WO2016124143A1 - 抗菌肽wy-21及其应用 - Google Patents

抗菌肽wy-21及其应用 Download PDF

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WO2016124143A1
WO2016124143A1 PCT/CN2016/073390 CN2016073390W WO2016124143A1 WO 2016124143 A1 WO2016124143 A1 WO 2016124143A1 CN 2016073390 W CN2016073390 W CN 2016073390W WO 2016124143 A1 WO2016124143 A1 WO 2016124143A1
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lysine
antimicrobial peptide
peptide
antibacterial
phenylalanine
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French (fr)
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汪以真
易宏波
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浙江大学
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Priority to EP16746138.3A priority Critical patent/EP3255059B1/en
Priority to US15/164,028 priority patent/US9475843B2/en
Publication of WO2016124143A1 publication Critical patent/WO2016124143A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the field of biomedical technology, in particular to the antibacterial peptide WY-21 and its application.
  • Antimicrobial peptides also known as antimicrobial Peptides (AMP) are important components of the body's innate immunity and are the first barrier against the invasion of foreign pathogens by many organisms.
  • Antibacterial peptides have broad-spectrum antibacterial activity, and mainly play a role in destroying bacterial cell membranes, and are not susceptible to drug resistance.
  • antibacterial peptides also have a variety of biological functions: stimulate cell proliferation, activate the immune system, anti-virus, anti-obesity, anti-inflammatory response. Because of its unique mechanism of action and extensive biological functions, it has attracted the attention of the scientific community as a molecular template for designing new antibacterial and anti-infective preparations. Therefore, antibacterial peptides are an important breakthrough in the development of new and highly effective antibiotic substitutes.
  • the ⁇ -helical antibacterial peptide is the most widely distributed antimicrobial peptide.
  • the hydrophilic residue and the hydrophobic residue are located on both sides of the helix, thus exhibiting a structure-dependent amphiphilic structure.
  • Spiral antimicrobial peptides generally do not have significant structural features in the aqueous phase, but form a typical alpha helical structure in a lipid-rich cell membrane environment.
  • Such antibacterial peptides mainly exert a bactericidal action by destroying the bacterial cell membrane, leading to the stepwise degradation of electrolytes or other signal substances on the membrane, and the bacteria are less likely to develop drug resistance.
  • the natural antimicrobial peptides have immunogenicity, low antibacterial activity, poor stability, strong cytotoxicity, and high hemolysis rate, which limits their use as antibacterial drugs. Therefore, the search for antibacterial peptides with small molecular weight, strong antibacterial activity, high safety and good stability is a key factor in the partial replacement of antibiotics by antibacterial peptides.
  • the technical problem to be solved by the present invention is to provide a safe and efficient antimicrobial peptide WY-21 and its application.
  • the present invention provides an antibacterial peptide WY-21, the entire sequence of which is: valine-lysine-phenylalanine-phenylalanine-arginine-lysine-bright -lysine-lysine-serine-valine-lysine-glutamate-lysine-isoleucine-glycine-lysine-glutamate-phenylalanine-lay -arginine. As shown in the sequence table NO1.
  • the antibacterial peptide WY-21 is an artificially designed synthetic ⁇ -helical cationic polypeptide comprising 21 amino acid residues, a molecular weight of 2624.2 Da, an isoelectric point of 10.79, and a net charge. It is +8.
  • the invention also discloses the use of the above antibacterial peptide WY-21: preparation of a therapeutic Gram-negative bacteria or Gram-positive A broad-spectrum antibacterial agent for bacterial infection.
  • the invention also discloses the use of the above antibacterial peptide WY-21: preparing a drug which exerts an immunoregulatory action to reduce inflammation.
  • Gram-negative bacteria include E.coli ATCC25922, E.coli K 88, E.coli K 12, E.coli EPEC O78:K80, E.coli EPEC O144:K26, E.coli EPEC O44:K74, S. choleraesuis CMCC50020, S. typhimurium CMCC50013, S. enteritidis CMCC50041, P. aeruginosa CMCC27853; Gram-positive bacteria include S. aureus ATCC25923, S. epidermidis ATCC 12228.
  • the method and amount of the antibacterial peptide WY-21 of the present invention can be referred to the use method and amount of the conventional antibacterial peptide (especially the antibacterial peptide having broad-spectrum antibacterial properties against Gram-negative bacteria or Gram-positive bacteria infection), for example, Reference may be made to the polypeptides PG-1 and CP-1 and the like.
  • the antibacterial peptide WY-21 designed and synthesized by the invention is an alpha helix cationic polypeptide, and has the characteristics of short peptide chain length, good stability and convenient artificial synthesis.
  • the amino acid sequence of the antimicrobial peptide WY-21 was searched by the NCBI protein database, and no identical polypeptide was found.
  • the antibacterial peptide WY-21 has the advantages of high net charge, wide antibacterial spectrum and strong bactericidal action. It has the characteristics of low hemolytic activity and small cytotoxicity, and can exert immunoregulatory effects to reduce inflammation.
  • Figure 1 is a graph comparing the hemolysis rate of porcine erythrocytes with different solubility of antimicrobial peptide WY-21. The above results are the average of 6 independent replicate experiments.
  • Figure 2 is a graph showing the cytotoxicity of different solubility antimicrobial peptides WY-21 against porcine PMBCs, LDH is the lactate dehydrogenase, and the above results are the average of 6 independent replicate experiments.
  • Fig. 3 is a graph showing the mitigation effect of LPS-induced macrophage inflammation in the control group, the WY-21 group, the LPS group, and the LPS+WY-21 group.
  • Figure 4 is a graph comparing the hemolysis rate of porcine red blood cells with antimicrobial peptides PG-1 of different solubility.
  • Figure 5 is a graph showing the alleviation effect of CP-1 on LPS-induced macrophage inflammation.
  • the preparation of the antimicrobial peptide WY-21 was carried out by solid phase chemical synthesis using an automatic peptide synthesizer from the C-terminus to the N-terminus according to the amino acid sequence described in the Summary. It was purified by HPLC reverse phase column chromatography. Purified antibiotic The peptide BW-21 was identified by high performance liquid chromatography HPLC method with purity higher than 95%. The molecular weight was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), which was basically consistent with the theoretical molecular weight. The amino acid sequence structure was determined by an automatic amino acid sequencer.
  • the synthetic antibacterial peptide WY-21 contains 21 amino acid residues with a molecular weight of 2624.2Da.
  • the complete sequence is: valine-lysine-phenylalanine-phenylalanine-arginine-lysine -leucine-lysine-lysine-serine-valine-lysine-glutamate-lysine-isoleucine-glycine-lysine-glutamate-phenylalanine - Lysine-arginine. As shown in the sequence table NO1.
  • the antimicrobial peptide WY-21 has a small molecular weight, good stability, a high positive charge content, and a small total hydrophilicity index.
  • the antibacterial activity of the antimicrobial peptide WY-21 is shown in Table 2.
  • WY-21 MIC indicates the minimum inhibitory concentration of the antimicrobial peptide WY-21, and the above results are the average of 6 independent replicate experiments.
  • the antimicrobial peptide WY-21 had strong antibacterial activity against Gram-positive bacteria and Gram-negative bacteria tested.
  • the heparin anticoagulated porcine anterior vena cava blood and RPMI1640 medium were mixed 1:1, centrifuged with Ficoll-Paque lymphocyte separation solution, and the ring-shaped milky white mononuclear cell layer (PMBCs) was taken up to collect the lowest erythrocytes. .
  • the red blood cells were washed with 1 x PBS solution until the supernatant was not red.
  • the washed red blood cells and 1 ⁇ PBS were mixed in a volume ratio of 1:89 to prepare a red blood cell working solution for hemolysis rate measurement.
  • hemolysis rate (%) 100% * ( ⁇ OD test - ⁇ OD negative control) / ( ⁇ OD positive control - ⁇ OD negative control).
  • the antibacterial peptide WY-21 has a low hemolysis rate on porcine red blood cells, and the hemolysis rate is less than 5% at a concentration of 512 ⁇ g/mL.
  • the isolated and purified PBMCs were diluted to 1 ⁇ 10 6 cells/mL with medium RMPI1640, 96-well plates were seeded at 90 uL per well, and a background control (100 ⁇ L RMPI1640 medium) and a low-level control well (90 ⁇ L cell suspension + 10 ⁇ L) were set. PBS) and high level control wells (90 ⁇ L cell suspension + 10 ⁇ L 20% Triton X-100).
  • the 96-well plate was statically cultured in a 37 ° C, 5% CO 2 incubator for 2 h; 10 uL of the corresponding concentration of the antibacterial peptide WY-21 was added to make the final concentration of the antibacterial peptide WY-21 512 ⁇ g / mL, 256 ⁇ g / mL, 128 ⁇ g, respectively. /mL, 64 ⁇ g/mL, 32 ⁇ g/mL, 16 ⁇ g/mL, 8 ⁇ g/mL, 6 replicates per treatment. After incubating in the incubator for 24 hours, add 10 ⁇ L of 20% TritonX-100 to the positive control wells, and mix by pipetting and continue to culture for 15 min.
  • LDH release rate (%) 100% * ( ⁇ OD test - ⁇ OD negative control) / ( ⁇ OD negative control - ⁇ OD positive control).
  • the LDH release rate of the antimicrobial peptide WY-21 porcine peripheral monocytes was less than 10% at a concentration lower than 256 ⁇ g/mL, and the results showed that the antimicrobial peptide WY-21 had low cytotoxicity.
  • Porcine macrophages (3D4/2 cell line) were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS), and cultured in a 37 ° C cell incubator at a concentration of 5% CO 2 to about 80-90%. The cells fuse to form a single cell layer, count cells, and plate. The cells were inoculated to a 6-well plate at 1 ⁇ 10 6 cells/well, RPMI-1640 complete medium was added, cultured to about 80% confluence, and replaced with serum-free RPMI-1640 medium (2 ml). Porcine macrophages were treated with antibacterial peptide WY-21 (20 ⁇ g/mL) and E. coli LPS (1 ⁇ g/mL).
  • FBS fetal bovine serum
  • the groups were set as follows: control group, WY-21 group, LPS group, LPS+WY-21 group (antibacterial)
  • the peptide WY-21 was 20 ⁇ g/mL
  • the Escherichia coli LPS was 1 ⁇ g/mL).
  • Six replicate wells were set in each group and cells were harvested after 24 hours of culture.
  • Total RNA was extracted from Trizol, cDNA was obtained by reverse transcription, and the relative expression of IL-6, IL-8 and IL-22 was quantitatively determined by fluorescence.
  • the internal reference gene was GAPDH.
  • LPS treatment of macrophages caused a significant increase in the expression levels of inflammatory cytokines IL-6, IL-8, and IL-22, while antibacterial peptide WY-21 treatment attenuated LPS-induced inflammation and decreased IL- 6.
  • Comparative Example 1 The polypeptides LL-37 and Cathelicidin-BF15 which were similar to WY-21 of the present invention were searched and searched by the NCBI protein database, and the polypeptides PG-1 and CP- which were found to have strong antibacterial activity were also found in the literature. 1. The above antibacterial peptide and antibiotic were tested as in Examples 2 to 5.
  • sequence of the polypeptide is as follows:
  • Antibacterial peptide LL-37 Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu Ser;
  • Antibacterial peptide C-BF15 Val Lys Arg Phe Lys Lys Phe Phe Arg Lys Leu Lys Lys Ser Val;
  • Antibacterial peptide PG-1 Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val Gly Arg;
  • Antibacterial peptide CP-1 Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys Arg Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg.
  • test results are as follows: As shown in Table 3, LL-37 and Cathelicidin-15 have certain antibacterial activity, but the antibacterial activity is not as strong as the antimicrobial peptide WY-21, and the antibacterial activity of PG-1 and CP-1 and the antimicrobial peptide WY-21 similar. However, PG-1 has a high hemolysis rate and is much safer than the antimicrobial peptide WY-21 (see Figure 4). At the same time, CP-1 did not relieve the inflammation caused by LPS, and the effect of exerting immune regulation of inflammation was far less than that of antimicrobial peptide WY-21 (see Figure 5).

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Abstract

本发明公开了一种抗菌肽WY-21，其全序列为：缬氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-精氨酸-赖氨酸-亮氨酸-赖氨酸-赖氨酸-丝氨酸-缬氨酸-赖氨酸-谷氨酸-赖氨酸-异亮氨酸-甘氨酸-赖氨酸-谷氨酸-苯丙氨酸-赖氨酸-精氨酸。该抗菌肽WY-21的用途是：制备治疗革兰氏阴性菌或者革兰氏阳性菌感染的广谱抗菌药物；制备发挥免疫调控作用从而降低炎症的药物。

Description

抗菌肽WY-21及其应用 技术领域
本发明属于生物医学技术领域，特别是涉及抗菌肽WY-21及其应用。
背景技术
抗生素滥用造成耐药性细菌的产生，也会造成抗生素在畜产品中残留，带来食品安全问题。由于耐药性问题的严重化，越来越多的抗生素对病原菌丧失了杀伤作用。同时长期使用抗生素会破坏影响肠道的稳态，引起肠道菌群紊乱，从而破坏机体的先天免疫。因此，寻找安全、高效的抗菌药物成为了研究热点。
抗菌肽，又叫抗微生物肽(Antimicrobial Peptides，AMP)，是机体先天免疫的重要组成成分，是许多生物体抵抗外来致病菌侵袭的第一道屏障。抗菌肽具有广谱抗菌杀菌活性，且主要通过破坏细菌细胞膜发挥作用，不容易产生耐药性。同时抗菌肽还具有多样的生物学功能：刺激细胞增殖，激活免疫系统，抗病毒，抗肥胖，抗炎症反应。由于其独特作用机制与广泛生物学功能引起了科学界的关注，将其作为设计新型抗菌、抗感染制剂的分子模板。所以说抗菌肽是研发新型高效抗生素替代品的重要突破。
α-螺旋抗菌肽是分布最广泛的抗菌肽，当形成α螺旋结构时通常亲水性残基和疏水性残基分别位于螺旋的两侧，因此呈现结构依赖性的两亲结构。螺旋抗菌肽在水相中一般不具有明显的结构特征，但在富含脂类的细胞膜环境下形成典型的α螺旋结构。此类抗菌肽主要通过破坏细菌细胞膜发挥杀菌作用，导致膜上电解质或其他信号物质的逐级降解等，细菌不易产生耐药性。然而，天然的抗菌肽部分存在免疫原性、抗菌活性低，稳定性差，细胞毒性强，溶血率高等问题限制了其作为抗菌药物的推广应用。因此，寻找分子量小、抗菌活性强、安全性高、稳定性好的抗菌肽是实现抗菌肽部分替代抗生素的关键因素。
发明内容
本发明要解决的技术问题是提供一种安全高效的抗菌肽WY-21及其应用。
为了解决上述技术问题，本发明提供了一种抗菌肽WY-21，其全序列为：缬氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-精氨酸-赖氨酸-亮氨酸-赖氨酸-赖氨酸-丝氨酸-缬氨酸-赖氨酸-谷氨酸-赖氨酸-异亮氨酸-甘氨酸-赖氨酸-谷氨酸-苯丙氨酸-赖氨酸-精氨酸。为序列表NO1所示。
作为本发明的抗菌肽WY-21的改进：所述的抗菌肽WY-21为人工设计合成的α螺旋阳离子多肽，包含21个氨基酸残基，分子量为2624.2Da，等电点为10.79，净电荷为+8。
本发明还同时公开了上述抗菌肽WY-21的用途：制备治疗革兰氏阴性菌或者革兰氏阳性 菌感染的广谱抗菌药物。
本发明还同时公开了上述抗菌肽WY-21的用途：制备发挥免疫调控作用从而降低炎症的药物。
备注说明：革兰氏阴性菌包括E.coli ATCC25922、E.coli K 88、E.coli K 12、E.coli EPEC O78:K80、E.coli EPEC O144:K26、E.coli EPEC O44:K74、S.choleraesuis CMCC50020、S.typhimurium CMCC50013、S.enteritidis CMCC50041、P.aeruginosa CMCC27853；革兰氏阳性菌包括S.aureus ATCC25923、S.epidermidis ATCC12228。
本发明的抗菌肽WY-21的使用方法和用量可参照常规抗菌肽(特别是对革兰氏阴性菌或者革兰氏阳性菌感染具有广谱抗菌性能的抗菌肽)的使用方法和用量，例如可参照多肽PG-1和CP-1等。
本发明所设计合成的抗菌肽WY-21为α螺旋阳离子多肽，具有肽链长度较短、稳定性较好、人工合成方便的特点。将抗菌肽WY-21的氨基酸序列经NCBI蛋白数据库搜寻比对，未发现有相同的多肽。抗菌肽WY-21具有净电荷高、抗菌谱广、杀菌作用强等优点，同时具有溶血活性低和细胞毒性小、能发挥免疫调控作用从而降低炎症等特点。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是不同溶度的抗菌肽WY-21对猪红细胞的溶血率对比图，以上结果为6次独立重复实验平均值。
图2是不同溶度抗菌肽WY-21对猪PMBCs的细胞毒性对比图，LDH表示乳酸脱氢酶，以上结果为6次独立重复实验平均值。
图3是对照组、WY-21组、LPS组、LPS+WY-21组对LPS引起的巨噬细胞炎症的缓解作用对比图。
图4是不同溶度的抗菌肽PG-1对猪红细胞的溶血率对比图。
图5是CP-1对LPS引发的巨噬细胞炎症的缓解作用图。
具体实施方式
下面的实施例进一步说明本发明的实质性内容，但本发明的内容不仅仅局限于此。
实施例1、抗菌肽WY-21的制备
抗菌肽WY-21的制备利用固相化学合成法，根据发明内容中所述的氨基酸序列，用自动多肽合成仪从C端到N端逐一进行合成。通过HPLC反相柱层析脱盐纯化。纯化的抗 菌肽WY-21用高效液相色谱HPLC方法鉴定其纯度，纯度高于95％。用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)测定其分子量，与理论分子量基本一致，用自动氨基酸测序仪测定氨基酸序列结构。
人工合成的抗菌肽WY-21包含21个氨基酸残基，分子量为2624.2Da，其全序列为：缬氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-精氨酸-赖氨酸-亮氨酸-赖氨酸-赖氨酸-丝氨酸-缬氨酸-赖氨酸-谷氨酸-赖氨酸-异亮氨酸-甘氨酸-赖氨酸-谷氨酸-苯丙氨酸-赖氨酸-精氨酸。为序列表NO1所示。
抗菌肽WY-21的理化参数如表1所示。
表1、抗菌肽WY-21的理化参数
Figure PCTCN2016073390-appb-000001
由表1可知抗菌肽WY-21分子量较小，稳定性较好，正电荷含量高，总亲水性指数小。
实施例2、抗菌肽WY-21的抗菌实验(MIC)
细菌接种于MH琼脂平板，倒置培养至长出单菌落。挑取单菌落接种于3mL MH肉汤培养基中，恒温震荡培养至菌液浑浊。将菌悬液30μL转接至3mL新鲜MH肉汤培养基中，恒温震荡培养至OD600＝0.5左右；取菌悬液10μL转接至10mL新鲜MH肉汤培养基中涡旋混匀，此时细菌数需在1×105-5×105CFU/mL，用于MIC的测定。在96孔圆底板中加入90μL菌悬液，再逐一加入10μL相应浓度的二倍梯度稀释的抗菌肽溶液，使终浓度分别为256、128、64、32、16、8、4、2、1、0.5、0.25μg/mL；另外阳性对照为只加100μL菌悬液，阴性对照 为100μL MH肉汤培养基；每个处理6个重复；37℃生化培养箱中培养24小时；培养完后观察各孔底部是否有细菌沉淀产生，无肉眼可见细菌沉淀的最小浓度可判定为抗菌物质的MIC。
抗菌肽WY-21的抗菌活性见表2。
表2、抗菌肽WY-21的抗菌活性
Figure PCTCN2016073390-appb-000002
表2中，WY-21MIC表示抗菌肽WY-21的最小抑菌浓度，以上结果为6次独立重复实验平均值。
如表2所示，抗菌肽WY-21对受试的革兰氏阳性菌和革兰氏阴性菌具有较强的抗菌活性。
实施例3、抗菌肽WY-21的溶血活性
将肝素抗凝的猪前腔静脉血液和RPMI1640培养基按1：1混匀，利用Ficoll-Paque淋巴细胞分离液离心后，吸取环状乳白色单核细胞层(PMBCs)待用，收集最底层红细胞。用1×PBS溶液清洗红细胞至上清液没有红色。将冲洗好的红细胞和1×PBS按照1：89的体积比混匀制成用于溶血率测定的红细胞工作液。
在96孔板中加入90uL红细胞工作液，加入10uL抗菌肽WY-21的二倍梯度稀释液，使抗菌肽WY-21终浓度为512、256、128、64、32、16、8、4μg/mL，阳性对照使用Triton X-100 (终浓度为1％)，阴性对照加入10uL 1×PBS，每个处理6个重复。细胞培养箱中培养24h后，1500rpm离心20min。小心吸取上清至新96孔培养板中，用酶标仪测定OD414nm、OD546nm，计算ΔOD值。红细胞溶血率的计算公式为：溶血率(％)＝100％*(ΔOD试验-ΔOD阴性对照)/(ΔOD阳性对照-ΔOD阴性对照)。
抗菌肽WY-21的对猪血红细胞的溶血活性结果见图1。
如图1所示，抗菌肽WY-21对猪血红细胞的溶血率低，在512μg/mL浓度时，溶血率低于5％。
实施例4、抗菌肽WY-21的细胞毒性
将分离纯化的PBMCs用培养基RMPI1640稀释至1×106个/mL，按照每孔90uL接种96孔板，同时设背景对照(100μL RMPI1640培养基)，低水平对照孔(90μL细胞悬液+10μL PBS)和高水平对照孔(90μL细胞悬液+10μL 20％的TritonX-100)。将96孔板于37℃，5％CO2培养箱中静置培养2h；加入10uL相应浓度的抗菌肽WY-21，使抗菌肽WY-21终浓度分别为512μg/mL、256μg/mL、128μg/mL、64μg/mL、32μg/mL、16μg/mL、8μg/mL，每个处理6个重复。培养箱中培养24h后，向阳性对照孔中加入10μL 20％的TritonX-100，并用移液器吹打混匀，继续培养15min；培养结束后，1500rpm离心10min；离心后自各孔中小心吸取60uL细胞上清转移到透明平底96孔培养板相应孔中；每孔加入稀释好的LDH检测试剂30uL，室温避光震荡培养30min，酶标仪测定OD492nm、OD900nm，计算ΔOD值。
LDH释放率按下面公式计算：LDH释放率(％)＝100％*(ΔOD试验-ΔOD阴性对照)/(ΔOD阴性对照-ΔOD阳性对照)。
抗菌肽WY-21对猪外周单核细胞的LDH释放率结果见图2。
如图2所示，在浓度低于256μg/mL时，抗菌肽WY-21猪外周单核细胞的LDH释放率都小于10％，结果表明抗菌肽WY-21的细胞毒性低。
实施例5、抗菌肽WY-21发挥免疫调控作用缓解炎症
猪巨噬细胞(3D4/2细胞系)培养于含10％胎牛血清(FBS)的RPMI-1640完全培养基中，5％CO2浓度的37℃细胞培养箱中培养至约80～90％细胞融合形成单细胞层，细胞计数，铺板。按1×106个/孔接种至6孔板，加入RPMI-1640完全培养基，培养至约80％融合，更换为无血清RPMI-1640培养基(2ml)。用抗菌肽WY-21(20μg/mL)和大肠杆菌LPS(1μg/mL)处理猪巨噬细胞，组别设置如下：对照组、WY-21组、LPS组、LPS+WY-21组(抗菌肽WY-21为20μg/mL，大肠杆菌LPS为1μg/mL)。每组设置六个重复孔，培养24小时后收集细胞。用Trizol提取细胞总RNA，反转录获得cDNA,荧光定量测定IL-6、IL-8、IL-22的相对表达 量，内参基因为GAPDH。
抗菌肽WY-21对LPS引起的巨噬细胞炎症的缓解作用结果见图3。
如图3所示，LPS处理巨噬细胞，引起炎症因子IL-6、IL-8、IL-22的表达量显著升高，而抗菌肽WY-21处理可以缓解LPS引发的炎症，降低IL-6、IL-8、IL-22的表达量。
对比例1、通过NCBI蛋白数据库搜寻比对所找到的与本发明的WY-21较类似的多肽LL-37和Cathelicidin-BF15，同时文献查找到的抗菌活性较强的多肽PG-1和CP-1，将上述抗菌肽和抗生素进行如同实施例2～实施例5的检测。
多肽的序列具体如下：
抗菌肽LL-37：Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu Ser；
抗菌肽C-BF15:Val Lys Arg Phe Lys Lys Phe Phe Arg Lys Leu Lys Lys Ser Val；
抗菌肽PG-1:Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val Gly Arg；
抗菌肽CP-1：Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys Arg Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg。
检测结果如下：如表3所示，LL-37和Cathelicidin-15具有一定的抗菌活性，但是抗菌活性不如抗菌肽WY-21强，PG-1和CP-1的抗菌活性与抗菌肽WY-21相近。然而PG-1的溶血率很高，安全性远低于抗菌肽WY-21(见图4)。同时CP-1对LPS引发的炎症没有缓解作用，发挥免疫调控炎症的效果远不如抗菌肽WY-21(见图5)。
表3、4种抗菌肽的抗菌活性(MIC，μg/ml)
Figure PCTCN2016073390-appb-000003
Figure PCTCN2016073390-appb-000004
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Figure PCTCN2016073390-appb-000005

Claims (4)

  1. 一种抗菌肽WY-21，其特征在于：其全序列为：缬氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-精氨酸-赖氨酸-亮氨酸-赖氨酸-赖氨酸-丝氨酸-缬氨酸-赖氨酸-谷氨酸-赖氨酸-异亮氨酸-甘氨酸-赖氨酸-谷氨酸-苯丙氨酸-赖氨酸-精氨酸。
  2. 根据权利要求1所述的抗菌肽WY-21，其特征在于：所述抗菌肽WY-21为人工设计合成的活性多肽，包含21个氨基酸残基，分子量为2624.2Da，等电点为10.79，净电荷为+8。
  3. 如权利要求1或2所述的抗菌肽WY-21的用途，其特征在于：制备治疗革兰氏阴性菌或者革兰氏阳性菌感染的广谱抗菌药物。
  4. 如权利要求1或2所述的抗菌肽WY-21的用途，其特征在于：制备发挥免疫调控作用从而降低炎症的药物。
PCT/CN2016/073390 2015-02-06 2016-02-03 抗菌肽wy-21及其应用 WO2016124143A1 (zh)

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