CN116041476B - 一种衍生自猪肝脏表达抗菌肽malk及其制备方法和应用 - Google Patents
一种衍生自猪肝脏表达抗菌肽malk及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种衍生自猪肝脏表达抗菌肽MALK及其制备方法和应用,属于农业畜牧兽医应用领域，其序列如SEQ ID No.1所示。本发明通过截取猪肝脏表达Hepcidin的N端23个氨基酸，得到含有1个净正电荷，疏水值为0.904的多肽，将多肽中Leu强疏水面进行优化，形成(RL)3交替结构；再将肽链L‑S中的S和S‑L中的L均替换为K以提高净正电荷含量，然后将其C端酰胺化，N端乙酰化；本发明的抗菌肽对革兰氏阳性菌和革兰氏阴性菌都具有高效的杀菌作用，而且具有较低的溶血活性和真核细胞毒性，使其具备成为抗生素替代物的发展潜力。
Description
技术领域
本发明属于农业畜牧兽医应用领域，具体涉及一种衍生自猪肝脏表达抗菌肽MALK及其制备方法和应用。
背景技术
目前，中国的畜牧业正朝着规模化、集约化方向快速发展。在此背景下，抗生素在预防动物疾病、调节免疫、促进生长等方面的作用尤为重要。由此造成抗生素的滥用和抗生素残留，继而引发细菌耐药性、环境污染等诸多问题。因此，绿色、安全、高效、无残留、不易产生耐药性的新型抗菌剂的研发已迫在眉睫。抗菌肽是一类具有广谱抗菌活性、结构多样性的短肽，通常由12-30或更多的氨基酸残基组成。尽管不同抗菌肽段之间在氨基酸含量、长度和结构上有相当大的差异，但它们通常是带正电性和疏水性氨基酸残基的。大多数抗菌肽通过物理性吸附并迅速渗透和破坏细菌膜的基本成分来杀死细菌。猪肝脏表达的抗菌肽Hepcidin，其肽链长度较长，抗菌活性较差，不利于作为抗菌药物使用。
发明内容
基于以上不足之处，本发明的目的在于公开一种衍生自猪肝脏表达抗菌肽MALK，使其具备高效杀菌活性和低毒性的优点，具有开发成为抗生素替代物的潜力。
本发明的目的是这样实现的：一种衍生自猪肝脏表达抗菌肽MALK，其氨基酸序列如SEQ ID No.1所示，其C端酰胺化，N端乙酰化。
本发明的另一目的是提供如上所述的一种衍生自猪肝脏表达抗菌肽MALK的制备方法，如下：通过截取猪肝脏表达Hepcidin的N端23个氨基酸，得到含有1个净正电荷，疏水值为0.904的多肽MALS，其氨基酸序列为：MALSVQIRAACLLLLLLVSLTAG；将6个Leu组成的强疏水面进行优化，形成(RL)3交替结构；再将肽链中双Ala替换成双Leu以进一步回调疏水性，然后分别将靠近肽链N端的Leu-Ser序列中的Ser和靠近C端的Ser-Leu序列中的Leu替换为Lys以提高净正电荷含量，然后将其C端酰胺化，N端乙酰化，以进一步提高净正电荷和稳定性，最后得到含有7个净正电荷，疏水值为0.513的多肽，其序列如SEQ ID No.1所示；然后采用固相化学合成法和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；再经过杀菌活性的测定、溶血活性的测定、真核细胞毒性的测定，最终命名为抗菌肽MAKL。
本发明的另一目的是提供如上所述的一种衍生自猪肝脏表达抗菌肽MAKL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，所述的革兰氏阴性菌为大肠杆菌。
进一步的，如上所述的应用，所述的革兰氏阳性菌为金黄色葡萄球菌。
本发明的有益效果及优点：本发明通过截取的方法简化了衍生自猪肝脏表达抗菌肽Hepcidin，保留其活性中心，提高了抗菌肽在细菌细胞和哺乳动物细胞之间的选择性。对抗菌肽MALK进行杀菌活性和溶血活性检测，发现不但对大肠杆菌、金黄色葡萄球菌等6个菌种具有良好的杀灭作用，而且具有较低的溶血毒性和真核细胞毒性，抗菌肽MALK在128μM的浓度下仅造成6.12％的红细胞溶血，未能引起10％的红细胞溶血，抗菌肽MALK处理的小鼠巨噬细胞RAW264.7的存活率达到80％以上，提高了其成为抗生素替代物的发展潜力。
附图说明
图1为本发明的抗菌肽MALK的高效液相色谱图。
图2为本发明的抗菌肽MALK的基质辅助激光解吸/电离飞行时间质谱图。
图3为本发明抗菌肽MALK的杀菌活性图。
图4为本发明抗菌肽MALK和蜂毒素ME的溶血活性图。
图5为本发明抗菌肽MALK和蜂毒素ME的细胞毒性图。
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
抗菌肽的设计
通过截取猪肝脏表达抗菌肽Hepcidin的N端23个氨基酸，得到含有1个净正电荷，疏水值为0.904的多肽，命名为MALS，其氨基酸序列为：MALSVQIRAACLLLLLLVSLTAG；将6个Leu组成的强疏水面进行优化，形成(RL)3交替结构；得出的氨基酸序列为：MALSVQIRAACRLRLRLVSLTAG；再将肽链中双Ala替换成双Leu以进一步回调疏水性，然后分别将靠近肽链N端的Leu-Ser序列中的Ser和靠近C端的Ser-Leu序列中的Leu替换为Lys以提高净正电荷含量，然后将其C端酰胺化，N端乙酰化以进一步提高净正电荷和稳定性，最后得到含有7个净正电荷，疏水值为0.513的多肽，命名为MALK。抗菌肽的序列如表1所示。
表1抗菌肽MALK的氨基酸序列。
实施例2：
固相化学合成法合成抗菌肽
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0.2M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、杀菌活性的测定：以PBS为稀释液，将12.5μl的浓度为16μM的抗菌肽MALK加入到987.5μl的PBS中，取上述溶液100μl置于1.5ml EP管内，然后添加等体积的待测菌液，孵育2h。随后，取10μl孵育完成的菌液加入990μl的PBS，震荡后取10μl稀释液均匀涂布在固体培养基MHA上。阳性对照组为未经过抗菌肽处理的细菌样品。37℃恒温培养12-18h，计算细菌存活率。通过图3可以看出，抗菌肽MALK对革兰氏阴性菌(E.coli 25922、E.coli UB1005和E.coli K99)和革兰氏阳性菌(S.aureus 29213、S.aureus 12228和S.aureus MRSA43300)均表现出较高的杀菌活性。
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 PBS溶液中，3000rpm离心10min，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；孵育结束后取出，4℃、3000rpm下离心10min；取出上清液用酶标仪在570nm处测光吸收值。其中50μL红细胞加50μL PBS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，检测结果见图4。通过图4可以看出，抗菌肽MALK在检测范围内未表现出溶血活性，该抗菌肽在128μM浓度下造成仅6.12％的红细胞溶血，未能引起10％的红细胞溶血，与对照组蜂毒素ME呈显著性差异。
3、真核细胞毒性的测定：采用MTT法检测小鼠巨噬细胞RAW264.7的细胞毒性。
(1)培养基的准备及细胞的培养：将DMEM(培养基)与胎牛血清按9:1混合配置完全培养基，并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。
(2)待测细胞的处理：无菌PBS清洗并重悬细胞3次，并用0.25％胰蛋白酶溶液对细胞消化处理，使其在瓶底脱落，用完全培养基冲洗，获得单个细胞悬液，同时在96孔板中填入终浓度约为2×104的50μL细胞悬液。
(3)抗菌肽处理：96孔板第一孔内加入10μL抗菌肽MALK并倍比稀释，96孔板1-10孔内加入50μL稀释后的细胞悬液，第11孔加50μL完全培养基，第12孔加100μL完全培养基，恒温培养4h；
(4)毒性检测：取50μL 5mg/mL MTT溶液加入96孔板，继续培养3-4h后，加入150μLDMSO，酶标仪OD570nm处测定吸光度，检测结果见图5。通过图5可以看出，抗菌肽MALK在检测范围内未表现出对小鼠巨噬细胞RAW264.7的毒性，与对照组蜂毒素ME呈现显著性差异，抗菌肽MALK处理的小鼠巨噬细胞RAW264.7的存活率达到80％以上，提高了其成为抗生素替代物的发展潜力。
Claims (3)
1.一种衍生自猪肝脏表达抗菌肽MAKL，其特征在于，其氨基酸序列如SEQ ID No.1所示，其C端酰胺化，N端乙酰化。
2.根据权利要求1所述的一种衍生自猪肝脏表达抗菌肽MAKL的制备方法，其特征在于，方法如下：通过截取猪肝脏表达Hepcidin的N端23个氨基酸，得到含有1个净正电荷，疏水值为0.904的多肽MALS，其氨基酸序列为：MALSVQIRAACLLLLLLVSLTAG；将6个Leu组成的强疏水面进行优化，形成(RL)3交替结构；再将肽链中双Ala替换成双Leu以进一步回调疏水性，分别将靠近肽链N端的Leu-Ser序列中的Ser和靠近C端的Ser-Leu序列中的Leu替换为Lys以提高净正电荷含量，然后将其C端酰胺化，N端乙酰化以进一步提高净正电荷和稳定性，最后得到含有7个净正电荷，疏水值为0.513的多肽，其序列如SEQ ID No.1所示；然后采用固相化学合成法和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；再经过杀菌活性的测定、溶血活性的测定、真核细胞毒性的测定，最终命名为抗菌肽MAKL。
3.根据权利要求1所述的一种衍生自猪肝脏表达抗菌肽MAKL在制备治疗大肠杆菌和/或金黄色葡萄球菌感染性的疾病的药物中的应用。
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