CN117285598A - 抗胞内革兰氏阳性菌感染的纳米肽f3ft及其制备方法和应用 - Google Patents
抗胞内革兰氏阳性菌感染的纳米肽f3ft及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种抗胞内革兰氏阳性菌感染的纳米肽F3FT及其制备方法和应用,属于生物技术领域,纳米肽F3FT的氨基酸序列如SEQ ID No.1所示。制备方法:选择人免疫缺陷病毒的转录反式激活因子的第47‑57位序列片段TAT11,再其N端侧放置3个连续的苯丙氨酸。最后在N末端连接9‑芴基甲氧基羰基。本发明的纳米肽F3FT对具有胞内生存能力的革兰氏阳性菌具有强效的抗菌活性,而且细胞毒性低。此外,纳米肽F3FT能高效穿透巨噬细胞并降低巨噬细胞内的金黄色葡萄球菌的存活率,且主要通过破坏细菌膜发挥抗菌作用,在抗胞内革兰氏阳性菌感染方面具有极高的应用潜力。
Description
技术领域
本发明属于生物技术领域,具体涉及一种抗胞内革兰氏阳性菌感染的纳米肽F3FT及其制备方法和应用。
背景技术
由金黄色葡萄球菌、单增李斯特菌、结核分枝杆菌等革兰氏阳性菌引起的胞内感染非常难以治疗。这些细菌具有在宿主细胞内生存和复制的独特机制,增加了全球与传染病相关的发病率和死亡率。由于部分抗生素细胞渗透性差,在受感染的宿主细胞内的有效浓度低,导致抗菌活性差,使细胞内细菌感染更难根除。虽然有些抗生素被用于治疗细胞内细菌感染,但细菌耐药性也在不断增加,从而降低或消除了这些抗生素的疗效。
同属于膜活性肽的抗菌肽和细胞穿透肽有望成为对抗胞内细菌感染的潜在抗菌剂,然而绝大多数的抗菌肽和细胞穿透肽都不能兼具抗菌和穿透这两项胞内抗菌必需的能力。此外,随着纳米技术的进步,多肽的自组装被报道具有增强抗菌活性、提高稳定性等优势。在这种情况下,研发具有抗菌能力和细胞穿透能力的纳米肽是抗胞内细菌感染的重要策略。
发明内容
基于以上不足之处,本发明的目的在于提供一种抗胞内革兰氏阳性菌感染的纳米肽F3FT,解决了多数抗菌肽和细胞穿透肽对胞内革兰氏阳性菌感染治疗效果差的问题。
本发明所采用的技术方案如下:一种抗胞内革兰氏阳性菌感染的纳米肽F3FT,其氨基酸序列如SEQ ID No.1所示,其N末端接枝9-芴基甲氧基羰基基团。
进一步的,所述的纳米肽F3FT,其特征在于,其分子式如式(I)所示,
进一步的,将如上所述的纳米肽F3FT溶解在PBS缓冲液后静置24h能够自组装成纳米结构。
本发明还提供如上所述的一种抗胞内革兰氏阳性菌感染的纳米肽F3FT的制备方法,如下:选择人免疫缺陷病毒的转录反式激活因子(Transactivator of transcription,TAT)的第47-57位序列片段TAT11,以提供抗菌活性所需的正电荷和胞内杀菌所需的细胞穿透能力。在序列片段TAT11的N端侧放置3个连续的苯丙氨酸(Phenylalanine),以提供抗菌活性所需的疏水性。最后在N末端接枝9-芴基甲氧基羰基(9-fluorenylmethyloxycarbonyl,Fmoc)基团,以驱动自组装形成纳米结构,得到多肽,其氨基酸序列如SEQ ID No.1所示,采用固相化学合成法合成所述的多肽,再将所述的多肽经高效液相色谱进行纯化,再经过抑菌活性测定、细胞毒性的测定、细胞穿透能力测定和胞内杀菌活性的测定,最后命名为纳米肽F3FT。
本发明的另一目的是提供一种抗胞内革兰氏阳性菌感染的纳米肽F3FT在制备治疗革兰氏阳性菌感染性疾病的药物中的应用。
进一步的,如上所述的应用中所述的革兰氏阳性菌为单增李斯特菌、表皮葡萄球菌或金黄色葡萄球菌。
本发明具有如下优点及有益效果:本发明的纳米肽F3FT对常见的金黄色葡萄球菌、单增李斯特菌、粪肠球菌和表皮葡萄球菌等常见的具有胞内感染能力的革兰氏阳性菌具有很强的抗菌活性;对巨噬细胞几乎没有毒性;能够高效穿透巨噬细胞并且有效杀灭巨噬细胞内的金黄色葡萄球菌,此外主要通过破坏细菌膜发挥抗菌作用,具有治疗胞内革兰氏阳性菌感染的应用潜力。
附图说明
图1为纳米肽F3FT的质谱图。
图2为纳米肽F3FT的高效液相色谱图。
图3为纳米肽F3FT的透射电子显微镜纳米表征图。
图4为纳米肽F3FT的细胞毒性。
图5为纳米肽F3FT的细胞穿透能力。
图6为纳米肽F3FT的胞内抗菌活性。
图7为扫描电子显微镜下纳米肽F3FT的抗菌机理图。
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
纳米肽的设计
选择人免疫缺陷病毒的转录反式激活因子(Transactivator of transcription,TAT)的第47-57位序列片段(TAT11),以提供抗菌活性所需的正电荷和胞内杀菌所需的细胞穿透能力。随后再其N端侧放置3个连续的苯丙氨酸(Phenylalanine,F),以提供抗菌活性所需的疏水性。最后在N末端连接9-芴基甲氧基羰基(9-fluorenylmethyloxycarbonyl,Fmoc)以驱动自组装形成纳米结构,由此设计的自组装纳米肽命名为F3FT。
纳米肽F3FT的序列如表1所示。
表1纳米肽的序列
分子式如式(I)所示,
实施例2
固相化学合成法合成纳米肽
一、固相合成:
1.树脂溶涨:将Fmoc-Arg(Pbf)-WangResin树脂放入反应管中,加DCM(15mL/g),振荡30min。
2.接第一个氨基酸:通过沙芯抽滤掉溶剂,加入3倍摩尔过量的Fmoc-Arg(Pbf)-OH氨基酸和缩合剂Pybop,再加入3倍摩尔过量的DIEA,最后加入DMF溶解,振荡30min。
3.脱保护:去掉DMF,加20%哌啶DMF溶液(15mL/g),5min,去掉,再加20%哌啶DMF溶液(15ml/g),15min。
4.检测:(1)脱保护检测:抽掉哌啶溶液,取十几粒树脂,加入茚三酮,KCN,苯酚溶液各一滴,105℃-110℃加热5min,树脂变深蓝色。(2)缩合检测:抽掉DMF溶液,取十几粒树脂,加入茚三酮,KCN,苯酚溶液各一滴,105℃-110℃加热5min,树脂变透明白色。
5.洗:DMF(10mL/g)两次,DCM(10mL/g)两次,DMF(10mL/g)两次。
6.缩合:保护氨基酸Fmoc-Fmoc-Arg(Pbf)-OH三倍过量,Pybop三倍过量,均用尽量少DMF溶解,加入反应管,立刻加入DIEA(3倍过量),反应30min。
7.洗:DMF(10mL/g)两次,DCM(10mL/g)两次,DMF(10mL/g)两次。
8.重复2-7步操作,从右到左依次连接序列中的主链氨基酸。
9.最后一个氨基酸Fmoc-Phe-OH连接后,不脱保护。
10.收缩:DMF(10mL/g)两次,DCM(10mL/g)两次,甲醇(10mL/g)两次,抽干10min。11.从树脂上切割多肽:配制切割液(10/g),TFA 94.5%;水2.5%;EDT 2.5%;TIS1%。切割时间:120min。12.吹干洗涤:将裂解液用氮气尽量吹干,用乙醚洗六次,然后常温挥干,得到粗品。
二、纯化鉴定:
1.检测粗品MS:取少量粗品,溶解后使用LC-MS,确定分子量(如图1所示)与表1中的理论分子量基本一致后再进行纯化。
2.纯化:使用高效液相色谱仪纯化多肽,制备得到纯度>95%的多肽(如图2所示)。
实施例3
纳米肽的透射电子显微镜表征
将纳米肽溶解在PBS缓冲液后静置约24h,把样品转移到300目的铜载网上,然后用1%的磷钨酸负染30s。然后将其风干后在透射电子显微镜下观察。图3纳米肽F3FT的透射电子显微镜纳米表征图,从图中可以看出F3FT能够形成均匀的短纳米纤维结构。
实施例4
纳米肽的抑菌活性测定
采用标准微肉汤稀释法测定肽的最小抑菌浓度(Minimuminhibitoryconcentration,MIC)。将对数期的细菌稀释至~2×105CFU/mL。将50μl制备完成的不同浓度的纳米肽和同等体积的细菌悬液添加到96孔板各孔中,同时设置阴性对照(仅培养基)和阳性对照(细菌和培养基),然后将96孔板置于37℃恒温培养箱18~20小时。用酶标仪在492nm(OD492)处测定吸光度值,确定最小抑菌浓度。进行3次独立重复试验,每个重复2个平行。结果见表2。
表2纳米肽F3FT的最小抑菌浓度(μM)
通过表2可以看出,纳米肽F3FT对于金黄色葡萄球菌、单增李斯特菌、表皮葡萄球菌、粪肠球菌等多种具有胞内生存能力的革兰氏阳性菌均表现出很强的抑菌活性,最小抑菌浓度在2-4μM。
实施例5
纳米肽的细胞毒性的测定
将小鼠巨噬细胞RAW 264.7稀释至~2×105cells/mL,将50μL稀释后的细胞加入到96孔板1-11列孔中,并在含5% CO2的37℃细胞培养箱中继续培养至细胞完全贴壁生长。在新96孔板中,将50μl制备完成的不同浓度的纳米肽加入1-10列。11列中加入50μL相应的细胞培养基,12列中加入100μL相应的细胞培养基。将上述新96孔板各孔中的纳米肽或培养基转移至含有细胞的96孔板相应的孔中,在含有5% CO2的37℃细胞培养箱中培养4h。接下来,在每孔中加入25μL的MTT(0.5mg/mL)继续培养3h。最后,弃去上清,并向每个孔中加入150μL的DMSO以溶解甲瓒晶体。11列和12列分别为阳性对照和阴性对照。用酶标仪在波长为570nm处测定吸光度值。共进行3次独立重复试验。细胞存活率计算公式如下:细胞存活率(%)=[(样品OD570—阴性对照OD570)/(阳性对照OD570—阴性对照OD570)]×100%
图4为纳米肽F3FT对RAW 264.7细胞的细胞毒性,在4-32μM浓度范围内纳米肽F3FT处理后RAW 264.7细胞的存活率仍然保持在85%以上,表明纳米肽F3FT的细胞毒性低,具有良好的生物相容性。
实施例6
纳米肽的细胞穿透能力测定
将小鼠巨噬细胞RAW 264.7稀释至~2×105cells/mL,将1mL稀释后的细胞加入到24孔板,并在含5% CO2的37℃细胞培养箱中继续培养至细胞完全贴壁生长。然后加入FITC标记的纳米肽F3FT在37℃处理2h,加入0.4%的台盼蓝淬灭细胞外的荧光,然后用PBS洗2-3遍后收集细胞,随后用流式细胞仪分析细胞。
图5为纳米肽F3FT的细胞穿透能力,在16μM的浓度下纳米肽F3FT能穿透进入超过95%的小鼠巨噬细胞,表明其具有高效的细胞穿透能力。
实施例7
纳米肽的胞内杀菌活性的测定
将小鼠巨噬细胞RAW 264.7稀释至~2×105cells/mL,将1mL稀释后的细胞加入到24孔板,并在含5% CO2的37℃细胞培养箱中继续培养至细胞完全贴壁生长。然后将对数生长期的金黄色葡萄球菌29213添加到24孔板(感染复数=10)在37℃感染1h,加入庆大霉素(100μg/mL)继续在37℃孵育1h以彻底清除胞外细菌,然后用PBS洗2-3遍,再加入制备完成的纳米肽(对照加入PBS)在37℃处理4h,然后用PBS洗2-3遍,使用Triton X-100裂解细胞15min,之后稀释后涂布于MHA平板,在37℃培养箱过夜培养后计数。
图6为纳米肽F3FT的胞内抗菌活性,16μM的F3FT对RAW 264.7细胞内的金黄色葡萄球菌29213的杀伤率约在90%以上,表明其具有优异的抗胞内细菌活性。
实施例8
纳米肽的抗菌机理
将金黄色葡萄球菌29213接种于MHB培养基中,37℃,220rpm过夜培养后,转移到新的MHB培养基中,直至达到指数生长阶段。将在指数生长阶段的金黄色葡萄球菌29213离心弃去MHB培养基,收集剩余细菌,用PBS(10mM,pH=7.4)洗三次并用其重新悬浮细菌至OD600=0.2。将抗菌肽(对照未经抗菌肽处理)与细菌在37℃孵育1h,然后离心收集细菌,加入600μL戊二醛(2.5%)重悬菌体,在4℃过夜固定。用不同浓度的乙醇(50%,70%,90%和100%)对样品进行脱水处理。用1:1的乙醇和叔丁醇的混合液和叔丁醇分别置换15min。将样品用冷冻干燥仪干燥后,用导电胶布粘在样品板上,用镀膜仪在样品表面镀金属膜。最后用扫描电子显微镜采集图像。
图7为扫描电子显微镜图,与对照相比,经过纳米肽F3FT处理后的金黄色葡萄球菌29213的菌膜表面有大量的纳米肽F3FT,菌膜出现褶皱、凹陷、破裂,而未经处理的细菌膜表明光滑、无破损,说明纳米肽F3FT的抗菌机理是通过破坏细菌膜杀死细菌。
综上所述,纳米肽F3FT能够自组装形成纳米结构,对常见的金黄色葡萄球菌、单增李斯特菌、粪肠球菌、表皮葡萄球菌等常见的胞内细菌具有很强的抗菌活性,对RAW 264.7细胞细胞几乎没有毒性,能够高效穿透RAW 264.7细胞并对RAW 264.7细胞内的金黄色葡萄球菌具有优异的杀菌效果,而且通过破坏细菌膜杀死细菌,具有极高的应用潜力。
Claims (6)
1.一种抗胞内革兰氏阳性菌感染的纳米肽F3FT,其特征在于,其氨基酸序列如SEQ IDNo.1所示,其N末端接枝9-芴基甲氧基羰基基团。
2.根据权利要求1所述的一种抗胞内革兰氏阳性菌感染的纳米肽F3FT,其特征在于,其分子式如式(I)所示,
3.根据权利要求1或2所述的一种抗胞内革兰氏阳性菌感染的纳米肽F3FT,其特征在于:将所述的纳米肽F3FT溶解在PBS缓冲液后静置24h能够自组装成纳米结构。
4.根据权利要求1或2所述的一种抗胞内革兰氏阳性菌感染的纳米肽F3FT的制备方法,其特征在于,制备方法如下:选择人免疫缺陷病毒的转录反式激活因子的第47-57位序列片段TAT11,在其N端侧放置3个连续的苯丙氨酸,以提供抗菌活性所需的疏水性,最后在N末端接枝9-芴基甲氧基羰基基团,以驱动自组装形成纳米结构,进而得到多肽,其氨基酸序列如SEQ ID No.1所示,采用固相化学合成法合成所述的多肽,再将所述的多肽经高效液相色谱进行纯化,再经过抑菌活性测定、细胞毒性的测定、细胞穿透能力测定和胞内杀菌活性的测定,最后命名为纳米肽F3FT。
5.根据权利要求1或2所述的一种抗胞内革兰氏阳性菌感染的纳米肽F3FT在制备治疗革兰氏阳性菌感染性疾病的药物中的应用。
6.根据权利要求5所述的应用,所述的革兰氏阳性菌为单增李斯特菌、表皮葡萄球菌或金黄色葡萄球菌。
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