.87。
上述抗菌肽在制备治疗革兰氏阴性菌或者革兰氏阳性菌感染的广谱抗菌药物中的应用。
上述抗菌肽在制备抗菌剂中的应用。
本发明的抗菌肽不但对大肠杆菌、枯草杆菌和表皮葡萄球菌等革兰氏阴性菌和阳性菌都有明显的抑制作用，而且具有很低的溶血活性和细胞毒性，稳定性好，抗菌活性强，具有高效广谱抑菌作用，可作为抗生素替代品使用。
附图说明
图1为抗菌肽AP16-A的质谱图；
图2为抗菌肽AP16-A和蜂毒肽对大肠杆菌的抑菌曲线图；
图3为抗菌肽AP16-A和蜂毒肽对枯草杆菌的抑菌曲线图；
图4为抗菌肽AP16-A和蜂毒肽对表皮葡萄球菌的抑菌曲线图；
图5为抗菌肽AP16-A和蜂毒肽的溶血活性测定结果图；
图6为抗菌肽AP16-A和蜂毒肽的MTT法细胞毒性测定结果图；
图7为抗菌肽AP16-A的小鼠急性毒性试验测定结果图；
图8为抗菌肽AP16-A的小鼠急性毒性试验体重变化图；
图9为抗菌肽AP16-A37℃热处理后的HPLC图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明。
HIV-1的胞外近膜区域(Ac-665WASLWNWFNITNW LWYIK683-NH2)具有膜活化的特性并且此区域包含HIV-1的广谱中和抗体4E10和2F5表位。根据抗菌肽的作用机理和氨基酸组成的特点，本发明在4E10表位的基础上在其C-末端添加一个含三个赖氨酸的水溶性尾巴，以增加多肽的正电性和水溶性，并在此基础上进行一系列的多肽改造。本实施方式中共有七个抗菌肽，其一级机构和分子量如表1所示，其一级结构的氨基酸序列如表2所示。
表1抗菌肽的一级结构和分子量
表2抗菌肽一级结构的氨基酸序列
本实施例中涉及的抗菌肽均委托杭州中肽有限公司合成，涉及的大肠杆菌购自Takara，枯草杆菌和表皮葡萄球菌购自ATCC(美国模式菌种收集中心)，涉及到其他化学试剂及药品均通过商业途径获得。
(1)多肽AP16及其衍生物的抗菌实验
A.最小杀菌浓度(MBC)的测定
分别将革兰氏阴性菌株大肠杆菌(E.coli DH5α)、革兰氏阳性菌株枯草杆菌(B.subtilis ATCC 6633)和表皮葡萄球菌(S.epidermidis ATCC12228)培养至对数生长期，5000rpm离心10分钟后用磷酸钾缓冲液重悬，再次离心，5000rpm离心10分钟，用磷酸钾缓冲液重悬，将菌株稀释至1×106-2×106CFU/ml，待用。在96孔板内，分别将多肽P16及其衍生物用磷酸钾缓冲液按1:2逐级稀释，浓度为256μg/ml-1μg/ml，每孔100μl，再每孔加入100μl稀释好的以上三种菌株之一，空白孔内加入100μl的磷酸钾缓冲液和100μl已准备好的菌株，同时每个多肽浓度做两个复孔，37℃静止培养2h。将每孔内的液体吹匀，同一多肽浓度的两个孔内的液体加入到一个新的EP管内，混匀后取出100μl涂平板，37℃倒置过液培养。第二天查菌斑数并记录，完全没有菌斑的平板对应的最小多肽浓度为最小杀菌浓度。
表3中用最小杀菌浓度值(MBC)表征抗菌肽的抑菌能力，MBC值愈小则说明抑菌能力愈强。由表3可以看出，AP16-A的抑菌活性最好，最小杀菌浓度在2-16μg/ml之间。后续研究选取AP16-A作为主要的研究对象，将多肽AP16-A经过电喷雾质谱法分析，在质谱图(图1)中显示的分子量2181.63D与理论分子量2181.6D基本一致，多肽AP16-A的等电点为9.87，多肽AP16-A的纯度大于98％。
表3多肽AP16及其衍生物的最小杀菌浓度
B.抑菌活性的测定
将革兰氏阴性菌株大肠杆菌(E.coli DH5α)、革兰氏阳性菌株枯草杆菌(B.subtilis ATCC 6633)和表皮葡萄球菌(S.epidermidis ATCC12228)培养至对数生长期，5000rpm离心10分钟后用磷酸钾缓冲液重悬，再次离心，5000rpm离心10分钟，用磷酸钾缓冲液重悬，将菌株稀释至1×106-2×106CFU/ml，待用。在96孔板内，将多肽AP16-A用磷酸钾缓冲液按1:2逐级稀释，浓度为256μg/ml-1μg/ml，每孔100μl，再每孔加入100μl稀释好的以上三种菌株之一，空白孔内加入100μl的磷酸钾缓冲液和100μl的已准备好的菌株，同时每个多肽浓度做两个复孔，37℃静止培养2h。将每孔内的液体吹匀，同一多肽浓度的两个孔内的液体加入到一个新的EP管内，混匀后取出100μl到装有900μl液体LB培养基的EP管内，按1:10逐级稀释至1:103，每个稀释度各取出100μl涂平板，37℃倒置过液培养。第二天查菌斑数并记录，每个平板上的菌斑数乘以10再乘以稀释倍数为抗菌肽作用后每毫升内未被杀死的细菌数量。
测定结果如图2-4所示，多肽浓度为1/2最小杀菌浓度时，使得超过99％的大肠杆菌和表皮葡萄球死亡，超过99.99％的枯草杆菌死亡，在1/4最小杀菌浓度时，超过99％的枯草杆菌死亡。
(2)细胞毒性研究
抗菌肽的研究面临的很重要的问题就是对宿主细胞的毒性，抗菌肽必须能够 区分哺乳动物细胞和细菌细胞。
A.溶血活性实验
新鲜的豚鼠红细胞用生理盐水洗2次，3000rpm，离心30分钟，4％重悬到生理盐水中。在96孔板内按1:2将多肽AP16-A进行逐级稀释，蜂毒肽作为对照组。每孔100μl，再每孔加入100μl豚鼠红细胞，37℃培养1h。3000rpm，离心30min，转移150ul上清至新的96孔板内，450nm测OD值。用生理盐水和1％Triton X-100作为阴性和阳性对照，1％Triton X-100能够使豚鼠红细胞全部溶血。溶血百分比计算如下：
溶血百分比％＝[(OD多肽-OD生理盐水)/(OD Triton-OD生理盐水)]×100％
检测结果如图5所示。
蜂毒肽是由26个氨基酸残基组成的多肽，分子量2840，其一级结构的氨基酸序列SEQ NO:8为：Gly Ile Gly Ala Val Leu Lys Val Leu Tyr Tyr Gly Leu Pro Ala LeuIle Ser Trp Ile Lys Arg Lys Arg Gln Gln。
同天然存在的抗菌肽蜂毒肽相比，在最小杀菌浓度时AP16-A几乎没有溶血活性，然而蜂毒肽几乎造成超过72％的豚鼠红细胞溶血。
B.MTT测定细胞毒性
hCMEC/D3细胞(1×104)接种于96孔板内，每孔100μl，过夜培养至汇合率达到80％。多肽1:2逐级稀释后加入细胞培养液中，每孔100μl，培养3h，阴性对照孔内加入100μl的细胞和100μl的空的RPMI-1640培养基。5mg/ml的3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)溶液20μl加入到细胞培养液中，避光孵育2小时。弃去培养基，甲臜晶体用150μl二甲亚砜(DMSO)溶液溶解，检测490nm处光密度(OD)。测定结果如图6所示，同蜂毒肽相比较，AP16-A随着浓度的增加没有表现出细胞毒性。
C.小鼠体内毒性检测(极性毒性试验)
4-6周龄，18-20g的BALB/c雌鼠，随机分组，每组六只，腹腔注射200mg和400mg多肽AP16-A，空白对照组注射等体积的生理盐水。记录七天内的死亡率和体重变化。由图7可知，七天内没有小鼠死亡，且由图8可看出，同对照组相比，体重也没有明显的变化。
(3)热稳定性检测
用水将多肽溶解，分装于不同的EP管中，放于37℃，0h，3h，6h，12h，24h，2d，3d，9d，15d分别收集样品放于-80℃保存。将样品进行HPLC和抑菌试验检测。由表4可知，AP16-A经过在37℃下15天的处理后没有明显的降解，抑菌浓度和未经37℃处理的样品一致。由图9所示，从HPLC的检测结果可以看出经过15d的37℃的处理，没有明显的杂峰出现。
表4抗菌肽AP16-A 37℃下不同时间的热稳定性检测
Claims (2)
1.由SEQ ID NO：2所示的氨基酸序列组成的抗菌肽在制备治疗革兰氏阴性菌或者革兰氏阳性菌感染的广谱抗菌药物中的应用。
2.由SEQ ID NO：2所示的氨基酸序列组成的抗菌肽在制备抗菌剂中的应用。
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