CN107446026B - 一组具有抗临床多重耐药菌作用的小肽及其衍生物和应用 - Google Patents
一组具有抗临床多重耐药菌作用的小肽及其衍生物和应用 Download PDFInfo
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Abstract
一组具有抗临床多重耐药菌作用的小肽及其衍生物和应用,包括序列如SEQ ID NO.1‑6所示的小肽。该抗菌肽及其衍生物,降解后成为氨基酸被人体吸收利用,无抗生素残留。用固相合成法制备具有较高抗耐药菌活性的抗菌肽及其衍生物,制备方法简单,合成工艺和产品质量容易控制,适合于大规模工业化生产;同时,对环境友好,无任何有害物质产生。
Description
技术领域
本发明涉及一组多肽及其衍生物,具体地说涉及一组具有抗临床多重耐药菌作用的小肽及其衍生物和应用。
背景技术
抗生素的发现在人类医学史上具有重要的意义,它的使用挽救了无数的生命,使人的预期寿命增加了十年以上。但是随着人们对抗生素的广泛使用,细菌也逐渐适应并对其产生了耐药性。虽然目前临床上使用的抗生素有上百种之多,但都属于化学小分子,新研制的抗生素也一般是其结构修饰物,很难解决细菌对抗生素耐药的问题。随着耐药菌问题的出现,新型的抗耐药菌的抗生素的研发已成为国际上广泛关注的问题。
自然界中的许多生物,如昆虫、两栖类动物、植物和哺乳类动物都会产生保护机体的具有抗菌活性的小分子肽。这些抗菌肽能与生物体的细胞膜发生作用,从而达到杀死细菌、真菌和病毒的作用。昆虫抗菌肽都带有不同数量的正电荷,其作用机制在于,它所带的正电荷能与细菌细胞膜的磷脂双分子层的负电荷结合,从而影响膜上的离子通道,增加通透性,使细菌死亡,同时抗菌肽还可以和膜上的脂多糖结合,减轻由细菌感染引起的临床症状。
目前国际上已经报道的抗菌肽有数百种,其抗菌范围很广,对革兰氏阳性菌、革兰氏阴性菌、真菌和病毒都有抑制作用,由于抗菌肽是利用生物体自身免疫机制而达到抗菌作用,与抗生素的作用有很大不同,故其对目前临床上的耐药菌仍具有杀菌作用,被国际上认为是一类具有广阔应用前景的新型抗菌药。但是直接利用天然抗菌肽作为抗菌药物的尝试很少。主要原因有:很多天然抗菌肽的抗菌活性不高,而且收率低;很多抗菌肽有很强的溶血副作用。如从蜜蜂毒液中提取的蜂毒肽,它具有很强的抗菌活性,但同时也具有溶血副作用,而使蜂毒肽作为抗菌药物的应用受到很大的限制。
发明内容
解决的技术问题:本发明提供一组具有抗临床多重耐药菌作用的小肽及其衍生物和应用。具体来说,是通过固相合成的方法,合成了一组具有较高抗耐药菌活性、较低溶血副作用的肽及其C端氨基化衍生物。同时,将这些抗菌肽应用于制备治疗耐药性革兰氏阳性菌和革兰氏阴性菌的药物中。
技术方案:一组具有抗临床多重耐药菌作用的小肽,包括序列如SEQ ID NO.1-6所示的小肽。
上述一组具有抗临床多重耐药菌作用的小肽的衍生物,该组小肽的衍生物为,在如SEQ ID NO.1-6所示小肽的甲硫氨酸的羧基端修饰有氨基。
上述一组具有抗临床多重耐药菌作用的小肽,其特征在于组成上述结构的小肽的氨基酸是L型或者D型异构体。
上述一组小肽及其衍生物在制备具有抗临床多重耐药菌作用药物中的应用。
一种食品、化妆品、饲料的添加剂,有效成分为上述一组小肽及其衍生物。
1、Gly-Ser-Lys-Lys-Pro-Val-Pro-Ile-Ile-Tyr-Cys-Gln-Arg-Arg-Thr-Gly-Lys-Cys-Gln-Arg-Met(T1);
单字母表达:GSKKPVPIIYCQRRTGKCQRM
2、Gly-Ser-Lys-Lys-Pro-Val-Pro-Ile-Ile-Tyr-Cys-Asn-Arg-Arg-Ser-Gly-Lys-Cys-Gln-Arg-Met(T2);
单字母表达:GSKKPVPIIYCNRRSGKCQRM
3、Gly-Ser-Lys-Lys-Pro-Val-Pro-Ile-Ile-Tyr-Cys-Asn-Arg-Arg-Thr-Ala-Lys-Cys-Gln-Arg-Met(T3);
单字母表达:GSKKPVPIIYCNRRTAKCQRM
4、Gly-Ser-Lys-Lys-Pro-Val-Pro-Ile-Ile-Tyr-Cys-Gln-Arg-Arg-Ser-Gly-Lys-Cys-Gln-Arg-Met(T4);
单字母表达:GSKKPVPIIYCQRRSGKCQRM
5、Gly-Ser-Lys-Lys-Pro-Val-Pro-Ile-Ile-Tyr-Cys-Gln-Arg-Arg-Thr-Ala-Lys-Cys-Gln-Arg-Met(T5);
单字母表达:GSKKPVPIIYCQRRTAKCQRM
6、Gly-Ser-Lys-Lys-Pro-Val-Pro-Ile-Ile-Tyr-Cys-Asn-Arg-Arg-Ser-Ala-Lys-Cys-Gln-Arg-Met(T6);
单字母表达:GSKKPVPIIYCNRRSAKCQRM
7、T1-NH2(T7);
8、T2-NH2(T8);
9、T3-NH2(T9);
10、T4-NH2(T10);
11、T5-NH2(T11);
12、T6-NH2(T12)。
式中的Gly为甘氨酸,Ser为丝氨酸,Lys为赖氨酸,Pro为脯氨酸,Val为缬氨酸,Tyr为酪氨酸,Cys为半胱氨酸,Asn为天冬酰胺,Arg为精氨酸,Thr为苏氨酸,Gln为谷氨酰胺,Met为甲硫氨酸,Met-NH2为羧基进行氨基化修饰甲硫氨酸。
有益效果:本发明提供了一组抗菌肽及其衍生物,降解后成为氨基酸被人体吸收利用,无抗生素残留。用固相合成法制备具有较高抗耐药菌活性的抗菌肽及其衍生物,制备方法简单,合成工艺和产品质量容易控制,适合于大规模工业化生产;同时,对环境友好,无任何有害物质产生。本发明涉及的抗菌肽及其衍生物不仅能杀灭非耐药细菌,对耐药性细菌也具有强烈的抑杀作用,一次用药能同步杀灭耐药和非耐药菌,有效解决临床传统抗生素的选择问题。命名为T1~T6的6种抗菌肽对大部分临床分离的革兰氏阴性耐药性细菌具有强烈的抑杀作用,MIC小于20μg/mL,但是对革兰氏阳性菌无明显作用,MIC均大于500μg/mL;经C端氨基化修饰后,得到的衍生物T7~T12小肽的抗革兰氏阴性菌活性提高2~20倍,同时对革兰氏阳性细菌也具有显著抑菌效果,对常见的临床革兰氏阳性细菌抑菌MIC在2~32μg/mL之间。具有强烈的内毒素中和作用。败血症病人经抗生素治疗后,大量内毒素释放,引起严重的临床症状(如高热、低血压等),是病人高死亡率的原因之一,本产品能有效中和细菌内毒素,有效提高病人的生存率。溶血实验和急性局部刺激实验表明,6种抗菌肽及其衍生物的浓度在2.5mg/mL时均未发生溶血反应,产品安全性较高;具有较低的急性毒性刺激反应。
具体实施方式
以下结合实施例,具体说明本发明。
实施例1
抗菌肽及其衍生物的固相合成
通过以FMOC(9-芴甲氧羰基)为保护基团的固相合成法合成抗菌肽。用95%三氟乙酸、2.5%水和2.5%三异丙甲硅烷(TIA)将其从树脂上裂解。在乙醚反复沉淀后,通过反相高效液相色谱纯化多肽。纯化中采用C18反相柱:以含0.05%三氟乙酸的0%~60%乙腈为流动相,3mL/min的流速进行梯度洗脱。再将多肽以1mg/mL的浓度溶解在氧化缓冲液(100mmol/L醋酸铵,pH8.5)中,并在室温下不断搅拌3d,使其充分氧化折叠形成二硫键。最终用反相HPLC纯化至95%以上,冻干备用。C端氨基化的多肽衍生物也采用本法合成,末端甲硫氨酸采用商品化的氨基化M-NH2。HPLC/Mass结果显示,合成的小肽及其衍生物分子量和理论分子量一致。
实施例2
抗菌活性的测定
用灭菌生理盐水配制浓度为6mg/mL多肽样品溶液。试验用菌种分别接种于营养肉汤,于37℃培养24小时,临用前用无菌生理盐水作1:105倍稀释,取12支细菌培养管并编号,于第1管中加入营养肉汤1.8mL,其余11管中均加入1.0mL肉汤。于第1管加入0.2mL多肽溶液,混匀后取出1.0mL加入到第2管中,如此反复,依次稀释到第12管,每管各加菌液0.2mL,轻轻振摇均匀,置37℃培养24小时。以无菌生长的最低多肽浓度,即为抑制细菌生长的最低样品浓度(MIC)。表1为实施例1中制备的抗菌肽衍生物对几种菌的最低抑菌浓度。
表1抗菌肽衍生物最低抑菌浓度MIC(mg/mL)
实施例3
多肽溶血活性的测定:
将人血红细胞悬浮在磷酸盐缓冲液(pH7.4)中,得到红细胞悬浮液(5%v/v)。将多肽溶解在磷酸盐缓冲液中,配成约5mg/mL储备液,取14支1.5mL离心管,于第1支离心管中加1mL多肽储备液,其余各管中加0.5mL磷酸盐缓冲液,从第1管中取出0.5mL多肽储备液加至第2管中,用微量混合仪混合均匀,再从第2管中取出0.5mL溶液加至第3管中混合均匀,以此类推,即以倍比稀释法依次稀释到第14管,弃去0.5mL,各管补加0.5mL配好的5%血红细胞悬浮液至终体积1.0mL,轻轻摇匀,37℃恒温箱中保温60min后,于4000rpm离心10分钟,取上清液在414nm下比色,以红细胞悬浮在磷酸盐缓冲液中为空白,以红细胞悬浮在1%TritonX-100中为100%溶血。溶血百分率用下式计算:
定义溶血百分率为50%时的多肽浓度为半数溶血剂量(HC50)。结果表明,所有抗菌肽及其衍生物在2.5mg/mL时未见溶血。
实施例4
兔眼结膜局部刺激实验
取健康家兔6只,实验前先检查眼结膜血管、角膜透明度及眼分泌物等状况,选用正常者供试。将供试药物用生理盐水配制成等渗。取实施例1中制备的抗菌肽各0.1mL(5mg/mL)滴入左眼结膜囊中,停留2min;右眼滴入同量生理盐水作为对照。使用放大镜观察给药后1、24、48、72h的眼部情况。结果显示,至72h时,兔眼角膜无浑浊,虹膜正常,结膜无充血水肿,无分泌物出现。表明药液对兔眼无急性刺激作用。
实施例5
抗菌肽对内毒素的中和作用
抗菌肽用无热源水配置成200μg/mL,倍比稀释,稀释品中分别加入1EU/mL的内毒素,37℃反应30min,测定内毒素含量,以没有加入抗菌肽的内毒素作对照。结果显示,抗菌肽以浓度依赖的方式中和内毒素,半数抑制浓度分别为:T1,20μg/mL;T2,17.5μg/mL,T3,20μg/mL;T4,15μg/mL;T5,25μg/mL;T6,17.5μg/mL;T7,12.5μg/mL;T8,10μg/mL;T9,5μg/mL;T10,7.5μg/mL;T11,12μg/mL;T12,10μg/mL。
实施例6
将90只SPF级Wistar雄性大鼠(体重为180±20g)随机分成3大组,每组30只,分别为生理盐水对照组、肺炎克雷伯氏菌标准菌株组、临床分离的全耐药肺炎克雷伯氏菌野生菌株组。每组又分为高、中、低3个剂量组,每组10只。取单菌落,用MH培养基扩增后调整细菌浓度,按2×1010个细菌/只分别注射大鼠腹腔。造模后按实验分组尾静脉注射不同浓度的抗菌肽及其衍生物,在不同时段考察模型动物死亡率。如表2结果所示,抗菌肽衍生物能明显降低标准菌株和野生耐药菌株引起的败血症模型动物的死亡率,24小时半数抑制浓度分别为:T1,12.5mg/Kg;T2,10mg/Kg;T3,12.5mg/Kg;T4,20mg/Kg;T5,10mg/Kg;T6,12.5mg/Kg;T7,7.5mg/Kg;T8,5mg/Kg;T9,12.5mg/Kg;T10,10mg/Kg;T11,10mg/Kg;T12,15mg/Kg。
表2多粘菌素和小肽单独或联合使用对绿脓杆菌败血症模型小鼠死亡率的影响
SEQUENCE LISTING
<110> 东南大学
<120> 一组具有抗临床多重耐药菌作用的小肽及其衍生物和应用
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
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Gly Ser Lys Lys Pro Val Pro Ile Ile Tyr Cys Gln Arg Arg Thr Gly
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Gly Ser Lys Lys Pro Val Pro Ile Ile Tyr Cys Gln Arg Arg Thr Ala
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Gly Ser Lys Lys Pro Val Pro Ile Ile Tyr Cys Asn Arg Arg Ser Ala
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Claims (1)
1.如SEQ ID NO.1-6所示小肽的衍生物在制备具有抗临床多重耐药菌作用药物中的应用,所述衍生物为所示小肽的甲硫氨酸的羧基端修饰有氨基。
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CN102807610A (zh) * | 2012-09-05 | 2012-12-05 | 东南大学 | 抗菌肽及其在制备抗耐药菌药物中的应用 |
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JP2011072294A (ja) * | 2009-10-02 | 2011-04-14 | Hokkaido Univ | 新規抗菌ペプチド |
CN102807610A (zh) * | 2012-09-05 | 2012-12-05 | 东南大学 | 抗菌肽及其在制备抗耐药菌药物中的应用 |
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