CN116143877A - 基于cation-π跨链交互作用的β发卡抗菌肽及其制备方法和应用 - Google Patents
基于cation-π跨链交互作用的β发卡抗菌肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种基于cation‑π跨链交互作用的高效低毒β发卡抗菌肽及其制备方法。抗菌肽WRKFPG,其序列如序列表1所示。其制备方法为单点突变,选择赖氨酸对β‑发夹结构抗菌肽WRRFPG中C端第四位与色氨酸跨链互作的精氨酸进行取代,使赖氨酸和精氨酸与色氨酸共同形成cation‑π跨链作用,形成化学结构稳定和生物学功能稳定的β‑发卡结构的抗菌肽。抗菌肽既保证了抗菌活性,又降低了溶血活性,治疗指数达到228.11,具有成为抗生素替代产品的潜力。
Description
技术领域
本发明属于生物技术领域,具体涉及一种基于cation-π跨链交互作用的β发卡抗菌肽及其制备方法和应用。
背景技术:
由于抗生素的滥用导致耐药菌滋生,故开发抗生素的替代品以抵抗病原菌的入侵迫在眉睫。抗菌肽(AMPs)广泛存在于生物体内,可以协助机体抵御病原微生物的侵害,是生物体先天性防御***中的重要组成成分。人们普遍认为,抗菌肽通过膜损伤机制,破坏微生物细胞膜或细胞壁的完整性,不易产生耐药性。抗菌肽是由20~50个氨基酸残基组成的一类肽类物质。其具有广谱抑菌活性,并且安全、稳定、无毒副作用。现在已知AMPs在微生物、植物和无脊椎动物或更复杂的两栖动物和哺乳动物等多物种中都存在表达,作为宿主免疫防御的第一道防线用以抵抗入侵的病原体。
β-折叠肽通过二硫键来稳定而呈反平行折叠结构,其中分子量较大的肽中也可能包含较小的螺旋片段。这些二硫键的存在,在目前化学合成的实施中,合成难度和合成成本都比较高,严重影响多肽快捷廉价的制备。近年来,利用色氨酸的芳香族侧链与精氨酸的正电性形成多肽分子内跨链的cation-π作用,取代了半胱氨酸形成的二硫键,同时缩短多肽序列长度,提供足够的正电荷和疏水性以形成β-发夹的两亲结构,解决β-发夹结构抗菌肽序列过长、合成困难、合成成本高的问题。但是在保证其抗菌活性的同时,溶血活性依然偏高。其毒副作用对抗菌肽的应用造成了较大的障碍。
发明内容
基于以上不足之处,本发明的目的是提供一种基于cation-π跨链交互作用的β-发卡抗菌肽WRKFPG,其高效低毒,解决了现有的抗菌肽溶血活性偏高、毒副作用较大的问题。
本发明所采用的技术方案如下:一种基于cation-π跨链交互作用的β-发卡抗菌肽WRKFPG,所述的抗菌肽WRKFPG以PG为转角单元,通过色氨酸与精氨酸和赖氨酸跨链交互共同作用形成β发卡结构的力,其氨基酸序列如SEQ ID No.1所示,并且其C端酰胺化。
本发明的另一目的是提供一种基于cation-π跨链交互作用的β-发卡抗菌肽WRKFPG的制备方法,以β-发卡两亲肽排布基础,得到含色氨酸W与精氨酸R和单赖氨酸K跨链交互作用和PG转角单元的抗菌肽模板XWYKYPGXWYRY-NH2,当X=R,Y=F时,得到多肽,其氨基酸序列如SEQ ID No.1所示,再经过固相化学合成法合成,通过抗菌活性的测定和溶血活性的测定,最终命名为抗菌肽WRKFPG。
本发明的另一目的是提供一种基于cation-π跨链交互作用的β-发卡抗菌肽WRKFPG在制备治疗革兰氏阳性菌或/和革兰氏阴性菌引起的感染性疾病的药物中的应用。
本发明的有益效果及优点:通过本方法制备的抗菌肽,在不含半胱氨酸对形成的二硫键条件下,形成稳定的β-发卡结构,氨基酸序列长度仅有12,且在单赖氨酸取代与色氨酸进行cation-π跨链互作的精氨酸后,保持良好抑菌活性的基础上降低了溶血活性。对得到的抗菌肽进行抗菌和溶血活性检测,发现WRKFPG对大肠杆菌、绿脓杆菌、鼠伤寒沙门氏菌、金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌等多种菌株有高效的抑制作用,在检测范围内未表现出溶血活性,其治疗指数达到228.11。综上所述,WRKFPG是一种具有较高应用价值的抗菌肽,具有高效低毒的优点。
附图说明
图1为抗菌肽WRKFPG的高效液相色谱图;
图2为抗菌肽WRKFPG的质谱图。
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
抗菌肽的设计
抗菌肽WRKFPG的氨基酸序列为:
以β-发卡两亲肽排布为基础,通过色氨酸与单赖氨酸和精氨酸进行cation-π跨链交互作用作为辅助PG转角单元形成β发卡结构的力,设计出模板XWYKYPGXWYRY-NH2,X为正电荷氨基酸,Y为疏水性氨基酸,当X=R,Y=F时,命名为WRKFPG。序列如表1所示。
表1衍生肽的氨基酸序列
WRKFPG的序列长度为12,转角单元为PG单元,将肽WRRFPG的N端第四个氨基酸取代为带有正电荷且对膜破坏能力较小的赖氨酸。该方法使抗菌肽WRKFPG具有高效抑菌活性的同时具有较低的溶血活性。
实施例2
固相化学合成法合成WRKFPG抗菌肽(由上海生工生物工程股份有限公司合成)
1、抗菌肽的制备从C末端到N末端逐一进行,通过多肽合成仪来完成。首先将Fmoc-X(X是每个抗菌肽的C末端第一个氨基酸)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末端第二个氨基酸);按照这个程序依次从C末端合成到N末端,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***,-20℃沉淀3h,析出白色粉末物,以2500g离心10min,收集沉淀,再用无水***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pH 7.4)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,质谱图中显示的分子量(如图1、2所示)与表1中的理论分子量基本一致,抗菌肽的纯度大于95%。
实施例3:抗菌肽抗菌活性的测定
1、抗菌活性的测定:利用微量肉汤稀释法测定几种抗菌肽的最小抑菌浓度。细菌在220rpm恒定摇动下于37℃孵育过夜,然后转移至新的MHB直至生长的对数期。将50μL细菌培养物(~106CFU/mL)添加到50μL含有不同浓度抗菌肽的BSA中,在37℃下孵育18h。在18h孵育结束时观察并测量492nm的吸光度,在视觉和分光光度法下未观察到微生物生长的抗菌肽浓度为最小抑菌浓度。含微生物细胞的肉汤用作阴性对照,未接种的肉汤用作阳性对照。测试进行3次重复,每个重复两个平行。
检测结果见表2。
表2抗菌肽的抑菌活性(μM)
通过表2可以看出,WRKFPG对于革兰氏阴性和阳性菌均表现出较高的抑菌活性。
2、溶血活性的测定:健康人的新鲜血液1mL,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;lh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50μL PBS作为阴性对照;50μL红细胞加50μL 0.1%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。检测结果见表3。
表3抗菌肽溶血活性的测定及治疗指数
通过表3可以看出,WRKFPG在检测范围内未表现出溶血活性,其治疗指数达到228.11。与已知的抗菌肽WRRFPG相比,降低了溶血活性;与已知的抗菌肽WKKFPG相比,提高了抗菌活性。
3、稳定性的测定:评估血清,酸性和碱性条件对抗菌活性的影响,肽在不同浓度的血清水平(50%,100%)和不同pH水平(2、12)下孵育4小时,测定抑菌活性。检测结果见表4。
表4.抗菌肽WRKFPG在血清和不同pH条件下对大肠杆菌25922和金黄色葡萄球菌29213的抑菌活性(μM)
根据实验结果可以看出,WRKFPG在50%和100%血清及极酸极碱环境中依旧保持着良好的抑菌活性。
以上结果显示,以单赖氨酸取代与色氨酸跨链作用的精氨酸,使赖氨酸和精氨酸共同与色氨酸形成cation-π跨链交互作用,以PG为转角单元设计出的抗菌肽WRKFPG具有良好的抗菌活性和更低的溶血活性以及较好的稳定性,表明其具有较强的替代抗生素的潜力。
Claims (3)
1.一种基于cation-π跨链交互作用的高效低毒β-发卡抗菌肽WRKFPG,其特征在于,所述的抗菌肽WRKFPG以PG为转角单元,通过色氨酸W与精氨酸R和单赖氨酸K跨链交互共同作用形成β发卡结构的力,其氨基酸序列如SEQ ID No.1所示,其C端酰胺化。
2.根据权利要求1所述的一种基于cation-π跨链交互作用的高效低毒β-发卡抗菌肽WRKFPG的制备方法,其特征在于,方法如下:以β-发卡两亲肽排布基础,得到含色氨酸W与精氨酸R和单赖氨酸K跨链交互作用和PG转角单元的抗菌肽模板XWYKYPGXWYRY-NH2,当X=R,Y=F时,得到多肽,其氨基酸序列如SEQ ID No.1所示,再经过固相化学合成法合成,通过抗菌活性的测定和溶血活性的测定,最终命名为抗菌肽WRKFPG。
3.根据权利要求1所述的一种基于cation-π跨链交互作用的高效低毒β-发卡抗菌肽WRKFPG在制备治疗革兰氏阳性菌或/和革兰氏阴性菌引起的感染性疾病的药物中的应用。
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