CN111533781B - 非特异受体结合型真菌靶向抗菌肽及其制备方法和应用 - Google Patents
非特异受体结合型真菌靶向抗菌肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种非特异受体结合型真菌靶向抗菌肽及其制备方法和应用。该抗菌肽P19，其序列如SEQ ID No.1所示。制备方法：利用R语言统计分析天然抗菌肽库中对细菌或真菌具有活性的抗菌肽相关结构功能参数，对比相关参数，利用抗真菌抗菌肽的高频氨基酸组成及高频电荷数，疏水性，结构特性筛选得到最佳的氨基酸组成，对比各肽链抗菌活性及菌株选择性，得到了通过氨基酸调整序列疏水性及两亲性控制抗菌肽P19。该抗菌肽在制备治疗真菌感染性疾病药物中应用。本抗菌肽只针对真菌菌株，尤其是白色念珠球菌具有较强的杀灭作用，而对其他的细菌菌株无影响，表现出较精准的靶向特异性。
Description
技术领域
本发明属于生物技术领域，具体涉及一种非特异受体结合型真菌靶向抗菌肽及其制备方法和应用。
背景技术
白色念珠菌作为条件性致病菌所导致的畜禽消化道真菌病及真菌性乳房炎等将严重影响畜禽生产的经济效益，然而，相比于原核生物的细菌病，仍有大量诸如抗生素之类的医药产品能够成功破坏细菌细胞而不会损害正常人细胞，真菌病由于真菌细胞结构与人类真核细胞相似使得杀死真菌细胞的治疗方法在不损害正常人体细胞的情况下更加困难使得大多数化学处理方法对真菌和宿主细胞都有毒性无法应用。目前商业上有四种类型的用于抗真菌感染的治疗方法，但是，抗真菌药物的负面影响不可忽视，虽然这些副作用大多数都不严重，比如水样腹泻，腹泻，胃痛或者恶心以及少数的过敏反应。此外，抗真菌药的使用还会破坏肠道和阴道中的常见微生物平衡，使得有害微生物大量繁殖造成病变。
发明内容
基于以上不足之处，本发明的目的在于公开一种非特异受体结合型真菌靶向抗菌肽，该抗菌肽对真菌，尤其是白色念珠球菌具有选择性杀灭效果，而对其它微生态菌群影响较小。
本发明的目的通过如下技术方法实现：一种非特异受体结合型真菌靶向抗菌肽P19，其序列如SEQ ID No.1所示。
本发明的另一目的在于公开如上所述的抗菌肽P19的制备方法，如下：利用R语言统计分析天然抗菌肽库中对细菌或真菌具有活性的抗菌肽相关结构功能参数，对比相关参数，利用抗真菌抗菌肽的高频氨基酸组成及高频电荷数，疏水性，结构特性筛选得到最佳的氨基酸组成：Ala,Cys,Ser,Leu,Ile,Phe，经过抗菌活性测定，并针对真菌的靶向杀菌效果，选择氨基酸Ser和Phe，得到抗菌肽P19的序列，如序列表SEQ ID No.1所示。
本发明的另一目的在于公开如上所述的抗菌肽P19在制备治疗真菌感染性疾病药物中应用。
本应用的优选是所述的真菌为白色念珠球菌。
本发明具有如下优点及有益效果：本抗菌肽P19抑菌作用机理为抗菌肽通过作用于细菌细胞膜表面，使其通透性增加，从而使细胞膜表面破碎导致其死亡，本抗菌肽P19只针对真菌菌株，尤其是白色念珠球菌具有较强的杀灭作用，而对其他的细菌菌株无影响，表现出较精准的靶向特异性，而且具有较高的细胞存活率。体内模拟环境稳定性结果说明设计所得抗菌肽具有较高的应用潜力。综上所述，是一种具有较高应用价值的靶向抗菌肽。
附图说明
图1是本发明的抗菌肽的细胞毒性结果图，
图2是本发明的抗菌肽处理后四种菌株的存活率。
图3是本发明的抗菌肽的液相色谱图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
利用R语言统计分析天然抗菌肽库中真菌抗菌肽及广谱抗菌肽的氨基酸序列特征，对比两类抗菌肽的各参数组成，尤其是氨基酸组成，筛选得到最佳的氨基酸组成,即Ala,Cys,Ser,Leu,Ile；在此基础上，将氨基酸以色氨酸为中心的对称结构(++hyh W hyh++),(h,疏水性氨基酸；+,正电荷氨基酸；y,亲水性氨基酸)中；以获得低毒，高效不易引起机体免疫调节紊乱的短链靶向抗菌肽序列。
表1抗菌肽的氨基酸序列
实施例2
固相化学合成法合成信息素标记抗菌肽
1、抗菌肽的制备从C端到N端逐一进行，通过多肽合成仪来完成。首先将Fmoc-X(X是每个抗菌肽的C端第一个氨基酸)接入到Wang树脂，然后脱去Fmoc基团后得到X-Wang树脂；再将Fmoc-Y-Trt-OH(9-芴甲氧羧基-三甲基-Y，Y为每个抗菌肽C端第二个氨基酸)；按照这个程序依次从C端合成到N端，直至合成完毕，得到脱去Fmoc基团的侧链保护的树脂；
2、在上述得到的肽树脂中，加入切割试剂，20℃避光下反应2h，过滤；沉淀TFA(三氟乙酸)洗涤，将洗液与上述滤液混合，旋转蒸发仪浓缩，再加入10倍左右体积的预冷无水乙醚，-20℃沉淀3h，析出白色粉末物，以2500g离心10min，收集沉淀，再用无水乙醚洗涤沉淀，真空干燥，得到多肽，其中切割试剂由TFA、水和TIS(三异丙基氯硅烷)按照质量比95:2.5:2.5混合而成；
3、使用0.2mol/L硫酸钠(磷酸调节至pH7.5)进行柱平衡30min，用90％乙腈水溶液溶解多肽，过滤，C18反相常压柱，采用梯度洗脱(洗脱剂为甲醇和硫酸钠水溶液按照体积比为30:70～70:30混合)，流速为1mL/min，检测波为220nm，收集主峰，冻干；再利用反相C18柱进一步纯化，洗脱液A为0.1％TFA/水溶液；洗脱液B为0.1％TFA/乙腈溶液，洗脱浓度为25％B～40％B，洗脱时间为12min，流速为1mL/min，再同上收集主峰，冻干；
4、抗菌肽的鉴定：将上述得到的抗菌肽经过电喷雾质谱法分析，抗菌肽的纯度大于95％。
实施例3
抗菌肽活性的测定
1、抗菌活性的测定：将抗菌肽配置成为一定储存液以备使用。利用微量肉汤稀释法测定几种抗菌肽的最小抑菌浓度。以含0.2％BSA的0.01％乙酸(细菌)及加入MOPS的RPMI1640(真菌)作为稀释液，使用二倍稀释法依次配置系列梯度的抗菌肽溶液。取上述溶液100μL置于96孔细胞培养板中，然后分别添加等体积的待测菌液(～105个/mL)于各孔中。分别设置阳性对照(含有菌液而不含有抗菌肽)和阴性对照(既不含菌液也不含肽)。37℃恒温培养20h(真菌为28℃培养48h)，以肉眼未见孔底部有混浊现象的即为最小抑菌浓度。结果如表1-3所示。
表1针对细菌抗菌肽的抑菌活性
表2针对真菌抗菌肽的抑菌活性
表3生理盐环境下抗菌肽对白色念珠球菌的抑菌活性
| 抗菌肽 | 对照 | NaCl | KCl | MgCl<sub>2</sub> | NH<sub>4</sub>Cl | ZnCl<sub>2</sub> | FeCl<sub>3</sub> | pH＝6 |
| P19 | 2 | 32 | 4 | 4 | 4 | 4 | 4 | 4 |
通过表1-3可以看出，设计得到的靶向抗菌肽P19具有较强的针对真菌的选择性杀灭作用，并表现出体内盐环境的抗菌稳定性。
2、溶血活性的测定：采集人的新鲜血液1mL，肝素抗凝后溶解到2mLPBS溶液中，1000g离心5min，收集红细胞；用PBS洗涤3遍，再用10mL PBS重悬；取50μL红细胞悬液与50μL用PBS溶解的不同浓度的抗菌肽溶液混合均匀，在37℃培养箱内恒温孵育1h；l h后取出，4℃、1000g离心5min；取出上清液用酶标仪在570nm处测光吸收值；每组取平均值，并比较分析。其中50μL红细胞加50μLPBS作为阴性对照；50μL红细胞加50μL0.1％Tritonx-100作为阳性对照。最小溶血浓度是抗菌肽引起10％溶血率时的抗菌肽浓度。结果如图1所示。如图可以看出，P19在最大测定浓度128μM下引起的溶血率仍低于5％，说明该抗菌肽具有较低的细胞毒性。
以上结果表明，本发明设计得到的靶向抗菌肽P19可特异性杀灭真菌，对乳酸杆菌、葡萄球菌等无作用。同时表现出较低的细胞毒性，具有开发成靶向抗真菌药物的潜力。
序列表
<110> 东北农业大学
<120> 非特异受体结合型真菌靶向抗菌肽及其制备方法和应用
<140> 202010234600X
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Arg Arg Phe Ser Phe Trp Phe Ser Phe Arg Arg-NH2
1 5 10
Claims (4)
1.一种非特异受体结合型真菌靶向抗菌肽P19，其特征在于，其序列如SEQ ID No.1所示。
2.根据权利要求1所述的一种非特异受体结合型真菌靶向抗菌肽P19的制备方法，其特征在于：利用R语言统计分析天然抗菌肽库中对细菌或真菌具有活性的抗菌肽相关结构功能参数，对比相关参数，利用抗真菌抗菌肽的高频氨基酸组成及高频电荷数，疏水性，结构特性筛选得到最佳的氨基酸组成：Ala,Cys,Ser,Leu,Ile,Phe，经过抗菌活性测定，并针对真菌的靶向杀菌效果，选择氨基酸Ser和Phe，得到抗菌肽P19的序列，如序列表SEQ ID No.1所示。
3.根据权利要求1所述的一种非特异受体结合型真菌靶向抗菌肽P19在制备治疗念珠菌感染性疾病的药物中的应用。
4.根据权利要求3所述的应用，其特征在于：所述的念珠菌为白色念珠球菌。
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