CN116693606A - 抗菌多肽与应用和细菌生长抑制剂、抗炎药物或保健品 - Google Patents
抗菌多肽与应用和细菌生长抑制剂、抗炎药物或保健品 Download PDFInfo
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Abstract
本发明涉及一种源于大曲的具有对以大肠杆菌和金黄色葡萄球菌为代表的革兰氏阴性菌和革兰氏阳性菌有抑制活性的多肽。其氨基酸序列为Leu‑Gln‑Pro‑His‑Gln‑Ile‑Ala‑Gln‑Leu。多肽LQPHQIAQL对革兰氏阴性菌和革兰氏阳性菌具有良好的抑制活性，作为天然安全的抗菌剂在制备预防和/或降低由大肠杆菌和金黄色葡萄球菌引起的肠道感染疾病的药物和/或保健品方面具有良好的应用前景。
Description
技术领域
本发明涉及一种来源于大曲的抗菌肽LQPHQIAQL及其在制备细菌生长抑制剂、抗炎药物和/或保健品中的应用。
背景技术
抗生素是一种由微生物所产生的特殊的次生代谢有机物，低浓度使用时即可通过阻碍细菌的核酸形成、抑制细菌蛋白质合成、改变细菌的细胞膜通透性以及干扰细菌细胞壁的合成等方式对细菌的生长和代谢起抑制作用。抗生素由于其显著的抗菌效果被广泛应用，然而过度使用也导致了细菌耐药性的产生，使得细菌感染性疾病的的治疗愈发困难。
抗菌肽(antimicrobial peptides,AMPs)作为一种新型的抗生素，来源非常广泛，具有10-50个氨基酸，强阳离子性，两亲性，以及广谱抑菌性等特点。相比于传统的抗生素，抗菌肽的最大优势为：抗菌肽主要是通过静电引力结合到细菌细胞膜上，凭借疏水作用与磷脂结合插入细胞质膜，使细菌细胞膜结构破坏。这种外力作用基本不会使微生物产生耐药性。然而，目前抗菌肽的应用仍然面临着两大难题：首先是天然抗菌肽的抗菌活性相对较低，其次是抗菌肽的稳定性、细胞毒性难以调控。因此。开发安全、稳定、低细胞毒性的抗菌肽具有重要意义。
大曲是微生物以谷物为底物高温发酵而成，其中源于谷物蛋白的抗菌肽是食源性的，天然安全，作为抗菌药物具有良好的潜质。因此，本发明从大曲中筛选出一种源于大曲底物小麦蛋白的抗菌肽，可以作为一种制备预防和/或降低由大肠杆菌和金黄色葡萄球菌引起的肠道感染疾病的药物和/或保健品进行研究。
发明内容
本发明的目的是提供一种源于大曲底物小麦蛋白的抗菌肽LQPHQIAQL在制备预防和/或降低由大肠杆菌和金黄色葡萄球菌引起的肠道感染疾病的药物和/或保健品中的应用。
为实现上述目的，本发明以所述多肽LQPHQIAQL为抑制微生物生长活性的有效成份。
其具有序列表SEQ ID NO：1中氨基酸序列，多肽LQPHQIAQL为抗大肠杆菌和金黄色葡萄球菌药物的活性成份，其中可添加药物学上可接受的载体或辅料。
具有对大肠杆菌和金黄色葡萄球菌抑制活性的多肽化合物LQPHQIAQL，氨基酸序列为Leu-Gln-Pro-His-Gln-Ile-Ala-Gln-Leu。分子量1047.22Da，白色粉末状，易溶于水，对大肠杆菌和金黄色葡萄球菌的生长具有很强的抑制作用。
本发明与现有技术相比，具有如下有益效果：
本发明从大曲中获得并确定了上述多肽的结构，首次验证了上述多肽具有较好的抑菌活性，在制备预防和/或降低由大肠杆菌和金黄色葡萄球菌引起的肠道感染疾病的药物和/或保健品方面具有良好的应用前景。
附图说明
图1为多肽LQPHQIAQL的抑菌结果图，其中图1(A)为多肽LQPHQIAQL抑制大肠杆菌的实验结果图；图1(B)为多肽LQPHQIAQL抑制金黄色葡萄球菌的实验结果图。
具体实施方式
实施例1多肽LQPHQIAQL的制备及鉴定
采用LC-MS/MS与bottom-up蛋白质组学技术相结合的方法。以市售酱香型高温大曲(RH-02，泸州瑞华生物制曲有限公司)为原料，经过水和乙醇的梯度提取，离心，超滤及LC-MS/MS分析，结合构效关系特征，筛选对大肠杆菌和金黄色葡萄球菌具有抑制作用的肽段。
其具体方法如下：
(1)样品制备
1g大曲样品与20mL超纯水混合，37℃，200rpm振荡2h，4℃、15000×g转速下离心15min，取出上清液(记为上清液A)，沉淀(记为沉淀A)加入20mL 50％的乙醇-水溶液，37℃，200rpm振荡2h，4℃、15000×g转速下离心15min，取出上清液(记为上清液B)，沉淀(记为沉淀B)加入20mL乙醇，37℃，200rpm振荡2h，4℃、15000×g转速下离心15min，取出上清液(记为上清液C)。上清液A、B、C分别用截留分子量为10000Da的超滤膜超滤，获得的相应滤液A、B、C分别用C18-SPE柱(Waters Oasis HLB SPE)除盐。除盐后将样品冻干，-80℃保存，获得相应的冻干保存的样品A、B、C。
(2)LC-MS/MS分析
冻干保存的样品A、B、C分别复溶于0.1％(v/v)FA-H2O溶液中，LTQ-OrbitrapVelos(双分压线性阱和静电场轨道阱组合式高分辨质谱)对样品进行质谱分析。上样量分别为2μg，液相色谱的流动相A为含0.1％甲酸的水溶液(v/v)，流动相B为含0.1％甲酸的乙腈(v/v)。梯度洗脱程序如下：
0-80min，7％-27％B(v/v)；80-95min，27％-40％B；95-97min，40％-90％B；97-107min，90％B；107-109min，90％-0％B；109-126min，0％B，流速60μL/min
质谱离子源为ESI，离子传输温度250℃，喷雾电压2.2kV，归一化碰撞能量35％，一级谱分辨率60000，扫描范围400-2000m/z，数据采集模式为数据依赖模式(DDA)。选取一级谱中20个最强的母离子进行二级碎裂，动态排除设置为:重复计数，2；重复时间,30s；排除时间为60秒。
(3)数据检索
RAW文件使用MaxQuantTM(v.1.5.3.30)软件在小麦(wheat，379个蛋白)数据库(http://www.uniprot.org/)中进行检索。搜库参数如下：不设置酶切、最大漏切数和固定修饰，可变修饰设置为甲硫氨酸的氧化(+15.9949Da)。母离子的质量容忍偏差为20ppm，碎片离子为0.5Da。控制PSM假阳性率(FDR)<1％的多肽视为有效数据进行分析。实验中共鉴定到来源于39个蛋白的475条多肽。由于抗菌肽通常具有以下特点，如通常含有疏水性氨基酸、碱性氨基酸；具有强阳离子性，两亲性等。因此，结合抗菌肽的构效关系从鉴定结果中筛选出部分潜在的抗菌肽，结果如表1所示：
表1部分潜在抗菌肽鉴定结果
(4)多肽LQPHQIAQL性质分析
多肽LQPHQIAQL来源于小麦胚乳中的主要储藏蛋白——麦谷蛋白(P10386，f260-268)肽段等电点为6.74，分子质量为1047.22Da。通过在线工具ExPASy获取该多肽的生物学信息表明，该多肽呈中性，不稳定系数为66.51，脂肪族氨基酸指数和亲水性分别为141.11和-0.156，证明该多肽具有较强的疏水性及两亲性。
SEQ ID No.1的信息
(a)序列特征
*长度：9氨基酸
*类型：氨基酸
*链型：线性单链
(b)分子类型：蛋白
序列描述：
SEQ ID No.1
LQPHQIAQL
实施例2多肽LQPHQIAQL的抑菌活性检测
选择三条潜在的抗菌肽LQPHQIAQL、IFWGIPALLK和AAFSPVSLHSALSLLAAGAGS委托南京杰肽生物科技有限公司通过固相法合成，纯度分别为95.97％、99.73％和95.31％。以革兰氏阳性菌大肠杆菌、革兰氏阴性菌金色葡萄球菌为靶标菌种，考察三条潜在抗菌肽的抗菌活性。
(1)菌株及复苏
大肠杆菌(Escherichia coli K12)和金黄色葡萄球菌(Staphylococcus aureus)均以甘油保藏法保存于-80℃。实验前用接种环将一环大肠杆菌接种到5mL液体LB培养基中，一环金黄色葡萄球菌接种到5mL液体TSB培养基中，于37℃、200rpm条件下培养12h使菌株复苏。
12h后取100μL大肠杆菌菌液转移至5mL液体LB培养基中，取100μL金黄色葡萄球菌菌液转移至5mL液体TSB培养基中，再次于37℃、200rpm条件下培养12h进行复苏。重复此复苏操作2-3次，使菌株恢复活力。
(2)培养基
抗菌实验使用直径为90mm的圆形培养皿。培养基为LB固体培养基和TSB固体培养基，两种培养基中分别均含质量浓度1.5％琼脂。LB固体培养基混入终浓度为1×105CFU/mL的大肠杆菌，TSB固体培养基混入终浓度为1×105CFU/mL的金黄色葡萄球菌。将混有菌液的培养基分别倒入六个不同的圆形培养皿中(大肠杆菌和金黄色葡萄球菌分别各自倒入三个培养皿)，培养皿中培养基的装填高度为4mm，密封后于4℃保存，得六个平板。
(3)抗菌实验
分别在六个平板上制作出3个直径3mm的孔，孔间距为3cm。六个平板分成三组，将一个含有大肠杆菌的平板和一个含有金黄色葡萄球菌的平板为一组，于每组的二个平板中的二个孔中每孔加入用50μL无菌水溶解的1.5mg相同标肽(三条标肽，三组实验)，各进行两次平行实验。第三个孔中以无菌水作空白对照，同样加入50μL。加样后的平板于4℃冰箱中静置4h，待孔中样液完全吸收扩散后取出，在37℃恒温培养箱中倒置培养12h，观察并测量记录抑菌圈大小。
(4)实验结果
三条标肽对大肠杆菌和金黄色葡萄球菌的抑菌圈直径见表2。多肽IFWGIPALLK和AAFSPVSLHSALSLLAAGAGS对大肠杆菌没有抑制效果，对金黄色葡萄球菌仅有微弱的抑制效果；而LQPHQIAQL对大肠杆菌和金黄色葡萄球菌均有抑制效果，且对大肠杆菌的抑制效果略优于金黄色葡萄球菌。该结果说明多肽LQPHQIAQL对革兰氏阴性菌和革兰氏阳性菌均具有抑制活性，且对革兰氏阴性菌的抑制活性更高。
图1显示了多肽LQPHQIAQL对大肠杆菌和金黄色葡萄球菌的抑制效果。
表2多肽LQPHQIAQL的抑菌效果
序列表
<110> 中国科学院大连化学物理研究所
<120> 抗菌多肽与应用和细菌生长抑制剂、抗炎药物或保健品
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Leu Gln Pro His Gln Ile Ala Gln Leu
1 5
Claims (8)
1.一种抗菌肽，其特征在于：所述多肽为LQPHQIAQL，具有序列表SEQ ID NO：1中氨基酸序列；该多肽的氨基酸序列具体为Leu-Gln-Pro-His-Gln-Ile-Ala-Gln-Leu。
2.一种权利要求1所述多肽在制备大肠杆菌和/或金黄色葡萄球菌生长抑制剂中的应用。
3.一种权利要求1所述多肽在制备预防和/或治疗由大肠杆菌和/或金黄色葡萄球菌引起的肠道细菌感染疾病的药物和/或保健品中的应用。
4.按照权利要求2或3所述的应用，其特征在于：其是以多肽LQPHQIAQL为活性成份，其中可添加药物学上或食品上可接受的载体或辅料。
5.一种大肠杆菌和/或金黄色葡萄球菌生长抑制剂，其特征在于：其以权利要求1所述多肽为活性成份。
6.按照权利要求5所述的大肠杆菌和/或金黄色葡萄球菌生长抑制剂，其特征在于：其以权利要求1所述多肽为活性成份，其中可添加药物学上或食品上可接受的载体或辅料。
7.一种预防和/或治疗由大肠杆菌和/或金黄色葡萄球菌引起的肠道细菌感染疾病的药物和/或保健品，其特征在于：其以权利要求1所述多肽为活性成份。
8.按照权利要求7所述的药物和/或保健品，其特征在于：其以权利要求1所述多肽为活性成份，其中可添加药物学上或食品上可接受的载体或辅料。
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