CN112851762A - 一种人乳内源性抗菌多肽及其在制备抗炎药物中的应用 - Google Patents
一种人乳内源性抗菌多肽及其在制备抗炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种来源于人乳内源对以大肠杆菌和金黄色葡萄球菌为代表的革兰氏阳性菌和革兰氏阴性菌有抑制活性的多肽化合物AAEVYGTERQPRTHYYAV，其氨基酸序列为Ala‑Ala‑Glu‑Val‑Tyr‑Gly‑Thr‑Glu‑Arg‑Gln‑Pro‑Arg‑Thr‑His‑Tyr‑Tyr‑Ala‑Val。多肽AAEVYGTERQPRTHYYAV具有对革兰氏阳性菌和革兰氏阴性菌的广泛抑制活性，作为预防和/或降低新生儿由微生物引起的肠道感染疾病的药物和/或保健品或者新型婴儿配方食品的添加剂具有良好的应用前景。
Description
技术领域
本发明涉及一种来源于人乳的内源性抗菌多肽AAEVYGTERQPRTHYYAV及其应用。
背景技术
抗生素是能抵抗致病微生物的药物，是抗菌消炎药中最大的一类。抗生素是由细菌、真菌或其他微生物在生活过程中所产生的物质，具有抑制或杀灭细菌、真菌等致病微生物的作用，因此作为抗感染药物广泛地应用于各种感染性疾病。抗生素在治疗感染中的显著效果也导致了细菌耐药性，使部分感染性疾病的治疗变得愈加困难。抗菌肽作为一种新型的抗生素，具有广谱的抗菌活性，不会轻易使微生物产生耐药性，具有广阔的应用前景。
抗菌肽在自然界中广泛分布，作为被广泛关注的一种新型抗生素，越来越多的抗菌肽在微生物、动植物体内被发现。目前解析的抗菌肽构效关系中有两个较清晰的特征，分别是“带正电荷的阳离子”和“两亲性序列结构”。然而抗菌肽作为抗菌药物仍然面临许多待解决的问题：部分阳离子抗菌肽可以对哺乳动物的细胞产生较大的毒性作用；两亲性结构则要求氨基酸序列中有一定数量的碱性氨基酸，而碱性氨基酸易被蛋白酶水解的特性使得其作为药物进入人体后发挥效果大大受限。因此，开发安全、稳定、低细胞毒性的抗菌肽具有重要意义。
母乳作为婴儿最佳的天然食物，是丰富的蛋白质和多肽资源，兼顾抗菌肽的优势及人乳优质安全的蛋白来源两个特质。人乳中含有多种与免疫相关的蛋白质。人乳腺中的内源性酶对蛋白质有较强的水解作用，而来源于人乳中稳定存在的内源性抗菌肽，其本身的存在就解决了抗菌肽进入人体后易被进一步水解的问题，作为抗菌药物具有良好的潜质。因此，从人乳内源性多肽出发进一步寻找具有抑菌活性的肽会为临床上对婴幼儿抗感染的治疗提供新的治疗手段。
发明内容
本发明的目的是提供一种内源性抗菌多肽AAEVYGTERQPRTHYYAV，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/或保健品或者新型婴儿配方食品中的应用。
为实现上述目的，本发明以所述多肽AAEVYGTERQPRTHYYAV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AAEVYGTERQPRTHYYAV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AAEVYGTERQPRTHYYAV的氨基酸序列为Ala-Ala-Glu-Val-Tyr-Gly-Thr-Glu-Arg-Gln-Pro-Arg-Thr-His-Tyr-Tyr-Ala-Val。分子量2111.30Da，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：
本发明从人乳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，因此作为预防和/或降低新生儿由微生物引起的肠道感染疾病的药物和/或保健品或者新型婴儿配方食品的添加剂具有良好的应用前景。
附图说明
图1为多肽AAEVYGTERQPRTHYYAV的抑菌结果图,其中左面为A.多肽AAEVYGTERQPRTHYYAV抑制大肠杆菌的实验结果图；右面为B.多肽AAEVYGTERQPRTHYYAV抑制金属葡萄球菌的实验结果图.
具体实施方式
实施例1
人乳内源肽的制备及鉴定。
(1)样品制备
取200μL人乳原液以水稀释10倍,水浴95℃条件下5min变性，以截留分子量10K Da的超滤管在14000×g、20℃条件下超滤20min，管中收集的超滤液为内源肽。
(2)Triple TOFTM 6600+MS分析
内源肽样品按如下步骤以C18柱(Waters Oasis HLB SPE柱)除盐：商品化的C18柱用1.5mL甲醇活化后加入1.5mL 0.1％(V/V)TFA-H2O溶液平衡，将样品以0.1％(V/V)TFA-H2O溶液复溶后加入C18柱中，以1.5mL 80％(V/V)ACN/0.1％(V/V)TFA-H2O溶液进行洗脱，收集洗脱液分装冻干于-80℃保存。上述步骤处理得到的内源肽样品以0.1％(V/V)FA-H2O复溶，样品浓度0.4mg/ml，所有样品以相同的8μL体积上样，进行Triple TOFTM 6600+MS分析，进样量3.2μg。使用Triple TOF 6600质谱仪(AB SCIEX，USA)，以信息依赖获取(IDA)模式进行nanoLC-MS/MS分析。预处理柱C18，分析柱：CHROMXP C18,3UM,150X0.3MM COLUMN。串联质谱以正离子数据依赖模式采集获取，质谱扫描设定为一个全扫描荷质比(m/z)范围350-1250的离子，accumulation time:0.25s，并对其中最强的40个离子进行离子碰撞解离(CID)，再进行MS/MS扫描，扫描范围在m/z 100-1500，accumulation time:0.05s。
使用Waters Nano ACQUITY UPLC系统运输流动相。流动相A为含0.1％甲酸的水溶液，流动相B为含0.1％甲酸和98％乙腈的水溶液。梯度洗脱程序如下：0-1min，4％B；1-56min，4％-18％B；56-70min，18％-40％B；70-70.5min，40％-80％B；70.5-75.5min，80％B；75.5-76min，80％-4％B；76-80min，4％B，流速5μL/min。
(3)数据检索
Xcalibur采集的*.wiff文件用Thermo Proteome Discoverer Daemom(v1.4)AB_SCIEX_MS_Data_Converter转换成*.MGF格式，之后用Mascot 2.5.1软件在人(human，蛋白质数目为20185)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50ppm，碎片离子为0.2Da。导出肽段时控制假阳性率(FDR)<1％，对导出结果Score>20的肽段视为有效数据进行分析。用maxquant软件在人的库中label-free检索，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)。
(4)LC-MS/MS得到目标肽段信息
在人乳内源肽样品中鉴定到一千条以上的肽段信息(具体肽段信息在补充表1中有示例给出)，其中约50％以上来源于β-酪蛋白，有约2％的肽段来源于乳铁蛋白。多肽AAEVYGTERQPRTHYYAV在分析的多例人乳内源肽样品中均被鉴定到，说明其在人乳中稳定存在。来源于人乳内源的多肽AAEVYGTERQPRTHYYAV蛋白质前体为人乳铁蛋白(f97-114)，肽段等电点为6.80，分子质量为2111.30Da，具有18个氨基酸，较短的序列及较低的相对分子质量，结合质谱鉴定结果表明该序列可以稳定存在不易继续被蛋白酶酶解。通过在线工具获取SEQ ID NO：1所示氨基酸序列的生物学信息表示，该肽段的不稳定系数为68.16，脂肪族氨基酸指数和亲水性分别为48.89和-0.900。
SEQ ID No.1的信息
(a)序列特征
*长度：18氨基酸
*类型：氨基酸
*链型：单链
(b)分子类型：蛋白
序列描述：
SEQ ID No.1
AAEVYGTERQPRTHYYAV
实施例2多肽的抗菌活性
委托上海杰肽生物科技有限公司通过固相法合成多肽AAEVYGTERQPRTHYYAV，纯度为95.14％。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，考察多肽AAEVYGTERQPRTHYYAV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℃，实验前将两种菌分别接种到液体LB培养基和TSB培养基中，于37℃条件下培养12h使菌种复苏，重复上述复苏操作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。双层培养基，下层以15mL 2％(m/V)的琼脂水溶液做支撑，上层分别加入5mL含0.7％(m/V)琼脂、菌浓度为1×104CFU/mL的LB培养基和TSB培养基，将制备好的两种培养基密封后于4℃保存。
(3)抗菌实验
在平板上层制作出直径2μm的孔，各孔间距约3.5cm。合成的标肽分装成5mg/支，每支加入50μL无菌水，完全溶解后向孔中加入标肽水溶液35μL，每孔实际加入标肽量3.5mg。以水作空白对照，加样体积与标肽样品同为35μL，加样后的平板于4℃冰箱中静置3h，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，观察是否有抑菌圈出现，测量抑菌圈大小并记录。
(4)实验结果
对大肠杆菌和金黄葡萄球菌的抑菌圈直径见表1和图1，表中列出了目标肽段的抑菌圈直径，结果表明多肽AAEVYGTERQPRTHYYAV对革兰氏阴性菌和革兰氏阳性菌均有抑制活性，对金黄色葡萄球菌的生长抑制活性略高于大肠杆菌。
表1多肽AAEVYGTERQPRTHYYAV的抑菌结果
“-”表示没有抑菌活性。
补充表1鉴定到的肽段信息
序列表
<110> 中国科学院大连化学物理研究所
<120> 一种人乳内源性抗菌多肽及其在制备抗炎药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ala Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr
1 5 10 15
Ala Val
Claims (4)
1.一种人乳内源性抗菌多肽，其特征在于：所述多肽为AAEVYGTERQPRTHYYAV，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Ala-Ala-Glu-Val-Tyr-Gly-Thr-Glu-Arg-Gln-Pro-Arg-Thr-His-Tyr-Tyr-Ala-Val。
2.一种权利要求1所述多肽在制备革兰氏阳性细菌和革兰氏阴性细菌的生长抑制剂或抗胃肠道感染药物中的应用。
3.按照权利要求2所述的应用，其特征在于：所述微生物生长抑制剂或抗感染药物是以多肽AAEVYGTERQPRTHYYAV为活性成份，其中可添加药物学上可接受的载体或辅料。
4.按照权利要求2所述的应用，其特征在于：所述多肽在制备预防和/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/或保健品或者新型婴儿配方食品中的应用。
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| WO2007106951A1 (en) * | 2006-03-23 | 2007-09-27 | Agriculture Victoria Services Pty Limited | Antimicrobial protein |
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| WO2007106951A1 (en) * | 2006-03-23 | 2007-09-27 | Agriculture Victoria Services Pty Limited | Antimicrobial protein |
| WO2014060610A2 (en) * | 2012-10-19 | 2014-04-24 | Katholieke Universiteit Leuven, K.U. Leuven R&D | Antimicrobial peptides |
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