CN111658762A - 天然宿主防御肽Hc-CATH的应用 - Google Patents
天然宿主防御肽Hc-CATH的应用 Download PDFInfo
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Abstract
本发明涉及天然宿主防御肽Hc‑CATH的应用，具体涉及天然宿主防御肽Hc‑CATH在制备抗水产致病菌药物中的应用、在制备水产养殖动物免疫调节剂中的应用。本发明进一步公开了天然宿主防御肽Hc‑CATH在制备水产养殖动物饲料中的应用。以大口黑鲈为实验动物的研究结果表明，Hc‑CATH能够杀死多种常见水产致病菌，调节大口黑鲈免疫功能，提高大口黑鲈对细菌感染的抵抗力，提高大口黑鲈的存活率。Hc‑CATH在水产养殖领域具有广泛的应用前景。
Description
技术领域
本发明涉及多肽应用领域，尤其涉及一种天然宿主防御肽Hc-CATH的应用。
背景技术
抗生素是指由微生物产生的在低浓度下能够抑制或杀死其它微生物的化学物质。抗生素由于具有促进动物生长、提高饲料利用率、预防和治疗动物疫病的作用，曾在养殖业发展中发挥了重要作用。
但是，随着抗生素在养殖业中的大量使用，特别是不科学的滥用，细菌耐药性和药物残留等问题日益突出。由于抗生素残留超标等问题，我国养殖业产品出口量仅占生产量的0.9％-1.2％，严重影响我国养殖业经济的发展。因此研发新型高效抗菌抗感染、无残留的新型抗生素替代药物日益重要。
CN103665111A公开了青环海蛇抗菌肽Hc-CATH的改造体及其制备方法和应用，其具有革兰氏阴性菌、革兰氏阳性菌的抗菌活性，但是同一多肽对不同菌株的抗菌活性不能一概而论，开发Hc-CATH的其他领域的用途能更充分的发挥该多肽的价值。
发明内容
为解决上述技术问题，本发明的目的是提供天然宿主防御肽Hc-CATH的应用，本发明公开了Hc-CATH对水产致病菌的抗菌作用及对水产动物的免疫调节作用，其可在水产养殖领域作为水产养殖动物饲料使用。
本发明的第一个目的是公开天然宿主防御肽Hc-CATH在制备抗水产致病菌药物中的应用，Hc-CATH的氨基酸序列如SEQ ID No.1所示。
进一步地，水产致病菌包括溶藻弧菌、鳗弧菌、巴西弧菌、霍乱弧菌、河流弧菌、哈维氏弧菌、副溶血弧菌、灿烂弧菌、创伤弧菌、嗜水气单胞菌、温和气单胞菌和维氏气单胞菌中的一种或几种。
进一步地，Hc-CATH对水产致病菌的最小抑菌浓度为2.34-9.38μg/mL。
本发明的第二个目的是公开天然宿主防御肽Hc-CATH在制备水产养殖动物免疫调节剂中的应用，Hc-CATH的氨基酸序列如SEQ ID No.1所示。
进一步地，免疫调节剂用于治疗水产致病菌引起的炎症。
进一步地，免疫调节剂用于抑制水产养殖动物体内水产致病菌感染引起的促炎因子TNF-α和IL-1β的基因表达且提高细胞趋化因子IL-8基因的表达水平。
进一步地，免疫调节剂的剂量为1-100mg/kg。优选地，免疫调节剂的剂量为10-20mg/kg。
进一步地，水产养殖动物为鱼类。优选地，水产养殖动物为大口黑鲈、草鱼、鲢鱼、鳙鱼、青鱼、鳜鱼、牙鲆、大菱鲆、美国红鱼、石斑鱼。
优选地，炎症由水产致病菌副溶血弧菌、鳗弧菌、霍乱弧菌、灿烂弧菌和嗜水气单胞菌中的一种或几种所引起。
本发明的第三个目的是公开天然宿主防御肽Hc-CATH在制备水产养殖动物饲料中的应用，Hc-CATH的氨基酸序列如SEQ ID No.1所示。
进一步地，水产养殖动物饲料用于抗水产致病菌和/或调节水产养殖动物的免疫功能。
进一步地，水产养殖动物为鱼类。优选地，水产养殖动物为大口黑鲈、草鱼、鲢鱼、鳙鱼、青鱼、鳜鱼、牙鲆、大菱鲆、美国红鱼、石斑鱼。
本发明中，天然宿主防御肽Hc-CATH来源于爬行类动物青环海蛇。
借由上述方案，本发明至少具有以下优点：
本发明公开了天然宿主防御肽Hc-CATH对常见水产致病菌具有广谱高效的抗菌作用，杀菌作用迅速，能够调节水产养殖动物免疫功能，提高水产养殖动物对细菌感染的抵抗力，提高水产养殖动物的存活率，在水产养殖领域具有广泛的应用前景。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1图示了Hc-CATH对大口黑鲈脾脏细胞因子表达的影响；
图2图示了Hc-CATH对大口黑鲈细菌感染后存活率的影响。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1 Hc-CATH的制备
(1)、用自动多肽合成仪(433A,Applied Biosystems)合成来源于爬行类动物青环海蛇Hc-CATH的全序列，通过HPLC反相柱层析脱盐纯化。
(2)、分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)。
(3)、纯化的Hc-CATH用高效液相色谱HPLC方法鉴定其纯度，分子量测定采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)，等电聚焦电泳测定等电点，用自动氨基酸测序仪测定氨基酸序列结构。
本发明的天然宿主防御肽Hc-CATH的氨基酸序列如SEQ ID No.1所示。其由30个氨基酸组成，分子量为3628.5Da，等电点为12.61，其中所有氨基酸均为L-型。
实施例2 Hc-CATH对常见水产致病菌的抗菌活性检测
(1)、分别挑取保存于斜面上的试验菌株(水产致病菌)均匀涂布于营养肉汤固体培养基(nutrient broth medium，英国OXOID公司)平板上，将经过灭菌的0.5cm直径的滤纸片置于培养基表面，滴加溶解于灭菌去离子水的2mg/mL的Hc-CATH样品溶液10μL，于37℃倒置培养18-20小时，观察抑菌圈形成与否。若样品具有抗菌活性，则会在滤纸片周围形成清晰透明的抑菌圈，抑菌圈越大表明样品抗菌活性越强。
(2)、Hc-CATH最小抑菌浓度(Minimum Inhibitory Concentration)测定(2倍稀释法)：
选择上步实验中具有抑菌圈的菌株进行MIC测定实验。试验菌株接种到NB液体培养基(英国OXOID公司)中，37℃振荡培养到对数生长期，而后用新鲜NB液体培养基将培养至对数生长期的培养液稀释到2×105cfu/mL待用。
按照表1中的稀释方法，在无菌96孔板各孔中预先加入100μLNB液体培养基，然后在第一孔中加入100μL用NB液体培养基稀释到一定浓度的经0.22μm孔滤膜过滤的的Hc-CATH样品溶液，混匀后取100μL加入第2孔，依次倍比稀释(参见表1)，自第9孔吸出100μL弃去，第10孔系对照管，其中含有阳性对照硫酸新霉素。
表1稀释方法
将96孔板放置37℃缓慢振荡培养18小时，于600nm波长处测定光吸收。最小抑菌浓度为看不见细菌生长的最低样品浓度，结果如表2所示。
表2.Hc-CATH对水产致病菌抗菌活性
由表2可见，Hc-CATH对表2中的水产致病菌均表现出极强的抗菌活性，MIC值处于2.34-9.38μg/mL的范围，和阳性对照硫酸新霉素抗菌活性相当。
实施例3 Hc-CATH杀菌速度测定
副溶血弧菌用NB液体培养基(英国OXOID公司)在37℃培养12小时，然后用新鲜的NB液体培养基稀释成106CFU/mL的菌悬液。将溶解于灭菌的去离子水中的Hc-CATH样品加入到菌悬液中，使终浓度为5×MIC。将加入Hc-CATH样品的菌液放置于37℃培养箱中震荡培养，分别在0、15、30、60、120和180分钟取50μL菌液稀释1000倍，然后取50μL稀释的菌液涂布到NB固体培养基平板上，37℃培养箱培养过夜后菌落计数。该实验用硫酸新霉素作为阳性对照，灭菌的去离子水作为阴性对照。
结果如表3所示，Hc-CATH对副溶血弧菌的杀菌速度迅速，在30分钟内即可杀死所有细菌，快于阳性对照硫酸新霉素的杀菌速度。
表3 Hc-CATH对副溶血弧菌的杀菌速度
实施例4 Hc-CATH对大口黑鲈免疫系统调节活性测定
以下实施例中所用大口黑鲈购自苏州吴江养殖场，在实验室暂养1周后，挑选规格整齐、健康的个体(1-2g/条)随机分组饲养于整理箱中，每组20条。实验共设2个组：细菌感染组，10mg/kg Hc-CATH组。
副溶血弧菌用NB液体培养基(英国OXOID公司)在37℃培养12小时，然后用PBS洗涤2次并稀释成1×108CFU/mL的菌悬液。大口黑鲈腹腔注射稀释好的菌悬液(20μL/条)，注射细菌后立即注射用PBS溶解的多肽样品(10mg/kg)。于0、6、12、24小时从每一组中随机选取大口黑鲈3条，处死后取脾脏组织，用TRIzol reagent(Life Technology,USA)提取组织总RNA，用PrimeScript 1st Strand cDNA Synthesis Kit(Takara,Japan)逆转录合成cDNA一链，用SYBR Premix Ex TaqTM II(Tli RNaseH Plus)two-step qRT-PCR kit(Takara,Japan)检测促炎因子TNF-α、IL-1β和细胞趋化因子IL-8的表达。用β-actin基因作为内参。所用引物如表4所示：
表4 qRT-PCR引物
结果如图1所示，在细菌感染后6和12小时，10mg/kg Hc-CATH能够显著抑制大口黑鲈体内副溶血弧菌感染引起的促炎因子TNF-α和IL-1β的基因表达，说明Hc-CATH具有较好的体内抗炎活性。与此同时，与细菌感染组相比，10mg/kg Hc-CATH显著提高了大口黑鲈体内细胞趋化因子IL-8基因的表达水平，说明Hc-CATH具有较好的免疫调节作用，能够通过诱导细胞趋化因子的表达来调节免疫细胞向感染部位的趋化，从而起到抗感染的效果。
实施例5 Hc-CATH对大口黑鲈细菌感染后存活率的影响
以下实施例中所用大口黑鲈购自苏州吴江养殖场，在实验室暂养1周后，挑选规格整齐、健康的个体(1-2g/条)随机分组饲养于整理箱中，每组20条。实验共设3个组：空白组、细菌感染组、10mg/kg Hc-CATH组。
副溶血弧菌用NB液体培养基(英国OXOID公司)在37℃培养12小时，用PBS缓冲液洗涤两次并重悬成1×108CFU/mL的菌悬液备用。大口黑鲈腹腔注射稀释好的菌悬液(40μL/条)，注射细菌后立即注射用PBS溶解的多肽样品(10mg/kg)。注射结束后大口黑鲈继续暂养96小时，观察活力，计算最终存活率。
结果如图2所示，Hc-CATH能够为大口黑鲈提供有效的免疫保护作用。注射10mg/kg的Hc-CATH能够显著提高细菌感染后大口黑鲈的存活率。
综上，本发明以大口黑鲈为实验动物的研究结果表明，Hc-CATH能够杀死多种常见水产致病菌，调节大口黑鲈免疫功能，提高大口黑鲈对细菌感染的抵抗力，提高大口黑鲈的存活率。Hc-CATH在水产养殖领域具有广泛的应用前景。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
序列表
<110>苏州大学
<120> 天然宿主防御肽Hc-CATH的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> PRT
<213> （人工序列）
<400> 1
Lys Phe Phe Lys Arg Leu Leu Lys Ser Val Arg Arg Ala Val Lys Lys
1 5 10 15
Phe Arg Lys Lys Pro Arg Leu Ile Gly Leu Ser Thr Leu Leu
20 25 30
Claims (10)
1.天然宿主防御肽Hc-CATH在制备抗水产致病菌药物中的应用，所述Hc-CATH的氨基酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用，其特征在于：水产致病菌包括溶藻弧菌、鳗弧菌、巴西弧菌、霍乱弧菌、河流弧菌、哈维氏弧菌、副溶血弧菌、灿烂弧菌、创伤弧菌、嗜水气单胞菌、温和气单胞菌和维氏气单胞菌中的一种或几种。
3.根据权利要求1所述的应用，其特征在于：所述Hc-CATH对水产致病菌的最小抑菌浓度为2.34-9.38μg/mL。
4.天然宿主防御肽Hc-CATH在制备水产养殖动物免疫调节剂中的应用，所述Hc-CATH的氨基酸序列如SEQ ID No.1所示。
5.根据权利要求4所述的应用，其特征在于：所述免疫调节剂用于治疗水产致病菌引起的炎症。
6.根据权利要求4所述的应用，其特征在于：所述免疫调节剂的剂量为1-100mg/kg。
7.根据权利要求4所述的应用，其特征在于：水产养殖动物为鱼类。
8.天然宿主防御肽Hc-CATH在制备水产养殖动物饲料中的应用，所述Hc-CATH的氨基酸序列如SEQ ID No.1所示。
9.根据权利要求8所述的应用，其特征在于：所述水产养殖动物饲料用于抗水产致病菌和/或调节水产养殖动物的免疫功能。
10.根据权利要求8所述的应用，其特征在于：水产养殖动物为鱼类。
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