CN116253782B - 一种广谱抗菌肽ktr及其应用 - Google Patents
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Abstract
本发明公开了一种新型广谱抗菌肽KTR及其应用,抗菌肽KTR,其氨基酸序列为RIKTRTWRLALRWLKL,包括16个氨基酸残基,理论分子量为2110.63Da,等电点为12.48,净电荷为+6,属于碱性抗菌肽。抑菌实验表明,抗菌肽KTR具有良好的广谱抗菌活性,其显著提高了对多种革兰氏阴性菌和革兰氏阳性菌的抑制效果,MIC值为2.4~9.5μM。安全评价实验结果显示KTR具有较低的溶血活性,同时其对人源肝细胞LO2和小鼠成纤维细胞NIH3T3的毒性较低。因此,本发明提供的抗菌肽KTR有望成为一种绿色安全的新型抗生素代替药物。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种基于理性分子设计理念获得的新型广谱抗菌肽KTR及其应用。
背景技术
目前,传统抗生素已在治疗细菌感染性疾病方面取得了显著效果,但由于人们对抗生素的滥用导致细菌耐药性逐渐增强,严重威胁人类的生命健康,因此越来越多的国家开始寻求绿色安全的抗生素替代物。因其抗菌活性高、抗菌范围广、分子量小、结构简单且不易产生耐药性等特性,天然抗菌肽已成为目前最具潜力的抗生素替代物之一。
天然抗菌肽是一类广泛存在于生物体内的小分子活性多肽,同时也是机体防御细菌感染的主要物质之一。抗菌肽有两个共同的物理特征即阳离子性和疏水性,阳离子性可促进抗菌肽与带负电的细菌细胞膜之间的静电相互作用,可有效减弱其与哺乳动物细胞的结合能力,从而降低细胞毒性;疏水性则可促进抗菌肽与细菌细胞膜中脂质的相互作用,从而提高抗菌效果。与抗生素相比,抗菌肽具有广谱的抑菌活性以及独特的膜损伤或胞内杀菌机制。因此,研究抗菌肽对于开发新型抗生素代替药物具有十分重要的意义。
虽然天然抗菌肽具有许多优点,但其也存在某些明显的不足。许多天然抗菌肽的抑菌活性较低、稳定性较差、毒性较高,对哺乳动物体细胞溶血活性较高等,不能满足实际应用中的需求。因此针对上述问题,研究者通过对天然抗菌肽的物理化学参数(即净电荷,疏水性,两亲性等)进行改造,可有效改善天然抗菌肽的缺点,以适应不同应用场景的需要。因此,对现有天然抗菌肽进行修饰,以期获得抗菌活性更高、毒性更低的抗菌肽,已成为目前抗菌肽领域研究的热点之一。
发明内容
本发明针对上述天然抗菌肽存在的缺陷,提供一种基于氨基酸理性分子设计策略改造得到的广谱抗菌肽KTR及其应用。
为达到上述目的,本发明提供了一种广谱抗菌肽KTR,以鹰嘴豆球蛋白(Legumin)源天然抗菌肽Leg2为模板,通过氨基酸理性分子设计策略改造得到的新型广谱抗菌肽KTR,其氨基酸序列(如SEQ ID NO.1所示)为:Arg-Ile-Lys-Thr-Arg-Thr-Trp-Arg-Leu-Ala-Leu-Arg-Trp-Leu-Lys-Leu,用单字母表示为:RIKTRTWRLALRWLKL。
所述抗菌肽KTR以鹰嘴豆源天然抗菌肽Leg2为模板,通过氨基酸理性分子设计理念改造制得。其中所述天然抗菌肽Leg2的氨基酸序列为:Arg-Ile-Lys-Thr-Val-Thr-Ser-Phe-Asp-Leu-Pro-Ala-Leu-Arg-Trp-Leu-Lys-Leu,用单字母表示为:RIKTVTSFDLPALRWLKL,如(如SEQ ID NO.2所示)。
进一步的,上述新型广谱抗菌肽KTR,包括16个氨基酸残基,理论分子量为2110.63Da,等电点为12.48,净电荷为+6,属于碱性抗菌肽。
本发明还提供了一种抗菌肽的抗菌应用,即所述新型广谱抗菌肽KTR在抑制多种革兰氏阴性菌和/或革兰氏阳性菌中的应用。
进一步的,上述新型广谱抗菌肽KTR表现出广谱且高效的抗菌活性,其对革兰氏阴性菌如大肠杆菌(ATCC 35150)、大肠杆菌(ATCC 8739)、鼠伤寒沙门氏菌(ATCC 14028)、肺炎克雷伯菌(ATCC 13883)和铜绿假单胞菌(ATCC 10145);对革兰氏阳性菌如金黄色葡萄球菌(ATCC 25923)和耐甲氧西林金黄色葡萄球菌(ATCC 43300)等多种不同菌株均表现出优异的抗菌活性。
进一步的,所述广谱抗菌肽KTR在抑制革兰氏阴性菌的应用中,其对大肠杆菌(ATCC 35150)、大肠杆菌(ATCC 8739)、鼠伤寒沙门氏菌(ATCC14028)、肺炎克雷伯菌(ATCC13883)和铜绿假单胞菌(ATCC 10145)的最小抑菌浓度分别为4.7μM、4.7μM、4.7μM、2.4μM和9.5μM。
进一步的,所述广谱抗菌肽KTR在抑制革兰氏阳性菌的应用中,其对金黄色葡萄球菌(ATCC 25923)和甲氧西林耐药金黄色葡萄球菌(ATCC43300)的最小抑菌浓度分别为9.5μM和7.1μM。
所述抗菌肽KTR在4.7~150.4μM浓度范围内,其对大鼠红细胞的溶血率<20%。
所述抗菌肽KTR在18.9~303.2μM浓度范围内,LO2和NIH3T3的细胞存活率均>80%。
所述的广谱抗菌肽KTR在制备治疗革兰氏阳性菌和革兰氏阴性菌感染的广谱抗菌药物中的应用。
本发明的技术优势在于:
一、本发明以鹰嘴豆源天然抗菌肽Leg2为模板,通过氨基酸理性分子设计策略,成功构建出一条具有广谱抗菌活性的新型抗菌肽KTR,通过生物学参数预测其理化性质得知KTR主要为α-螺旋结构,理论分子量为2110.63Da,等电点为12.48,净电荷为+6,本发明的提出对抗菌肽的设计和优化具有重要的指导意义。
二、本发明通过测定抗菌肽KTR的抗菌活性发现,与Leg2相比,理性分子设计得到的新型抗菌肽KTR可显著提高对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、肺炎克雷伯菌、铜绿假单胞菌等多种菌株的抑菌或杀菌效果。
三、本发明提供的新型抗菌肽KTR具有较低的溶血活性和细胞毒性。具体来说,在4.7~150.4μM浓度范围内,KTR对大鼠红细胞的溶血率<20%;在19.8~316.0μM浓度范围内,LO2和NIH3T3的细胞存活率均>80%。因此,抗菌肽KTR可作为一种绿色安全的抗菌物质应用于食品、医药和农业生产等相关微生物控制领域中。
附图说明
图1为抗菌肽Leg2与KTR的二级结构预测图;
图2为不同MIC浓度下抗菌肽KTR对大鼠红细胞的溶血率;
图3为抗菌肽KTR对LO2和NIH3T3细胞存活率的影响;
图4为抗菌肽Leg2对LO2和NIH3T3细胞存活率的影响。
具体实施方式
为了更加清楚、完整地描述本发明的目的与方案内容,下面将结合具体的实施例及图表对本发明的技术方案进行详细介绍。本发明所描述的具体实施例仅用以解释本发明,并不代表本发明所限定的权利保护范围,本发明具体的权利保护范围以权利要求书所述内容为准。
实施例1
天然抗菌肽Leg2是来源于鹰嘴豆球蛋白(Legumin)的一种小分子活性多肽,其主要由18个氨基酸组成,理论分子量为2157.63Da,净电荷为+3。Leg2对多种革兰氏阳性菌和革兰氏阴性菌均具有抑菌活性,但相比于现有的抗菌肽,其抑菌活性相对较低且细胞毒性较高。因此,本发明通过理性分子设计策略对抗菌肽Leg2进行氨基酸的替换或删减、增加正电荷氨基酸数量、改变疏水性比例以及提高α-螺旋结构比例等方面的修饰,以期增强其抑菌活性,降低细胞毒性,同时提高生物安全性。具体方案如下:
Leg2中由于丝氨酸(S)与脯氨酸(P)存在较大的侧链基团,即丝氨酸侧链庞大的CH2-OH基团和脯氨酸侧链庞大的N-CH2基团使得其氨基酸残基构象受到限制,可能会影响抗菌肽α-螺旋结构的形成,因此通过删减抗菌肽Leg2序列中的丝氨酸(S)与脯氨酸(P)可提高α-螺旋结构比例;同时,Leg2序列中的酸性氨基酸天冬氨酸(D)会影响抗菌肽的净电荷,因此采用带正电荷的精氨酸(R)取代Leg2序列中9位带负电荷的天冬氨酸(D),以提高抗菌肽的正电荷数量;此外,采用色氨酸(W)取代Leg2序列中8位的苯丙氨酸(F)有望提高抗菌肽与细菌细胞膜的相互作用;而采用丙氨酸(A)取代Leg2序列中5位的缬氨酸(V),则可改变抗菌肽的疏水性进而降低其细胞毒性。根据上述技术方案成功设计出一种新型广谱抗菌肽KTR,通过分析软件ExPASy和Heliquest,对抗菌肽KTR的理化性质如分子量、电荷数、等电点、疏水性比例、疏水力矩(两亲性)和不稳定指数进行分析,并通过I-TASSER软件对抗菌肽KTR可能形成的二级结构进行预测。
天然抗菌肽Leg2以及改造后得到的新型抗菌肽KTR的理化性质预测结果对比如表1所示。与天然抗菌肽Leg2相比,通过理性分子设计获得的抗菌肽KTR,其正电荷与疏水氨基酸比例均有所提高,这有利于促进抗菌肽KTR与细菌细胞膜之间的相互作用,提高抑菌活性,同时抗菌肽KTR的不稳定指数下降,疏水力矩(两亲性)有所提高,这有利于其更加稳定的与细菌细胞膜发生作用,进而提高抗菌活性。此外,二级结构预测结果(图1)表明抗菌肽Leg2的二级结构以无规则卷曲结构为主,并在C端形成少量α-螺旋结构。而在新型抗菌肽KTR中,其α-螺旋结构比例与Leg2相比明显增加,这可能有助于提高抗菌肽KTR的抑菌活性。
表1抗菌肽Leg2与KTR理化性质预测结果对比表
实施例2
本实施例用于测定新型广谱抗菌肽KTR的抑菌活性。
利用微量稀释法测定抗菌肽KTR的最小抑菌浓度(MIC):选择大肠杆菌ATCC35150、大肠杆菌ATCC 8739、鼠伤寒沙门氏菌ATCC 14028、肺炎克雷伯菌ATCC 14028、铜绿假单胞菌ATCC 10145、金黄色葡萄球菌ATCC 25923和耐甲氧西林金黄色葡萄球菌ATCC43300等7种菌株作为测试菌。
将上述菌种分别接种于Muller-Hinton肉汤(MHB)液体培养基中进行活化培养,在37℃、转速180rpm的恒温摇床中振荡培养至对数生长期,然后用新鲜的MHB液体培养基将上述菌液稀释至约1×106CFU/mL,待用。
在无菌96孔板中加入100μL上述预先准备好的菌悬液,其次逐一向96孔板中加入100μL含有不同浓度KTR和Leg2的MHB液体培养基,最后将96孔板放置于37℃恒温培养箱中静置培养24h。用酶标仪检测在波长600nm时各孔的吸光度值(OD600),将最小抑菌浓度确定为吸光度值(OD600)<0.1的最低抗菌肽浓度。
KTR最小杀菌浓度(MBC)的测定:根据上述MIC的测定结果,从吸光度值<0.1的孔内吸取100μL菌液加入PCA琼脂平板内(每孔菌液混匀后吸取),使用涂布棒涂布均匀后,将其倒置于37℃恒温培养箱中培养18~24h,观察PCA平板上的菌落生长情况。未出现菌落生长的最低浓度则为抗菌肽KTR对该菌种的最小杀菌浓度。
MIC结果如表2所示,本发明提供的新型抗菌肽KTR表现出高效且广谱的抑菌活性,其对不同大肠杆菌(ATCC 35150、ATCC 8739)的MIC均为4.7μM,对鼠伤寒沙门氏菌的MIC为4.7μM,对铜绿假单胞菌(ATCC10145)的MIC为9.5μM,为对肺炎克雷伯菌的MIC为2.4μM,对不同金黄色葡萄球菌(ATCC 25923、ATCC 43300)的MIC为7.4~9.9μM,与鹰嘴豆天然抗菌肽Leg2相比较,抗菌肽KTR对多种细菌的MIC值均有所降低,表明KTR显著提高了对多种细菌的抑制效果。
同时,MBC结果显示,Leg2在370.8μM或463.5μM浓度下仍不能杀死细菌,而KTR在较低浓度就可起到杀菌的效果,其对不同大肠杆菌的MBC为7.1~23.7μM,对鼠伤寒沙门氏菌与铜绿假单胞菌的MBC均为23.7μM,对肺炎克雷伯菌的MBC为11.8μM,对不同金黄色葡萄球菌的MBC为11.8~47.4μM。与Leg2相比,抗菌肽KTR具有高效的杀菌活性,有望成为一种新型抗生素替代物。
综上所述,上述抗菌实验结果表明本发明成功基于理性分子设计策略设计出一种新型的广谱抗菌肽。
表2抗菌肽Leg2与KTR的抑菌活性测定结果
实施例3
本实施例用于测定新型抗菌肽KTR的溶血活性。
具体方案如下:将SD大鼠血浆加入PBS反复吹打混匀后,在4℃、3000g下离心10min去除上清液,收集沉淀红细胞。将红细胞用PBS稀释成5%的红细胞溶液,然后取0.5mL上述红细胞溶液,分别加入0.5mL不同浓度(1,2,4,8,16和32倍MIC)的KTR溶液。同时,以1%Triton X-100和PBS分别作为阳性对照和阴性对照。在37℃恒温培养1h后,将红细胞在3000g下离心10min。在波长570nm处用酶标仪测量上清液中释放的血红蛋白的吸光度值,通过以下公式计算抗菌肽溶血率。
溶血实验结果表明(图2):在1-32倍MIC浓度范围内,随着抗菌肽KTR浓度增加,SD大鼠红细胞的溶血率逐渐升高,但抗菌肽KTR对大鼠红细胞的溶血率仍低于20%,说明本发明提供的新型抗菌肽KTR对红细胞的渗透性较小,具有较低的溶血活性,可作为安全的生物抗菌药物应用的食品、医学等领域。
实施例4
本实施例用于测定广谱抗菌肽KTR的对人源肝细胞LO2和小鼠胚胎成纤维细胞NIH3T3的细胞毒性。细胞毒性采用MTT法进行测定,具体步骤如下:
细胞复苏:取液氮中冻存的肝细胞LO2和小鼠胚胎成纤维细胞NIH3T3,迅速置于37℃恒温水浴锅中,摇动至融化,吹打均匀后移入10mL离心管,加入3mL DMEM培养基,混匀,1000rpm离心3min,弃去上清液,加入8mL DMEM培养基,吹打均匀后移入细胞培养瓶中,置于37℃,5%CO2的细胞培养箱中过夜培养。
传代:取出细胞培养瓶,吸走DMEM培养基,加入1mL PBS冲洗2次,加入1mL 0.25%胰酶,置于37℃,5%CO2的细胞培养箱中消化60-90s,加入3mL新鲜DMEM培养基终止消化,吹洗瓶壁使细胞完全脱落,转入4mL离心管,1000rpm离心3min,弃去上清液,在离心管中加入3mL DMEM培养基,吹打均匀后,取1mL转入装有6mL DMEM培养基的细胞培养瓶中,置于37℃,5%CO2的细胞培养箱中培养48h,可根据细胞状态隔天进行换液,保证复苏细胞的活力。
铺板:取出细胞培养瓶,吸掉DMEM培养基,加入1mL PBS冲洗2次(洗去血清),使用1mL 0.25%胰酶消化肝细胞LO2和小鼠胚胎成纤维细胞NIH3T3,1000rpm离心3min后,收集细胞,调整细胞悬液浓度,接种于96孔板,每孔100μL,使细胞个数维持在5000个/孔,置于37℃、5%CO2的细胞箱中培养过夜,使细胞贴壁生长。
加样:取出接种有细胞的96孔板,吸去废旧DMEM培养基,加入含有不同浓度(19.0~303.2μM)抗菌肽KTR的新鲜DMEM培养基,每个浓度设3-5个平行,每孔体积100μL,对照组加入相同体积的不含抗菌肽的新鲜DMEM培养基,置于37℃、5%CO2细胞培养箱中继续培养24h。
加MTT染色:培养24h后,取出96孔板,每孔加入20μL 5mg/mL的MTT溶液,放入细胞培养箱中继续避光培养4h。
溶解结晶,测定吸光度:吸掉孔内的DMEM培养基,再向每孔加入100μL二甲基亚砜(DMSO),置于37℃摇床、低速振荡10min,使结晶物充分溶解后,采用酶联免疫检测仪,测定490nm处吸光值。
由图3数据可知,在18.9~303.2μM的测试浓度范围内,KTR对LO2和NIH3T3细胞的增殖及活性均不会产生明显的抑制作用,即细胞存活率均大于80%,说明抗菌肽KTR对两种细胞的毒性较低,因此不会对正常细胞产生毒性威胁,生物安全性可靠。
综上所述,本发明提供的新型抗菌肽KTR具有抑菌范围广,毒性低等优点,可作为一种绿色安全的抗生素代替物进行深入研究和开发利用。
以上所述实施例并不用于限制本发明,按照本发明的技术方案内容,在不脱离本发明上述基本技术思想前提下,本发明还可以对其中部分技术特征做出其它多种形式的修改、替换或变更,均落在本发明权利保护范围之内。
Claims (4)
1.一种广谱抗菌肽KTR,其特征在于,所述广谱抗菌肽KTR的氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述的广谱抗菌肽KTR在制备治疗革兰氏阳性菌或革兰氏阴性菌感染的广谱抗菌药物中的应用,其特征在于,所述革兰氏阴性菌为大肠杆菌、鼠伤寒沙门氏菌、肺炎克雷伯菌或铜绿假单胞菌;
所述革兰氏阳性菌为金黄色葡萄球菌或耐甲氧西林金黄色葡萄球菌。
3.根据权利要求2所述的应用,其特征在于,所述革兰氏阴性菌为大肠杆菌ATCC35150、大肠杆菌ATCC 8739、鼠伤寒沙门氏菌ATCC 14028、肺炎克雷伯菌ATCC 13883或铜绿假单胞菌ATCC 10145。
4.根据权利要求2所述的应用,其特征在于,所述革兰氏阳性菌为金黄色葡萄球菌ATCC25923或耐甲氧西林金黄色葡萄球菌ATCC 43300。
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利用亮氨酸和赖氨酸设计新型α-螺旋抗菌肽;王良;马清泉;单安山;董娜;吕银凤;;微生物学通报;20140220(第02期);全文 * |
棉花种子活力劣变的差异蛋白质组学研究;王志军;叶春秀;李有忠;赵曾强;孙国清;谢宗铭;;西北植物学报;20171115(第11期);全文 * |
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