CN114409754B - 一种微电压方法协同抗菌肽pv-q5及其用途 - Google Patents
一种微电压方法协同抗菌肽pv-q5及其用途 Download PDFInfo
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- CN114409754B CN114409754B CN202111659224.XA CN202111659224A CN114409754B CN 114409754 B CN114409754 B CN 114409754B CN 202111659224 A CN202111659224 A CN 202111659224A CN 114409754 B CN114409754 B CN 114409754B
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
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Abstract
本发明公开了一种微电压方法协同抗菌肽PV‑Q5及其用途。微电压方法协同抗菌肽PV‑Q5的氨基酸序列如SEQ ID NO:5所示,其具有抗菌的用途。该抗菌肽在微电压协同作用下对副溶血性弧菌的抑菌效果有明显增强。本发明为后续进一步研究抗菌肽作为食品防腐剂开发奠定了基础。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种微电压方法协同抗菌肽PV-Q5及其用途。
背景技术
食品是人类赖以生存和发展的物质基础,食品安全不仅关乎广大人民群众的身体健康和切身利益,同时也关系到国家经济的稳步发展和社会的繁荣昌盛。致病性微生物是引起食源性疾病的重要因素,近年来,每年全球近15亿患食源性疾病的人群中,有70%是因为食品中的致病性微生物污染所导致。致病性微生物所引起的食源性疾病正严重威胁着人民的身体健康,阻碍社会经济发展。副溶血性弧菌是广泛存在于海产品中的食源性细菌之一,也是引起细菌性胃肠炎的主要原因。
微电压是一种通过外加弱电流激发电极反应以刺激微生物细胞,为提高细胞生长和活性提供了一条有效途径。目前,这一技术已在酵母发酵体系和生物脱氮体系中得到初步应用。通过研究发现,微电压刺激能够改变细菌细胞的增长,提高细胞增长速度。另一方面,现已有报道表明微电压亦可对微生物细胞施加负面影响,如在生物膜体系中施加微电压可显著提升灭菌剂的杀菌效果,影响细胞的生长代谢,改变细胞膜的通透性。然而,微电压的手段并不能够灭活病原微生物,但可以增效其他的抑菌方式来抑制细菌生长。
抗菌肽由生物体内免疫防御体系产生,是一类具有抵抗外源性病原菌的多肽类活性物质。由于细菌的外膜含有较多带负电荷的分子,如脂多糖、磷壁酸等,阳离子抗菌肽可通过静电吸引与膜成分相互作用,从而与细菌细胞膜结合,产生抑菌效果。自然界中的抗菌肽以动物源抗菌肽为主,因其具有广谱高效抗菌活性、细胞选择性及不易产生耐药性等特点,一向被认为是抗生素的理想替代品。在无脊椎动物的先天免疫系统中,抗菌肽是一种重要的免疫效应分子。
发明内容
本发明的目的在于提供一种微电压方法协同抗菌肽PV-Q5。
为实现上述目的,本发明提供一种抗菌肽PV-Q5,其特征在于,所述抗菌肽PV-Q5的氨基酸序列如SEQ ID NO:5所示。
本发明还保护所述抗菌肽PV-Q5用于抗菌的用途。
进一步,所述菌是指细菌。
进一步,所述细菌是指副溶血性弧菌和大肠杆菌。
进一步,所述抗菌是指抑制和/或杀灭副溶血性弧菌、大肠杆菌。
进一步,所述抗菌在微电压辅助处理下,具有增强的效果。
本发明还保护所述抗菌肽PV-Q5具有改变菌细胞膜表面通透性的用途。
本发明还保护所述抗菌肽PV-Q5用于制备抗菌药物、食品防腐剂和水产动物养殖饲料的用途。
本发明还保护一种抗菌药物,其特征在于,含有所述抗菌肽PV-Q5。
本发明还保护一种食品防腐剂,其特征在于,含有所述抗菌肽PV-Q5。
本发明预测并合成了对副溶血性弧菌具有弱抗菌活性的抗菌肽PV-Q5。然而,在通电的辅助下,肽PV-Q5的抗菌效果有了很大的提高,并对其协同作用机制进行了进一步的探讨。
本发明的目的在于提供一种微电压的辅助方法,并研究其对抗菌肽PV-Q5抑菌增效作用及其应用,通过微电压辅助增效抗菌肽对副溶血性弧菌的研究,为寻找新的食品添加剂杀菌技术供实验依据,促进我国食品行业健康、持续发展。
为了解决上述目的,本发明采用的技术方案如下:
抗菌肽PV-Q5,其氨基酸序列QVRNFPRGSAASPSALASPR,如SEQ ID NO:5所示。
以凡纳滨对虾(Penaeus vannamei)为目标寻找其序列中的抗菌肽存在可行的理论依据。因此,首先利用质谱和生物信息学对凡纳滨对虾的蛋白序列进行筛选,然后发现了一条对副溶血性弧菌具有抑制作用的抗菌肽序列QVRNFPRGSAASPSALASPR,命名PV-Q5,分子量为2068.1Da。
抗菌肽PV-Q5可以改变细菌细胞膜的通透性,并抑制细胞膜生成,降低细菌的存活率。
本发明的抗菌肽可以采用本领域技术人员已知的方法合成,例如固相合成,并采用本领域技术人员已知的方法进行纯化,例如高效液相色谱法。
实施本发明,具有如下有益效果:
本发明以副溶血性弧菌为研究对象,并通过质谱鉴定和生物信息学筛选,合成一个全新氨基酸序列的多肽PV-Q5。研究不同处理的抗菌肽PV-Q5对副溶血性弧菌的抑菌活性;并利用透射电镜观察抗菌肽PV-Q5对副溶血性弧菌和通电条件下的破坏程度;最后对抗菌肽PV-Q5的二级结构和三维模型进行验证。实验结果表明,微电压辅助处理的抗菌肽PV-Q5对副溶血性弧菌具有强烈的抑制作用。它的抑菌机理是与细菌细胞膜相互作用后改变其细胞膜的通透性,从而使得细菌走向凋亡。本发明为抗菌肽PV-Q5作为食品防腐剂提供了实验依据。
附图说明
图1为本发明抗菌肽PV-Q5的质谱图。
图2为本发明抗菌肽PV-Q5单独处理对副溶血性弧菌最低抑制浓度(MIC)测定对照图。
图3为本发明抗菌肽PV-Q5协同微电压处理对副溶血性弧菌最低抑制浓度(MIC)测定对照图。
图4为本发明抗菌肽PV-Q5对副溶血性弧菌孵育和微电压条件的时间杀伤测定曲线。
图5为本发明抗菌肽PV-Q5作用副溶血性弧菌的透射电镜观察图。
图6为本发明抗菌肽PV-Q5对副溶血性弧菌膜通透性变化折线图。
图7为本发明抗菌肽PV-Q5在SDS溶液和PBS溶液中的二级结构变化图。
图8为本发明抗菌肽PV-Q5的三维结构预测图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。在下面的实施例中,如未明确说明,“%”均指重量百分比。
实施例1:凡纳滨对虾的高效液相色谱-质谱联用技术(UPLC-MS)
使用色谱仪Nano Aquity UPLC system(Waters Corp.)对分子量小于3kDa的对凡纳滨对虾虾液进行梯度洗脱,色谱条件:进样量:5.0μL。色谱柱:C18分析色谱柱,长度25cm,内径75μm。流动相:A:0.1%甲醇水溶液;B:乙腈。结合搜索软件在MAXQUANT v1.6.5.0在数据库中得到了10个蛋白质序列,对所得肽段进行质谱鉴定,肽段PV-Q5质谱结果如图1所示。
实施例2:凡纳滨对虾中抗菌肽的生物信息学筛选
通过高效液相色谱-质谱结果(质谱条件设置为翘气速率:40mL/min;辅助气速率:10mL/min;喷雾电压:3.0kV;毛细管温度:300℃、S-lens:50%;HCD:27%。扫描模式为正离子,Full ms→ddms2。一级扫描:分辨率70000,范围350~1600m/z;二级扫描:分辨率17500,fixed first mass 120m/z。Dynamic exclusion:10.0s)鉴定出10条肽段序列,肽段的片段平均长度为14.5,分子量在973-2116Da范围。利用在线软件APD3对10条肽段的电荷、疏水性进行分析,最终如表1所示,筛选出一条分子量为2068.1Da、疏水性为35%、电荷为+3、氨基酸序列为QVRNFPRGSAASPSALASPR(SEQ ID NO:5)的多肽进行化学合成(由北京中科亚光生物科技有限公司合成),并对其抑菌活性进行验证。
表1:凡纳滨对虾LCMS所得肽段分析结果表
实施例3:最低抑制浓度(MIC)测定
将副溶血性弧菌(ATCC 17802)在37℃培养12h至对数生长期,在0.01M pH 7.2磷酸盐缓冲液中稀释至106-7CFU/mL。将抗菌肽PV-Q5溶于磷酸盐缓冲中,再等体积与菌液混合后,分别进行静置、微电压(10V)处理。将在37℃培养箱中静置2小时的作为空白对照组,将在37℃培养箱中静置通入10V的微电压处理2小时的作为样品组,培养2小时后涂平板,平板在37℃培养箱中静置过夜。如图2和图3所示,图2为本发明抗菌肽PV-Q5单独处理对副溶血性弧菌最低抑制浓度(MIC)测定对照图,其中,图2和图3的A:空白组抗菌肽浓度500μg/mL;B:空白组抗菌肽浓度125μg/mL;C:空白组抗菌肽浓度62.5μg/mL;D:空白组抗菌肽浓度31.25μg/mL。E:空白组抗菌肽浓度15.62μg/mL。图3是抗菌肽PV-Q5培养箱静置2h协同微电流(10V)对副溶血性弧菌最低抑制浓度(MIC)测定图。可以看出,抗菌肽PV-Q5在培养箱中2小时后对副溶血性弧菌的MIC为31.25μg/mL、抗菌肽PV-Q5经过微电压处理2小时后对副溶血性弧菌的MIC为15.62μg/mL。
实施例4:时间杀伤曲线测定
将副溶血性弧菌在37℃培养12h至对数生长期,在0.01M pH 7.2磷酸盐缓冲液中稀释至106-7CFU/mL。取MIC浓度肽PV-Q5等体积与菌混合后在37℃分别进行静置、微电压(10V)处理。将在37℃培养箱中静置的作为空白对照组,将在37℃培养箱中静置通入10V的微电压的作为样品组。每隔30分钟分别取出空白组和样品组涂平板,37℃培养过夜后记录菌落总数。结果见图4,图4为时间杀伤曲线为副溶血性弧菌。(●)为抗菌肽PV-Q5在孵育处理下的时间杀伤曲线,(▲)为抗菌肽PV-Q5在微电压处理下的时间杀伤曲线。由结果可知,无论是抗菌肽PV-Q5单独处理副溶血性弧菌,或微电压辅助处理抗菌肽PV-Q5对副溶血性弧菌时的作用效果,均在2小时内达到顶峰,随后趋于平缓,但微电压辅助处理抗菌肽PV-Q5明显优于抗菌肽PV-Q5单独处理。
实施例5:透射电镜分析
以106-7CFU/mL的细菌在37℃下用2×MIC的抗菌肽PV-Q5处理2小时,然后在2700g下离心10min,用磷酸盐缓冲液(pH 7.2)洗涤两次。用1%的锇酸固定后,用95%乙醇脱水,然后丙酮处理20min。样品在70℃下烘烤24h,在铜网格上制备70-90nm的薄片,然后用柠檬酸铅和乙酸铀酯染色。用H-7650透射电子显微镜观察和捕获超微结构。
以未处理的细菌细胞作为空白对照组,将抗菌肽PV-Q5单独作用副溶血性弧菌2小时和抗菌肽PV-Q5作用副溶血性弧菌同时通入微电压(10v)作用2小时为样品组。为探究抗菌肽PV-Q5和抗菌肽PV-Q5协同微电压对细菌超微结构的影响,使用透射电镜观察其中的变化。结果见图5,抗菌肽PV-Q5作用副溶血性弧菌的透射电镜图,其中A为:副溶血性弧菌空白对照组;B为抗菌肽PV-Q5处理2h后的副溶血性弧菌;C为抗菌肽PV-Q5微电压处理2h后的副溶血性弧菌。可以看出:空白对照组显示组织分布均匀,无渗漏,表面细胞膜光滑(A)。用抗菌肽PV-Q5处理后,副溶血性弧菌的细胞膜出现一些模糊和不规则,细胞内溶质渗漏(B)。然而抗菌肽PV-Q5协同微电压处理后细菌细胞内溶质渗漏、细胞破裂(C)。这进一步证实了在微电压的条件下能够辅助抗菌肽PV-Q5通过破坏细胞膜的方式进行杀菌。
实施例6:抗菌肽PV-Q5对细菌细胞膜通透性的影响
为了研究抗菌肽PV-Q5对副溶血性弧菌通透性的影响,将细菌置于1×MIC浓度的抗菌肽PV-Q5下培养,观察其对细菌细胞膜通透性的影响。具体操作如下:通过离心收集副溶血性弧菌细胞,再重悬于以乳糖为唯一碳源的M9培养基中,37℃摇床培养至OD600nm<0.4。将混合物加入96孔平底板中,在37℃下孵育,然后加入0.5mg/mL的ONPG(2-硝基苯基-β-D-吡喃半乳糖苷)混匀后进行摇床培养观察并测定其在(0-8h)的OD42nm0的变化,如图6所示。图6是抗菌肽PV-Q5作用副溶血性弧菌的细胞膜通透性图。
抗菌肽PV-Q5作用副溶血性弧菌后能明显观察到副溶血性弧菌的细胞膜通透性增加。细胞壁(膜)结构完整对维持细胞形态与胞内环境有重要作用,受损时可引起细胞通透性增强甚至出现不可逆孔洞,造成胞内离子及生物大分子物质泄露并诱导细胞凋亡。
实施例7:圆二色谱测定抗菌肽PV-Q5的二级结构
用Chirascan V100圆二色谱仪在25℃下以100nm/min的扫描速度测定了肽的平均残基摩尔椭圆度。将抗菌肽PV-Q5溶于25mM十二烷基硫酸钠(SDS)中,溶液最终浓度为0.20mg/mL。抗菌肽PV-Q5在0.01M pH 7.2磷酸盐缓冲液(PBS)中的情况下同一处理作为对照。
CD光谱可以检测抗菌肽在不同溶液中的二级结构。抗菌肽PV-Q5在0.01M pH7.2PBS和0.20mg/mL SDS溶液中的CD光谱如图7所示。图7是抗菌肽PV-Q5圆二色谱图。抗菌肽PV-Q5在PBS体系中波长200nm附近有负的吸收峰,符合无规则卷曲的特征吸收峰,其二级结构为无规则卷曲;抗菌肽PV-Q5在SDS体系中时,波长190nm处附近有正的吸收峰、200nm和220nm处附近有负的吸收峰,符合α-螺旋的特征吸收。表明PV-Q5在模拟细胞膜环境中,以α-螺旋结构存在。
α-螺旋中的亲水侧链和疏水侧链分别处于螺旋的两侧,这一特殊结构会促进生物膜和多肽片段相互作用。当抗菌肽与细菌的细胞膜结合时,α-螺旋会相互聚集,在细胞膜表面形成孔洞,造成细胞质密度降低、内容物流出,细胞死亡。
实施例8:抗菌肽PV-Q5的三维结构预测
利用在线结构预测服务器I-Tasser对凡纳滨对虾抗菌肽PV-Q5的三维结构进行预测。得到凡纳滨对虾中抗菌肽PV-Q5三维结构如图8所示。图8是抗菌肽PV-Q5的三维结构预测图。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
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SEQUENCE LISTING
<110> 集美大学
<120> 一种微电压方法协同抗菌肽PV-Q5及其用途
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Claims (7)
1.一种抗菌肽PV-Q5,其特征在于,所述抗菌肽PV-Q5的氨基酸序列如SEQ ID NO:5所示。
2.权利要求1所述抗菌肽PV-Q5用于制备抗菌药物、食品防腐剂和水产动物养殖饲料的用途;所述菌是指副溶血性弧菌。
3.如权利要求2所述的用途,其特征在于,所述抗菌是指抑制和/或杀灭副溶血性弧菌。
4.如权利要求2所述的用途,其特征在于,所述抗菌在微电压辅助处理下,具有增强的效果。
5.权利要求1所述抗菌肽PV-Q5具有制备改变副溶血性弧菌细胞膜表面通透性药物的用途。
6.一种抗菌药物,其特征在于,含有权利要求1所述抗菌肽PV-Q5。
7.一种食品防腐剂,其特征在于,含有权利要求1所述抗菌肽PV-Q5。
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CN112898386A (zh) * | 2021-03-02 | 2021-06-04 | 集美大学 | 一种大黄鱼肌球蛋白重链抗菌肽lcmhc及其应用 |
CN113087771A (zh) * | 2021-04-25 | 2021-07-09 | 集美大学 | 一种南美白对dna结合抗菌肽vpdb40及其应用 |
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CN112898386A (zh) * | 2021-03-02 | 2021-06-04 | 集美大学 | 一种大黄鱼肌球蛋白重链抗菌肽lcmhc及其应用 |
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Jingyi Dai等.Antibacterial Activity and Mechanism of Peptide PV-Q5 against Vibrio parahaemolyticus and Escherichia coli, Derived from Salt-Fermented Penaeus vannamei.Foods.2023,第12卷(第9期),1804. * |
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