CN117285599A - 一种抗胞内细菌的细胞穿透型抗菌肽5vt及其制备方法和应用 - Google Patents
一种抗胞内细菌的细胞穿透型抗菌肽5vt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细菌的细胞穿透型抗菌肽5VT及其制备方法和应用,属于生物技术领域。抗菌肽5VT的氨基酸序列如SEQ ID No.1所示。制备方法:通过在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的末端标记5个连续的缬氨酸,设计得到抗胞内细菌的细胞穿透型抗菌肽5VT,然后通过固相合成法合成。本发明的抗菌肽对具有胞内生存能力的细菌具有强效的抗菌活性,包括鼠伤寒沙门氏菌、单增李斯特菌和金黄色葡萄球菌,而且细胞毒性低。抗菌肽5VT能够高效穿透巨噬细胞并能够降低巨噬细胞内细菌的存活率,通过破坏细菌膜发挥抗菌作用,在抗胞内细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细菌的细胞穿透型抗菌肽5VT及其制备方法和应用。
背景技术
许多细菌病原体已进化出在哺乳动物细胞内生存的能力,这类细菌被称为胞内细菌,例如,沙门氏菌、单增李斯特菌、结核分歧杆菌、金黄色葡萄球菌等。胞内细菌不仅能够逃避机体宿主免疫系统的攻击,而且还能在宿主细胞内生存和复制,造成严重的持续慢性感染。而大部分的传统抗生素对哺乳动物细胞的渗透性不佳以及细胞内有限的累积浓度,此外不断发展的细菌耐药性也使得抗生素可能在不久的将来彻底失去活性。
抗菌肽作为宿主免疫系统的重要组成部分,具有抗细菌、抗真菌、免疫调节等多种生物学活性,而且以膜破坏为主的多重抗菌机理不易使细菌产生耐药性,所以具有替代抗生素的巨大潜力。但是,大多数抗菌肽对哺乳动物细胞的穿透性较差,使其无法成为对抗胞内细菌的高效抗菌剂。因此,研发具有高效穿透能力和强效抗菌活性的抗菌肽是对抗细胞内细菌的有效途径。
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细菌的细胞穿透型抗菌肽5VT,解决了抗菌肽对细胞内细菌抑菌活性不佳的问题。
本发明所采用的技术方案如下:一种抗胞内细菌的细胞穿透型抗菌肽5VT,其氨基酸序列如SEQ ID No.1所示。
进一步的,抗菌肽5VT的分子式如式(I)所示,
本发明的另一目的是提供如上所述的一种抗胞内细菌的细胞穿透型抗菌肽5VT的制备方法,如下:通过选择源自人免疫缺陷病毒的转录反式激活因子(Transactivator oftranscription,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。随后通过疏水性氨基酸末端标记技术在序列片段TAT11的N-末端放置5个连续的缬氨酸(Valine,V),以提供抗菌活性所需的疏水性,由此设计得到多肽,其氨基酸序列如SEQ ID No.1所示;然后采用固相化学合成法得到多肽,将所述的多肽经过质谱鉴定及反相高效液相色谱纯化,再经过抑菌活性测定、细胞毒性的测定和细胞穿透能力测定,最终命名为细胞穿透型抗菌肽5VT。
本发明的另一目的是提供如上所述的一种抗胞内细菌的细胞穿透型抗菌肽5VT在制备治疗革兰氏阳性菌和/或革兰氏阴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,所述的革兰氏阴性菌为沙门氏菌、大肠杆菌或绿脓杆菌。
进一步的,如上所述的应用,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。
本发明具有如下优点及有益效果:本发明的细胞穿透型抗菌肽5VT对常见的沙门氏菌、单增李斯特菌、金黄色葡萄球菌等常见的胞内细菌具有很强的抗菌活性;对巨噬细胞几乎没有毒性,而且能够高效穿透巨噬细胞并对巨噬细胞内的沙门氏菌和金黄色葡萄球菌具有优异的杀菌效果;此外,主要通过破坏细菌膜发挥抗菌作用,具有抗细胞内细菌的应用潜力。
附图说明
图1为抗菌肽5VT的质谱图。
图2为抗菌肽5VT的高效液相色谱图。
图3为抗菌肽5VT的细胞毒性。
图4为抗菌肽5VT的细胞穿透能力。
图5为抗菌肽5VT的胞内抗菌活性。
图6为扫描电子显微镜下抗菌肽5VT对S.typhimurium 14028的抗菌机理图。
图7为扫描电子显微镜下抗菌肽5VT对S.aureus 29213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
抗菌肽的设计
通过选择源自人免疫缺陷病毒的转录反式激活因子(transactivator oftranscription,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。随后通过疏水性氨基酸末端标记技术在TAT11的N-末端放置5个连续的缬氨酸(Valine,V),以提供抗菌活性所需的疏水性。由此设计得到的细胞穿透型抗菌肽命名为5VT。
抗菌肽5VT的氨基酸序列如下:
表1抗菌肽的氨基酸序列
抗菌肽5VT的分子式如式(I)所示,
实施例2
固相化学合成法合成抗菌肽
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.最后一个氨基酸连接后,脱去保护基。
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),TFA94.5%;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,得到纯度>95%的多肽(如图2所示)。
实施例3
抗菌肽的抑菌活性测定
采用标准微肉汤稀释法测定肽的最小抑菌浓度(MIC)。将对数期的细菌稀释至~2×105CFU/mL。将50μl不同浓度的肽(肽的终浓度为1-128μM)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,每个重复两个平行。结果见表2。
表2抗菌肽5VT的最小抑菌浓度(μM)
通过表2可以看出,抗菌肽5VT对于鼠伤寒沙门氏菌、大肠杆菌、绿脓杆菌、金黄色葡萄球菌、单增李斯特菌、表皮葡萄球菌等多种细菌均表现出很强的抑菌活性,最小抑菌浓度在1-8μM。
实施例4
抗菌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1-10列孔中的多肽浓度分别稀释至128-0.5μM,体积为50μL。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孔板各孔中的肽转移至含有细胞的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3为抗菌肽5VT对小鼠巨噬细胞RAW 264.7的细胞毒性,在4-64μM浓度范围内抗菌肽5VT对小鼠巨噬细胞的存活率没有影响,仍然保持在90%以上,表明抗菌肽5VT具有良好的生物相容性。
实施例5
抗菌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标记的抗菌肽5VT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4为抗菌肽的细胞穿透能力,在16μM的浓度下抗菌肽5VT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6
抗菌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S.typhimurium 14028或者S.aureus 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,再加入不同浓度的抗菌肽(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5为抗菌肽5VT的胞内抗菌活性,32μM的5VT对小鼠巨噬细胞内的鼠伤寒沙门氏菌和金黄色葡萄球菌的杀伤率在90%以上,表明其具有优异的抗胞内细菌活性。
实施例7
抗菌肽的抗菌机理
将S.typhimurium 14028或者S.aureus 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S.typhimurium 14028或者S.aureus 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6和图7分别为抗菌肽5VT对S.typhimurium 14028、S.aureus 29213的扫描电子显微镜图,与对照相比,经过抗菌肽5VT处理后的S.typhimurium 14028和S.aureus 29213的菌膜都出现褶皱、破裂,而未经处理的细菌膜表明光滑、无破损,说明抗菌肽5VT的抗菌机理是通过破坏细菌膜杀死细菌。
综上所述,抗菌肽5VT对常见的鼠伤寒沙门氏菌、单增李斯特菌、金黄色葡萄球菌等常见的胞内细菌具有很强的抗菌活性,能够高效穿透巨噬细胞并对胞的沙门氏菌和金黄色葡萄球菌具有优异的杀菌效果,对巨噬细胞几乎没有毒性,而且通过破坏细菌膜杀死细菌,具有极高的应用潜力。
Claims (6)
1.一种抗胞内细菌的细胞穿透型抗菌肽5VT,其特征在于,其氨基酸序列如SEQ IDNo.1所示。
2.根据权利要求1所述的一种抗胞内细菌的细胞穿透型抗菌肽5VT,其特征在于,其分子式如式(I)所示,
3.根据权利要求1或2所述的一种抗胞内细菌的细胞穿透型抗菌肽5VT的制备方法,其特征在于,方法如下:通过选择源自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,在序列片段TAT11的N-末端放置5个连续的缬氨酸,以提供抗菌活性所需的疏水性,得到多肽,其氨基酸序列如SEQ ID No.1所示;然后采用固相化学合成法得到多肽,将所述的多肽经过质谱鉴定及反相高效液相色谱纯化,再经过抑菌活性测定、细胞毒性的测定和细胞穿透能力测定,最终命名为细胞穿透型抗菌肽5VT。
4.根据权利要求1或2所述的一种抗胞内细菌的细胞穿透型抗菌肽5VT在制备治疗革兰氏阳性菌和/或革兰氏阴性菌感染性疾病的药物中的应用。
5.根据权利要求4所述的应用,所述的革兰氏阴性菌为沙门氏菌、大肠杆菌或绿脓杆菌。
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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