CN114380893B - 一种腌制凡纳滨对虾抗菌肽ec-v10及其用途 - Google Patents
一种腌制凡纳滨对虾抗菌肽ec-v10及其用途 Download PDFInfo
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- CN114380893B CN114380893B CN202111682365.3A CN202111682365A CN114380893B CN 114380893 B CN114380893 B CN 114380893B CN 202111682365 A CN202111682365 A CN 202111682365A CN 114380893 B CN114380893 B CN 114380893B
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- antibacterial peptide
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Abstract
本发明公开了一种腌制凡纳滨对虾抗菌肽EC‑V10及其用途。腌制凡纳滨对虾抗菌肽EC‑V10的氨基酸序列如SEQ ID NO：10所示，其具有抗菌的用途。其抑菌机制是通过改变细菌细胞膜表面的通透性，使得细胞内容物质流出，细胞凋亡。可在预防或抑制由于副溶血性弧菌、大肠杆菌等致病性微生物污染的低盐腌渍水产品和凡纳滨对虾养殖虾病中应用。为后续进一步研究腌制凡纳滨对虾抗菌肽作为低盐腌渍水产品中的防腐剂和水产动物养殖饲料开发奠定了基础。
Description
技术领域
涉及生物技术领域，尤其涉及一种腌制凡纳滨对虾抗菌肽EC-V10及其用途。
背景技术
副溶血性弧菌和大肠杆菌是低盐腌渍海产品和凡纳滨对虾养殖中常见的，具有耐盐性的致病性微生物。由于其对盐度的耐受性强、传播途径广、危害性大而在食品卫生和凡纳滨对虾养殖中上存在较大的安全隐患。据世界卫生组织(WHO)统计，全球每年发生食源性疾病数十亿人，发达国家每年约有三分之一人次感染食源性疾病，其中70％是由于致病性微生物造成。因此，副溶血性弧菌和大肠杆菌这类致病性微生物已经成为全球范围严重危害食品安全的主要因素。
过去几年，致病性微生物的防治都是通过传统抗生素发挥作用，但最近由于抗生素的滥用使得许多致病性微生物都产生了耐药性等问题。而抗菌肽(AntimicrobialPeptides,AMPs)是一种从多种动物、植物和微生物中分离出来的小分子多肽类物质，在毒性、致敏性、抗菌性等方面都优于抗生素，且结构复杂且缺乏特异性，很难产生细菌耐药性。因此，抗菌肽在动物饲料、食品防腐等领域都具有广阔的应用前景。
凡纳滨对虾(Penaeus vannamei)具有生长速率快、耐高温及养殖区域广等优点，是世界上养殖产量最大的三大对虾之一,也是中国对虾养殖的优势种类,占中国对虾养殖总产量的80％以上。凡纳滨对虾在遭受细菌和病毒等病原体的侵害时，主要依靠先天免疫进行抵抗。在先天免疫过程中会产生一种重要的免疫效应因子，即抗菌肽。
腌制加工一直以来都是水产品传统的加工方式，一些研究学者通过研究发现腌制加工后得到了许多不同的活性物质。例如：Patricia Castellano通过质谱等技术，在腌制过的西班牙火腿中鉴定出了1条对单增李斯特菌具有天然抑菌效果的多肽。
发明内容
本发明的目的在于提供一种腌制凡纳滨对虾抗菌肽EC-V10。
为实现上述目的，本发明提供一种腌制凡纳滨对虾抗菌肽EC-V10，其特征在于，所述抗菌肽EC-V10的氨基酸序列如SEQ ID NO：10所示。
本发明还保护所述抗菌肽EC-V10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。
本发明还保护所述抗菌肽EC-V10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EC-V10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EC-V10。
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EC-V10。
本发明还保护一种水产动物养殖饲料，其特征在于，含有所述抗菌肽EC-V10。
本发明合成了对副溶血性弧菌、大肠杆菌具有抗菌活性的带正电荷性抗菌肽EC-V10，使其对副溶血性弧菌、大肠杆菌等致病性微生物产生更好的抑制效果，并对其抑菌机制进行了进一步的探讨。
通过腌制凡纳滨对虾鉴定出的抗菌肽EC-V10对副溶血性弧菌、大肠杆菌抑菌活性的研究，为寻找新的低盐腌渍水产品中的防腐剂和水产动物养殖饲料提供实验依据，促进我国食品行业的健康、持续发展。
本发明以腌制凡纳滨对虾为研究对象，通过高效液相色谱-质谱联用技术(LCMS)质谱后鉴定出10条多肽片段，然后利用APD3对所得的肽进行筛选，筛选出肽EC-V10可能具有抑菌效果。研究EC-V10对副溶血性弧菌、大肠杆菌等致病菌在低盐条件下(盐度＜5％)的抑菌活性，并以大肠杆菌为例利用透射电镜观察EC-V10对其破坏程度，最后对抗菌肽的二级结构和三维模型进行验证。实验结果表明，抗菌肽EC-V10对副溶血性弧菌、大肠杆菌均产生了强烈的抑制作用。其抑菌机制是通过改变细菌细胞膜表面的通透性，使得细胞内容物质流出，细胞凋亡。
附图说明
图1为本发明抗菌肽EC-V10的质谱图。
图2为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)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EC-V10在低盐条件下(盐度＜5％)对大肠杆菌最低抑制浓度(MIC)测定测定对照图。
图4为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)对副溶血性弧菌时间杀伤曲线测定结果图。
图5为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)对大肠杆菌时间杀伤曲线测定结果图。
图6为本发明抗菌肽EC-V10作用大肠杆菌的透射电镜观察图。
图7为本发明抗菌肽EC-V10对大肠杆菌细胞通透性变化折线图。
图8为本发明抗菌肽EC-V10在SDS溶液和PBS溶液中的二级结构图。
图9为本发明抗菌肽EC-V10三维结构预测图。
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。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
实施例1：腌制凡纳滨对虾的高效液相色谱-质谱联用技术
将凡纳滨对虾(Penaeus vannamei)在30％盐浓度下，20℃自然发酵15天。腌渍期间每天搅拌，在第15天时取凡纳滨对虾样品，放适量蒸馏水，去盐后离心，离心完成过3kd超滤膜。
使用色谱仪Nano Aquity UPLC system(Waters Corp.)进行分析，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在数据库中搜索到10个蛋白质序列，对所得肽段进行质谱鉴定，抗菌肽EC-V10质谱结果如图1所示。
实施例2：腌制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，再利用在线服务器APD3筛选腌制凡纳滨对虾中可能存在抗菌效果的肽段，并对10条肽段进行疏水性、电荷分析(如表1所示)，最终筛选出氨基酸序列VNQLPIHPIGFYIYK(SEQ ID NO:10)的肽段进行化学合成(由北京中科亚光生物科技有限公司合成)，并验证其抑菌活性。抗菌肽一般认为电荷在+2-+9之间，疏水性在30％-60％之间。
表1：腌制凡纳滨对虾LCMS所得肽段分析结果表
| 名称 | 多肽的序列 | 分子量(Da) | 净电荷 | 疏水性 | 序列号 |
| EC-V1 | AKDDLAEALR | 1100.6 | -1 | 50％ | SEQ ID NO:1 |
| EC-V2 | TREQLAEEK | 1102.6 | -1 | 22％ | SEQ ID NO:2 |
| EC-V3 | VGVKAPGIIPR | 1105.7 | +2 | 45％ | SEQ ID NO:3 |
| EC-V4 | SKYESEGVAR | 1124.6 | 0 | 20％ | SEQ ID NO:4 |
| EC-V5 | GYSFTTTAER | 1131.5 | 0 | 20％ | SEQ ID NO:5 |
| EC-V6 | HQGVMVGMGQK | 1170.6 | +2 | 36％ | SEQ ID NO:6 |
| EC-V7 | DSYVGDEAQSK | 1197.5 | -2 | 18％ | SEQ ID NO:7 |
| EC-V8 | ELTFQSDEDK | 1210.5 | -3 | 20％ | SEQ ID NO:8 |
| EC-V9 | SEEEVHNLQK | 1211.6 | -1 | 20％ | SEQ ID NO:9 |
| EC-V10 | VNQLPIHPIGFYIYK | 1800.9 | +2 | 40％ | SEQ ID NO:10 |
实施例3：最低抑制浓度(MIC)测定
最低抑菌浓度(MIC)是指在37℃孵育过夜后，从微量滴定板上看不到细菌生长的抗菌肽最低浓度。在低盐条件下(盐度＜5％)将副溶血性弧菌(ATCC 17802)、大肠杆菌(ATCC25922)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EC-V10溶于磷酸盐缓冲中，37℃等体积与菌混合后分别进行孵育2h。如图2、图3所示，图2为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中，A：抗菌肽浓度1000μg/mL；B：抗菌肽浓度500μg/mL；C：抗菌肽浓度250μg/mL；D：抗菌肽浓度125μg/mL；E：抗菌肽浓度62.5μg/mL。图3为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)对大肠杆菌最低抑制浓度(MIC)测定测定对照图。其中，A：抗菌肽浓度1000μg/mL；B：抗菌肽浓度500μg/mL；C：抗菌肽浓度250μg/mL；D：抗菌肽浓度125μg/mL；E：抗菌肽浓度62.5μg/mL。F：抗菌肽浓度31.25μg/mL。可以看出，抗菌肽EC-V10孵育2h后，在低盐条件下对副溶血性弧菌的最低抑菌浓度(MIC)为62.5μg/mL、对大肠杆菌的最低抑菌浓度(MIC)为31.25μg/mL。
实施例4：时间杀伤曲线测定
在低盐条件下(盐度＜5％)将副溶血性弧菌、大肠杆菌在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1xMIC抗菌肽EC-V10在37℃等体积与菌混合后进行孵育5h处理，每隔1h取样涂平板，37℃培养过夜后记录菌落总数。结果见图4和图5。图4为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)对副溶血性弧菌时间杀伤曲线测定结果图。图5为本发明抗菌肽EC-V10在低盐条件下(盐度＜5％)对大肠杆菌时间杀伤曲线测定结果图。由结果可知，抗菌肽EC-V10在低盐条件下对副溶血性弧菌、大肠杆菌均在2h开始产生明显效果；对副溶血性弧菌弧菌而言，2h内的作用效果显著，随后继续呈下降趋势，在3h趋于平缓(图4)；大肠杆菌也在前2h产生明显的下降趋势(图5)。说明抗菌肽EC-V10在低盐条件下对副溶血性弧菌、大肠杆菌均产生了较强的抑菌效果，对于大肠杆菌而言效果明显。因此，以大肠杆菌为例进一步进行抑菌机制的验证。
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EC-V10处理2h，然后在2700g下离心10min，用0.01M pH 7.2磷酸盐缓冲液洗涤两次。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。
用透射电镜观察了抗菌肽EC-V10对大肠杆菌超微结构的影响。结果见图6。图6为本发明抗菌肽EC-V10作用大肠杆菌的透射电镜观察图，其中，A：大肠杆菌空白对照组(即没有使用抗菌肽EC-V10处理)；B：抗菌肽EC-V10处理2h后的大肠杆菌。未经抗菌肽EC-V10处理的大肠杆菌细胞膜和细胞壁光滑、形态规则，胞内物质充实致密、无渗漏(A)。而用抗菌肽EC-V10处理后，大肠杆菌细胞膜和细胞壁出现膜模糊和不规则，细胞膜出现溶解，胞内物质外泄(B)。表明对细胞膜的破坏作用是抗菌肽EC-V10的抑菌机制之一。
实施例6：抗菌肽EC-V10对细菌细胞膜通透性的影响
为了研究抗菌肽EC-V10对细胞膜通透性的影响，以大肠杆菌为例，将大肠杆菌置于1xMIC的抗菌肽EC-V10下培养，观察其对细菌细胞膜通透性的影响。具体操作如下：通过离心收集大肠杆菌细胞，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，然后与1xMIC浓度等体积的抗菌肽EC-V10混合。将混合物加入96孔平底板中，在37℃下分别进行孵育2h，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OD420nm的变化(图7)。图7为本发明抗菌肽EC-V10对大肠杆菌细胞通透性变化折线图。
细胞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。当抗菌肽EC-V10作用于副溶血性弧菌后，菌液的OD值随时间的延长而增大，且在第一小时OD值达到最高，之后的5小时内，且菌液的OD值都显著高于空白组(control组)。这表明添加抗菌肽EC-V10对副溶血性弧菌的细胞内膜产生了一定的作用，导致其细胞内膜渗透性增大，通透性增强。
实施例7：圆二色谱测定抗菌肽EC-V10二级结构
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EC-V10溶于25mM十二烷基硫酸钠(SDS)中，溶液最终浓度为0.20mg/mL，然后将溶液加入到1mm石英比色皿中，从190～250nm两次扫描其光谱。再将抗菌肽EC-V10溶于0.01M pH 7.2磷酸盐缓冲液(PBS)中，使用上述方式扫描其光谱，结果如图8所述。图8为本发明抗菌肽EC-V10在SDS溶液和PBS溶液中的二级结构图。
可以看出，抗菌肽EC-V10在190～210nm之间出现了一个负的特征峰，表面其二级结构是无规则卷曲结构。研究其结构，可以发现抗菌肽EC-V10(VNQLPIHPIGFYIYK)其中含有两个脯氨酸、一个甘氨酸，同时表现出对副溶血性弧菌、大肠杆菌产生较强的抗菌作用，故推测其抗菌效果可能来源于其中的特定性氨基酸。
实施例8：抗菌肽EC-V10的三维结构预测
利用在线结构预测服务器Swiss-Model对腌制凡纳滨对虾抗菌肽EC-V10的三维结构进行预测。根据GMQE(Global Model Quality Estimation)和QMEAN(Qualitative ModelEnergy Analysis)两项指标得到其三维结构如图9所示。图9为本发明抗菌肽EC-V10三维结构预测图。
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。
SEQUENCE LISTING
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Claims (9)
1.一种腌制凡纳滨对虾抗菌肽EC-V10，其特征在于，所述抗菌肽EC-V10的氨基酸序列如SEQ ID NO：10所示。
2.权利要求1所述抗菌肽EC-V10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。
3.如权利要求2所述的用途，其特征在于，所述菌是指细菌。
4.如权利要求3所述的用途，其特征在于，所述细菌是指副溶血性弧菌和大肠杆菌。
5.如权利要求3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、大肠杆菌。
6.权利要求1所述抗菌肽EC-V10具有制备改变菌细胞膜表面通透性药物的用途。
7.一种抗菌药物，其特征在于，含有权利要求1所述抗菌肽EC-V10。
8.一种食品防腐剂，其特征在于，含有权利要求1所述抗菌肽EC-V10。
9.一种水产动物养殖饲料，其特征在于，含有权利要求1所述抗菌肽EC-V10。
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