CN117264040A - 一种拟穴青蟹新型抗菌肽Scyreptin及其应用 - Google Patents
一种拟穴青蟹新型抗菌肽Scyreptin及其应用 Download PDFInfo
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Abstract
本发明公开了一种拟穴青蟹新型抗菌肽Scyreptin及其应用,其氨基酸序列如SEQ ID NO.01所示。本发明由30个氨基酸组成,分子式为C153H243N46O40S4,分子量为3494道尔顿,其带有5个正电荷,根据氨基酸残基电荷预测该抗菌肽等电点为10.05,具有很好的水溶性,是一种带有正电荷的阳离子多肽,具有长度短、易合成、抗菌活性高且稳定等特点,在医药食品等领域具有很好应用前景。
Description
技术领域
本发明属于海洋分子生物学技术领域,具体涉及一种拟穴青蟹新型抗菌肽Scyreptin及其应用。
背景技术
自青霉素发现以来,抗生素在治疗由病原性细菌、真菌等感染引起的疾病方面发挥着不可替代的作用,但随着抗生素滥用导致的全球范围内细菌耐药性,对人类和动物健康都带来了严重威胁。针对细菌耐药性问题,寻找抗生素的有效替代品已成为全球的研究趋势。
抗菌肽(AMPs)是存在于动物、植物和微生物等的先天性免疫因子,在抵御外源性病原微生物入侵感染中发挥重要免疫作用。抗菌肽除可直接抑杀菌外,在生物体内还具有重要的免疫调节作用,包括免疫细胞的募集和激活、诱导抗炎细胞因子、抑制促炎细胞因子、促进伤口愈合和血管的生成等。除此之外,研究发现抗菌肽不易产生细菌耐药性,被誉为未来最有效的抗生素替代品。目前APD3数据库中收录的约3500余种抗菌肽,约2600种来自于动物,70余种来源于甲壳类动物。迄今所报道的大多数抗菌肽是阳离子多肽,其抗菌机制是阳离子肽的表面正电荷与细菌表面带负电荷的组分(脂多糖和磷壁酸)通过静电作用非特异的相互作用,然后通过抗菌肽的两亲性插入细菌的膜结构致其完整性破坏而导致细菌死亡。
发明内容
本发明目的在于提供一种拟穴青蟹新型抗菌肽Scyreptin。
本发明的另一目的在于提供上述拟穴青蟹新型抗菌肽Scyreptin的应用。
本发明的技术方案如下:
一种拟穴青蟹新型抗菌肽Scyreptin,其氨基酸序列如SEQ ID NO.01所示。
上述拟穴青蟹新型抗菌肽Scyreptin在制备抗细菌组合物中的应用。
在本发明的一个优选实施方案中,所述拟穴青蟹新型抗菌肽Scyreptin对金黄色葡萄球菌、粪肠球菌、屎肠球菌、大肠埃希氏菌、铜绿假单胞菌和鲍曼不动杆菌具有抑制和杀灭作用。
一种抗细菌组合物,其有效成分包括上述拟穴青蟹新型抗菌肽Scyreptin。
在本发明的一个优选实施方案中,其有效成分为上述拟穴青蟹新型抗菌肽Scyreptin。
上述拟穴青蟹新型抗菌肽Scyreptin在制备抗真菌组合物中的应用。
在本发明的一个优选实施方案中,所述拟穴青蟹新型抗菌肽Scyreptin对新型隐球菌、禾谷镰孢、尖孢镰孢、赭曲霉、黑曲霉和腐皮镰孢具有抑制和杀灭作用。
一种抗真菌组合物,其有效成分包括上述拟穴青蟹新型抗菌肽Scyreptin。
在本发明的一个优选实施方案中,其有效成分为上述拟穴青蟹新型抗菌肽Scyreptin。
本发明的有益效果是:
1、本发明由30个氨基酸组成,分子式为C153H243N46O40S4,分子量为3494道尔顿,其带有5个正电荷,根据氨基酸残基电荷预测该抗菌肽等电点为10.05,具有很好的水溶性,是一种带有正电荷的阳离子多肽,具有长度短、易合成、抗菌活性高且稳定等特点,在医药食品等领域具有很好应用前景。
2、本发明对铜绿假单胞菌、金黄色葡萄球菌、大肠埃希氏菌、尖孢镰孢菌、腐皮镰孢菌、禾谷镰孢、黑曲霉菌、和新型隐球菌具有良好抗菌效果,对斑马鱼ZF4细胞和人肾上皮细胞HEK-293T无细胞毒性。
附图说明
图1为本发明实施例3中拟穴青蟹抗菌多肽Scyreptin对鲍曼不动杆菌和铜绿假单胞菌的杀菌动力学曲线。
图2至6为本发明实施例4中拟穴青蟹抗菌多肽Scyreptin抑制霉菌孢子萌发实验图;其中,图2为腐皮镰孢菌,图3为禾谷镰孢,图4为黑曲霉,图5为尖孢镰孢菌,图6为赭曲霉。上述Scyreptin终浓度为A:0μM;B:3μM;C:6μM;D:12μM;E:24μM;F:48μM;G:96μM。
图7为本发明实施例5中拟穴青蟹抗菌多肽Scyreptin与黑曲霉、赭曲霉、腐皮镰孢和尖孢镰孢菌作用后扫描电镜观察图;其中,A:黑曲霉;B:黑曲霉+24μM Scyreptin;C:赭曲霉;D:赭曲霉+48μM Scyreptin;E:腐皮镰孢;F:腐皮镰孢+12μM Scyreptin;G:尖孢镰孢菌;H:尖孢镰孢菌+12μM Scyreptin。
图8为本发明实施例6中拟穴青蟹抗菌多肽Scyreptin对鲍曼不动杆菌和铜绿假单胞菌抗菌活性热稳定性图;横坐标为时间(h),纵坐标为OD600值。
图9为本发明实施例6中MTS-PMS法检测拟穴青蟹抗菌多肽Scyreptin细胞毒性实验图。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1:
本实施例的拟穴青蟹新型抗菌多肽Scyreptin的氨基酸序列为:
Met-Arg-Ala-Leu-Tyr-Pro-Glu-Ser-Phe-Lys-Ser-Lys-Val-Ala-Met-Tyr-Ser-Gly-Ala-Trp-Cys-Gly-Cys-Arg-Pro-Arg-Thr-Arg-Gln-Leu(SEQ ID NO.01,MRALYPESFKSKVAMYSGAWCGCRPRTRQL)
本实施例委托生工生物工程(上海)有限公司以固相合成方法合成获得纯度达95%以上的拟穴青蟹抗菌多肽Scyreptin,并提供多肽分子量、HPLC等检测信息,相关理化参数如表1所示。
表1抗菌多肽Scyreptin理化参数
由表1可知本实施例的拟穴青蟹抗菌多肽Scyreptin分子量小、稳定性较好,具有很强的水溶性,是一种带有正电荷的阳离子多肽。
实施例2
本实施例涉及到的菌株有:金黄色葡萄球菌(Staphylococcus aureus)、大肠埃希氏菌(Escherichia coli)、铜绿假单胞菌(Pseudomonas Aeruginosa)、嗜水气单胞菌(Aeromonas hydrophila)、新型隐球菌(Cryptococcus neoformans)、禾谷镰孢(Fusariumgraminearum)、尖孢镰孢(Fusarium oxysporum)、赭曲霉(Aspergillus ochraceus)、黑曲霉(Aspergillus niger)、腐皮镰孢(Fusarium solani)。菌株均购自中国科学院微生物研究所菌种保藏中心,由本实验室保种贮藏。
具体方法如下:
(1)将保种的细菌金黄色葡萄球菌、大肠埃希氏菌、铜绿假单胞菌和嗜水气单胞菌菌株划线于营养肉汤平板;将新型隐球菌划线于YPD平板;禾谷镰孢、尖孢镰孢、赭曲霉、黑曲霉和腐皮镰孢接种于马铃薯葡萄糖平板,细菌于37℃或者28℃条件下倒置培养过夜,真菌28℃培养1-7d;
(2)挑取细菌单克隆于MH液体培养基中,37℃,200rpm培养至对数生长期;离心收集细菌,用10mM磷酸钠缓冲液(pH=7.4)将细菌重悬,最后用MH液体培养基稀释细菌,使得菌体的最终浓度为5×105cfu/mL;
(3)用10mM磷酸钠缓冲液(pH=7.4)将真菌孢子从斜面上冲下,用马铃薯葡萄糖液体培养基和磷酸钠缓冲液混合液稀释丝状真菌孢子,利用血球计数板对孢子计数,调整孢子浓度,使得真菌孢子最终浓度为5×104个/mL。
(4)将已合成的Scyreptin粉末溶于无菌Milli-Q水,倍比稀释蛋白浓度至1μM、3μM、6μM、12μM、24μM、48μM、96μM;
(5)在96孔无菌培养板上,每种待测菌设置空白对照组、阴性对照组和待测实验组,每组设置三个平行:
a空白对照组:50μL待测蛋白样品和50μL培养基
b阴性对照组:50μL无菌Milli-Q水和50μL菌悬液
c待测实验组:50μL待测蛋白样品和50μL菌悬液
将96孔培养板置于37℃或28℃培养箱中,培养18-24h,观察待测实验组中MIC结果;将待测实验组吹打混匀后,吸取适量的菌液涂布于MH平板上,于37℃或28℃倒置培养过夜,观察MBC结果。
拟穴青蟹抗菌多肽Scyreptin MIC、MBC观察结果如表2所示,Scyreptin抗菌谱广,对细菌和真菌都有较强的杀菌活性。
表2拟穴青蟹抗菌多肽Scyreptin的抗菌活性
注:MIC:最小抑菌浓度(μM),用a-b表示;a:肉眼可见菌体生长的最高蛋白浓度;b:肉眼未见菌体生长的最低蛋白浓度;
MBC:最小杀菌浓度(μM),杀死99.9%细菌浓度。
实施例3
本实施例选取铜绿假单胞菌和鲍曼不动杆菌作为待测菌,对拟穴青蟹抗菌多肽Scyreptin的杀菌动力学进行测定。
具体方法如实施例2中所述的抗菌活性测定方法一致,抗菌肽分别和被测细菌共孵一定时间后,不同时间点取共孵的混合液梯度稀释后涂布平板,静置培养过夜,进行菌落计数。
杀菌指数是指经过一定时间共孵后,待测实验组的克隆数与阴性对照组克隆数的比值,用百分比表示(参见图1)。
青蟹抗菌肽Scyreptin对铜绿假单胞菌和鲍曼不动杆菌的杀菌动力学曲线如图1所示,Scyreptin具有快速杀菌的作用,12μM的Scyreptin在15min和5min内即可杀灭铜绿假单胞菌和鲍曼不动杆菌。
实施例4
本实施例选取腐皮镰孢、禾谷镰孢、黑曲霉、尖孢镰孢、赭曲霉作为待测菌,观察拟穴青蟹抗菌肽Scyreptin对各霉菌孢子萌发的影响。
具体方法如实施例2中所述的抗菌活性测定方法一致。将等体积各浓度Scyreptin与各霉菌孢子于96孔细胞培养板混匀,置于28℃培养箱中,静置培养24h,在光学显微镜下观察霉菌孢子萌发情况(参见图2-6)。
当Scyreptin终浓度为12μM时即能有效的抑制腐皮镰孢(图2)和尖孢镰孢(图5)孢子的萌发;当Scyreptin终浓度为24μM时即能有效的抑制禾谷镰孢(图3)孢子的萌发;当Scyreptin终浓度为48μM时即能有效的抑制赭曲霉(图6)和黑曲霉(图4)孢子的萌发。
实施例5
本实施例选取黑曲霉、赭曲霉、腐皮镰孢、尖孢镰孢和铜绿假单胞菌作为待测菌株,扫描电镜样品的制备按以下步骤进行:
(1)如实施例2所述制备黑曲霉、赭曲霉、腐皮镰孢、尖孢镰孢孢子悬液至5×106个/ml,铜绿假单胞菌5×107cfu/mL,冰上放置备用。
(2)用无菌纯水溶解多肽Scyreptin,并调整蛋白浓度为24μM、48μM、96μM,冰上放置备用。
(3)悬液和蛋白等体积混合后在适宜温度孵育适宜时间。
(4)6000g离心5min收集孢子和菌体,加入1ml戊二醛固定液,4℃固定2h后,离心去上清,PBS清洗一次后离心收集菌体。
(5)去除大部分上清,留约10μL液体,将剩余悬液滴在玻片上,4℃静置过夜。
(6)PBS清洗一次。
(7)30%和50%乙醇分别脱水5min,70%和80%乙醇分别脱水10min,95%乙醇脱水15min,100%(v/v)乙醇脱水两次,每次脱水15min。
(8)送临界点干燥后,喷金后用扫描电子显微镜观察及拍照。
如图7所示,正常状态下黑曲霉(图7A)和赭曲霉(图7C)孢子近圆形,抗菌肽使得黑曲霉(图7B)孢子出现皱缩内容物外泄,赭曲霉(图7D)孢子出现明显的破裂;腐皮镰孢(图7E)和尖孢镰孢(图7G)在正常状态下外表平滑,抗菌肽使得腐皮镰孢(图7F)和尖孢镰孢(图7H)出现破裂表面有外泄的内容物凸起。正常状态下铜绿假单胞菌(图7I)菌体表面规则饱满,抗菌肽使得铜绿假单胞菌(图7J)表面收缩,膜完整性破坏,有泡状物附着在菌体周围,甚至有细胞内容物渗漏。
实施例6
本实施例选取铜绿假单胞菌和鲍曼不动杆菌作为待测菌,对拟穴青蟹抗菌肽Scyreptin的抗菌活性热稳定性进行测定。
具体方法如实施例2中所述的抗菌活性测定类似。调整Scyreptin终浓度至1倍MBC(铜绿假单胞菌:12μM,鲍曼不动杆菌:6μM),分别在100℃沸水中水浴不同时间后置于冰上备用。将Scyreptin或者无菌DPBS分别与待测菌共同孵育,在0h、12h、24h、36h、48h时用酶标仪测定OD600的值。如图8所示,在沸水浴处理30min后对鲍曼不动杆菌和铜绿假单胞菌仍然有较好的抗菌活性。
实施例7
本实施例选取斑马鱼成纤维细胞(ZF4)和人肾上皮细胞(HEK-293T),对拟穴青蟹抗真菌多肽Spamptin72-91细胞毒性进行测定,具体方法如下:
(1)收集生长状态良好的斑马鱼成纤维细胞(ZF4)和人肾上皮细胞(HEK-293T),调整细胞浓度为103-104个/mL,在96孔细胞培养板中每孔加入100μL细胞悬液,置于28℃和37℃,0.5%CO2条件下静置培养。
(2)吸出培养基,加入含有不同浓度Scyreptin的相应培养基,置于28℃和37℃,0.5%CO2条件下静置培养24h。
(3)加入20μL MTS-PMS溶液后避光孵育3h后,使用酶标仪测得OD492值,评价Scyreptin的细胞毒性(参见图9)。
结果如图9所示,在96μM浓度条件下,拟穴青蟹抗真菌多肽Scyreptin与ZF4细胞、HEK-293T细胞共孵24h后,与对照组相比实验组细胞存活率均在95%以上,说明拟穴青蟹抗菌多肽Scyreptin无细胞毒性。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (9)
1.一种拟穴青蟹新型抗菌肽Scyreptin,其特征在于:其氨基酸序列如SEQ ID NO.01所示。
2.权利要求1所述的拟穴青蟹新型抗菌肽Scyreptin在制备抗细菌组合物中的应用。
3.如权利要求2所述的应用,其特征在于:所述拟穴青蟹新型抗菌肽Scyreptin对金黄色葡萄球菌、粪肠球菌、屎肠球菌、大肠埃希氏菌、铜绿假单胞菌和鲍曼不动杆菌具有抑制和杀灭作用。
4.一种抗细菌组合物,其特征在于:其有效成分包括权利要求1所述的拟穴青蟹新型抗菌肽Scyreptin。
5.如权利要求4所述的一种抗细菌组合物,其特征在于:其有效成分为权利要求1所述的拟穴青蟹新型抗菌肽Scyreptin。
6.权利要求1所述的拟穴青蟹新型抗菌肽Scyreptin在制备抗真菌组合物中的应用。
7.如权利要求6所述的应用,其特征在于:所述拟穴青蟹新型抗菌肽Scyreptin对新型隐球菌、禾谷镰孢、尖孢镰孢、赭曲霉、黑曲霉和腐皮镰孢具有抑制和杀灭作用。
8.一种抗真菌组合物,其特征在于:其有效成分包括权利要求1所述的拟穴青蟹新型抗菌肽Scyreptin。
9.如权利要求8所述的一种抗真菌组合物,其特征在于:其有效成分为权利要求1所述的拟穴青蟹新型抗菌肽Scyreptin。
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