WO2021068478A1 - 一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物 - Google Patents

一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物 Download PDF

Info

Publication number
WO2021068478A1
WO2021068478A1 PCT/CN2020/085729 CN2020085729W WO2021068478A1 WO 2021068478 A1 WO2021068478 A1 WO 2021068478A1 CN 2020085729 W CN2020085729 W CN 2020085729W WO 2021068478 A1 WO2021068478 A1 WO 2021068478A1
Authority
WO
WIPO (PCT)
Prior art keywords
helicobacter pylori
polypeptide
biofilm
honey
composition
Prior art date
Application number
PCT/CN2020/085729
Other languages
English (en)
French (fr)
Inventor
张孝林
蒋安民
汪建飞
金光明
张钦元
Original Assignee
安徽科技学院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 安徽科技学院 filed Critical 安徽科技学院
Publication of WO2021068478A1 publication Critical patent/WO2021068478A1/zh
Priority to ZA2022/04622A priority Critical patent/ZA202204622B/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a polypeptide and a polypeptide composition that inhibit and eliminate the formation of biofilm by Helicobacter pylori.
  • Helicobacter pylori is one of the most commonly infected pathogens in humans, especially in developing countries. Its infection rate can even reach 90%. Many infected people often carry the bacteria for life. It has been determined that Helicobacter pylori infection and chronic active gastritis, gastric ulcers, ten Diodenal ulcers and gastric cancer are related. The World Health Organization has identified its Class I carcinogens (definitely carcinogenic) in 1994. Although most infected people are asymptomatic carriers, they can still cause the above-mentioned diseases. Therefore, all people with Helicobacter pylori infection have a sense of anxiety about being infected by Helicobacter pylori and hope to eliminate Helicobacter pylori colonized in the stomach.
  • Commonly used treatment methods are: 2 kinds of antibiotics + a proton pump inhibitor "triple” therapy or 2 kinds of antibiotics + a proton pump inhibitor + a bismuth "quadruple” therapy, the time from the original 1 week , Gradually increase to the current 10d and 2 weeks.
  • the purpose of the present invention is to provide a reagent or composition made by a polypeptide designed by itself and its application, and the honey and the polypeptide have the effect of synergistically enhancing the inhibition and elimination of the biofilm produced by Helicobacter pylori.
  • the polypeptide provided by the present invention has a good inhibition rate and clearance rate on the biofilm formed by Helicobacter pylori, and can play a role in the acidic environment of the stomach and the presence of proteases.
  • the present invention is realized as follows:
  • the embodiments of the present invention provide a designed polypeptide whose amino acid sequence is as SEQ ID NO. 1, 6, 13, 15 or 21.
  • the polypeptides shown in SEQ ID NO. 1, 6, 13, 15 and 21 are scientifically and rationally designed by the inventors.
  • the N-terminal has a positively charged amino acid, which is conducive to the contact of the polypeptide with the negatively charged biofilm of bacteria.
  • the hydrophobic amino acid at the C-terminus it is beneficial for the peptide to penetrate biological membranes.
  • Experimental research results show that these 5 peptides have good inhibition and clearance rates for Helicobacter pylori (including Helicobacter pylori that have not formed biofilms or have formed biofilms), and they still have a good inhibition rate and clearance rate in the acidic environment of the stomach and the presence of proteases.
  • the polypeptides shown in ID NO. 1 and 15 have outstanding inhibition and clearance rates for Helicobacter pylori. These 5 peptides can be used to prepare reagents or compositions for inhibiting and eliminating the biofilm produced by Helicobacter pylori, providing new means and methods for the clinical use of antibiotics to improve their therapeutic effects and reduce the production of Helicobacter pylori to antibiotic-resistant strains .
  • the embodiments of the present invention provide applications of the polypeptides described in the foregoing embodiments in compositions or reagents for inhibiting and eliminating the production of biofilms by Helicobacter pylori.
  • the embodiment of the present invention provides the application of the combination of the polypeptide and honey described in the foregoing embodiment in the preparation of a composition or reagent for inhibiting and eliminating the biofilm produced by Helicobacter pylori.
  • the Helicobacter pylori has the property of producing biofilms.
  • the Helicobacter pylori is Helicobacter pylori that forms a biofilm to reduce the sensitivity of Helicobacter pylori to antibiotics.
  • the embodiments of the present invention provide a reagent or composition for inhibiting and eliminating the production of biofilm by Helicobacter pylori, which contains the polypeptide described in the foregoing embodiment.
  • the polypeptide content is 10 ⁇ g/mL or more; preferably 10-90 ⁇ g/mL, and more preferably 80 ⁇ g/mL.
  • the amount of the polypeptide used alone to inhibit Helicobacter pylori biofilm production is preferably 5-80 ⁇ g/mL, and more preferably 80 ⁇ g/mL.
  • the reagent or composition also contains honey, and the amount of honey used alone to inhibit Helicobacter pylori biofilm formation is 10%-50% (w/v) preferably, and more preferably 40% (w /v).
  • the research of the present invention found that the combination of honey and the aforementioned polypeptides can greatly reduce the concentration of polypeptides required to inhibit and eliminate Helicobacter pylori biofilms, and at the same time, the concentration required for honey to inhibit and eliminate Helicobacter pylori biofilms is greatly reduced.
  • the ability of inhibiting and eliminating Helicobacter pylori to produce biofilm has been strengthened, and the two have a synergistic effect.
  • the reagent or composition is of great value in the treatment of Helicobacter pylori, because Helicobacter pylori colonizes the gastric mucosa, and the emptying of the stomach causes the concentration of the drug in the stomach to drop quickly. If it is at a lower concentration, it can Play the role of inhibiting and eliminating Helicobacter pylori biofilm production, so that it is easier to achieve the effective drug concentration in the gastric mucosa.
  • the polypeptide content is 4-5 ⁇ g/mL, and the honey mass volume percentage content is 9-11%.
  • Polypeptide and honey can play a better synergistic effect in a suitable concentration range.
  • the research of the present invention shows that when the polypeptide content is 4-5 ⁇ g/mL and the honey content is 9-11%, it can produce a better effect on Helicobacter pylori.
  • the biofilm has a high inhibition rate and a high clearance rate for the formed biofilm Helicobacter pylori.
  • the honey is linden honey.
  • the embodiments of the present invention provide a composition for inhibiting and eliminating the production of biofilm produced by Helicobacter pylori, which includes the polypeptide described in the foregoing embodiment, or the reagent or composition made from honey described in the foregoing embodiment .
  • Figure 1 shows the test results of Experimental Example 2.
  • FIG. 1 shows the test results of Experimental Example 3.
  • FIG. 3 shows the test results of Experimental Example 4.
  • Figure 4 shows the test results of Experimental Example 5.
  • Figure 5 shows the test results of Experimental Example 6.
  • amino acid sequence of the polypeptide provided in this example is shown in SEQ ID NO. 1, and the specific sequence is shown in Table 1 (Group 1).
  • amino acid sequence of the polypeptide provided in this example is shown in SEQ ID NO. 6, and the specific sequence is shown in Table 1 (group 6).
  • amino acid sequence of the polypeptide provided in this example is shown in SEQ ID NO. 13, and the specific sequence is shown in Table 1 (group 13).
  • amino acid sequence of the polypeptide provided in this example is shown in SEQ ID NO. 15, and the specific sequence is shown in Table 1 (group 15).
  • the amino acid sequence of the polypeptide provided in this example is shown in SEQ ID NO. 21, and the specific sequence is shown in Table 1 (group 21).
  • the polypeptides provided in Examples 1-5 were determined through an online analysis tool: https://web.expasy.org/peptide_cutter to confirm that these polypeptides would not be degraded by pepsin. Simulate the composition of gastric juice in vitro (according to the literature APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2006, p.
  • Each liter of artificial gastric juice contains 8.3g peptone, 3.5g glucose, 2.05g sodium chloride, 0.6g potassium dihydrogen phosphate, 0.11g calcium chloride, potassium chloride 0.37g, 0.05g of bile, 0.1g of lysozyme, 13.3mg of protease, adjust the pH to 2.5 with 6N hydrochloric acid, and then filter and sterilize with a 0.22 ⁇ m filter membrane. After the polypeptide was treated in the artificial gastric juice for 2 hours, the biological activity and structure of the polypeptide remained unchanged.
  • polypeptides provided in the embodiments of the present invention can be obtained by conventional chemical synthesis methods or genetic engineering techniques.
  • the anti-Helicobacter pylori reagent or composition provided in this embodiment contains any one of the polypeptides in embodiments 1-5 and honey.
  • control groups were set up at the same time.
  • the polypeptide sequences of the control groups are shown in Table 1. There are 28 groups of polypeptides, group 1, group 6, group 13, 15 and group 21 are the polypeptides provided in Examples 1-5, and the other groups are control polypeptides.
  • the Helicobacter pylori SS1 strain was cultured in Helicobacter pylori liquid medium at 37°C under microaerobic conditions for 24 hours.
  • the bacteria were in the logarithmic growth phase. Centrifuge at 3000r/min for 10 min. Collect the precipitate and renew the Helicobacter pylori liquid medium. Suspend the bacterial precipitation, determine the concentration of the bacterial solution, and adjust the bacterial solution to a final concentration of 5 ⁇ 105 CFU/mL. Add 100 ⁇ L of bacterial solution to 87 (28 peptides ⁇ 3 parallel groups + 1 control ⁇ 3 parallel groups) wells of a 96-well sterile enzyme-labeled culture plate.
  • each well Add 100 ⁇ L of saline as a control.
  • the remaining 84 wells are provided with 3 parallel holes for each peptide.
  • the final concentration of peptides in each well is 40 ⁇ g/mL.
  • Microaerobic at 37°C After culturing for 72 hours, the supernatant of each well was carefully discarded, and each well was washed twice with 100 ⁇ L of saline to wash away the suspended cells, and the remaining biofilm was fixed with 100 ⁇ L of methanol for 15min, each Add 100 ⁇ L of 1% crystal violet to the well and dye for 5min. The dye is discarded.
  • Helicobacter pylori broth according to the above method, add 100 ⁇ l of 5 ⁇ 105CFU/mL Helicobacter pylori broth to a 96-well sterile enzyme-labeled culture plate, then add 100 ⁇ l of normal saline to each well, and culture at 37°C under microaerobic conditions After 72 hours, the Helicobacter pylori biofilm was formed, the supernatant was discarded, and the supernatant was washed twice with 200 ⁇ l of normal saline, and then 200 ⁇ l of 40 ⁇ g/mL peptide was added to each well. In the control group, only 200 ⁇ l of normal saline was added to each well as a control.
  • Method According to the test method for detection of Helicobacter pylori biofilm formation and clearance, it was determined for 1 hour, 1.5 hours, 2 hours, 2.5 The clearance rate of H. pylori biofilm by peptides in hours and 3 hours. The result is shown in Figure 2
  • Figure 4 shows that the removal rate of 40% concentration of honey on the formed Helicobacter pylori biofilm reached 31.9% after 3 hours, indicating that the use of honey alone has a certain clearing effect on the formed Helicobacter pylori biofilm.
  • Inhibition rate and 1.5 hours in a short time (according to the gastric emptying time is 2-3 hours and the gastric emptying test after 1.5 hours, the drug concentration becomes very small, so 1.5 hours is selected as the concentration of investigation, and 10% (w /v) honey and 5 ⁇ g/mL peptide data are based on the test results of gastric emptying drug residues in the gastric mucosa) the clearance rate of the formed Helicobacter pylori biofilm.
  • the composition of honey and a lower concentration of polypeptides found that the composition has a lower inhibition rate on Helicobacter pylori biofilm formation than when used alone.
  • Polypeptide 5 ⁇ g/mL (inhibition rate 12.5%) and a lower concentration of honey10 %(w/v) (inhibition rate 8.9%) has a significant enhancement effect on the sum of 21.4% inhibition rate of Helicobacter pylori biofilm formation, reaching 28.7% (Figure 5A). It shows that the composition has a synergistic enhancement effect on inhibiting the formation of Helicobacter pylori biofilm.
  • the low concentration composition of honey and peptides of 10% (w/v) and 5 ⁇ g/mL can eliminate the biofilm formed by Helicobacter pylori with a clearance rate of 10.8% (Figure 5B) .
  • Example 4 The lower concentration of the polypeptide of Example 4 and the honey combination were tested for their effect on the elimination of Helicobacter pylori biofilm formation after 10 consecutive times.
  • Method Determine the clearance rate of the formed Helicobacter pylori biofilm by the composition of a lower concentration of 10% (w/v) honey and a lower concentration of polypeptide 5 ⁇ g/mL according to the above-mentioned Helicobacter pylori biofilm formation and clearance rate detection test method 1.5 hours after adding the composition, discard the composition, add the composition again for 1.5 hours, discard the composition, continue for 10 times, simulate in vitro the clearance rate of biofilm after oral composition twice a day for 5 consecutive days.
  • the low-concentration honey and polypeptide composition act continuously for 10 times. After 1.5 hours each time, the clearance rate of the biofilm formed by Helicobacter pylori can reach 98.6% (C in Figure 5).
  • This example uses low-concentration honey In the case of repeated multiple times, the combination with polypeptide has a good effect on removing the biofilm produced by Helicobacter pylori.
  • the invention provides a good means and method for improving the efficacy of antibiotics in the later clinical stage.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Insects & Arthropods (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Husbandry (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物，多肽其氨基酸序列如SEQ ID NO.1、6、13、15或21所示。该多肽具有抑制和清除幽门螺杆菌形成生物膜的作用，且该多肽不包含在胃酸性环境和蛋白酶条件下降解的氨基酸残基，在胃酸性环境和蛋白酶存在时仍保持生物活性。可以减少幽门螺杆菌耐药菌菌株的产生，提高临床使用抗生素治疗效果。

Description

一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物 技术领域
本发明涉及生物技术领域，具体而言，涉及一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物。
背景技术
幽门螺杆菌是人类最普遍被感染的病原菌之一，尤其在发展中国家其感染率甚至达到90%，很多感染者经常终身带菌，已经确定幽门螺杆菌感染与慢性活动性胃炎，胃溃疡，十二指肠溃疡，胃癌发生具有相关性，世界卫生组织在1994年就已明确其Ⅰ类致癌原（肯定致癌作用）。虽然大多数感染者是无症状的携带者，但仍然可引起上述的疾病发生。因此，所有的幽门螺杆菌感染者对身体被幽门螺杆菌感染存在焦虑感，都希望能清除胃中定植的幽门螺杆菌。常用的治疗方法是：2种抗生素+一种质子泵抑制剂的“三联”疗法或2种抗生素+一种质子泵抑制剂+一种铋剂的“四联”疗法，时间从原来的1周，逐渐增加到现在的10d和2周。
技术问题
但流行病学调查发现，幽门螺杆菌在我国的人群中的感染率不但没有下降反而略有升高，原因是幽门螺杆菌对多种抗生素产生耐药性，导致治愈率逐渐下降，幽门螺杆菌具有传染性，治愈的人群可能重新感染。现在越来越多的研究发现，幽门螺杆菌形成生物膜导致其可以生存在蔬菜水果和自来水中，人们在生食蔬菜和水果和饮用自来水时导致幽门螺杆菌传播和感染。幽门螺杆菌形成生物膜导致幽门螺杆菌对抗生素的敏感性下降，甚至可以抵抗1000倍的悬浮培养的细菌的最小抑菌浓度的药物浓度。抗生素如果没能有效杀灭幽门螺杆菌，反而诱导幽门螺杆菌对现有抗生素产生耐药性。因此，抑制幽门螺杆菌生成生物膜和清除幽门螺杆菌已生成的生物膜，对幽门螺杆菌防治具有重大的意义和价值。
研究发现很多细菌都能形成生物膜对自己生存的外界生存压力条件包括抗生素起保护作用，幽门螺杆菌也是如此，但近年来人们发现很多阳离子多肽确实可以发挥抑制或消除细菌生成生物膜或已生成的生物膜作用。但缺乏对幽门螺杆菌生物膜生成的抑制和消除的明确抗菌肽。更主要的原因要抑制和消除胃中定植的幽门螺杆菌多肽必须是在胃酸性环境和蛋白酶存在时仍具有稳定性，不被胃蛋白酶降解。
鉴于此，特提出本发明。
技术解决方案
本发明的目的在于提供多肽是自己设计的多肽及其应用、蜂蜜和多肽之间具有相互协同增强抑制和消除幽门螺杆菌生成生物膜作用制成的试剂或组合物。本发明提供的多肽对幽门螺杆菌形成的生物膜具有较好的抑制率和清除率，其可以在胃酸性环境和蛋白酶存在下发挥作用。
有益效果
本发明是这样实现的：
第一方面，本发明实施例提供一种设计的多肽，其氨基酸序列如SEQ ID NO.1、6、13、15或21所示。
SEQ ID NO.1、6、13、15和21所示的多肽为发明人经过科学合理设计而成，其N端具有带正电荷的氨基酸，利于多肽与细菌带负电荷的生物膜接触，另外，在C端的疏水性氨基酸存在下，有利于多肽穿透生物膜。实验研究结果显示，这5种肽对幽门螺杆菌（包括未生成生物膜或已生成生物膜的幽门螺杆菌）都具有较好的抑制率和清除率，且在胃酸性环境和蛋白酶存在时仍具有稳定性，不被胃蛋白酶降解，尤其是SEQ ID NO.1和15所示的多肽对幽门螺杆菌的抑制率和清除率较为突出。这5种肽可以用于制备成抑制和消除幽门螺杆菌生成生物膜的试剂或组合物，为临床使用抗生素提高其治疗效果，减少幽门螺杆菌对抗生素耐药菌株的产生提供新的手段和方法。
容易理解到，对本领域技术人员而言，在不付出创造性的劳动下，在上述公开的多肽序列的基础上进行常规的单个或多个氨基酸的替换、缺失、两端延长或缩短等，并取得与上述公开的多肽序列等同的抗幽门螺杆菌效果，这样的多肽序列也是属于本发明的保护范围。
第二方面，本发明实施例提供前述实施方式所述的多肽在抑制和消除幽门螺杆菌生成生物膜的组合物或试剂中的应用。
第三方面，本发明实施例提供前述实施方式所述的多肽与蜂蜜组合在制备抑制和消除幽门螺杆菌生成生物膜的组合物或试剂中的应用。
在可选的实施方式中，所述幽门螺杆菌具有生成生物膜特性。
在可选的实施方式中，所述幽门螺杆菌为形成了生物膜降低幽门螺杆菌对抗生素的敏感性的幽门螺杆菌。
第四方面，本发明实施例提供一种抑制和消除幽门螺杆菌生成生物膜的试剂或组合物，其含有前述实施方式所述的多肽。
在可选的实施方式中，所述多肽含量为10μg/mL以上；优选为10-90μg/mL，进一步优选为80μg/mL。
单独使用多肽抑制幽门螺杆菌生成生物膜所用量进行5-80μg/mL优选，进一步优选为80μg/mL。
在可选的实施方式中，所述试剂或组合物还含有蜂蜜，单独使用蜂蜜抑制幽门螺杆菌生成生物膜所用量进行10%-50%(w/v)优选，进一步优选为40%(w/v)。
本发明的研究发现，将蜂蜜与前述的多肽组合，可以大大降低多肽抑制和消除幽门螺杆菌生物膜所需的浓度，同时蜂蜜抑制和消除幽门螺杆菌生物膜所需的浓度也大大降低。抑制和消除幽门螺杆菌生成生物膜的能力得到加强，二者起了相互协同作用。
该试剂或组合物在幽门螺杆菌治疗中应用具有重大的价值，因为幽门螺杆菌定植在胃黏膜，胃的排空导致药物在胃中的浓度下降很快，如果在较低的浓度下就能发挥抑制和消除幽门螺杆菌生成生物膜作用，这样在胃黏膜处更容易达到有效药物浓度的要求。
在可选的实施方式中，所述多肽含量为4-5μg/mL，所述蜂蜜质量体积百分比含量为9-11%。
多肽与蜂蜜在合适的浓度范围下可以起到更好的协同作用，本发明的研究显示，多肽含量为4-5μg/mL和蜂蜜含量为9-11%的组合时，其对幽门螺杆菌生成生物膜有较高抑制率，对已生成的生物膜幽门螺杆菌具有较高的清除率。
在临床实际中由于胃的排空和胃的黏液层存在即使口服较大浓度的多肽和蜂蜜，最终能与幽门螺杆菌生物膜直接接触的多肽浓度和蜂蜜浓度是很小的，这需探究一种具有协同作用组合物，在较低浓度条件下就能发挥很好的消除已生成的幽门螺杆菌生物膜的作用。又因为，蜂蜜和多肽在胃中滞留时间不是很长，因此，多肽必须在较短的时间就能发挥很好清除幽门螺杆菌生物膜作用。
在可选的实施方式中，所述蜂蜜为椴树蜜。
第五方面，本发明实施例提供一种抑制和消除幽门螺杆菌生成生物膜生成的组合物，其包括前述实施方式所述的多肽，或前述实施方式所述的蜂蜜制成的试剂或组合物。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实验例2的检测结果。
图2为实验例3的检测结果。
图3为实验例4的检测结果。
图4为实验例5的检测结果。
图5为实验例6的检测结果。
本发明的实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供的多肽，其氨基酸序列如SEQ ID NO.1所示，具体序列见表1（组1）。
实施例2
本实施例提供的多肽，其氨基酸序列如SEQ ID NO.6所示，具体序列见表1（组6）。
实施例3
本实施例提供的多肽，其氨基酸序列如SEQ ID NO.13所示，具体序列见表1（组13）。
实施例4
本实施例提供的多肽，其氨基酸序列如SEQ ID NO.15所示，具体序列见表1（组15）。
实施例5
本实施例提供的多肽，其氨基酸序列如SEQ ID NO.21所示，具体序列见表1（组21）。实施例1-5提供的多肽通过在线分析工具：https://web.expasy.org/peptide_cutter，确定这些多肽不会被胃蛋白酶降解。在体外在模拟胃液成分（根据文献APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2006, p. 5384–5395）具体：每升人工胃液含䏡蛋白胨8.3g、葡萄糖3.5g、氯化钠2.05g、磷酸二氢钾0.6g、氯化钙0.11g、氯化钾0.37g、胆汁0.05g、溶菌酶0.1g、蛋白酶13.3mg，用6N的盐酸调pH 至2.5，然后用孔径0.22μm的滤膜过滤除菌。多肽在上述人工胃液中处理2小时后，多肽前后生物活性和结构未发生变化。
需要说明的是，本发明实施例提供的多肽可以通过常规的化学合成方法或基因工程技术获得。
实施例6
本实施例提供的抗幽门螺杆菌的试剂或组合物，其含有实施例1-5中的任意一种多肽，以及蜂蜜。
实验例1
检测实施例1-5的多肽对幽门螺杆菌生物膜生成的抑制率，同时设置23个对照组，对照组的多肽序列见表1，共28组多肽，组1、组6、组13、组15和组21为实施例1-5提供的多肽，其他组为对照组多肽。
方法：
幽门螺杆菌SS1菌株在微需氧的条件下37℃在幽门螺杆菌液体培养基中培养24小时，细菌处在对数生长期，3000r/min离心10min，收集沉淀，幽门螺杆菌液体培养基重新悬浮细菌沉淀，确定菌液浓度，把菌液调整到最终浓度为5×105CFU/mL。在96孔的无菌酶标培养板的87（28条多肽×3平行组+1对照组×3平行组）孔中分别加入100μL的菌液，在对照组设有平行的3孔，每孔加100μL生理盐水做对照，剩下84孔每种多肽设有平行3孔，在每孔加100μL浓度为80μg/mL的多肽，最终每孔多肽的浓度为40μg/mL，在37℃微需氧的条件下，培养72小时，每孔的上清液被小心弃去，用100μL的生理盐水洗每孔2次，洗去悬浮生长的细胞，留下的生物膜用100μL 的甲醇固定15min，每孔加1%的结晶紫100μL染色5min，染料被弃去，每孔用200μL的无菌水洗2次，平板在37℃干燥30min.每孔加33%的乙酸100μL轻微的搅拌混合均匀。然后在595nm处测吸光度。多肽对幽门螺杆菌生物膜生成的抑制率=对照品孔的平均吸光度值-每个多肽的平均吸光度值/对照品孔的平均吸光度值×100%。
按照上述的方法获得幽门螺杆菌菌液，在96孔无菌酶标培养板中加入5×105CFU/mL幽门螺杆菌菌液100μl然后在每孔加生理盐水100μl，在微需氧和37℃培养72小时，形成幽门螺杆菌生物膜，弃去上清液，用200μl的生理盐水洗2次，再在每孔加40μg/mL的多肽200μl，对照组每孔只加200μl生理盐水做对照。在微需氧和37℃条件下，150r/min 震荡培养4小时，按照上述的方法测生物膜量，每个样品设置3组求平均值。多肽对幽门螺杆菌已生成生物膜的清除率=对照品孔的平均吸光度值-每个多肽的平均吸光度值/对照品孔的平均吸光度值×100%
结果见表1。
表1 实施例1-5提供的多肽对幽门螺杆菌生物膜生成的抑制率和清除率
分组 多肽序列 抑制率（%） 清除率（%） 分组 多肽序列 抑制率（%） 清除率（%）
1 KRIVQRIKDVIR 75.8 56.4 15 KRIVQRIKDIKP 86.8 67.5
2 KRIVQRIKDVGP 1.6 0.6 16 KRIVQRIKDIIF 0.5 0
3 KRIVQRIKDTAP 1.3 0.4 17 KRIVQRIKDIEP 1.8 0.4
4 KRIVQRIKDSVP 1.5 0.7 18 KRIVQRIKDIAP 0.3 0
5 KRIVQRIKDRVP 0.3 0 19 KRIVQRIKDIAE 0.7 0
6 KRIVQRIKDRRR 20.6 10.7 20 KRIVQRIKDGPI 1.9 0.3
7 KRIVQRIKDPSP 0.8 0 21 KRIVQRIKDGKV 12.6 7.2
8 KRIVQRIKDPGP 1.7 0.2 22 KRIVQRIKDGGP 1.8 0.2
9 KRIVQRIKDMPP 7.5 0.8 23 KRIVQRIKDGGM 7.7 0.3
10 KRIVQRIKDMNP 6.3 0.7 24 KRIVQRIKDGEG 8.9 0.9
11 KRIVQRIKDIVP 1.9 0.2 25 KRIVQRIKDERP 6.8 0.7
12 KRIVQRIKDIVQ 0.1 0 26 KRIVQRIKDADA 11.8 0.9
13 KRIVQRIKDIRI 60.7 28.1 27 KRIVQRIKDAAP 10.2 1.3
14 KRIVQRIKDIPP 3.8 0.5 28 KRIVQRIKDAEP 4.8 0.7
从表1可以看出，实施例1-5提供的多肽对幽门螺杆菌生物膜生成的抑制率和清除率都比较高，明显高于对照组的多肽（ P<0.05）；其中，值得注意的是，实施例1的多肽（组1）其抑制率和清除率分别达到75.8%和56.4%，实施例4提供的多肽（组15）其抑制率和清除率分别达到86.8%和67.5%，效果突出。
实验例2
验证不同浓度的多肽对幽门螺杆菌生物膜生成的抑制或清除作用
方法：按照上述幽门螺杆菌生物膜生成抑制率检测试验方法分别测定5μg/mL、10μg/mL、20μg/mL、40μg/mL、80μg/mL的多肽对幽门螺杆菌生物膜生成的抑制率。
结果见图1，可以看出，实施例4的多肽（KRIVQRIKDIKP）在不同浓度下对幽门螺杆菌生物膜生成都具有清除作用，并随着多肽的浓度增大，对生物膜生成的抑制率不断增加，当多肽的浓度达到80μg/mL时，对幽门螺杆菌生物膜生成的抑制率达到98.2%，几乎接近100%。
实验例3
验证80μg/mL实施例4的多肽在不同时间下对幽门螺杆菌已生成生物膜的清除作用方法：按照上述幽门螺杆菌生物膜生成清除率检测试验方法测定1小时，1.5小时，2小时，2.5小时、3小时多肽对已生成幽门螺杆菌生物膜的清除率。结果见图2
结果见图2。临床中多肽在胃中因胃的排空，多肽在胃中的滞留的时间不是很长，最长4小时，这样，随时时间的推移胃中多肽的浓度已经不是很高，因此，对前3个小时80μg/mL的多肽对幽门螺杆菌已生成的生物膜的清除率进行考察。图2显示，在第3小时实施例4的多肽（KRIVQRIKDIKP）对已生成的幽门螺杆菌生物膜的清除率达到56.8%，可以说明，在较短时间内，实施例4的多肽表现出非常好的对幽门螺杆菌的清除率。
实验例4
检测不同浓度蜂蜜对幽门螺杆菌生物膜生成的抑制作用方法：本发明用的是椴树蜜。按照上述幽门螺杆菌生物膜生成抑制率检测试验方法测定10%(w/v) 、20%(w/v) 、30%(w/v) 、40%(w/v) 、50%(w/v) 的多肽对幽门螺杆菌生物膜生成的抑制率。结果见图3
图3结果显示蜂蜜在开始随着蜂蜜浓度的增加，对幽门螺杆菌生成的生物膜抑制率不断增加，但随后随着蜂蜜浓度的增加，蜂蜜对幽门螺杆菌生物膜生成的抑制率并不显著增加，原因可能是：蜂蜜浓度的增加，蜂蜜的流动性减少，影响蜂蜜对细菌的作用。
实验例5
检测40%浓度蜂蜜在不同时间段下对幽门螺杆菌生物膜生成的清除作用方法：按照上述幽门螺杆菌生物膜生成清除率检测试验方法测定1小时，1.5小时，2小时，2.5小时、3小时蜂蜜对已生成幽门螺杆菌生物膜的清除率。结果见图4。
图4显示，在3小时后 40%浓度的蜂蜜对已生成的幽门螺杆菌生物膜的清除率达到31.9%，说明，单独使用蜂蜜表现对已生成的幽门螺杆菌生物膜具有一定清除作用。
实验例6
检测实施例4的较低浓度的多肽与蜂蜜组合对幽门螺杆菌生物膜生成抑制和清除作用
方法：按照上述幽门螺杆菌生物膜生成抑制率和清除率检测试验方法测定较低浓度10%(w/v)的蜂蜜与较低浓度多肽5μg/mL的组合物对幽门螺杆菌生成生物膜的抑制率和在较短时间1.5小时（依据胃的排空时间是2-3小时和胃排空试验在1.5小时后药物浓度变得很小，因此选择1.5小时作为考察浓度，且10%(w/v)的蜂蜜和5μg/mL的多肽数据是基于胃排空药物在胃黏膜残留量试验结果）对已生成幽门螺杆菌生物膜的清除率。
结果见图5。
在临床实际中由于胃的排空和胃的黏液层存在即使口服较大浓度的多肽和蜂蜜，最终能与幽门螺杆菌生物膜直接接触的多肽浓度和蜂蜜浓度是很小的，这需探究一种具有协同作用组合物，在较低浓度条件下就能发挥很好的抑制和消除的幽门螺杆菌生成生物膜的作用。又因为，蜂蜜和多肽在胃中滞留时间不是很长，胃的排空时间是2-3小时。因此，在较短的时间1.5小时就能发挥很好清除幽门螺杆菌生物膜作用，其临床应用价值更大。为临床使用抗生素提高其的治疗效果，减少幽门螺杆菌对抗生素耐药菌株的产生提供新的手段和方法。
本实验例应用蜂蜜和多肽较低浓度的组合物发现，该组合物对幽门螺杆菌生物膜生成的抑制率比单独应用较低用多肽5μg/mL（抑制率12.5%）和较低浓度蜂蜜10%(w/v)（抑制率8.9%）对幽门螺杆菌生成生物膜的抑制率之和21.4%有显著增强作用达到28.7%（图5中A）。说明组合物在对抑制幽门螺杆菌生物膜生成有协同增强作用。在第1.5小时，蜂蜜和多肽10%(w/v)和5μg/mL的低浓度组合物，对幽门螺杆菌已生成的生物膜的清除率可达到10.8%的清除率（图5中B）。
实验例7
检测实施例4的较低浓度的多肽与蜂蜜组合连续10次后对幽门螺杆菌生物膜生成的清除作用。
方法：按照上述幽门螺杆菌生物膜生成清除率检测试验方法测定较低浓度10%(w/v)的蜂蜜与较低浓度多肽5μg/mL的组合物对已生成幽门螺杆菌生物膜的清除率，在加入组合物1.5小时后，倒掉组合物，再次加入组合物1.5小时候，倒掉组合物，连续进行10次，体外模拟连续5天每天2次口服组合物后对生物膜的清除率。
结果见图5。
低浓度的蜂蜜和多肽组合物连续作用10次，每次1.5小时后，其对幽门螺杆菌已生成的生物膜的清除率可达到98.6%（图5中C）本实施例利用低浓度的蜂蜜和多肽组合物在反复多次的情况下，对幽门螺杆菌生成的生物膜有很好的清除作用。该发明为后期临床提高抗生素的疗效提供一个很好手段和方法。
 工业实用性
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种多肽，其特征在于，其氨基酸序列如SEQ ID NO.1、6、13、15或21所示。
  2. 权利要求1所述的多肽在制备抑制和消除幽门螺杆菌生成生物膜的组合物或试剂中的应用。
  3. 权利要求1所述的多肽与蜂蜜组合在制备抑制和消除幽门螺杆菌生成生物膜的组合物或试剂中的应用。
  4. 根据权利要求2或3所述的应用，其特征在于，所述幽门螺杆菌为形成了生物膜降低幽门螺杆菌对抗生素的敏感性的幽门螺杆菌。
  5. 一种抑制和消除幽门螺杆菌生成生物膜的试剂或组合物，其特征在于，其含有权利要求1所述的多肽。
  6. 根据权利要求5所述的抑制和消除幽门螺杆菌生成生物膜的试剂或组合物，其特征在于，所述多肽含量为10μg/mL以上；优选为10-90μg/mL，进一步优选为80μg/mL。
  7. 根据权利要求5所述的抑制和消除幽门螺杆菌生成生物膜的试剂或组合物，其特征在于，所述多肽试剂或组合物还含有蜂蜜；
    优选地，所述多肽含量为4-5μg/mL，所述蜂蜜质量体积百分比含量（w/v）为9-11%。
  8. 根据权利要求6或7所述的试剂或组合物，其特征在于，所述蜂蜜为椴树蜜。
  9. 一种抑制和消除幽门螺杆菌生成生物膜的药物，其特征在于，其包括权利要求1所述的多肽，或权利要求5-8任一项所述的试剂或组合物。
PCT/CN2020/085729 2019-10-12 2020-04-20 一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物 WO2021068478A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
ZA2022/04622A ZA202204622B (en) 2019-10-12 2022-04-25 Polypeptide and polypeptide composition for inhibiting and eliminating biofilm formed by helicobacter pylori

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910966789.9 2019-10-12
CN201910966789.9A CN110590914B (zh) 2019-10-12 2019-10-12 一种抑制和消除幽门螺杆菌生物膜生成的蜂蜜和多肽组合物

Publications (1)

Publication Number Publication Date
WO2021068478A1 true WO2021068478A1 (zh) 2021-04-15

Family

ID=68866682

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/085729 WO2021068478A1 (zh) 2019-10-12 2020-04-20 一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物

Country Status (3)

Country Link
CN (1) CN110590914B (zh)
WO (1) WO2021068478A1 (zh)
ZA (1) ZA202204622B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113999285A (zh) * 2021-12-02 2022-02-01 浙江大学 抗菌七肽及其应用
CN114478742A (zh) * 2022-03-10 2022-05-13 安徽科技学院 一种抗幽门螺杆菌活性多肽及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110590914B (zh) * 2019-10-12 2022-07-19 安徽科技学院 一种抑制和消除幽门螺杆菌生物膜生成的蜂蜜和多肽组合物

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1878789A (zh) * 2003-10-21 2006-12-13 加利福尼亚大学董事会 人凯萨林菌素抗微生物肽
WO2011083344A2 (en) * 2010-01-11 2011-07-14 Isogenica Ltd Antimicrobial peptides
CN108434438A (zh) * 2018-06-22 2018-08-24 安徽科技学院 抗菌肽在制备治疗幽门螺杆菌病的药物中的用途以及药物组合物
CN110317248A (zh) * 2019-06-05 2019-10-11 遵义医科大学珠海校区 一种人工合成抗菌肽及其设计方法与应用
CN110590914A (zh) * 2019-10-12 2019-12-20 安徽科技学院 一种抑制和消除幽门螺杆菌生物膜生成的蜂蜜和多肽组合物

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100935031B1 (ko) * 2007-08-31 2010-01-06 조선대학교산학협력단 헬리코박터 파일로리균의 리보좀 단백질 ｌ１유래의 유사체펩타이드로부터 풀림구조를 절단한 양이온 증가 항생펩타이드 및 항균 및 항진균용 조성물
JP2010538982A (ja) * 2007-09-11 2010-12-16 モンドバイオテック ラボラトリーズ アクチエンゲゼルシャフト 単独でまたはｇｌｐ−１と組み合わせて用いる、治療剤としてのインスリンｃ−ペプチドの使用
CN107556372A (zh) * 2017-10-23 2018-01-09 安徽科技学院 具有抗幽门螺旋杆菌活性的抗菌肽以及包含该抗菌肽的融合蛋白

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1878789A (zh) * 2003-10-21 2006-12-13 加利福尼亚大学董事会 人凯萨林菌素抗微生物肽
WO2011083344A2 (en) * 2010-01-11 2011-07-14 Isogenica Ltd Antimicrobial peptides
CN108434438A (zh) * 2018-06-22 2018-08-24 安徽科技学院 抗菌肽在制备治疗幽门螺杆菌病的药物中的用途以及药物组合物
CN110317248A (zh) * 2019-06-05 2019-10-11 遵义医科大学珠海校区 一种人工合成抗菌肽及其设计方法与应用
CN110590914A (zh) * 2019-10-12 2019-12-20 安徽科技学院 一种抑制和消除幽门螺杆菌生物膜生成的蜂蜜和多肽组合物

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SURESH MANEESHA K.; BISWAS RAJA; BISWAS LALITHA: "An update on recent developments in the prevention and treatment ofStaphylococcus aureusbiofilms", INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, URBAN UND FISCHER, DE, vol. 309, no. 1, 1 January 1900 (1900-01-01), DE, pages 1 - 12, XP085579663, ISSN: 1438-4221, DOI: 10.1016/j.ijmm.2018.11.002 *
WINDHAM IAN H, SERVETAS STEPHANIE L, WHITMIRE JEANNETTE M, PLETZER DANIEL, HANCOCK ROBERT E W, MERRELL D SCOTT: "Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 84, no. 14, 1 July 2018 (2018-07-01), XP055798795, DOI: 10.1128/AEM.003 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113999285A (zh) * 2021-12-02 2022-02-01 浙江大学 抗菌七肽及其应用
CN113999285B (zh) * 2021-12-02 2023-02-14 浙江大学 抗菌七肽及其应用
CN114478742A (zh) * 2022-03-10 2022-05-13 安徽科技学院 一种抗幽门螺杆菌活性多肽及其应用

Also Published As

Publication number Publication date
ZA202204622B (en) 2022-06-29
CN110590914A (zh) 2019-12-20
CN110590914B (zh) 2022-07-19

Similar Documents

Publication Publication Date Title
WO2021068478A1 (zh) 一种抑制和消除幽门螺杆菌形成生物膜的多肽和多肽组合物
Yuan et al. A novel membrane-disruptive antimicrobial peptide from frog skin secretion against cystic fibrosis isolates and evaluation of anti-MRSA effect using Galleria mellonella model
Lee et al. Adverse effect of a presenilin-1 mutation in microglia results in enhanced nitric oxide and inflammatory cytokine responses to immune challenge in the brain
CN115715781A (zh) 抗微生物疗法
MIYOSHI Vibrio vulnificus infection and metalloprotease
JP2021509809A (ja) コロナウイルス感染症の治療または予防に使用するためのプロテアーゼ活性を有するペプチド
Xu et al. Recombinant Trichinella pseudospiralis serine protease inhibitors alter macrophage polarization in vitro
Crew et al. NeutroPhase® in chronic non-healing wounds
CN112625109A (zh) 一种绿海龟抗菌肽的改造体抗菌肽c-cm6及其制备方法和应用
CN112625108A (zh) 一种绿海龟抗菌肽的改造体抗菌肽c-cm5及其制备方法和应用
Kulshrestha et al. Beneficial effects of N-acetylcysteine on protease-antiprotease balance in attenuating bleomycin-induced pulmonary fibrosis in rats
di Cologna et al. Amyloid aggregation of Streptococcus mutans Cnm influences its collagen-binding activity
Ye et al. MSI-1 combats drug-resistant S. aureus by affecting bacterial viability and inhibiting carotenoid pigment production
CN108659102B (zh) 具有抗菌及抗炎活性的多肽化合物
Gleich Eosinophil granule proteins and bronchial asthma
Chen et al. Divergent roles of three cytochrome c in CTSB-modulating coelomocyte apoptosis in Apostichopus japonicus
CN114478742B (zh) 一种抗幽门螺杆菌活性多肽及其应用
KR101985663B1 (ko) 꽃게 유래의 항균 펩타이드 및 그의 용도
CN109602894B (zh) 一种天蚕素衍生肽的应用
TW202207909A (zh) 二硫龍治療SARS-CoV-2感染的用途
CN113999297A (zh) 一种抗菌肽hrNCM及其制备方法与应用
KR102098029B1 (ko) 키토산을 포함하는, 헬리코박터 파이로리 광역동 치료효과 증진용 조성물
CN108715601A (zh) 一种同时具有抗氧化和抑制Aβ42聚集特性的多肽及其应用与编码该多肽的基因
Li et al. Unraveling the mechanism of ethyl acetate extract from Prismatomeris connata YZ Ruan root in treating pulmonary fibrosis: insights from bioinformatics, network pharmacology, and experimental validation
Li et al. Characterization of a novel LTA/LPS-binding antimicrobial and anti-inflammatory temporin peptide from the skin of Fejervary limnocharis (Anura: Ranidae)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20875211

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20875211

Country of ref document: EP

Kind code of ref document: A1