CN114957430B - 一种拟穴青蟹抗菌多肽Sparepcin173-194及其应用 - Google Patents
一种拟穴青蟹抗菌多肽Sparepcin173-194及其应用 Download PDFInfo
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Abstract
本发明公开了一种拟穴青蟹抗菌多肽Sparepcin173‑194及其应用,其分子式C107H183N39O25S2,其氨基酸序列如SEQ ID NO.01所示。本发明具有广谱的抗细菌和抗霉菌活性,杀菌效率快,无细胞毒性,安全性高,可作为有效成分,应用于抑菌防霉剂和水产饲料,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,具体涉及一种拟穴青蟹抗菌多肽Sparepcin173-194及其应用。
背景技术
拟穴青蟹(Scylla paramamosain)简称青蟹,属于热带亚热带种类,分布广泛。因味道鲜美、营养价值丰富,目前已成为我国重要经济海水养殖蟹类之一。随着青蟹养殖规模逐年增加,养殖密度的增大,导致病害频繁爆发。为控制水产养殖中的病害问题,抗生素大量投入使用,但也带来了细菌耐药性、药物残留、环境污染等一系列问题。研发可有效抗细菌、真菌的药物或活性物质也势在必行。海洋无脊椎动物拟穴青蟹因其自身是开放系统且有多次蜕皮的脆弱发育阶段,易受微生物侵染,其主要依靠先天免疫因子来抵御外界环境胁迫,是开发高效抗病原微生物的新型抗菌肽的重要来源。目前拟穴青蟹中已发现的抗菌肽包括ALFs、Crustins、Scygonadin、SpPR-AMP1、SpHyastatin、arasin-likeSp、GRPSp、Sphistin、Scyreprocin、Sparamosin等。
抗菌肽(antimicrobial peptides,AMPs)是广泛存在于自然界中的一类小分子多肽,是先天性免疫系统的重要组成部分。作为重要先天性免疫因子对多种病原菌具有较强的抑杀作用且不易产生耐药性,并具有良好的热稳定性,在抵御外来病原入侵和免疫调节中发挥关键作用。相对于抗生素,抗菌肽具有抗菌谱广、不易产生耐药性,无有害残留等特点,被视为抗生素的有效替代品,在医药、养殖业、食品等多个领域都具有重要的潜在应用价值。因此,获得安全有效的新型抗菌肽具有重要的科学意义和应用价值。
发明内容
本发明目的在于提供一种拟穴青蟹抗菌多肽Sparepcin173-194。
本发明的另一目的在于提供上述拟穴青蟹抗菌多肽Sparepcin173-194的应用。
一种拟穴青蟹抗菌多肽Sparepcin173-194,其分子式为C107H183N39O25S2,其氨基酸序列如SEQ ID NO.01所示。
上述拟穴青蟹抗菌多肽Sparepcin173-194在制备抗细菌组合物中的应用。
一种抗细菌组合物,其有效成分包括上述拟穴青蟹抗菌多肽Sparepcin173-194。
在本发明的一个优选实施方案中,其对金黄色葡萄球菌、谷氨酸棒状杆菌、溶壁微球菌、屎肠球菌、粪肠球菌、鲍曼不动杆菌、铜绿假单胞菌、施氏假单胞菌、荧光假单胞菌和哈氏弧菌具有抑制或杀灭作用。
上述拟穴青蟹抗菌多肽Sparepcin173-194在制备防霉菌组合物中的应用。
一种防霉菌组合物,其有效成分包括上述拟穴青蟹抗菌多肽Sparepcin173-194。
在本发明的一个优选实施方案中,其对黑曲霉、烟曲霉和赭曲霉具有抑制或杀灭作用。
上述拟穴青蟹抗菌多肽Sparepcin173-194在制备水产饲料添加剂中的应用。
一种水产饲料添加剂,其有效成分包括上述拟穴青蟹抗菌多肽Sparepcin173-194。
在本发明的一个优选实施方案中,其对荧光假单胞菌、哈氏弧菌和霉菌具有抑制或杀灭作用。
本发明的有益效果是:
1、本发明的拟穴青蟹抗菌多肽Sparepcin173-194由22个氨基酸组成,分子式为C107H183N39O25S2,分子量为2480.00道尔顿,理论等电点为12.0,亲水性平均系数为0.141,是一种带有正电荷的阳离子多肽。
2、本发明的拟穴青蟹抗菌多肽Sparepcin173-194具有广谱抗菌活性,同时对正常哺乳动物细胞人肾上皮细胞和拟穴青蟹血细胞在高浓度下无细胞毒性。
3、本发明的拟穴青蟹抗菌多肽Sparepcin173-194抗菌效果强,杀菌效率快,其来源于甲壳动物,无细胞毒性,安全性高,可作为有效成分,应用于抑菌防霉剂和水产饲料,具有广泛的应用前景。
附图说明
图1为本发明实施例3中拟穴青蟹抗菌多肽Sparepcin173-194对金黄色葡萄球菌和鲍曼不动杆菌的杀菌动力学图,其中,横坐标为时间(min),纵坐标为杀菌指数(%)。
图2为本发明实施例4中拟穴青蟹抗菌多肽Sparepcin173-194对金黄色葡萄球菌和鲍曼不动杆菌抗菌活性的热稳定性图,其中,横坐标为时间(h),纵坐标为OD600值。
图3为本发明实施例5中拟穴青蟹抗菌多肽Sparepcin173-194处理金黄色葡萄球菌和鲍曼不动杆菌的扫描电镜观察图。其中,A为金黄色葡萄球菌,B为金黄色葡萄球菌+96μMSparepcin173-194,C为鲍曼不动杆菌,D为鲍曼不动杆菌+96μM Sparepcin173-194。
图4为本发明实施例6中拟穴青蟹抗菌多肽Sparepcin173-194抑制黑曲霉、烟曲霉、赭曲霉(从左至右)孢子萌发的实验图,其中,Sparepcin173-194从上至下的终浓度分别为:0μM、3μM、6μM、12μM和24μM。
图5为本发明实施例7中MTS-PMS法检测拟穴青蟹抗菌多肽Sparepcin173-194的细胞毒性实验图;其中,横坐标为抗菌多肽Sparepcin173-194的浓度(μM),纵坐标为细胞增殖率(%)。
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拟穴青蟹抗菌多肽Sparepcin173-194的制备
本发明拟穴青蟹抗菌多肽Sparepcin173-194的氨基酸序列为:
Arg-Arg-Gly-Leu-Met-Val-Arg-Arg-His-Asn-Val-Val-Ser-Ala-Ala-Val-Ala-Gly-Lys-Trp-Leu-Cys(SEQ ID NO.01)
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Sparepcin173-194。本实施例中的拟穴青蟹抗菌多肽Sparepcin173-194委托金斯瑞生物科技有限公司以固相合成方法合成获得并提供多肽分子量和HPLC等检测信息。Sparepcin173-194理化参数如表1所示:
表1Sparepcin173-194的理化参数
由表1可知Sparepcin173-194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Sparepcin173-194最小抑菌浓度(minimum inhibitionconcentration,MIC)和最小杀菌浓度(minimum bactericidal concentration,MBC)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)、溶壁微球菌(Micrococcus lysodeikticus)、屎肠球菌(Enterococcus faecium)、粪肠球菌(Enterococcus faecalis)、谷氨酸棒状杆菌(Corynebacterium glutamicum)、大肠埃希氏菌(Escherichia coli)、鲍曼不动杆菌(Acinetobacter baumannii)、铜绿假单胞菌(Pseudomonas Aeruginosa)、施氏假单胞菌(Pseudomonas stutzeri)、嗜水气单胞菌(Aeromonas hydrophila)、荧光假单胞菌(Pseudomonas fluorescens)、哈氏弧菌(Vibrioharveyi)、解藻弧菌(Vibrio alginolyticus)、黑曲霉(Aspergillus niger)、烟曲霉(Aspergillus fumigatus)和赭曲霉(Aspergillus ochraceus)。以上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、溶壁微球菌、屎肠球菌、粪肠球菌、谷氨酸棒状杆菌、大肠埃希氏菌、鲍曼不动杆菌、铜绿假单胞菌、施氏假单胞菌、嗜水气单胞菌、荧光假单胞菌涂布于营养肉汤平板上,在各适宜温度倒置培养18-24h;将哈氏弧菌和解藻弧菌涂布于2216平板上,于28℃倒置培养18-24h;黑曲霉、烟曲霉和赭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。用10mM磷酸钠缓冲液(pH=7.4)将细菌从斜面上冲下,调整菌悬液浓度。用MH液体培养基稀释细菌,用海水培养基稀释弧菌,使得菌体的最终浓度为5×105CFU/mL。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,利用血球计数板在光学显微镜下对孢子计数,调整孢子浓度,使得真菌孢子最终浓度为5×104个/mL。
(3)将已合成的Sparepcin173-194粉末分别用灭菌MilliQ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MilliQ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。
拟穴青蟹抗菌多肽Sparepcin173-194具有广谱抗菌活性,其中对谷氨酸棒状杆菌、溶壁微球菌、施氏假单胞菌、黑曲霉具有较强的杀菌效果,最小杀菌浓度<24μM;对烟曲霉和赭曲霉也有较好的抑杀效果,最小杀菌浓度为12~24μM;对金黄色葡萄球菌、粪肠球菌、屎肠球菌、鲍曼不动杆菌、铜绿假单胞菌、荧光假单胞菌、哈氏弧菌、烟曲霉具有良好的杀菌作用,最小杀菌浓度为<48μM。结果如表2所示:
表2拟穴青蟹抗菌多肽Sparepcin173-194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),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Sparepcin173-194杀菌动力学曲线
选取金黄色葡萄球菌和鲍曼不动杆菌作为待测菌,对拟穴青蟹抗菌多肽Sparepcin173-194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类似。调整Sparepcin173-194终浓度至杀菌浓度(金黄色葡萄球菌:48μM;鲍曼不动杆菌:48μM),Sparepcin173-194与被测细菌共孵一定的时间后,取6μL菌悬液稀释后取适量涂布于营养肉汤平板上,37℃培养1-2d进行克隆计数。灭菌MilliQ水与菌悬液共孵0h的样品作为阳性对照,取6μL经同样稀释倍数涂布于营养肉汤平板上的克隆数定义为100%,杀菌指数用经一定时间孵育的实验组的克隆数相对于阳性对照克隆数的百分比表示,如图1所示,Sparepcin173-194与金黄色葡萄球菌共孵30min可达到100%杀菌效果,与鲍曼不动杆菌仅共孵2min即可达到100%杀菌效果。
实施例4拟穴青蟹抗菌多肽Sparepcin173-194抗菌活性热稳定性
选取金黄色葡萄球菌和鲍曼不动杆菌作为待测菌,对拟穴青蟹抗菌多肽Sparepcin173-194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Sparepcin173-194终浓度至杀菌浓度(金黄色葡萄球菌:48μM;施鲍曼不动杆菌:48μM),将抗菌多肽Sparepcin173-194在100℃沸水中加热10min、20min、30min,置于冰上备用。将Sparepcin173-194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2所示,Sparepcin173-194经沸水浴30min后仍保留良好的抗菌活性。
实施例5扫描电镜观察Sparepcin173-194处理后细菌的形态结构变化
选取金黄色葡萄球菌和和鲍曼不动杆菌为待测菌株,扫描电镜样品的制备按以下步骤进行:
(1)活化菌种,待克隆长至合适大小,随机挑取3-5个克隆至对应的营养肉汤液体培养基,摇至对数生长期,测OD,离心去上清,用MH培养基重悬菌体,调至OD为0.1,放置于冰上备用。
(2)用无菌MilliQ溶解多肽粉末,并用0.22μM滤膜过滤,调整多肽终浓度为2倍MBC。分别设置实验组和对照组,混匀后于37℃共同孵育1h。离心去上清,用PBS清洗一次后收集菌体。
(2)用2.5%戊二醛重悬收集的菌体,4℃冰箱固定1.5h或更长时间。固定后的菌体用PBS洗三次后,制成高浓度悬液,滴至已切割好的载玻片上,黏附30min,用滤纸吸干后进行乙醇梯度脱水。
(3)30%-50%-70%-80%-90%-95%-100%-100%(v/v)乙醇逐级脱水,每级脱水10-15min。
(4)临界点干燥后,10mA电流,喷金60s;扫描电镜观察并拍片记录。
结果如图3所示,对照组细菌形态正常,结构完整,表面光滑无皱缩。经抗菌多肽Sparepcin173-194处理后的细菌,表面出现塌陷及褶皱,细菌破裂,胞内的内容物外泄。
实施例6拟穴青蟹抗菌多肽Sparepcin173-194作用后真菌孢子萌发的光学显微镜观察
选取烟曲霉、赭曲霉和黑曲霉作为待测菌,观察拟穴青蟹抗菌多肽Sparepcin173-194对真菌孢子萌发的影响。
具体方法如实施例2中所述的抗菌活性测定类似。调整Sparepcin173-194浓度为6μM、12μM、24μM和48μM,置于冰上备用;调整各真菌孢子最终浓度为5×104个/mL。将等体积各浓度Sparepcin173-194与各真菌孢子于96孔细胞培养板混匀,并设置空白对照组,置于28℃培养箱中,静置培养24h,在光学显微镜下观察真菌孢子萌发情况。如图4所示,Sparepcin173-194在浓度为24μM时可抑制黑曲霉、烟曲霉和赭曲霉孢子的萌发。
实施例7拟穴青蟹抗菌多肽Sparepcin173-194细胞毒性测定
选取人肾上皮细胞(HEK-293T)和拟穴青蟹正常血细胞,对拟穴青蟹抗菌肽Sparepcin173-194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,细胞计数板计数并用培养基将细胞浓度调至1×105个/mL,将细胞悬液混匀,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100μL用培养基梯度稀释抗菌多肽至浓度为3、6、12、24、48、96μM,置于适宜温度培养箱培养24h。
(3)每孔加入20μL MTS-PMS溶液后避光孵育2h后,用酶标仪测得OD492值,评价Sparepcin173-194的细胞毒性。
结果如图5所示,浓度高达96μM的Sparepcin173-194对人肾上皮细胞和拟穴青蟹血细胞不具毒性作用。
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。
序列表
<110> 厦门大学
厦门海嘉成生物科技有限公司
<120> 一种拟穴青蟹抗菌多肽Sparepcin173-194及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> 擬穴青蟹(Scylla paramamosain)
<400> 1
Ala Ala Gly Leu Met Val Ala Ala His Ala Val Val Ser Ala Ala Val
1 5 10 15
Ala Gly Leu Thr Leu Cys
20
Claims (8)
1.一种拟穴青蟹抗菌多肽Sparepcin173-194,其特征在于:其分子式为C107H183N39O25S2,其氨基酸序列如SEQ ID NO.01所示。
2.权利要求1所述的拟穴青蟹抗菌多肽Sparepcin173-194在制备抗细菌组合物中的应用,其特征在于:所述拟穴青蟹抗菌多肽Sparepcin173-194对金黄色葡萄球菌、谷氨酸棒状杆菌、溶壁微球菌、屎肠球菌、粪肠球菌、鲍曼不动杆菌、铜绿假单胞菌、施氏假单胞菌、荧光假单胞菌和哈氏弧菌具有抑制或杀灭作用。
3.一种抗细菌组合物,其特征在于:其有效成分包括权利要求1所述的拟穴青蟹抗菌多肽Sparepcin173-194,该拟穴青蟹抗菌多肽Sparepcin173-194对金黄色葡萄球菌、谷氨酸棒状杆菌、溶壁微球菌、屎肠球菌、粪肠球菌、鲍曼不动杆菌、铜绿假单胞菌、施氏假单胞菌、荧光假单胞菌和哈氏弧菌具有抑制或杀灭作用。
4.权利要求1所述的拟穴青蟹抗菌多肽Sparepcin173-194在制备防霉菌组合物中的应用,其特征在于:所述拟穴青蟹抗菌多肽Sparepcin173-194对黑曲霉、烟曲霉和赭曲霉具有抑制或杀灭作用。
5.一种防霉菌组合物,其特征在于:其有效成分包括权利要求1所述的拟穴青蟹抗菌多肽Sparepcin173-194,该拟穴青蟹抗菌多肽Sparepcin173-194对黑曲霉、烟曲霉和赭曲霉具有抑制或杀灭作用。
6.权利要求1所述的拟穴青蟹抗菌多肽Sparepcin173-194在制备水产饲料添加剂中的应用。
7.一种水产饲料添加剂,其特征在于:其有效成分包括权利要求1所述的拟穴青蟹抗菌多肽Sparepcin173-194。
8.如权利要求7所述的一种水产饲料添加剂,其特征在于:其对荧光假单胞菌、哈氏弧菌和霉菌具有抑制或杀灭作用。
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