CN112778424B - 嵌合肽r7及其应用 - Google Patents
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Abstract
本发明提供一种嵌合肽R7及其应用，嵌合肽R7是由脂多糖结合蛋白LBP14和抗菌肽L7之间通过Linker连接而成，其氨基酸序列如SEQ ID NO:1所示。本发明的嵌合肽R7对革兰氏阴性菌具有显著的抑制作用，其MIC值比母体肽L7的MIC值低，在小鼠毒血症模型的试验中能够显著提高小鼠的存活率，R7对致死剂量LPS攻毒小鼠的预防治愈率明显高于等当量的L7。本发明为新型靶向LPS的双功能可剪切抗菌药物创制提供理论参考依据。
Description
技术领域
本发明涉及生物医药领域，具体地说，涉及一种嵌合肽R7及其应用。
背景技术
抗菌肽是由生物有机体产生的前体经蛋白酶降解生成的具有生物学活性的小分子多肽，主要构成了机体防御的第一道防线。专抗革兰氏阴性菌抗菌肽对细菌的作用机制主要是以膜作为靶位点，通过静电力和受体介导的细胞膜作用力，引起膜通透性改变，降低细胞膜稳定性，或插入细胞膜中形成跨膜的孔洞，最终导致细菌内容物外泄而死亡。由于抗菌肽特殊的作用机制，使其不易产生耐药性，并且对许多临床耐药菌同样具有杀菌作用。因此，抗菌肽被认为可以替代传统抗生素，应用于饲料添加剂、医药卫生和食品防腐等领域。
发明内容
本发明的目的是提供一种新型的嵌合肽R7及其应用。
本发明构思如下：R7是通过可剪切Linker(RKRR)连接脂多糖结合蛋白LBP14和抗菌肽L7而形成的可剪切嵌合肽，在体外具有较强的杀灭革兰氏阴性菌活性以及中和LPS(脂多糖)的能力。LBP14是LPS结合蛋白(LPB)的核心区域，由14个氨基酸组成(LBP蛋白的86-99位氨基酸)，具有强力的中和LPS的能力。此外，L7是牛乳铁蛋白肽LfcinB的一条衍生肽，具有良好的抗革兰氏阴性菌活性。为了提高L7中和LPS的能力，通过可剪切Linker连接LBP14和L7形成可剪切嵌合肽R7。使其具有组成部分各自的生物学功能，既能够靶向杀伤细菌细胞、又能够中和LPS。通过对R7的抗菌活性、结合LPS能力、体外切割效率以及预防小鼠LPS攻毒的能力进行综合评价，为新型靶向LPS的双功能可剪切抗菌药物创制提供理论参考依据。
为了实现本发明目的，第一方面，本发明提供一种嵌合肽R7，由脂多糖结合蛋白LBP14和抗菌肽L7之间通过Linker连接而成。
其中，脂多糖结合蛋白LBP14、抗菌肽L7的氨基酸序列分别如SEQ ID NO:2-3所示。
优选地，所述Linker的氨基酸序列为RKRR。该Linker可以被存在于真核生物体内的弗林蛋白酶识别，并在第四位精氨酸(R)处切割开。
所述嵌合肽R7的氨基酸序列如SEQ ID NO:1所示。
可采用固相合成法合成嵌合肽R7。R7包含35个氨基酸残基，带14个正电荷，分子量为4477.41Da，等电点为12.85。
第二方面，本发明提供编码所述嵌合肽R7的核酸分子。
第三方面，本发明提供含有上述核酸分子的生物材料，所述生物材料包括但不限于重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
第四方面，本发明提供含有所述嵌合肽R7的广谱抗菌药物或组合物。
第五方面，本发明提供含有所述嵌合肽R7的防腐剂或杀菌剂。
第六方面，本发明提供所述嵌合肽R7的以下任一应用：
1)用于制备抗菌药物或组合物；
2)用于制备防腐剂；
3)用于制备杀菌剂。
所述菌包括革兰氏阳性菌和革兰氏阴性菌。
所述菌包括但不限于葡萄球菌属(Staphylococcus)、埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)细菌。
优选地，所述菌包括金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门氏菌(Salmonella typhimurium)、大肠杆菌(Escherichia coli)等。
本发明的嵌合肽R7对革兰氏阴性菌具有显著的抑制作用，相比与母体肽L7，其抑菌效果提高了2～4倍，且在体外可以被弗林蛋白酶切割成两个部分(L7和LBP14-RKRR)，切割效率呈时间依赖性。在小鼠毒血症模型的试验中，R7能够显著提高小鼠的存活率，R7(7μmol/kg)对致死剂量LPS攻毒小鼠的预防治愈率为100％，明显高于等当量的L7(治愈率40％)。
附图说明
图1为本发明较佳实施例中嵌合肽R7的体外剪切效率。
图2为本发明较佳实施例中嵌合肽R7中和LPS的能力。
图3为本发明较佳实施例中嵌合肽R7在血清中剪切结果。
图4为本发明较佳实施例中嵌合肽R7对毒血症小鼠的预防保护实验。
图5为嵌合肽R7的结构示意图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW，Molecular Cloning:a Laboratory Manual，2001)，或按照制造厂商说明书建议的条件。
实施例1嵌合肽R7的设计与合成
1、以L7母体肽序列(SEQ ID NO:3)为基础，通过Linker(RKRR)将脂多糖结合蛋白LBP14(SEQ ID NO:2)与L7连接起来。嵌合肽R7的结构如图5所示。
2、抗菌肽合成采用固相合成法，通过十二通道半自动多肽合成仪进行合成。反向高效液相色谱C18柱对合成肽进行纯化(纯度>90％)，ESI-MS质谱确认嵌合肽R7的分子量。R7包含35个氨基酸残基(SEQ ID NO:1)，带14个正电荷，分子量为4477.41Da，等电点为12.85。
实施例2嵌合肽R7的抑菌实验
本实施例中的病原菌均来自于中国兽医微生物菌种保藏管理中心(CVCC)或美国模式培养物集存库(ATCC)，具体菌株如表1所示。
抗菌肽的最小抑菌浓度(MIC，minimum inhibitory concentration)的测定参照临床和实验室标准协会(CLSI，Clinical andLaboratory Standards Institute)制定的方法(WIEGAND等，Agar and broth dilution methods to determine the minimalinhibitory concentration(MIC)of antimicrobial substances.Nature protocols,2008,3(2):163-175)，根据实际情况略有改动，具体如下：
将受试菌株的单菌落挑至MH液体培养基中，37℃ 250rpm振荡过夜培养活化后，转接至MH液体培养基中培养至对数生长期(OD600＝0.4～0.6)，然后制备成105CFU/mL的菌液，加入96孔无菌细胞培养板内，每孔90μL。
用PBS通过2倍倍比稀释法对抗菌肽进行稀释，每孔10μL抗菌肽，使其终浓度分别为64、32、16、8、4、2、1和0.5μM，阴性对照组为PBS代替抗菌肽的受试菌液，空白对照组为无菌MH培养基。每个处理三个平行样。
将培养板置于37℃恒温培养箱孵育16～18h，直至阴性对照孔出现肉眼可见的明显浑浊菌液，能够完全抑制细菌生长的最低浓度即为抗菌肽对受试菌株的MIC值。如出现跳孔或平行样间结果不一致情况，则重新测试。
结果如表1所示，嵌合肽R7对大肠杆菌、沙门氏菌的MIC值为16μM；对金黄色葡萄球菌的MIC值为32μM，说明R7对革兰氏阴性菌和阳性菌株均表现出不同程度的抑菌效果的提升。
表1嵌合肽R7的MIC值测定
实施例3嵌合肽R7的体外剪切
将50μg R7与4μL弗林蛋白酶(2,000U/mL，购自NEB，#P8077S)于25℃孵育65h，在不同时间点取200μL样品用高效液相色谱检测R7的切割效率。
结果如图1所示，孵育24h，弗林蛋白酶对R7的切割效率是36.23％，孵育65h的切割效率是61.84％。总体上，R7的切割效率呈现出时间依赖性。
实施例4嵌合肽R7在血清中剪切
对L7和R7的血清稳定性进行了测试，并进行了25-26的较小改动。简而言之，将R7添加到25％的牛血清中至终浓度为0.5mM；在37℃孵育2h后取250μL样品，并与20μl 15％三氯乙酸(TCA)混合。将样品在4℃下孵育15min，然后以12,000rpm离心10min。将上清通过基质辅助激光解吸/电离飞行时间(MALDI-TOF)质谱分析R7在血清中的切割。
结果如图3所示，将R7与牛血清孵育后，可以检测到L7(分子量为2133.132Da)，说明在血清中，R7确实可以释放出L7。
实施例5嵌合肽R7的体外中和LPS能力测定
将来自于大肠杆菌0111:B4(购自Sigma)的LPS(40μg/mL)和BODIPY-TR-尸胺(BC)(10μM)(购自Sigma)等比例混合在50mM Tris缓冲液(pH7.4)中，并添加到黑色96孔板(180μL/孔)。将不同浓度的肽(20μL)添加到各孔中，并在室温25℃(激发波长580nm，发射波长620nm)下通过荧光分光光度法检测。记录相对荧光的变化。
结果如图2所示，R7引发的荧光强度明显高于L7，说明R7中和LPS的能力强于L7。
实施例6嵌合肽R7对LPS诱导的小鼠败血症的预防效果
将15只小鼠(6周龄SPF级ICR小鼠，购自北京维通利华试验动物有限公司)分为3组，每组5只，在0小时腹腔注射7μmol/kg R7、L7或PBS(各200μl)。在6h注射13.5mg/kg的LPS200μl(购自Sigma)进行攻毒。连续7天观察小鼠存活情况，并绘制存活曲线。
小鼠存活率如图4所示。同剂量下嵌合肽R7保护小鼠的幸存率高于L7。R7和L7存活率分别为100％和40％。仅用PBS预防的小鼠在2天内全部死亡。因此，R7比L7具有更高的体内活性。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国农业科学院饲料研究所
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Claims (9)
1.嵌合肽R7，其特征在于，所述嵌合肽R7的氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述嵌合肽R7的核酸分子。
3.含有权利要求2所述核酸分子的生物材料，所述生物材料为重组DNA、表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
4.含有权利要求1所述嵌合肽R7的广谱抗菌药物或组合物。
5.含有权利要求1所述嵌合肽R7的防腐剂或杀菌剂。
6.权利要求1所述嵌合肽R7的以下任一应用：
1)用于制备抗菌药物或组合物；
2)用于制备防腐剂；
3)用于制备杀菌剂。
7.根据权利要求6所述的应用，其特征在于，所述菌包括革兰氏阳性菌和革兰氏阴性菌。
8.根据权利要求7所述的应用，其特征在于，所述菌包括葡萄球菌属(Staphylococcus)、埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)细菌。
9.根据权利要求8所述的应用，其特征在于，所述菌选自金黄色葡萄球菌(Staphylococcus aureus)、鼠伤寒沙门氏菌(Salmonella typhimurium)、大肠杆菌(Escherichia coli)。
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