CN110551191B - 一种低溶血活性的蜂毒肽及其应用 - Google Patents
一种低溶血活性的蜂毒肽及其应用 Download PDFInfo
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- CN110551191B CN110551191B CN201910854177.0A CN201910854177A CN110551191B CN 110551191 B CN110551191 B CN 110551191B CN 201910854177 A CN201910854177 A CN 201910854177A CN 110551191 B CN110551191 B CN 110551191B
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Abstract
本发明涉及一种低溶血活性的蜂毒肽及其应用。蜂毒肽作为一种优良的生物抗菌剂，是抗生素的最佳替代品之一，但蜂毒肽具有较强的红细胞溶血性，不适合直接注射入血，局部使用也存在诸多约束，如何在保留蜂毒肽抗菌能力的同时降低其溶血能力是蜂毒肽临床应用中亟待解决的问题。发明人根据野生型蜂毒肽序列，通过序列改进获得另一种蜂毒肽，在保留野生型蜂毒肽抗菌功能的基础上，同时获得低溶血能。本发明提供的蜂毒肽及其变体具有很好的临床应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种低溶血活性的蜂毒肽及其应用。
背景技术
随着抗生素的滥用，耐药菌问题已越发成为严重威胁到国民健康的重大问题。如耐甲氧西林金黄葡萄球菌(Methicillin-resistant Staphylococcus aureus，MRSA)，目前已具备了广谱耐药性，对所有与甲氧西林结构相近的β-内酰胺类和头孢类抗生素，以及氨基糖苷类、大环内酯类、四环素类、氟喹诺酮类、磺胺类、利福平均产生不同程度的耐药。MRSA同时也是医院院内感染的一种常见病原菌，其感染多见于外伤与手术，2016年，国内主要地区医院MRSA的平均检出率为38.4％，部分医院的检出率高达53.5％-75.3％，严重影响到了病人的康复过程，这对开发新型抗菌药物及材料提出了迫切要求。
蜂毒肽(Melittin)是蜂毒的主要成分，也是蜂毒中具有药理作用和生物学活性的主要组分。特定的氨基酸序列使蜂毒肽能够靶向细菌细胞壁，并对细菌细胞膜产生穿膜作用，故蜂毒肽具备广谱的抗菌性，对革兰氏阳性菌与阴性菌有较好的杀伤作用，是一种很好的抗生素替代产品。但蜂毒肽具有较强的红细胞溶血性，不适合直接注射入血，局部使用也存在诸多约束，这极大的限制了蜂毒肽的临床使用。如何在保留蜂毒肽抗菌能力的同时降低其溶血能力是蜂毒肽临床应用中亟待解决的问题。
本发明的目的在于，根据野生型蜂毒肽序列，通过反复的实验进行序列改进，获得另一种蜂毒肽，在保留野生型蜂毒肽抗菌功能的基础上，同时获得低溶血能力。
此外，在肿瘤细胞毒性试验中发现，本发明提供的蜂毒肽对肿瘤细胞也具有很好的杀伤作用。本发明提供的蜂毒肽具有很好的临床应用前景。
发明内容
本发明的目的在于提供一种蜂毒肽，蜂毒肽的序列为与SEQ NO ID.1有75％以上同源性的序列，所述SEQ NO ID.1序列为：GIGAVLKVLTTGLPALISWIKRKRQQ。
本发明的目的在于提供一种蜂毒肽，其序列为与SEQ NO ID.1有75％以上同源性的序列，其特征在于，在SEQ NO ID.1中的氨基酸上取代、缺失、添加一个或几个氨基酸形成与上述蜂毒肽具有相同功能的蜂毒肽的变体。
进一步，蜂毒肽的变体序列为与GIGAVLKVLTTGLPALISWIKKKKQQ(SEQ NO ID.2)有75％以上同源性的序列。
目前，蜂毒肽的有各种各样的修饰形式，如连接上脂肪酸、氨基酸氟化、酰化以及用D型氨基酸代替L型氨基酸等，通过不同的修饰，使得多肽的活性提高、毒性降低、制备工艺简化等。
进一步，上述蜂毒肽的N端被乙酰化修饰和/或C端被酰胺化修饰。
一种核苷酸片段，核苷酸片段编码上述蜂毒肽。
一种重组载体，所述载体含有上述核苷酸片段。
一种重组载体，所述载体表达上述蜂毒肽。
进一步，载体为原核表达载体或真核表达载体。
更进一步，载体为大肠杆菌表达载体、小球藻表达载体、杆状病毒介导的昆虫表达载体、酵母表达载体。
一种重组细胞，所述细胞含有上述重组载体。
含有上述的核苷酸片段的表达载体、重组微生物或转基因细胞系。
一种组合物，所述组合物含有上述蜂毒肽或其药学上可接受的盐及药学上可接受的载体。
所述药学上可接受的载体包括离子交换材料、氧化铝、硬脂酸铝、卵磷脂、自乳化药物传递系统(SEDDS)，如d-维生素E聚乙二醇1000琥珀酸酯、吐温或其他类似聚合介质等药物制剂用的表面活性剂、血清蛋白如人血清白蛋白、缓冲物质如磷酸盐、氨基乙酸、山梨酸、山梨酸钾、饱和植物脂肪酸部分甘油酯混合、水、盐、电解质如硫酸盐精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、硅胶、硅酸镁等。聚乙烯吡咯酮、纤维素物质、聚乙烯醇、羧甲基纤维素钠、聚丙烯酸酯、乙烯-聚氧乙烯-嵌段聚合物和羊毛脂、环糊精如α-、β-、γ-环糊精或其经化学修饰的衍生物如2-和3-羟丙基-β-环糊精等羟烷基环糊精及其可溶性衍生物。
上述蜂毒肽、蜂毒肽的变体、核苷酸片段、组合物、重组载体或重组细胞在制备抑菌和/或杀菌性能的产品或药物中的应用。
进一步，产品可以是医用耗材。
进一步，药物为预防或治疗感染的药物，优选的，所述的干扰有细菌、真菌、病毒、寄生虫等引起。
进一步，所述的菌为革兰氏阳性菌、革兰氏阴性菌、真菌、病毒或寄生虫。
进一步，所述的菌为金黄色葡萄球菌、大肠杆菌、副溶血弧菌、肠炎弧菌、铜绿假单胞菌、耐甲氧西林金黄色葡萄球菌、鲍曼不动菌或鲍曼不动临床分离全耐药菌。
金黄色葡萄球菌是人类的一种重要病原菌，隶属于葡萄球菌属，是革兰氏阳性菌的代表，可引起许多严重感染。大肠杆菌为革兰氏阴性短杆菌，一般多不致病，为人和动物肠道中的常居菌，在一定条件下可引起肠道外感染，某些血清型菌株的致病性强，引起腹泻，引起严重腹泻和败血症。副溶血弧菌，又称为肠炎弧菌，属于弧菌属，是一种常见的病原菌。肠炎弧菌是一种嗜盐性的革兰氏阴性菌，主要的栖息地在海水中，是引起食物中毒的主要病原菌之一，在沿海地区，每年都有相当多的病患因食用被肠炎弧菌污染的海产品而发生食物中毒。耐甲氧西林金黄葡萄球菌是医院院内感染的一种常见病原菌，其感染多见于外伤或手术后。鲍曼不动杆菌，又称鲍氏不动杆菌，属于革兰氏阴性菌，该菌是不动杆菌属细菌中在医院感染中常见的一种，也是水产养殖业动物的病原菌，它通常会引起菌血症，肺炎，脑膜炎，腹膜炎，心内膜炎，以及泌尿道和皮肤感染。鲍曼不动杆菌已经成为医院感染的主要来源，尤其是重症监护室，该病菌因为抗生素的滥用，导致鲍曼不动杆菌产生抗药性，变成“多重抗药性鲍曼不动杆菌”(鲍曼不动临床分离全耐药菌)。
上述蜂毒肽、蜂毒肽的变体、核苷酸片段、组合物、重组载体或重组细胞在制备抗肿瘤药物中的应用。
所述药物进一步包含药用辅料，包括填充剂(如无水乳糖、淀粉、乳糖珠粒和葡萄糖)、粘合剂(如微晶纤维素)、崩解剂(如交联羧甲基淀粉钠、交联羧甲基纤维素钠、低取代羟丙基纤维素和交联PVP)、润滑剂(如硬脂酸镁)、吸收促进剂、香味剂、甜味剂、稀释剂、赋形剂、润湿剂、溶剂、增溶剂和着色剂等。
所述药物的给药制剂可包括注射剂、霜剂、软膏剂、贴剂、喷雾剂等。
所述药物给药途径包括皮下、皮内、动脉内、静脉内、肌内、关节内、滑液内、胸骨内、鞘内、病灶内、颅内注射或输注，口服、局部、直肠、经鼻、经颊、阴道、舌下、皮内、粘膜、气管、尿道给药，通过吸入气雾、植入蓄积及针刺方式给药。
附图说明
图1是不同浓度下野生型蜂毒肽和蜂毒肽-K对SA29213的抗菌能力图；
图2是不同浓度下野生型蜂毒肽和蜂毒肽-K对USA300的抗菌能力图；
图3是不同浓度下野生型蜂毒肽和蜂毒肽-K对AbBAA747的抗菌能力图；
图4是不同浓度下野生型蜂毒肽和蜂毒肽-K对Ab1814516的抗菌能力图；
图5是不同浓度下野生型蜂毒肽和蜂毒肽-K对红细胞的溶血能力图；
图6是浓度为4ug/ml是野生型蜂毒肽和蜂毒肽-K对红细胞的溶血能力对比图；图7是不同浓度下野生型蜂毒肽和蜂毒肽-K对肿瘤细胞杀伤图。
具体实施方式
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测。
实施例1多肽设计及抗菌测试
1.多肽的设计
基于蜂毒肽的多肽序列，设计了蜂毒肽-K序列(K-Meli)：
蜂毒肽(Melittin)序列：GIGAVLKVLTTGLPALISWIKRKRQQ-NH2
蜂毒肽-K(K-Meli)序列：GIGAVLKVLTTGLPALISWIKKKKQQ-NH2
2.抗菌测试
对蜂毒肽-K进行最小抑菌浓度(MIC)测试，使用肉汤稀释法。
2.1 MIC板制备
将浓度为256mg/ml的样品溶液分别点入96孔聚苯乙烯板第一排孔中，每孔100ul，每种样品重复3次。后排每孔加入50ulLB液体培养基，使用排枪从第一排吸取50ul样品溶液，与第二排充分吹打混合，再吸取50ul加入与第三板吹打混合，如此依次进行两倍稀释，至最后一排将混合后的50ul溶液弃去，得到256、128、64、32、16、8、4、2ug/ml共8个浓度梯度的样品。另设1列只加100ul LB培养基的空白组，以及一列菌浓度为5×105CFU/mL而不含样品的对照组。
2.2接种物配制
实验使用了革兰氏阳性的金黄色葡萄球菌SA29213株，耐甲氧西林金黄色葡萄球菌USA300株，以及革兰氏阴性的鲍曼不动杆菌Ab BAA747标准株，鲍曼不动杆菌临床分离全耐药株Ab 1814516(北京市安贞医院检验科赠与)。细菌过夜培养，配制得0.5麦氏浊度标准的菌悬液，1:100稀释后，向样品实验组每孔加入50ul，此时1至8孔样品浓度分别为128、64、32、16、8、4、2、1ug/ml。
2.3观察统计实验数据
温箱培养24h，用酶标仪OD600测取96孔板中各孔洞吸光度值，绘制成图。
细菌生长率＝(OD实验组-OD空白组)/(OD对照组-OD空白组)
3.实验结果
由图1-4所示数据可知，两种蜂毒肽对革兰氏阳性菌及革兰氏阴性菌均均有较好的抑菌杀菌作用，当蜂毒肽浓度为32ug/ml时，对革兰氏阳性菌的杀伤作用接近100％，当蜂毒肽浓度为8ug/ml时，对革兰氏阴性菌的杀伤作用接近100％。在抗菌能力方面，两种抗菌肽变化不大，蜂毒肽-K对鲍曼不动耐药株的抑菌能力在低浓度是抑菌能力略好于野生型蜂毒肽。
实施例2红细胞溶血实验
1.实验试剂：
TBS缓冲液：605mg Tris(终浓度10mM TRIS)，4.4gNacl(150mM NaCl)，500mlddH2O，PH＝7.2；
红细胞裂解液：以TBS缓冲液稀释的0.2％Triton X-100。
2.实验步骤：
1)血样取自正常人血，离心力1000g，5min，离心，移除上清，加入等量TBS缓冲液，轻轻混匀，再次以1000g，5min离心，重复3-5次至上清澄清。
2)收集所得沉淀，1:50稀释在TBS缓冲液中，轻轻来回颠倒混匀，获得红细胞悬液。
3)96孔板加样：使用排枪加样稀释法使实验组的各样品溶液形成128、64、32、16、8、4、2、1ug/ml共8个浓度梯度，每孔100ul，再加入100ul红细胞悬液；空白组不加多肽样品，100ulTBS缓冲液+100ul红细胞悬液；对照组加入100u l红细胞裂解液以及100ul红细胞悬液。轻摇混匀，37℃培养1h。
4)将96孔板配好平离心，转速3700rpm，5min。
5)新取一个96孔板，吸取70ul离心后的96孔板上清依次加入新板，酶标仪测OD600含量，计算各样本溶血能力并绘制图表。
细胞存活率＝(OD实验组-OD空白组)/(OD对照组-OD空白组)
3.实验结果
由图5和图6数据可知，蜂毒肽-k的溶血能力比野生型蜂毒肽弱，在4ug/ml浓度时溶血率比野生型低了一半。总体来说，蜂毒肽-K具备成为广谱抗菌药物的潜力。
实施例3蜂毒肽-K对肿瘤细胞的毒性实验
1.实验试剂：
MTS细胞毒性染色试剂，购于promega公司。DMEM高糖细胞培养基，10％胎牛血清+90％DMEM配置，购于中国医学科学院细胞资源中心。
肿瘤细胞：SW620人结肠癌细胞。
2.实验步骤：
1)复苏SW620细胞，传代2代以上，待细胞生长稳定后，取一皿生长良好的细胞消化后平板计数，根据统计值调整细胞悬液浓度并加入96孔板铺板，一列只加培养基不加细胞作空白组，其他每孔细胞数在20000左右，37℃，5％二氧化碳培养箱过夜处理，12小时后细胞完成贴壁。
2)待细胞贴壁后，小心吸出96孔板中旧培养基，按照实验需求加入含有特定多肽浓度的新培养基，混合液加样前在EP管中配好。对照组不含多肽，实验组浓度梯度为128、64、32、16、8、4、2、1ug/ml，每组两个重复。培养箱中孵育24h。
3)选择490nm的波长，在酶联免疫检测仪上测定各孔吸光值，记录结果，绘制图表。
细胞活性＝(OD实验组-OD空白组)/(OD对照组-OD空白组)
3.实验结果
由图7可知，两种蜂毒肽对肿瘤细胞的杀伤作用均比较明显，野生型蜂毒肽在浓度为16ug/ml时，肿瘤细胞SW620的死亡率不到60％，蜂毒肽-K在浓度为16ug/ml时，肿瘤细胞SW620的死亡率超过80％，在32ug/ml浓度时肿瘤细胞SW620的死亡率是100％。
序列表
<110> 中国医学科学院基础医学研究所
<120> 一种低溶血活性的改良型蜂毒肽及其应用
<160> 2
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
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Claims (13)
1.一种蜂毒肽，其特征在于，所述蜂毒肽的氨基酸序列如SEQ NO ID.2所示。
2.根据权利要求1所述的蜂毒肽，其特征在于，蜂毒肽的N端被乙酰化修饰和/或C端被酰胺化修饰。
3.一种核苷酸片段，其特征在于，核苷酸片段编码权利要求1所述的蜂毒肽。
4.含有权利要求3所述的核苷酸片段的表达载体、重组微生物或转基因细胞系。
5.含有权利要求3所述的核苷酸片段的表达载体，其特征在于，载体为大肠杆菌表达载体、小球藻表达载体、杆状病毒介导的昆虫表达载体或酵母表达载体。
6.一种组合物，其特征在于，组合物含有权利要求1-2任意一项所述的蜂毒肽或其药学上可接受的盐及药学上可接受的载体。
7.权利要求1-2任意一项所述的蜂毒肽、权利要求3所述的核苷酸片段、权利要求4所述的表达载体、重组微生物或转基因细胞、权利要求5所述的表达载体、权利要求6所述的组合物在制备抑菌和/或杀菌性能的产品中的应用。
8.权利要求1-2任意一项所述的蜂毒肽、权利要求3所述的核苷酸片段、权利要求4所述的表达载体、重组微生物或转基因细胞、权利要求5所述的表达载体、权利要求6所述的组合物在制备抑菌和/或杀菌性能的药物中的应用。
9.根据权利要求7或8任意一项所述的应用，其特征在于，所述菌为革兰氏阳性菌或革兰氏阴性菌。
10.根据权利要求7或8任意一项所述的应用，其特征在于，所述菌为金黄色葡萄球菌、大肠杆菌、副溶血弧菌、肠炎弧菌、铜绿假单胞菌或鲍曼不动杆菌。
11.根据权利要求7或8任意一项所述的应用，其特征在于，所述菌为耐甲氧西林金黄色葡萄球菌或鲍曼不动杆菌临床分离全耐药菌。
12.权利要求1-2任意一项所述的蜂毒肽、权利要求3所述的核苷酸片段、权利要求4所述的表达载体、重组微生物或转基因细胞、权利要求5所述的表达载体、权利要求6所述的组合物在制备抗肿瘤药物中的应用，其特征在于，肿瘤为结肠癌。
13.根据权利要求8-12任意一项所述的应用，其特征在于，所述药物进一步包含药用辅料，所述药物辅料包括填充剂、粘合剂、崩解剂、润滑剂、吸收促进剂、香味剂、甜味剂、润湿剂、增溶剂和着色剂。
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