CN114014923B - 拟穴青蟹抗菌多肽Sp-LECin及其应用 - Google Patents
拟穴青蟹抗菌多肽Sp-LECin及其应用 Download PDFInfo
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Abstract
本发明公开了拟穴青蟹抗菌多肽Sp‑LECin及其应用。该抗菌多肽Sp‑LECin的分子式为C117H185N29O26S1,其氨基酸序列如SEQ ID NO.1所示。本发明的拟穴青蟹抗菌多肽Sp‑LECin具有抗细菌活性及抗真菌活性,抗菌效果好,抗菌谱广,杀菌速率快,其来源于甲壳动物,既可以应用于水产饲料添加剂,也可以研制成为防腐剂、抗菌防霉剂和抗菌药物等,具有广泛的应用前景。
Description
技术领域
本发明属于海洋分子生物学技术领域,具体涉及多肽及其应用。
背景技术
由于抗生素在医疗卫生和养殖业等领域的滥用,全球性抗生素污染问题日趋严峻,亟需开发新型抗耐药性细菌感染、可替代抗生素的治疗药物。抗菌肽(AntimicrobialPeptide,AMP)是存在于各种生物体中的一种重要的先天性免疫因子,具有抗细菌、真菌、病毒和寄生虫等免疫特性,因其不同于传统抗生素,不易产生耐药性,被科学家誉为“天然抗生素”。根据携带电荷性质的不同分为阳离子抗菌肽和阴离子抗菌肽,大多数抗菌肽属于阳离子抗菌肽,通常富含赖氨酸和精氨酸等带有正电荷及疏水性的氨基酸残基。抗菌肽在生物体内不仅具有抗菌功能,还能通过不同的途径调节机体的免疫反应,是机体先天性免疫系统中重要的组成部分。
目前抗菌肽库中收录的抗菌肽多数是基于已报道的抗菌肽序列、EST片段或结构域等分析获得的,新型抗菌肽的发现仍是少数。海洋动物由于其生存在复杂甚至极端的海洋环境,且以无脊椎动物居多,主要依靠先天性免疫系统来维持健康生存,抗菌肽是其先天性免疫的重要因子。甲壳动物等无脊椎动物缺乏获得性免疫,抗菌肽等免疫效应分子在外界病原微生物入侵时能够在体内迅速合成并释放以抵御感染,在无脊椎动物的先天性免疫系统中发挥重要的作用。
拟穴青蟹(Scylla paramamosain),简称青蟹,是我国东南沿海重要的养殖品种,具有较高的营养价值和经济价值。目前在青蟹中已经发现并成功分离了多种具有广谱抗微生物活性的抗菌肽,如ALFs、crustins、源于组蛋白H2A的Sphistin、在血细胞中高表达的SpHyastatin以及在青蟹生殖免疫中起重要作用的scygonadin和SCY2等。
发明内容
本发明的目的在于克服现有技术的不足之处,提供了拟穴青蟹抗菌多肽Sp-LECin及其应用。基于前期建立的人工感染青蟹转录组数据库,从中获得一条抗菌多肽,将其命名为Sp-LECin。该多肽Sp-LECin具有抗细菌及抗真菌活性,抗菌效果好,抗菌谱广,杀菌速率快,其来源于甲壳动物,既可以应用于水产饲料添加剂,也可以研制成为防腐剂、抗菌防霉剂和抗菌药物等,具有广泛的应用前景。
本发明解决其技术问题所采用的技术方案之一是:
一种拟穴青蟹抗菌多肽Sp-LECin,其氨基酸序列如SEQ ID NO.1所示,具体为:Gly-Cys-Val-Phe-Leu-Leu-Pro-Ala-Lys-Pro-His-Asn-Tyr-Lys-Lys-Val-Phe-Leu-Ser-Lys-Gly-Val。
所述拟穴青蟹抗菌多肽Sp-LECin由22个氨基酸组成,分子式为C117H185N29O26S1,分子量为2445.99道尔顿,其中含有4个带正电的氨基酸残基和0个带负电的氨基酸残基,根据氨基酸残基电荷预测该抗菌肽等电点为9.87,亲水性平均系数为0.25,是一种带有正电荷的阳离子多肽。
所述拟穴青蟹抗菌多肽Sp-LECin可以以固体(如粉末等)、液体(如水溶液等)等多种形式应用。
本发明解决其技术问题所采用的技术方案之二是:
上述的拟穴青蟹抗菌多肽Sp-LECin在制备抗菌剂中的应用。
本发明解决其技术问题所采用的技术方案之三是:
一种抗菌剂,所述抗菌剂包括氨基酸序列如SEQ ID NO.1所示的拟穴青蟹抗菌多肽Sp-LECin。
本发明所述的“抗菌”指的是抑制或杀灭革兰氏阳性菌、革兰氏阴性菌或霉菌中的至少一种。所述革兰氏阳性菌包括金黄色葡萄球菌、耐药金黄色葡萄球菌、表皮葡萄球菌、李斯特菌、屎肠球菌或粪肠球菌中的至少一种。所述革兰氏阴性菌包括铜绿假单胞菌、大肠埃希氏菌、施氏假单胞菌、弗式志贺氏菌、荧光假单胞菌、鲍曼不动杆菌或耐药鲍曼不动杆菌中的至少一种。所述霉菌包括黑曲霉或尖孢镰孢中的至少一种;用于抑制或杀灭霉菌时产生防霉作用,也可称为防霉剂。本发明的拟穴青蟹抗菌多肽Sp-LECin可以用于杀灭屎肠球菌、鲍曼不动杆菌、临床耐药鲍曼不动杆菌等。
本发明所述的“抗菌剂”可以是药物形式的抗菌剂,即作为抗菌药物,也可以是非药物形式的抗菌剂,例如作为消毒产品、或作为纺织品、塑料等产品的添加剂等。
本发明解决其技术问题所采用的技术方案之四是:
上述的拟穴青蟹抗菌多肽Sp-LECin在制备防腐剂中的应用。
本发明解决其技术问题所采用的技术方案之五是:
一种防腐剂,所述防腐剂包括氨基酸序列如SEQ ID NO.1所示的拟穴青蟹抗菌多肽Sp-LECin。
本发明的Sp-LECin具有抑制或杀灭多种微生物如革兰氏阳性菌、革兰氏阴性菌、霉菌等的抗菌防霉效果,因而也可作为防腐剂,防止由微生物引起的腐败变质等,可用于食品、医疗品、化妆品等领域。
本发明解决其技术问题所采用的技术方案之六是:
上述的拟穴青蟹抗菌多肽Sp-LECin在制备水产饲料添加剂中的应用。
本发明解决其技术问题所采用的技术方案之七是:
一种水产饲料添加剂,所述水产饲料添加剂包括氨基酸序列如SEQ ID NO.1所示的拟穴青蟹抗菌多肽Sp-LECin。
本发明的拟穴青蟹抗菌多肽Sp-LECin来源于甲壳动物且安全性好,可作为水产饲料添加剂使用,用于水产养殖中的抗菌。
本发明所涉及的设备、试剂、工艺、参数等,除有特别说明外,均为常规设备、试剂、工艺、参数等,不再作实施例。
本发明所列举的所有范围包括该范围内的所有点值。
本发明所述“大约”、“约”或“左右”等指的是所述范围或数值的±20%范围内。
本技术方案与背景技术相比,它具有如下优点:
1、本发明的拟穴青蟹抗菌多肽Sp-LECin对革兰氏阳性菌、革兰氏阴性菌、霉菌均有抗菌效果。此外,Sp-LECin对正常哺乳动物细胞如人正常肝细胞和人肾上皮细胞不具有细胞毒性作用。
2、本发明的拟穴青蟹抗菌多肽Sp-LECin,具有较强的抗细菌活性及抗真菌活性,抗菌效果好,抗菌谱广,杀菌速率快,其来源于甲壳动物,既可以应用于水产饲料添加剂,也可以研制成为防腐剂、抗菌防霉剂和抗菌药物等,具有广泛的应用前景。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为拟穴青蟹抗菌多肽Sp-LECin对屎肠球菌(A)和鲍曼不动杆菌(B)的杀菌动力学图;在图1中,横坐标为时间(min),纵坐标为杀菌指数(%)。
图2为拟穴青蟹抗菌多肽Sp-LECin对铜绿假单胞菌(A)和鲍曼不动杆菌(B)抗菌活性热稳定性图;在图2中,横坐标为时间(h),纵坐标为OD600值。
图3为拟穴青蟹抗菌多肽Sp-LECin抑制尖孢镰孢菌孢子萌发实验图:在图3中,Sp-LECin终浓度为A:0μM;B:24μM;C:48μM。
图4为拟穴青蟹抗菌多肽Sp-LECin抑制黑曲霉孢子萌发实验图:在图4中,Sp-LECin终浓度为A:0μM;B:24μM;C:48μM。
图5为拟穴青蟹抗菌多肽Sp-LECin与屎肠球菌作用后扫描电镜观察图;在图5中,A:屎肠球菌;B:屎肠球菌+24μM Sp-LECin。
图6为拟穴青蟹抗菌多肽Sp-LECin与鲍曼不动杆菌作用后扫描电镜观察图;在图6中,A:鲍曼不动杆菌;B:鲍曼不动杆菌+24μM Sp-LECin。
图7为MTS-PMS法检测拟穴青蟹抗菌多肽Sp-LECin对人正常肝细胞L02(A)和人肾上皮细胞293T(B)细胞毒性实验图;在图7中,横坐标为Sp-LECin蛋白浓度(μM),纵坐标为细胞增殖率(%)。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
实施例1拟穴青蟹抗菌多肽Sp-LECin的制备
所述拟穴青蟹抗菌多肽Sp-LECin的氨基酸序列为:
Gly-Cys-Val-Phe-Leu-Leu-Pro-Ala-Lys-Pro-His-Asn-Tyr-Lys-Lys-Val-Phe-Leu-Ser-Lys-Gly-Val(SEQ ID NO.1)
采用现有的固相化学合成的方法即可得到纯度达95%以上拟穴青蟹抗菌多肽Sp-LECin。本实施例中的拟穴青蟹抗菌多肽Sp-LECin委托金斯瑞(江苏)有限公司以固相合成方法合成获得,并提供多肽分子量、HPLC等检测信息。
抗菌多肽Sp-LECin理化参数如表1所示。
表1抗菌多肽Sp-LECin的理化参数
由表1可知Sp-LECin分子量小、稳定性较好,是一种带有正电荷的阳离子多肽。
实施例2拟穴青蟹抗菌多肽Sp-LECin的最小抑菌浓度(MIC:Minimum InhibitionConcentration)和最小杀菌浓度(MBC:Minimum Bactericidal Concentration)的测定
本实施例中所涉及到的菌株有:金黄色葡萄球菌(Staphylococcus aureus)、表皮葡萄球菌(Staphylococcus epidermidis)、屎肠球菌(Enterococcus faecium)、粪肠球菌(Enterococcus faecalis)、单核细胞增生李斯特菌(Listeria monocytogenes)、大肠埃希氏菌(Escherichia coli)、铜绿假单胞菌(Pseudomonas aeruginosa)、施氏假单胞菌(Pseudomonas stutzeri)、弗氏志贺氏菌(Shigella flexneri)、荧光假单胞菌(Pseudomonas fluorescens)、鲍曼不动杆菌(Acinetobacter baumannii)、尖孢镰孢(Fusarium oxysporum)、黑曲霉(Aspergillus niger)。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)将保种的金黄色葡萄球菌、耐药金黄色葡萄球菌、表皮葡萄球菌、单核细胞增生李斯特菌、屎肠球菌、粪肠球菌、大肠埃希氏菌、铜绿假单胞菌、施氏假单胞菌、弗氏志贺氏菌、荧光假单胞菌、鲍曼不动杆菌和耐药鲍曼不动杆菌涂布于营养肉汤平板上,在各适宜温度倒置培养18~24h;尖孢镰孢、黑曲霉涂布于马铃薯葡萄糖平板上,于28℃倒置培养1~7d。
(2)从各平板上挑取菌落接种于相应的培养基斜面上,细菌继续培养18~24h,霉菌继续培养1~7d。用10mM磷酸钠缓冲液(pH=7.4)将细菌和酵母真菌从斜面上冲下,调整菌悬液浓度。用MH液体培养基和磷酸钠缓冲液混合液稀释细菌,使得菌体的最终浓度为3.3×104CFU/mL。用10mM磷酸钠缓冲液(pH=7.4)将霉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孢子,利用血球计数板在光学显微镜下对孢子计数,调整孢子浓度,使得霉菌孢子最终浓度为5×104个/mL。
(3)将已合成的Sp-LECin粉末分别用灭菌MiliiQ水溶解,经过0.22μm滤膜过滤后,倍比稀释蛋白浓度至3μM、6μM、12μM、24μM、48μM、96μM,置于冰上备用。
(4)在96孔细胞培养板上,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
(5)将96孔细胞培养板置于28℃培养箱中,培养1~2d,观察待测实验组中MIC结果;将待测实验组吹打混匀后,吸取适量的菌液滴于相应固体培养基平板上,于适宜温度倒置培养1~2d,观察MBC结果。
拟穴青蟹抗菌肽Sp-LECin的MIC、MBC观察结果如表2所示:
表2拟穴青蟹抗菌肽Sp-LECin的MIC、MBC观察结果
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),用a~b表示。a:平板可见菌落生长的最高蛋白浓度;b:平板未见菌落生长的最低蛋白浓度
实施例3拟穴青蟹抗菌多肽Sp-LECin杀菌动力学曲线
选取屎肠球菌、鲍曼不动杆菌作为待测菌,对拟穴青蟹抗菌多肽Sp-LECin的杀菌动力学进行测定。
具体方法与实施例2中所述的抗菌活性测定类似。调整Sp-LECin终浓度至1倍MBC(屎肠球菌:24μM;鲍曼不动杆菌:12μM)。在共孵后10min、20min、30min、45min、60min、90min、120min,将96孔细胞培养板取空白对照组、阴性对照组、待测实验组混匀,吸取6μL屎肠球菌菌悬液稀释至600μL DPBS中,混匀后吸取40μL涂布至营养肉汤平板上,37℃倒置培养1~2d,记录屎肠球菌单克隆数量,计算杀菌指数。在共孵后5min、10min、20min、30min、45min、60min,将96孔细胞培养板取空白对照组、阴性对照组、待测实验组混匀,吸取40μL鲍曼不动杆菌菌悬液涂布至营养肉汤平板上,37℃倒置培养1~2d,记录鲍曼不动杆菌单克隆数量,计算杀菌指数。
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)。由图1可知拟穴青蟹抗菌多肽Sp-LECin对屎肠球菌、鲍曼不动杆菌具有良好的杀菌性能。
实施例4拟穴青蟹抗菌肽Sp-LECin抗菌活性热稳定性
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Sp-LECin的抗菌活性热稳定性进行测定。
具体方法与实施例2中所述的抗菌活性测定类似。调整Sp-LECin终浓度至1倍MBC(铜绿假单胞菌:24μM,鲍曼不动杆菌:12μM),分别在100℃沸水中水浴10min、20min、30min后置于冰上备用。将Sp-LECin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(参见图2)。由图2可知拟穴青蟹抗菌多肽Sp-LECin的热稳定性良好。
实施例5拟穴青蟹抗菌多肽Sp-LECin作用后霉菌孢子萌发显微镜观察
选取尖孢镰孢、黑曲霉作为待测菌,观察拟穴青蟹抗菌肽Sp-LECin对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类似。调整Sp-LECin蛋白浓度为24μM、48μM,置于冰上备用;调整各霉菌孢子最终浓度为5×104个/mL。将等体积各浓度Sp-LECin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3~4)。由图3~4可知拟穴青蟹抗菌多肽Sp-LECin可以抑制尖孢镰孢、黑曲霉的孢子萌发。
实施例6拟穴青蟹抗菌多肽Sp-LECin与细菌作用后的扫描电镜观察
选取屎肠球菌、鲍曼不动杆菌作为待测菌株,扫描电镜样品的制备按以下步骤进行:
(1)如实施例2所述制备屎肠球菌、鲍曼不动杆菌悬液(OD600=0.4)冰上放置备用。
(2)用灭菌纯水溶解合成肽Sp-LECin,并调整蛋白浓度为48μM,冰上放置备用。
(3)菌悬液和蛋白等体积混合后在适宜温度孵育适宜时间。其中屎肠球菌菌悬液与24μM Sp-LECin于37℃孵育60min;鲍曼不动杆菌菌悬液与24μM Sp-LECin于37℃孵育60min。
(4)加入等体积戊二醛固定液,4℃固定2h后,1000g离心10min。
(5)去尽上清,PBS清洗一次,1000g离心10min。
(6)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。
(7)PBS清洗一次。
(8)30%-50%-70%-80%-90%-95%-100%-100%(v/v)乙醇逐级脱水,每级脱水15min。
(9)用叔丁醇置换乙醇,4℃过夜冷冻;或者醋酸异戊酯置换乙醇后待干燥。
(10)叔丁醇脱水的用冷冻干燥仪冷冻干燥;醋酸异戊酯脱水的用临界点干燥仪干燥。
(11)喷金后用扫描电子显微镜观察及拍照(参见图5~6)。由图5~6可知拟穴青蟹抗菌多肽Sp-LECin可使屎肠球菌、鲍曼不动杆菌菌体破裂,可能是其发挥抗菌作用的机理之一。
实施例7拟穴青蟹抗菌多肽Sp-LECin细胞毒性测定
选取人肾上皮细胞(293T)和人正常肝细胞(L02),对拟穴青蟹抗菌肽Sp-LECin细胞毒性进行测定。
(1)收集生长状态良好的拟穴青蟹血细胞、人肾上皮细胞和人正常肝细胞,调整细胞浓度为1×105个/mL,将细胞均匀吹散,在96孔细胞培养板中每孔加入100μL细胞悬液,置于适宜温度培养箱培养至80%以上细胞贴壁。
(2)小心吸出培养基,加入含有不同浓度(0μM、3μM、6μM、12μM、24μM、48μM)Sp-LECin的培养基,置于适宜温度培养箱培养24h。
(3)加入20μL MTS-PMS溶液后避光孵育2h后,使用酶标仪测得OD492值,评价Sp-LECin的细胞毒性(参见图7)。由图7可知拟穴青蟹抗菌多肽Sp-LECin对人肾上皮细胞(293T)和人正常肝细胞(L02)无明显的细胞毒性,安全性较好。
以上所述,仅为本发明较佳实施例而已,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。
序列表
<110> 厦门大学
<120> 拟穴青蟹抗菌多肽Sp-LECin及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> 拟穴青蟹(Scylla paramamosain)
<400> 1
Gly Cys Val Phe Leu Leu Pro Ala Lys Pro His Asn Tyr Lys Lys Val
1 5 10 15
Phe Leu Ser Lys Gly Val
20
Claims (10)
1.一种拟穴青蟹抗菌多肽Sp-LECin,其特征在于:其氨基酸序列如SEQ ID NO.1所示。
2.权利要求1所述的拟穴青蟹抗菌多肽Sp-LECin在制备抗菌剂中的应用,所述抗菌剂抑制或杀灭革兰氏阳性菌、革兰氏阴性菌或霉菌中的至少一种;所述革兰氏阳性菌包括金黄色葡萄球菌、表皮葡萄球菌、李斯特菌、屎肠球菌或粪肠球菌中的至少一种;所述革兰氏阴性菌包括铜绿假单胞菌、大肠埃希氏菌、施氏假单胞菌、弗式志贺氏菌、荧光假单胞菌或鲍曼不动杆菌中的至少一种;所述霉菌包括黑曲霉或尖孢镰孢中的至少一种。
3.一种抗菌剂,其特征在于:所述抗菌剂包括氨基酸序列如SEQ ID NO.1所示的拟穴青蟹抗菌多肽Sp-LECin。
4.根据权利要求3所述的抗菌剂,其特征在于:所述抗菌指的是抑制或杀灭革兰氏阳性菌、革兰氏阴性菌或霉菌中的至少一种。
5.根据权利要求4所述的抗菌剂,其特征在于:所述革兰氏阳性菌包括金黄色葡萄球菌、表皮葡萄球菌、李斯特菌、屎肠球菌或粪肠球菌中的至少一种;所述革兰氏阴性菌包括铜绿假单胞菌、大肠埃希氏菌、施氏假单胞菌、弗式志贺氏菌、荧光假单胞菌或鲍曼不动杆菌中的至少一种。
6.根据权利要求4所述的抗菌剂,其特征在于:所述霉菌包括黑曲霉或尖孢镰孢中的至少一种。
7.权利要求1所述的拟穴青蟹抗菌多肽Sp-LECin在制备防腐剂中的应用。
8.一种防腐剂,其特征在于:所述防腐剂包括氨基酸序列如SEQ ID NO.1所示的拟穴青蟹抗菌多肽Sp-LECin。
9.权利要求1所述的拟穴青蟹抗菌多肽Sp-LECin在制备水产饲料添加剂中的应用。
10.一种水产饲料添加剂,其特征在于:所述水产饲料添加剂包括氨基酸序列如SEQ IDNO.1所示的拟穴青蟹抗菌多肽Sp-LECin。
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