CN112724198A - 一种抗耐甲氧西林金黄色葡萄球菌抗菌肽及其制备方法和应用 - Google Patents
一种抗耐甲氧西林金黄色葡萄球菌抗菌肽及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗耐甲氧西林金黄色葡萄球菌抗菌肽H5‑p9,所述抗菌肽H5‑p9由20个氨基酸残基组成,分子量2560.09Da,净电荷数为+8,等电点10.79,其氨基酸序列为丝氨酸‑酪氨酸‑谷氨酸‑精氨酸‑赖氨酸‑异亮氨酸‑天冬酰胺‑精氨酸‑组氨酸‑苯丙氨酸‑赖氨酸‑苏氨酸‑亮氨酸‑赖氨酸‑赖氨酸‑天冬酰胺‑亮氨酸‑赖氨酸‑赖氨酸‑赖氨酸‑NH2。本发明中的抗菌肽H5‑p9具有分子量小、人工合成简单、抗菌作用显著、对金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌作用效果好,溶血活性低的优点,具有广泛的应用前景。
Description
技术领域
本发明属于生物化学中的多肽药物技术领域,具体涉及抗菌肽H5-p9和该抗菌肽在制备抗菌药物中的用途。
背景技术
自从青霉素等抗生素被发现以来,针对细菌感染性疾病的治疗取得了根本性的改善,抗生素的使用拯救了无数人的生命,延长了人类的平均寿命。但随着抗生素的大规模使用或滥用,尤其是在发展中国家,导致抗药性细菌的产生和扩散,其中包括一些毒力很强的致病菌,如耐甲氧西林葡萄球菌与肺炎链球菌等。因此,寻找安全有效且不易产生耐药性的抗菌药物成为全世界科学家关注和努力的方向。
抗菌肽是当生物受到微生物侵入后机体迅速产生的高效、广谱的免疫分子,一般由12到100个氨基酸组成。抗菌肽广泛存在于不同类型生物体中,目前人们已从微生物、植物、两栖动物、海洋脊椎动物、哺乳动物甚至人体内鉴定出千余种抗菌肽。抗菌肽抗菌机理复杂,但大多数理论认为其机制涉及到抗菌肽的阳离子性和疏水性与带负电荷的微生物胞膜的作用,抗菌肽和细菌的细胞膜接触后,引起膜通透性改变,或在细菌细胞膜上形成跨膜的孔洞,最后导致细菌内容物外泄而死亡。因此,抗菌肽杀灭细菌的效率远远高于传统的抗生素,且不像抗生素在低浓度下抑制细菌的生长,抗菌肽对细菌的作用几乎都是致死性的,且不易产生耐药性。
抗菌肽的发现和快速发展为开发新型抗菌药物提供了巨大的资源库,也为解决临床耐药菌株问题提供了极大的可能性,在医药卫生、农业生产、食品工业等领域均有广泛的应用前景。随着人们对抗菌肽抗菌机理的进一步认识和新的抗菌肽的发现,人们不仅可以从生物体内直接分离纯化得到抗菌肽,也可以利用基因工程手段重组得到抗菌肽,又可以直接利用化学合成手段在短时间内合成大量小分子抗菌肽。
天然来源的抗菌肽部分会因为分子量较大存在免疫原性,抗菌活性低,对宿主细胞有细胞毒作用,或会引起溶血等方面限制了抗菌肽作为抗菌药物的推广应用。因此,寻找分子量更小,抗菌活性更强,尤其是不能存在溶血或细胞毒作用的抗菌肽已成为解决抗菌肽作为抗菌药物推广的最关键因素。近几年来,科学家们在寻找新型抗菌肽的同时也开始致力于对原有的天然抗菌肽进行结构改造或重新设计,如更换某些氨基酸残基或根据需要直接设计抗菌肽氨基酸的一级结构,以期获得活性更高、更有针对性且对宿主细胞无毒害作用的抗菌肽。
发明内容
本发明的目的在于提供一种新的具有药用价值的抗菌肽H5-p9。
本发明的另一目的在于提供上述抗菌肽在制备抗菌药物方面的用途。
本发明所述之抗菌肽H5-p9为人工设计合成的活性多肽,包含20个氨基酸残基,分子量2560.09Da,等电点10.79,其全序列为丝氨酸-酪氨酸-谷氨酸-精氨酸-赖氨酸-异亮氨酸-天冬酰胺-精氨酸-组氨酸-苯丙氨酸-赖氨酸-苏氨酸-亮氨酸-赖氨酸-赖氨酸-天冬酰胺-亮氨酸-赖氨酸-赖氨酸-赖氨酸-NH2。
本发明所述之抗菌肽H5-p9全序列的C-端酰胺化。
本发明中的抗菌肽在制备抗菌药或保健品或漱口水中的用途。
更进一步地,本发明提供了一种药物组合物,所述药物组合物包含上述抗耐甲氧西林菌抗菌肽H5-p9和药学上或美学上可接受的载体或稀释剂。本发明研究发现,上述抗耐甲氧西林菌抗菌肽H5-p9和含有H5-p9的组合物不仅能作为抗耐甲氧西林菌药物,也能应用于抗菌药物、保健品及漱口水等领域。
本发明的有益效果在于:
本发明中的抗菌肽H5-p9为人工合成,具有分子量小、人工合成方便,杀菌作用强等优点,此外抗菌肽H5-p9还具有极低溶血活性和真核细胞毒性的特点。
附图说明
图1为抗菌肽H5-p9的高效液相色谱(HPLC)纯度图谱。
图2抗菌肽H5-p9的抗菌活性,其中H5-p9MIC:即抗菌肽H5-p9的最小抑菌浓度,以上结果为三次独立重复实验平均值。
图3抗菌肽H5-p9溶血活性分析(小鼠血),其中LZ1:抗菌肽H5-p9;NC:生理盐水,其加样体积与样品组相同;阳性对照:Triton X-100,其加样体积与样品组相同,上述实验结果为三次独立实验的平均值。
图4抗菌肽H5-p9细胞毒性分析(胚胎肾细胞HEK),H5-p9:抗菌肽H5-p9;对照:生理盐水,其加样体积与样品组相同;上述实验结果为三次独立实验的平均值。
具体实施方式
根据下述实施例可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书所详细描述的本发明。
实施例1:抗菌肽H5-p9的制备
Ⅰ、抗菌肽H5-p9的化学合成方法:根据发明内容中所述的氨基酸序列,用自动多肽合成仪(433A,Applied Biosystems)合成其全序列,并通过HPLC反相柱层析脱盐纯化。
Ⅱ、电喷雾质谱法测定抗菌肽H5-p9分子量。
Ⅲ、纯化的抗菌肽H5-p9用HPLC方法(Welch XB C18 4.6*250mm)鉴定其纯度,分子量测定采用电喷雾质谱质谱,等电聚焦电泳测定等电点,用自动氨基酸测序仪测定氨基酸序列结构。
HPLC纯度鉴定结果如图1所示:抗菌肽H5-p9在9.060min处显示为单一对称峰。
抗菌肽H5-p9包含20个氨基酸残基,分子量2560.09Da,等电点10.79,其全序列为丝氨酸-酪氨酸-谷氨酸-精氨酸-赖氨酸-异亮氨酸-天冬酰胺-精氨酸-组氨酸-苯丙氨酸-赖氨酸-苏氨酸-亮氨酸-赖氨酸-赖氨酸-天冬酰胺-亮氨酸-赖氨酸-赖氨酸-赖氨酸-NH2(C-端酰胺化)。
实施例二:抗菌肽H5-p9的抗菌实验:
最小抑菌浓度(minimal inhibitory concentration,MIC):MIC为检测不到细菌生长的最低样品浓度。采用二倍稀释法,具体方法如下:
细菌接种于Luria-Bertani(LB)固体培养基上,37℃培养箱中倒置培养。待菌落长出后,用接种环挑取单菌落转接到LB液体培养基中,37℃培养箱震荡培养至对数生长期。在紫外分光光度计上检测菌液OD600,根据OD600=1×108CFU/ml将菌液用液体LB培养基稀释至2×105CFU/ml。在无菌96孔板各孔中预先加入100μL LB液体培养基,然后在第一孔中加入100μL稀释至一定浓度的经0.22μm微孔滤膜过滤除菌的抗菌肽样品,混匀后取100μL加入第2孔,依次倍比稀释,从第12孔吸出100μL弃去,至此二倍浓度梯度样品制备完毕。
向各孔中加入已稀释好的菌液100μL,混匀后于37℃缓慢震荡培养16h,测定600nm处的光吸收值。结果计算:取检测不到细菌生长的孔和与之相邻的有细菌生长的孔样品浓度之和的平均值作为样品最小抑菌浓度。
此外,白色念珠菌为真菌,培养使用的培养基为PDA培养基,其他条件类似。
结果如图2所示。
由图2可知,抗菌肽H5-p9对所受试菌株有非常强的杀伤作用,如对金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌的MIC值最低可达16μg/mL(6μM),说明抗菌肽H5-p9在极低浓度下就可抑制葡萄球菌的生长;抗菌肽H5-p9对口腔临床上分离的白色念球菌也有很好的杀菌效果,最低浓度可达到64μg/mL的浓度;对口腔有益菌血链球菌最小抑菌浓度大于64μg/mL说明抗菌肽H5-p9对革兰氏阳性的葡萄球菌效果显著,选择性好。
实施例三:抗菌肽H5-p9的溶血活性实验:
小鼠心脏采血,将所采集血液与阿氏液(Alsever Solution,8.0g柠檬酸钠,0.55g柠檬酸,20.5g葡萄糖,4.2g氯化钠,加去离子水至1L,调pH至6.1,高压灭菌后4℃保存)按1:1比例混合置于离心管中,1000rpm离心5min,生理盐水洗涤至上清液不再呈红色为止。将上述洗涤好的红细胞加生理盐水稀释成108浓度的悬浮液。上述稀释好的红细胞悬浮液与溶解于生理盐水的不同浓度的样品37℃保温30min,再于1000rpm离心5min,上清液于540nm测吸收值。阴性对照使用生理盐水,阳性对照使用Triton X-100。溶血活性与540nm吸收值成正比。
结果如图3所示。
由图3可知,抗菌肽H5-p9即使在640μg/mL的高浓度下也不会引起鼠血发生溶血现象。
实施例四:抗菌肽H5-p9的细胞毒实验:
人正常细胞胚胎肾细胞HEK 293于含有10%胎牛血清及双抗(青霉素和链霉素各100U/mL)的DMEM培养基培养至对数期,细胞用PBS缓冲液洗三遍后,用0.25%的胰蛋白酶消化下来,用新鲜DMEM培养基悬浮细胞,调整细胞密度至1*106个/mL,以每孔200μL的体积铺96孔板,等细胞贴壁后,加不同浓度的样品,在37℃,5%的二氧化碳条件下共培养24h,培养结束后,96孔细胞培养板每孔加入20μL 5mg/mL MTT溶液(用细胞培养PBS缓冲液配制),继续培养4h,用移液枪吸出孔中液体,每孔加入100μL DMSO,室温下轻摇10min,用酶标仪检测490nm波长的光吸收。
结果如图4所示。
由图4可知,即使在640μg/mL的浓度下抗菌肽H5-p9对人正常细胞胚胎肾细胞HEK293存在很低的细胞毒作用(约20%)。
综上所述,本发明中抗耐甲氧西林金黄色葡萄球菌抗菌肽H5-p9具有分子量小、人工合成简单、高效灭杀耐甲氧西林金黄色葡萄球菌、溶血活性低等优点。上述抗耐甲氧西林金黄色葡萄球菌抗菌肽H5-p9和含有H5-p9的组合物不仅能作为抗耐甲氧西林金黄色葡萄球菌药物,也能应用于口腔保健品及护肤品等领域。
Claims (5)
1.一种抗耐甲氧西林金黄色葡萄球菌抗菌肽H5-p9,其特征在于,所述的抗菌肽H5-p9包含20个氨基酸残基,分子量2560.09Da,等电点10.79,其全序列为丝氨酸-酪氨酸-谷氨酸-精氨酸-赖氨酸-异亮氨酸-天冬酰胺-精氨酸-组氨酸-苯丙氨酸-赖氨酸-苏氨酸-亮氨酸-赖氨酸-赖氨酸-天冬酰胺-亮氨酸-赖氨酸-赖氨酸-赖氨酸-NH2。
2.权利要求1所述的抗耐甲氧西林金黄色葡萄球菌抗菌肽H5-p9的制备方法,其特征在于,根据其氨基酸序列,用自动多肽合成仪合成其全序列,通过HPLC反相柱层析脱盐纯化。
3.权利要求1所述的抗耐甲氧西林金黄色葡萄球菌抗菌肽H5-p9在制备抗菌药物或口腔保健品或护肤品中的应用。
4.一种组合物,所组合物包括权利要求1所述的抗耐甲氧西林金黄色葡萄球菌抗菌肽H5-p9和药学上或美学上可接受的载体或稀释剂。
5.权利要求4所述的组合物在制备抗菌药物或口腔保健品或护肤品中的应用。
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