CN116375877B - 一种细胞穿透抗菌肽pw2及其制备方法和应用 - Google Patents
一种细胞穿透抗菌肽pw2及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种细胞穿透抗菌肽PW2及其制备方法和应用，属于农业畜牧兽医应用领域，细胞穿透抗菌肽PW2的序列如SEQ ID No.1所示；合成方法示意如下：通过成对Trp‑Trp基序与Pro排列来促进细胞穿透抗菌肽的抗菌活性，其序列为PWWPPWWP；衍生自HIV的细胞穿透肽TAT的氨基酸序列为RKKRRQRRR；两者通过三甘氨酸连接体GGG相连，并命名为PW2。本发明的细胞穿透抗菌肽PW2对革兰氏阴性菌和革兰氏阳性菌具有较高的抑菌活性，能够杀灭小鼠巨噬细胞RAW264.7内的金黄色葡萄球菌，使其具备成为饲用抗生素替代物的潜力。
Description
技术领域
本发明属于农业畜牧兽医应用领域，具体涉及一种细胞穿透抗菌肽PW2及其制备方法和应用。
背景技术
细胞内感染是慢性疾病的代表性杀手，病原体潜入细胞内可逃避机体免疫防御和清除的一系列作用，通常对宿主细胞毒性较低，以保证其长期隐匿于细胞内，因此在机体内不断生长和繁殖，并可在畜禽动物之间水平传播，在适当时机或当畜禽免疫力减弱时便发作，严重时可危及生命。细胞内感染病原体对畜牧生产造成了严重的危害，例如，无乳链球菌侵袭奶牛乳腺细胞后会造成奶牛泌乳量下降、乳品质降低等危害。猪伤寒沙门氏菌侵袭断奶仔猪肠道上皮细胞也会导致仔猪免疫力下降、降低采食量和生长性能。因此，解决细胞内感染在畜牧兽医领域具有极为广阔的市场应用前景。
虽然青霉素在目前是一种极为有效的抗感染药物，但是青霉素无法转导进入细胞内部来清除胞内细菌。此外，随着农业农村部194号公告的颁布，饲料中已全面禁用饲用抗生素。因此，迫切寻找一种能够清除胞内细菌的生物分子。抗菌肽是生物体内广泛存在的具有抗菌活性的小分子多肽，对真菌、病毒、寄生虫和癌细胞也具有杀伤作用。现需要一种能够清除细胞内细菌的高效细胞穿透抗菌肽，为饲用型抗菌肽的研发提供理论基础和技术支持。
发明内容
基于以上问题，本发明的目的在于公开一种细胞穿透抗菌肽PW2，该抗菌肽PW2是一种衍生自HIV的细胞穿透肽TAT和抗菌肽杂合形成的细胞穿透抗菌肽，其目的是能够穿透哺乳动物细胞并清除胞内病原体，克服青霉素无法解决的胞内感染的难题，使其拥有成为抗生素替代物的潜力。
本发明的目的是这样实现的：一种细胞穿透抗菌肽PW2，其氨基酸序列如SEQIDNo.1所示，其C端酰胺化。
本发明的另一目的是提供如上所述的一种细胞穿透抗菌肽PW2的制备方法，如下：通过成对Trp-Trp基序与Pro排列来促进细胞穿透抗菌肽的抗菌活性，其序列为PWWPPWWP；衍生自HIV的细胞穿透肽TAT的氨基酸序列为RKKRRQRRR；两者通过三甘氨酸连接体GGG相连；采用固相化学合成法得到肽树脂，将得到的肽树脂经过TFA切割后，得到多肽，其序列如SEQ ID No.1所示，经过反相高效液相色谱纯化和质谱鉴定后，即完成该多肽的制备；再经过抗菌活性检测、溶血活性的测定、细胞内杀菌能力的测定，最后命名为细胞穿透抗菌肽PW2。
本发明的另一目的是提供如上所述的一种细胞穿透抗菌肽PW2在制备治疗革兰氏阴性菌和/或革兰氏阳性菌感染性的疾病的药物中的应用。
进一步的，如上所述的应用，所述的抗菌肽PW2能够杀灭细胞内的金黄色葡萄球菌。
进一步的，如上所述的应用，所述的抗菌肽PW2能够杀灭小鼠巨噬细胞RAW264.7内的金黄色葡萄球菌。
本发明的有益效果及优点如下：本发明杂合了细胞穿透肽TAT和抗菌肽，对形成的细胞穿透抗菌肽PW2进行生物学活性检测，发现其不但对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌等6种菌种有高效的抑制作用，而且具备较低的溶血毒性，该细胞穿透抗菌肽在128μM的浓度下仅造成2.16％的红细胞溶血。另外，该细胞穿透抗菌肽具有杀灭小鼠巨噬细胞RAW264.7内金黄色葡萄球菌的能力，而青霉素无法杀灭胞内细菌，其已具备成为抗生素替代物和饲用型细胞穿透抗菌肽的发展潜力。
附图说明
图1为本发明的细胞穿透抗菌肽PW2的高效液相色谱图。
图2为本发明的细胞穿透抗菌肽PW2的基质辅助激光解吸/电离飞行时间质谱图。
图3为本发明的细胞穿透抗菌肽PW2和蜂毒素ME的溶血活性图。
图4为本发明的细胞穿透抗菌肽PW2和青霉素的细胞内杀菌图。
具体实施方式
下面根据说明书附图举例对本发明做进一步的说明:
实施例1
细胞穿透抗菌肽的设计
以成对Trp-Trp基序与Pro的排列，即PWWPPWWP的多肽，以及衍生自HIV的TAT，即RKKRRQRRR为细胞穿透肽，杂合得到细胞穿透抗菌肽，命名为PW2。细胞穿透抗菌肽的序列如表1所示。
表1细胞穿透抗菌肽的氨基酸序列。
PW2的正电荷数为+9，疏水值为0.181。该细胞穿透抗菌肽不仅可以抑制多种病原微生物的生长，而且对人血红细胞没有毒性。重要的是，该细胞穿透抗菌肽能够杀灭侵染进入小鼠巨噬细胞RAW264.7的金黄色葡萄球菌，具有开发为抗生素替代物的潜力。
实施例2：
固相化学合成法合成细胞穿透抗菌肽
1、细胞穿透抗菌肽的制备从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0.2M的硫酸钠(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，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：
1、抗菌活性的测定：利用微量肉汤稀释法测定细胞穿透抗菌肽PW2和蜂毒素ME的最小抑菌浓度。以2mg/ml的BSA(含0.01％乙酸)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(既不含菌液也不含抗菌肽)。37℃恒温培养14-18h，用酶标仪在492nm(OD492nm)处测定光吸收值，确定最小抑菌浓度。检测结果见表2。
表2细胞穿透抗菌肽PW2的抑菌活性
通过表2可以看出，PW2对于革兰氏阴性菌(E.coli 25922,S.typhimurium 14028和S.pullorum 7913)和革兰氏阳性菌(S.aureus 29213,S.aureus 12228和S.aureus43300)表现出较高的抑菌活性。
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2mL的PBS溶液中，3000rpm离心10min，收集红细胞；用PBS溶液洗涤3遍，再用10mL PBS溶液重悬；取50μL红细胞悬液与50μL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；随后在4℃，3000rpm下离心10min；取出上清液用酶标仪在570nm处测定光吸收值。其中50μL红细胞加50μLPBS溶液作为阴性对照，50μL红细胞加50μL的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，检测结果见图3。通过图3可以看出，PW2在检测范围内未表现出溶血活性，在128μM浓度下造成2.16％的红细胞溶血，未能引起10％的红细胞溶血，与对照组蜂毒素呈显著性差异。通过表3可以看出，PW2的选择指数高于具有强毒性的蜂毒素ME，表明PW2具备开发成为饲用型细胞穿透抗菌肽的潜力。
表3抗菌肽的MHC(μM)、GM(μM)和SI值
3、细胞内杀菌能力的测定：复苏液氮中的小鼠巨噬细胞RAW264.7，以细胞长满瓶底(80％-90％)为宜。使用无菌PBS溶液清洗并重悬细胞3次，并用胰酶消化液对细胞消化处理，使其在瓶底脱落，随后用完全培养基冲洗，获得单个细胞悬液，同时在24孔板中填入1.5mL终浓度约为2×105的细胞悬液。细胞悬液在37℃，5％ CO2的环境下培养24h，使其在孔板底贴壁。收集金黄色葡萄球菌悬浊液并调整至浓度OD600nm为0.4，随后取出500μl细菌悬浊液加入到24孔板的每个孔中。细胞在37℃，5％ CO2的环境下被细菌侵染1h，随后用无菌PBS溶液清洗3次，加入终浓度为100μg/mL的庆大霉素和终浓度为5μg/mL的青霉素，继续在37℃，5％ CO2的环境下孵育1h以杀灭细胞外未侵染的细菌。孵育结束后用无菌PBS溶液清洗3次，加入终浓度为16μM的细胞穿透抗菌肽PW2，以及终浓度为5μg/mL的青霉素作为对照组，继续孵育4h。待孵育结束后，用无菌PBS溶液清洗3次，加入0.025％的Tritonx-100裂解细胞。将裂解液均匀地涂布在MHA固体培养基上，在37℃下培养18-24h后计算细菌存活率。通过图4可以看出，经过细胞穿透抗菌肽PW2处理后，小鼠巨噬细胞RAW246.7内金黄色葡萄球菌的负载量降低到20％以下，而青霉素处理组几乎没有杀灭细菌，表明细胞穿透抗菌肽PW2具备杀灭细胞内病原体的能力。
Claims (4)
1.一种细胞穿透抗菌肽PW2，其特征在于，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所述的一种细胞穿透抗菌肽PW2的制备方法，其特征在于，方法如下：通过成对Trp-Trp基序与Pro排列来促进细胞穿透抗菌肽的抗菌活性，其序列为PWWPPWWP；衍生自HIV的细胞穿透肽TAT的氨基酸序列为RKKRRQRRR；两者通过三甘氨酸连接体GGG相连；采用固相化学合成法得到肽树脂，将得到的肽树脂经过TFA切割后，得到多肽，其序列如SEQ ID No.1所示，经过反相高效液相色谱纯化和质谱鉴定后，即完成该多肽的制备；再经过抗菌活性检测、溶血活性的测定、细胞内杀菌能力的测定，最后命名为细胞穿透抗菌肽PW2。
3.根据权利要求1所述的一种细胞穿透抗菌肽PW2在制备治疗革兰氏阴性菌和/或金黄色葡萄球菌感染性的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：所述的抗菌肽PW2能够杀灭小鼠巨噬细胞RAW264.7内的金黄色葡萄球菌。
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