WO2017101664A1 - 一种具有抑菌效果的多肽 - Google Patents

一种具有抑菌效果的多肽 Download PDF

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WO2017101664A1
WO2017101664A1 PCT/CN2016/107381 CN2016107381W WO2017101664A1 WO 2017101664 A1 WO2017101664 A1 WO 2017101664A1 CN 2016107381 W CN2016107381 W CN 2016107381W WO 2017101664 A1 WO2017101664 A1 WO 2017101664A1
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polypeptide
vibrio harveyi
antibacterial effect
seq
peptides
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PCT/CN2016/107381
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English (en)
French (fr)
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陈艺燕
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陈艺燕
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Publication of WO2017101664A1 publication Critical patent/WO2017101664A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the invention belongs to the technical field of active peptide screening application, and particularly relates to a polypeptide having a bacteriostatic effect.
  • a polypeptide is a compound in which two or more amino acids are linked by a peptide bond, and plays an important physiological role in a living body to exert a physiological function.
  • Bioactive peptides can be classified into endogenous bioactive peptides and exogenous bioactive peptides according to their sources. Exogenous active peptides are mostly present in proteins in specific amino acid sequence fragments, and are classified into lactopeptides, soybean peptides, corn peptides, protein peptides, aquatic peptides and the like according to their raw material sources.
  • the present invention also provides a polynucleotide, wherein the polynucleotide is a polynucleotide having a polypeptide of SEQ ID NO: 1;
  • polynucleotide is (GTTCCATTCGGATACACTCCAGCT) SEQ ID NO: 2.
  • the above polypeptide has an inhibitory effect on Vibrio harveyi and is used for preparing a product inhibiting Vibrio harveyi;
  • polypeptides can also be used as feed additives or antibacterial agents.
  • the invention separates and obtains a polypeptide having antibacterial effect from the enzymolysis product of scallop, and the use of the polypeptide as a feed additive can effectively increase the disease resistance of the cultured aquatic animal; and thus is used in the field of aquatic feed preparation.
  • Scallops are marine bivalve molluscs that are found in the intertidal zone to the deep sea.
  • the shell is fan-shaped, but the butterfly hinge is straight, and the ends of the butterfly hinge have wing-like protrusions.
  • the size is about 2.5 to 15 cm or more.
  • the shell is smooth or has radiating ribs.
  • the ribs are smooth, scaly or neoplastic, with bright red, purple, orange, yellow to white.
  • the lower shell is lighter and smoother.
  • the edge of the mantle has eyes and short tentacles. The tentacles can feel the change of water quality. When the shell is opened, it is located between the two shells.
  • Scallops are commonly found in sand or in fine seawater gravel, feeding on tiny organisms, collecting food particles by cilia and mucus and moving them into the mouth.
  • the degreasing operation was repeated 4 times, compared to 1:5 of 95% ethanol, degreased at 60 ° C for 4 h.
  • the residue after degreasing was vacuum dried at 40 ° C, pulverized, and sieved through 100 to obtain crude scallop protein, and the protein content thereof was determined to be 63.24%.
  • the enzymatic hydrolysate is subjected to ultrafiltration treatment with ultrafiltration membranes of different molecular weights, and the enzymatic hydrolysates of different molecular weight ranges are separately collected, and the activity of inhibiting the activity of Vibrio harveyi on the enzymatic hydrolysate is determined;
  • the enzymatic hydrolysate of the bacteria activity separates the single active peptide.
  • the product of the active peak was further separated and purified by high performance liquid chromatography.
  • the chromatographic conditions were as follows: the column was a reverse phase C18 bonded silica gel column (Agilent, ZORBAX SB-C18, 150 mm x 4.6 mm, 5 ⁇ m); mobile phase: A water (containing 1% trifluoroacetic acid), B acetonitrile, gradient wash The conditions are as follows; flow rate: 1.0 Ml/min, column temperature 25 ° C, detection wavelength is 214 nm, the main chromatographic peaks were collected, and lyophilized for short peptide samples. After further separation and purification by high-performance liquid phase, the active peptides were collected and freeze-dried, and then subjected to amino acid sequence analysis.
  • Amino acid sequence analysis was performed on the collected components using an amino acid sequence analyzer and a mass spectrometer.
  • the prepared active peptide fragment was subjected to amino acid sequence analysis and molecular weight determination by Edman degradation method and mass spectrometry.
  • the sequence of the polypeptide was ValProPhe GlyTyrThrProAla (SEQ ID NO: 1); the nucleotide sequence encoded by the sequence was SEQ ID NO: 2. .
  • the antibacterial activity of the synthetic peptide (SEQ ID NO: 1) against Vibrio harveyi was detected by punching and measuring the zone of inhibition, and Vibrio harveyi was cultured.
  • dilute the bacterial solution with PBS solution to a concentration of 10 7 to 10 8 /ml
  • apply 150 ⁇ l of the diluted bacterial solution to the plate and punch 5 holes on each plate with a 6 mm puncher.
  • a 2-fold gradient dilution of the polypeptide solution and 10 mg/ml of ampicillin were added to the wells as control, each 20 ⁇ l, three replicates at a time, and the inhibition zone was observed and measured after incubation for 12 hours in a 28 ° C incubator.
  • the minimum concentration of bacteriostatic is considered to be the minimum inhibitory concentration.
  • the antibacterial activity of the polypeptide was measured by colony counting method to detect the antibacterial activity of the polypeptide against Vibrio harveyi, and the Vibrio harveyi was cultured to logarithmic phase, and the bacterial solution was diluted with PBS solution to a concentration of 10 7 to 10 8 /ml, take 80 ⁇ l of the diluted bacterial solution, add 20 ⁇ l of different concentrations of the peptide solution to the plate, and 10mg/ml kanamycin as a control, each 100 ⁇ l, three repetitions each time, observe in the incubator at 37 °C for 12h And measure the inhibition zone.
  • the minimum concentration of bacteriostatic is considered to be the minimum inhibitory concentration.
  • the results showed that the minimal inhibitory concentration of the polypeptide against Vibrio harveyi was 0.07 mg/ml.
  • the above-mentioned screening and isolated polypeptide can be used as a feed additive to prepare a seawater biological feed, which can effectively prevent the reproduction of Vibrio harveyi in the culture water body and reduce the incidence of the cultured animal.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Fodder In General (AREA)

Abstract

一种具有抑菌效果的多肽，其氨基酸序列为SEQ ID NO:1，该多肽对哈维氏弧菌具有抑制效果，用于制备抑制哈维氏弧菌的制品。从扇贝的酶解产物中分离获得一种具有抑菌效果的多肽，用该多肽制成饲料添加剂，可以有效的增加养殖的水产动物的疾病抵抗力；从而用于水产饲料制备领域中。

Description

一种具有抑菌效果的多肽 技术领域
本发明属于活性肽筛选应用技术领域，具体涉及一种具有抑菌效果的多肽。
背景技术
多肽是两个或两个以上的氨基酸以肽键相连的化合物，在生物体内起重要生理作用，发挥生理功能。生物活性肽按照其来源可以分为内源性生物活性肽和外源性生物活性肽。外源性活性肽多以特定的氨基酸序列片段存在于蛋白质中，按其原料来源分为乳肽、大豆肽、玉米肽、蛋白肽、水产肽等。
近年来随着海水污染问题的加剧而导致陆地周边养殖环境的不断恶化，海水养殖病害频繁发生，给养殖业带来了严重影响，现在病害问题已经成为制约海水养殖产业发展的瓶颈之一。为了治疗及预防疾病，在传统养殖实践中通过在饲料中添加化学药物来实现，不但大大增加了养殖成本，而且导致了药物残留问题严重，该问题直接影响了养殖生物的商品质量，降低了成品价值，使养殖业遭受了更多的损失。同时大量使用抗生素类药物提高了致病微生物的抗药性，进而使其他的海水鱼类养殖业面临同样严峻的挑战；因此，开发高效绿色低成本的抗病方法是解决该问题的关键所在。
发明内容
本发明的目的是提供一种具有抑菌效果的多肽，从而弥补现有技术的不足。
本发明所提供的抑菌效果的多肽，包含有：
1)序列为ValProPhe GlyTyrThrProAla(SEQ ID NO:1)的多肽；
2)分别与1)中的多肽具有70％或以上同源性的多肽，该多肽的功能与1)中的多肽功能相同或相似。
本发明还提供一种多核苷酸，所述的多核苷酸是编码序列为SEQ ID NO：1多肽的多核苷酸；
该多核苷酸的一种序列为(GTTCCATTCGGATACACTCCAGCT)SEQ ID NO：2。
上述的多肽对哈维氏弧菌具有抑制效果，用于制备抑制哈维氏弧菌的制品；
上述的多肽还可以用作饲料添加剂或抗菌剂。
本发明从扇贝的酶解产物中分离获得一种具有抑菌效果的多肽，用该多肽制成饲料添加剂，可以有效的增加养殖的水产动物的疾病抵抗力；从而用于水产饲料制备领域中。
具体实施方式
下面结合实施例对本发明的方法做进一步说明。但实例仅限于说明，并不限于此。下列实例中未注明具体条件的实验方法，通常可按常规条件，如J.萨姆布鲁克(Sambrook)等编写的《分子克隆实验指南》中所述的条件，或按照制造厂商所建议的条件运行。
实施例1 多肽的筛选
1)扇贝蛋白酶解产物的制备
扇贝是海产双壳类软体动物，见于潮间带到深海。壳扇形，但蝶铰线直，蝶铰的两端有翼状突出。大小约2.5～15公分以上。壳光滑或有辐射肋。肋光滑、鳞状或瘤突状，色鲜红、紫、橙、黄到白色。下壳色较淡，较光滑。有个大闭壳肌。外套膜边缘生有眼及短触手，触手能感受水质的变化，壳张开时如垂帘状位于两壳间。扇贝常见于沙中或清净海水细砂砾中，取食微小生物，靠纤毛和黏液收集食物颗粒并移入口内。
1)酶解多肽的制备
将新鲜扇贝，清洗除去泥沙等杂物，再用蒸馏水浸泡清洗，去壳及足丝后，加入2倍量(处理后扇贝肉重量)水匀浆，将匀浆物冷冻干燥后，加入质量比1:5的95％乙醇，60℃脱脂4h，脱脂操作重复4次。将脱脂后的残留物40℃真空干燥后，粉碎，过100筛，得到扇贝粗蛋白，测定其蛋白含量为63.24％。取扇贝粗蛋白加入3倍量水，匀质5min后，预热至50℃，加入胰蛋白酶和胃蛋白酶进行搅拌酶解4h，100℃加热10min灭酶后，4000rpm/min离心15min，取上清液，60℃减压浓缩后冷冻干燥，得到扇贝蛋白酶解产物。
对酶解产物以不同分子量的超滤膜对其进行超滤处理，分别收集不同分子量范围的酶解液，对酶解液进行抑制哈维氏弧菌活性的测定；对具有抑制哈维氏弧菌活性的酶解液进行单一活性肽的分离。
2)多肽的分离纯化
使用Sephadex LH-20凝胶色谱柱层析方法来分离活性肽，取有抗氧化活性的酶解液用蒸馏水溶解成110mg/ml溶液，Sephadex LH-20凝胶色谱柱1.0cm×60cm进行分离纯化，流动相为蒸馏水，洗脱速度为0.6ml/min，220nm测定吸光度，收集各峰，冻干备用。扇贝蛋白酶解产物经Sephadex LH-20凝胶柱分离后得到5个峰。将每个峰再次进行抗氧化活性的分析，确定具有活性的峰
用高效液相色谱对具有活性的峰的产物进一步分离纯化。色谱条件如下：色谱柱为反相C18键合硅胶柱(Agilent,ZORBAX SB-C18,150mm×4.6mm,5μm)；流动相：A水(含1％的三氟乙酸)，B乙腈，梯度洗脱条件如下；流速：1.0 ml/min，柱温25℃，检测波长为214nm，收集主要色谱峰，冻干备用得到短肽样品。经过高效液相进一步分离纯化，收集活性肽冷冻干燥后，进行氨基酸序列分析。
利用氨基酸序列分析仪及质谱仪，对所收集所得组分进行氨基酸序列分析。利用Edman降解法和质谱分析，对所制备的活性肽段进行氨基酸序列分析和分子量测定，多肽的序列为ValProPhe GlyTyrThrProAla(SEQ ID NO:1)；其编码的核苷酸序列为SEQ ID NO：2。
实施例2 多肽的抑菌效果检测
采用打孔器打孔并测量抑菌圈的方法检测人工合成的多肽(SEQ ID NO:1)对哈维氏弧菌(Vibrio harveyi)的抑菌活活性，培养哈维氏弧菌(Vibrio harveyi)菌种至对数期，用PBS溶液稀释菌液至浓度为107～108个/ml，取稀释后的菌液150μl涂平板，用6mm打孔器在每个平板上打五个孔，孔中分别加入2倍梯度稀释的多肽溶液，以及10mg/ml的氨苄做对照，各20μl，每次三个重复，28℃培养箱培养12h后观察并测量抑菌圈。抑菌的最小浓度视为最低抑菌浓度。结果发现多肽对革兰氏阴性菌哈维氏弧菌有抑菌活性，观察到了明显的抑菌圈。
用菌落计数法测量抑菌效果的方法检测多肽对哈维氏弧菌的抑菌活活性，培养哈维氏弧菌至对数期，用PBS溶液稀释菌液至浓度为107～108个/ml，取稀释后的菌液80μl，加入20μl不同浓度的多肽溶液涂平板，以及10mg/ml的卡那霉素做对照，各100μl，每次三个重复，37℃培养箱培养12h后观察并测量抑菌圈。抑菌的最小浓度视为最低抑菌浓度。结果显示多肽对哈维氏弧菌的最小抑菌浓度为0.07mg/ml。
上述筛选分离的多肽能过用作饲料添加剂来制备海水生物的饲料，能够有效防止养殖水体中哈维氏弧菌的繁殖，减少了养殖动物的发病几率。

Claims (6)

  1. 一种多肽，其特征在于，所述的多肽包含有：
    1)序列为SEQ ID NO:1的多肽；
    2)分别与1)中的多肽具有70％或以上同源性的多肽，该多肽的功能与1)中的多肽功能相同或相似。
  2. 如权利要求1所述的多肽，其特征在于，所述的2)中的多肽与1)中的多肽具有90％或以上的同源性。
  3. 一种核苷酸，其特征在于，所述的核苷酸用于编码权利要求1或2所述的多肽。
  4. 如权利要求3所述的核苷酸，其特征在于，所述的核酸的序列为SEQ ID NO：2。
  5. 权利要求1所述的多肽在制备抑制哈维氏弧菌的制品中的应用。
  6. 如权利要求5所述的应用，其特征在于，所述的制品为饲料添加剂或抗菌剂。
PCT/CN2016/107381 2015-12-13 2016-11-26 一种具有抑菌效果的多肽 WO2017101664A1 (zh)

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CN105367625B (zh) * 2015-12-13 2019-01-01 广西洪旭农业科技有限公司 一种具有抑菌效果的多肽
CN105961915A (zh) * 2016-05-15 2016-09-28 卢美珍 一种具有杀菌、提高动物营养吸收能力的水产饲料添加剂
CN107586329B (zh) * 2017-11-03 2020-08-28 宁波大学 一种从银鲳中分离的多肽

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