WO2021134512A1 - 一种拟穴青蟹抗菌肽Scyreprocin新功能及其应用 - Google Patents

一种拟穴青蟹抗菌肽Scyreprocin新功能及其应用 Download PDF

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WO2021134512A1
WO2021134512A1 PCT/CN2019/130620 CN2019130620W WO2021134512A1 WO 2021134512 A1 WO2021134512 A1 WO 2021134512A1 CN 2019130620 W CN2019130620 W CN 2019130620W WO 2021134512 A1 WO2021134512 A1 WO 2021134512A1
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scyreprocin
scylla
amino acid
acid sequence
antimicrobial peptide
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PCT/CN2019/130620
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French (fr)
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王克坚
杨盈
陈芳奕
陈慧芸
彭会
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厦门大学
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Priority to PCT/CN2019/130620 priority Critical patent/WO2021134512A1/zh
Priority to CN201980065313.6A priority patent/CN112955460B/zh
Publication of WO2021134512A1 publication Critical patent/WO2021134512A1/zh
Priority to US17/747,360 priority patent/US20220296677A1/en

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • 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
    • 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 relates to a new application of the antibacterial peptide Scyreprocin of the pseudo-hole crab.
  • Cancer is one of the diseases with the highest morbidity and mortality in the world, and it is the second leading cause of death in the world.
  • effective but cheaper drug therapy is still the best choice for patients as first-line treatment.
  • chemotherapy is still a common treatment for advanced or metastatic cancers.
  • Traditional chemotherapeutic drugs often lack selectivity and usually target rapidly dividing cancer cells. They often inevitably cause damage to healthy cells and tissues. In severe cases, they will cause a series of rejection reactions (such as bone marrow suppression, intestinal mucosa). Inflammation, hair loss, etc.).
  • cationic antimicrobial peptides have bactericidal ability and no obvious toxicity to normal mammalian cells. In addition, they have a broad spectrum of cytotoxicity to cancer cells. As small molecule peptides, cationic antimicrobial peptides can enter tumor tissues more efficiently Thereby enhancing its activity or combining with chemotherapeutics to achieve the effect of enhancing the efficacy of chemotherapeutics.
  • the antibacterial peptide Scyreprocin is a natural active peptide that exists in the mud crab and has good antibacterial activity. At present, there is no report on the effect of Scyreprocin on cancer cells.
  • the anti-cancer activity of the anti-cancer activity of Scyreprocin is explored by using the anti-cancer peptide Scyreprocin to provide a basis for determining whether the anti-cancer activity of the anti-cancer peptide Scyreprocin has anti-cancer activity.
  • the foundation for the development of natural peptide anti-cancer drugs is explored by using the anti-cancer peptide Scyreprocin to provide a basis for determining whether the anti-cancer activity of the anti-cancer peptide Scyreprocin has anti-cancer activity.
  • the purpose of the present invention is to provide a new application of the antimicrobial peptide Scyreprocin of the pseudo-hole crab.
  • Another object of the present invention is to provide a new application of the antimicrobial peptide Scyreprocin for the above-mentioned scylla crab.
  • the amino acid sequence of the polypeptide possesses anti-cancer cell growth activity and autonomous membrane penetration activity.
  • the antimicrobial peptide Scyreprocin can be obtained with a purity of more than 85%.
  • An anti-cancer drug the active ingredient of which includes the above-mentioned scylla scylla antibacterial peptide Scyreprocin.
  • An anticancer composition the active ingredient of which comprises the above-mentioned scylla scylla antibacterial peptide Scyreprocin.
  • An anti-clinical tumor treatment plan the active ingredient of which comprises the above-mentioned scylla scylla antibacterial peptide Scyreprocin.
  • the antibacterial protein of the present invention is derived from mud crab, and its amino acid sequence is:
  • the molecular formula of Scyreprocin is C 396 H 636 N 106 O 127 S 4 and the molecular weight is 9107.258 Daltons.
  • Scyreprocin has no signal peptide sequence.
  • the total length of 84 amino acids contains 15 positively charged amino acid residues and 7 negatively charged amino acid residues.
  • the antimicrobial peptide is predicted based on the charge of the amino acid residues.
  • the electrical point is 9.61.
  • the average coefficient of hydrophilicity of the antimicrobial peptide is -0.968, which has strong water solubility and is a positively charged cationic antimicrobial peptide.
  • the antimicrobial peptide Scyreprocin can be obtained with a purity of more than 85% by using genetic engineering expression technology.
  • the new use of the antimicrobial peptide Scyreprocin of scylla of the present invention includes: having significant growth inhibitory activity on multiple human cancer cell lines.
  • the new use of the antimicrobial peptide Scyreprocin of the present invention includes: the polypeptide has the ability to penetrate the membrane autonomously, and can penetrate the cell membrane into cancer cells and non-cancer cells.
  • the scylla crab antibacterial peptide Scyreprocin of the present invention has no cytotoxicity to non-cancer cells and has specific anti-cancer activity.
  • the scylla crab antibacterial peptide Scyreprocin of the present invention has no hemolytic activity on human red blood cells, has good bio-friendliness, and can be safely used in intravenous injection dosing schemes.
  • Fig. 1 shows the effect of Scylla sinensis antimicrobial peptide Scyreprocin on the proliferation activity of different cells in Example 1 of the present invention.
  • Figure 2 shows the detection of different cell apoptosis effects caused by the antimicrobial peptide Scyreprocin of Scylla sinensis in Example 2 of the present invention.
  • the nucleus is blue (DAPI staining), and the positive signal of apoptosis is green (detected by TUNEL method).
  • Figure 3 shows the effect of the antimicrobial peptide Scyreprocin on the cloning ability of the cells in Example 3 of the present invention.
  • Figure 4 is the detection of the autonomous ability of the scylla scylla antimicrobial peptide Scyreprocin to penetrate the membrane in Example 4 of the invention.
  • the nucleus is blue (DAPI staining), and the positive signal of Scyreprocin protein is red (detected by cellular immunofluorescence).
  • Figure 5 shows the detection of the hemolysis rate of human red blood cells by the scylla scylla antimicrobial peptide Scyreprocin in Example 5 of the present invention.
  • a hemolysis rate of less than 5% is an acceptable safe hemolysis rate range.
  • antimicrobial peptide Scyreprocin used in the following examples are all products obtained by expression and purification of genetic engineering technology.
  • the cell lines involved in the following examples include: human cervical cancer cells (HeLa), human transitional bladder cancer cells (T24), mouse liver cells (AML12), human hepatocytes (L02), human non-small cell lung cancer cells (NCI-H460), human bladder cancer cells (Du145), human liver cancer cells (HepG2), human lung fibroblasts (HFL1), etc., all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • Example 1 Determination of the concentration of the antimicrobial peptide Scyreprocin in inhibiting the growth of cancer cells
  • HeLa human cervical cancer cells
  • T24 mouse liver cells
  • AML12 human liver cells
  • L02 human non-small cell lung cancer cells
  • NCI-H460 human non-small cell lung cancer cells
  • Du145 human Bladder cancer cells
  • HepG2 human liver cancer cells
  • the cells are cultured in the corresponding cell culture medium to a cell conjugation degree of 80-90%, they are trypsinized with EDTA and resuspended in the corresponding cell culture medium. The cell density is adjusted with the corresponding cell culture medium. The cells were seeded on the culture plate, and placed in a 37°C cell incubator for overnight culture under 5% CO 2 conditions.
  • the AQueous kit detects the level of cell proliferation.
  • the antimicrobial peptide Scyreprocin of the crab crab can significantly inhibit the proliferation of cancer cell lines, but has no significant effect on the proliferation of non-cancer cell lines. Therefore, the antimicrobial peptide Scyreprocin has good specific cancer cell growth inhibitory activity.
  • human non-small cell lung cancer cells NCI-H460
  • human transitional bladder cancer cells T24
  • human lung fibroblasts HFL1
  • the scylla scylla antimicrobial peptide Scyreprocin induces cancer cell apoptosis. Detection of death.
  • the cells are cultured in the corresponding cell culture medium to a cell conjugation degree of 80-90%, they are trypsinized with EDTA and resuspended in the corresponding cell culture medium. The cell density is adjusted with the corresponding cell culture medium. The cells were seeded on the culture plate, and placed in a 37°C cell incubator for overnight culture under 5% CO 2 conditions.
  • Positive control group add 100 ⁇ L of the corresponding cell culture medium, and treat the sample with DNase before detection.
  • Negative control group add 100 ⁇ L of the corresponding cell culture medium, without any treatment before the test.
  • Experimental group to be tested add 100 ⁇ L of the protein sample to be tested diluted with the corresponding cell culture medium.
  • Example 3 Determination of the ability of the antimicrobial peptide Scyreprocin to inhibit the growth of cancer cell clones
  • HeLa human cervical cancer cells
  • T24 human transitional bladder cancer cells
  • AML12 mouse liver cells
  • L02 human liver cells
  • NCI-H460 human non-small cell lung cancer cells
  • Du145 human Bladder cancer cells
  • HepG2 human liver cancer cells
  • the cells are cultured in the corresponding cell culture medium to a cell conjugation degree of 80-90%, they are digested with trypsin containing EDTA and resuspended in the corresponding cell culture medium, and the cell density is adjusted with the corresponding cell culture medium. Inoculate 500 cells in each well of the culture plate, place them in a 37°C cell incubator under 5% CO 2 and culture overnight until the cells adhere to the wall;
  • Negative control group solvent control group
  • Test group The test protein sample diluted with the corresponding cell culture solution (containing Scyreprocin at a final concentration of 5 ⁇ M);
  • HeLa human cervical cancer cells
  • T24 mouse liver cells
  • AML12 mouse liver cells
  • L02 human liver cells
  • NCI-H460 human non-small cell lung cancer cells
  • Du145 Human bladder cancer cells
  • HepG2 human liver cancer cells
  • the antimicrobial peptide Scyreprocin from the crab crabs can pass through the cell membranes of cancer cell lines and non-cancerous cell lines and enter the cytoplasm after incubation. Therefore, the antimicrobial peptide Scyreprocin has the properties of transmembrane.
  • human red blood cells were used as the test object to evaluate the hemolysis ratio of the antimicrobial peptide Scyreprocin.
  • Positive control group 100 ⁇ L MilliQ water plus 100 ⁇ L 4% red blood cell solution
  • Negative control group 100 ⁇ L normal saline plus 100 ⁇ L 4% red blood cell solution
  • Experimental group to be tested 100 ⁇ L of protein sample to be tested plus 100 ⁇ L of 4% red blood cell solution.
  • Hemolysis rate (%) (absorption of test sample-absorption of negative control)/(absorption of positive control-absorption of negative control) ⁇ 100%. A hemolysis rate exceeding 5% is regarded as hemolysis.
  • the present invention provides a new application of the antibacterial peptide Scyreprocin of the pseudo cave scylla.
  • the antibacterial protein of the present invention is derived from the mud crab, expressed and purified by genetic engineering technology.
  • the present invention discloses for the first time that the antibacterial peptide Scyreprocin not only has good antibacterial activity, but also has a membrane penetration function, has antibacterial activity against a variety of cancer cells, and has no cytotoxicity to human non-cancer cells, which proves that the antibacterial polypeptide has high efficiency.
  • the specific anti-cancer activity shows great application value, has good application prospects in the development and application of anti-tumor drug components, and has good industrial practicability.

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Abstract

一种拟穴青蟹抗菌肽Scyreprocin的新用途。抗菌蛋白来源于拟穴青蟹，采用基因工程技术表达、纯化获得。首次公开了所述抗菌肽Scyreprocin除具备良好的抗菌活性外，还具备穿膜功能，对多种癌细胞具有抑杀活性，同时对人非癌细胞无细胞毒性，证明该抗菌多肽具备高效特异性抗癌活性，显示出极大的应用价值，在作为抗肿瘤药物成分的研发及应用方面有良好的应用前景。提供了拟穴青蟹抗菌肽Scyreprocin的新用途，扩宽了Scyreprocin的应用范围，为Scyreprocin的应用提供了更多的可能性。

Description

一种拟穴青蟹抗菌肽Scyreprocin新功能及其应用 技术领域
本发明涉及一种拟穴青蟹抗菌肽Scyreprocin的新用途。
背景技术
癌症是全球发病率及死亡率最高的疾病之一，是全球范围内导致人口死亡的第二大因素。除了最近兴起的免疫疗法外，有效但更加便宜的药物治疗仍是患者作为一线治疗的最佳选择。尽管局部癌症能够通过手术直接移除或通过放射性疗法成功治疗，化疗依然是晚期或转移性癌症的常用治疗方式。传统的化疗药物往往缺乏选择性，通常针对快速分裂的癌细胞发挥作用，往往不可避免地对健康的细胞及组织造成伤害，严重的将会导致患者出现一系列排斥反应(如骨髓抑制、肠粘膜炎、脱发等等)。因此，开发不具备传统化疗药物的细胞毒性、不受常见癌细胞耐药机制限制影响的新型抗癌药物具有重要的科学、临床意义。大量研究表明，部分阳离子抗菌肽在具备杀菌能力的同时对正常哺乳动物细胞无明显毒性，此外，其对癌细胞具有广谱细胞毒性，作为小分子肽，阳离子抗菌肽能够较为高效地进入肿瘤组织从而增强其作用活性或与化疗药物联用达到增强化疗药物药效的作用。目前已有许多研究表明天然抗菌肽或其相应的人工合成衍生物具有抗癌活性，一部分具有较强活性的抗菌肽的抗癌机制逐步被揭示，并投入临床实验阶段，为将来抗菌肽作为抗癌活性物质在治疗人类癌症的相关应用提供相关数据。拟穴青蟹抗菌肽Scyreprocin是拟穴青蟹体内存在的天然活性肽，具有良好的抗菌活性。目前，尚无Scyreprocin对癌细胞的作用相关报道。
本发明通过以拟穴青蟹抗菌肽Scyreprocin对多株癌细胞进行体外抗癌活性试验，探讨Scyreprocin抑菌癌活性的强弱，为确定拟穴青蟹体抗菌肽Scyreprocin 是否具有抗癌活性提供依据，同时为研发天然肽类抗癌药物奠定基础。
发明内容
本发明目的在于提供一种拟穴青蟹抗菌肽Scyreprocin的新用途。
本发明的另一目的在于提供上述拟穴青蟹抗菌肽Scyreprocin新用途的应用。
本发明的技术方案之一：
一种拟穴青蟹抗菌肽Scyreprocin的新用途。所述多肽氨基酸序列具备抗癌细胞生长活性及自主穿膜活性。采用现有的基因工程重组蛋白表达技术即可获得纯度达85％以上的拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之二：
一种抗癌药物，其有效成分包括上述拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之三：
一种抗癌组合物，其有效成分包括上述拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之四：
一种抗临床肿瘤治疗方案，其有效成分包括上述拟穴青蟹抗菌肽Scyreprocin。
本发明的技术方案之五：
上述拟穴青蟹抗菌肽Scyreprocin在制备抗癌药物中的应用。
本发明的技术方案之六：
上述拟穴青蟹抗菌肽Scyreprocin在制备抗癌组合物中的应用。
本发明的技术方案之七：
上述拟穴青蟹抗菌肽Scyreprocin在临床肿瘤治疗方案中的应用。
本发明所述的抗菌蛋白来源于拟穴青蟹，其氨基酸序列为：
Figure PCTCN2019130620-appb-000001
Figure PCTCN2019130620-appb-000002
Scyreprocin的分子式为C 396H 636N 106O 127S 4，分子量为9107.258道尔顿。经SignalP4.1软件预测，Scyreprocin无信号肽序列，全长84个氨基酸中包含15个带正电荷的氨基酸残基和7个带负电荷的氨基酸残基，根据氨基酸残基电荷预测该抗菌肽等电点为9.61。该抗菌肽的亲水性平均系数为-0.968，具有较强的水溶性，是一种带有正电荷的阳离子抗菌肽。
采用基因工程表达技术方法即可获得纯度达85％以上的拟穴青蟹抗菌肽Scyreprocin。
本发明的有益效果是：
1、本发明所述拟穴青蟹抗菌肽Scyreprocin的新用途包括：对多株人类癌细胞具有显著的生长抑制活性。
2、本发明所述拟穴青蟹抗菌肽Scyreprocin的新用途包括：所述多肽具有自主穿膜能力，能够穿透细胞膜进入癌细胞与非癌细胞内。
3、本发明所述拟穴青蟹抗菌肽Scyreprocin对非癌细胞无细胞毒性，具有特异抗癌细胞活性。
本发明所述拟穴青蟹抗菌肽Scyreprocin对人血红细胞无溶血活性，具有良好的生物友好性，可安全用于静脉注射给药方案中。
附图说明
图1为本发明实施例1中拟穴青蟹抗菌肽Scyreprocin对不同细胞增殖活性的影响。
图2为本发明实施例2中拟穴青蟹抗菌肽Scyreprocin作用后引起不同细胞凋亡效应的检测。在图2中，细胞核呈蓝色(DAPI染色)，细胞凋亡阳性信号呈绿色(TUNEL法检测)。
图3为本发明实施例3中拟穴青蟹抗菌肽Scyreprocin作用对细胞成克隆能力的影响。
图4为本发明实施例4中拟穴青蟹抗菌肽Scyreprocin穿膜自主能力的检测。在图4中，细胞核呈蓝色(DAPI染色)，Scyreprocin蛋白阳性信号呈红色(细胞免疫荧光法检测)。
图5为本发明实施例5中拟穴青蟹抗菌肽Scyreprocin对人血红细胞溶血率的检测。溶血率小于5％为可接受的安全溶血率范围。
具体实施方式
以下通过具体实施方式对本发明的技术方案进行进一步的说明和描述。
下述实施例中所用拟穴青蟹抗菌肽Scyreprocin均为基因工程技术表达纯化获得的产物。
下述实施例涉及的细胞系有：人子宫颈癌细胞(HeLa)、人移行性膀胱癌细胞(T24)、小鼠肝细胞(AML12)、人肝细胞(L02)、人非小细胞肺癌细胞(NCI-H460)、人膀胱癌细胞(Du145)、人肝癌细胞(HepG2)、人肺成纤维细胞(HFL1)等，均购自中国科学院上海细胞库。
实施例1 拟穴青蟹抗菌肽Scyreprocin抑制癌细胞生长浓度的测定
本部分选取人子宫颈癌细胞(HeLa)、人移行性膀胱癌细胞(T24)、小鼠肝细胞(AML12)、人肝细胞(L02)、人非小细胞肺癌细胞(NCI-H460)、人膀胱癌细 胞(Du145)、人肝癌细胞(HepG2)作为受试细胞进行拟穴青蟹抗菌肽Scyreprocin抑制细胞增殖活性的测定。
(a)将细胞在对应细胞培养液中培养至细胞接合度80-90％后，用含EDTA的胰酶消化并用相应细胞培养液重悬，以相应细胞培养液调整细胞密度，在96孔细胞培养板中接种细胞，置于37℃细胞培养箱在5％CO 2条件下过夜培养。
(b)待每孔细胞生长到接合度约70-80％时，吸去细胞培养液，用相应细胞培养液倍比稀释Scyreprocin蛋白至5、10、20μM，同时设置溶剂对照组为阴性对照，每组设置3个平行样。
(c)蛋白与细胞孵育24小时后，以
Figure PCTCN2019130620-appb-000003
AQueous试剂盒检测细胞增殖水平。
结果表明，拟穴青蟹抗菌肽Scyreprocin可显著抑制癌细胞系细胞的增殖活力，而对非癌细胞系细胞的增殖无显著影响。因此，拟穴青蟹抗菌肽Scyreprocin具有良好的特异性癌细胞生长抑制活性。
实施例2 拟穴青蟹抗菌肽Scyreprocin诱导癌细胞凋亡的检测
本部分选取人非小细胞肺癌细胞(NCI-H460)、人移行性膀胱癌细胞(T24)、人肺成纤维细胞(HFL1)作为受试细胞，进行拟穴青蟹抗菌肽Scyreprocin诱导癌细胞凋亡的检测。
(a)将细胞在对应细胞培养液中培养至细胞接合度80-90％后，用含EDTA的胰酶消化并用相应细胞培养液重悬，以相应细胞培养液调整细胞密度，在96孔细胞培养板中接种细胞，置于37℃细胞培养箱在5％CO 2条件下过夜培养。
(b)待每孔细胞生长到接合度约70-80％时，吸去细胞培养液，用相应细胞培 养液倍比稀释Scyreprocin蛋白至1、5、10、20μM，如下设置阳性对照组、阴性对照组、待测实验组，每组设置2个平行样：
阳性对照组：加100μL相应细胞培养液，检测前使用DNase处理样品。
阴性对照组：加100μL相应细胞培养液，检测前不进行任何处理。
待测实验组：加100μL相应细胞培养液稀释的待测蛋白样品。
(c)将96孔细胞培养板置于37℃、5％CO 2条件下继续培养24h后，根据Roche试剂盒说明书用TUNEL法检测原位细胞凋亡情况。
结果表明：在1μM浓度下，拟穴青蟹抗菌肽Scyreprocin可引起癌细胞NCI-H460、T24凋亡，而对非癌细胞系细胞HFL1无影响。
实施例3 拟穴青蟹抗菌肽Scyreprocin抑制癌细胞克隆生长能力的测定
本部分选取人子宫颈癌细胞(HeLa)、人移行性膀胱癌细胞(T24)、小鼠肝细胞(AML12)、人肝细胞(L02)、人非小细胞肺癌细胞(NCI-H460)、人膀胱癌细胞(Du145)、人肝癌细胞(HepG2)作为受试细胞，评价拟穴青蟹抗菌肽Scyreprocin对不同细胞成克隆能力的影响。
(a)将细胞在对应细胞培养液中培养至细胞接合度80-90％后，用含EDTA的胰酶消化并用相应细胞培养液重悬，以相应细胞培养液调整细胞密度，在6孔细胞培养板中每孔接种500个细胞，置于37℃细胞培养箱在5％CO 2条件下过夜培养至细胞贴壁生长；
(b)吸去细胞培养液，如下设置阴性对照组及待测实验组，每组设置3个平行：
阴性对照组：即溶剂对照组；
待测实验组：以相应细胞培养液稀释的待测蛋白样品(含Scyreprocin终浓度为5μM)；
(c)蛋白与细胞孵育7天后，吸去孔中培养液，以HBSS洗涤细胞3次，每次5min以充分去除残留培养液及漂浮的细胞，加入4％(w/v)多聚甲醛，于室温下固定细胞1小时，以HBSS洗涤细胞3次，每次3min以去除残留的固定液；
(d)以结晶紫染液于室温下染色10-30分钟后，以HBSS充分洗涤去除多余的结晶紫，空气中晾干样品，以光学显微镜下观察，并计数总细胞数大于60个细胞的克隆，统计每个样品的合格克隆数进行分析。
结果表明，Scyreprocin处理后，癌细胞系细胞的成克隆能力显著下降，而非癌细胞系细胞的成克隆能力无显著变化。
实施例4 拟穴青蟹抗菌肽Scyreprocin穿膜能力的检测
本部分选取人人子宫颈癌细胞(HeLa)、人移行性膀胱癌细胞(T24)、小鼠肝细胞(AML12)、人肝细胞(L02)、人非小细胞肺癌细胞(NCI-H460)、人膀胱癌细胞(Du145)、人肝癌细胞(HepG2)作为受试细胞，进行拟穴青蟹抗菌肽Scyreprocin的穿膜能力检测。
(a)将细胞在对应细胞培养液中培养至细胞接合度80-90％后，用含EDTA的胰酶消化并用相应细胞培养液重悬，以相应细胞培养液调整细胞密度，在96孔细胞培养板中接种细胞，置于37℃细胞培养箱在5％CO 2条件下过夜培养；
(b)待每孔细胞生长到接合度约70-80％时，吸去细胞培养液，加入含终浓度为4μMScyreprocin的细胞培养液，同时设置溶剂对照为阴性对照组；
(c)蛋白与细胞孵育24小时后，吸去孔中培养液，以HBSS洗涤细胞3次， 每次5min以充分去除含有Scyreprocin的原培养液，加入4％(w/v)多聚甲醛，于室温下固定细胞1小时，再以HBSS洗涤细胞3次，每次3min以去除残留的固定液；
(d)以含0.1％Triton X-100的3％柠檬酸溶液于冰上透化细胞样品3min，再以HBSS洗涤样品3次，每次5min以去除透化液；
(e)以5％(w/v)BSA于室温封闭样品2小时后，以Scyreprocin特异性抗体及相应的荧光二抗标记胞内可能存在的Scyreprocin蛋白，上机前以DAPI染液对细胞核进行染色，利用荧光共聚焦显微镜观察。
结果表明，拟穴青蟹抗菌肽Scyreprocin可在孵育后无差别穿过癌细胞系及非癌细胞系细胞的细胞膜，进入胞质内部。因此，拟穴青蟹抗菌肽Scyreprocin具备穿膜特性。
实施例5 拟穴青蟹抗菌肽Scyreprocin对人类血红细胞溶血率的测定
本实施例采用人血红细胞作为受试对象，评价拟穴青蟹抗菌肽Scyreprocin对其的溶血比率。
(a)静脉取血5mL至含由抗凝的采血管中，离心并用生理盐水多次洗涤，除去表面白细胞，至上清液不成红色，弃上清后取1mL再用生理盐水定容至50mL，制成4％的红细胞溶液。
(b)将拟穴青蟹抗菌肽Scyreprocin用生理盐水倍比稀释至5、10、20μM。
(c)在96孔细胞培养板中，如下设置阳性对照组、阴性对照组及待测实验组，每组设置三个平行孔：
阳性对照组：100μL MilliQ水加100μL4％红细胞溶液；
阴性对照组：100μL生理盐水加100μL 4％红细胞溶液；
待测实验组：100μL待测蛋白样品加100μL 4％红细胞溶液。
(d)96孔细胞培养板于室温条件下静止3h，之后用11000rpm离心3min。小心吸出100μL于新的96孔细胞培养板上，用酶标仪在570nm波长检测吸光值。
溶血率(％)＝(待测样品吸收-阴性对照吸收)/(阳性对照吸收-阴性对照吸收)×100％。溶血率超过5％视为溶血。
结果表明，拟穴青蟹抗菌肽Scyreprocin在测试浓度下引起的人红细胞溶血率低于5％，即不会引起溶血，具有良好的生物安全性。
工业实用性
本发明提供了一种拟穴青蟹抗菌肽Scyreprocin的新用途。本发明所述的抗菌蛋白来源于拟穴青蟹，采用基因工程技术表达、纯化获得。本发明首次公开了所述抗菌肽Scyreprocin除具备良好的抗菌活性外，还具备穿膜功能，对多种癌细胞具有抑杀活性，同时对人非癌细胞无细胞毒性，证明该抗菌多肽具备高效特异性抗癌活性，显示出极大的应用价值，在作为抗肿瘤药物成分的研发及应用方面有良好的应用前景，具有良好的工业实用性。

Claims (12)

  1. 一种拟穴青蟹抗菌肽Scyreprocin，其氨基酸序列为：
    MKEDSNILDKTAKMTKQNKALLFTAGGAAAFMAGYYYYHCNYRNPAPKKSGSTTSQDKTDAQAVQSIPSPSGNKGKESKDPKVK其特征在于：所述多肽的氨基酸序列包括具有抗癌功能的氨基酸序列，所述多肽用于抑制癌细胞生长。
  2. 根据权利要求1所述一种拟穴青蟹抗菌肽Scyreprocin，其特征在于：所述多肽的氨基酸序列是人工合成的氨基酸序列或是以核苷酸为模板合成的氨基酸序列。
  3. 一种拟穴青蟹抗菌肽Scyreprocin，其氨基酸序列为：
    MKEDSNILDKTAKMTKQNKALLFTAGGAAAFMAGYYYYHCNYRNPAPKKSGSTTSQDKTDAQAVQSIPSPSGNKGKESKDPKVK，其特征在于：所述多肽的氨基酸序列包括具有穿膜功能的氨基酸序列，或在此基础上进行修饰所衍生的具有穿膜功能的氨基酸序列。
  4. 根据权利要求3所述的一种拟穴青蟹抗菌肽Scyreprocin，其特征在于：所述多肽的具备穿膜功能的氨基酸序列是人工合成的氨基酸序列或是以核苷酸为模板合成的氨基酸序列。
  5. 根据权利要求3及4所述的一种拟穴青蟹抗菌肽Scyreprocin，其特征在于：所述多肽的具备穿膜功能的氨基酸序列是具有自主穿膜功能的氨基酸序列。
  6. 一种抗肿瘤药物，其特征在于：其有效成分包括权利要求1至5中任一项权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  7. 一种抗肿瘤组合物，其特征在于：其有效成分包括权利要求1至5中任一项权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  8. 一种临床肿瘤治疗方案，其特征在于：其有效成分包括权利要求1至5中任一 权利要求所述的拟穴青蟹抗菌肽Scyreprocin。
  9. 如权利要求1至5中任一项权利要求所述的拟穴青蟹抗菌肽Scyreprocin在制备抗肿瘤组合物中的应用。
  10. 如权利要求1至5中任一权利要求所述的拟穴青蟹抗菌肽Scyreprocin在制备抗肿瘤药物中的应用。
  11. 如权利要求1至5中任一项权利要求所述的拟穴青蟹抗菌肽Scyreprocin在临床肿瘤治疗方案中的应用。
  12. 如权利要求1至5中所述的一种拟穴青蟹抗菌肽Scyreprocin的用途，其特征在于，所述抑制肿瘤为非小细胞肺癌、子宫颈癌、乳腺癌、肝癌、膀胱癌、前列腺癌。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114304021A (zh) * 2022-01-12 2022-04-12 宁波大学 一种通过调控环境因子促进拟穴青蟹幼蟹生长及蜕壳的方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113307855B (zh) 2021-06-25 2023-08-01 厦门大学 拟穴青蟹抗菌多肽Spampcin56-86及其应用
CN114716512B (zh) * 2021-06-28 2022-12-16 厦门大学 一种青蟹广谱抗菌肽Scyampcin44-63及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111205359A (zh) * 2018-11-21 2020-05-29 厦门大学 一种拟穴青蟹抗菌肽Scyreprocin及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2754453A1 (en) * 2013-01-11 2014-07-16 University College Dublin Conjugate of a cationic antimicrobial peptide and of a fatty acid for use as an anti-cancer agent
CN104004100B (zh) * 2014-06-13 2016-08-24 山西农业大学 猪抗菌肽NK-lysin融合蛋白及其编码基因与应用
US12011480B2 (en) * 2015-05-06 2024-06-18 Uti Limited Partnership Nanoparticle compositions for sustained therapy
WO2018049025A2 (en) * 2016-09-07 2018-03-15 The Broad Institute Inc. Compositions and methods for evaluating and modulating immune responses

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111205359A (zh) * 2018-11-21 2020-05-29 厦门大学 一种拟穴青蟹抗菌肽Scyreprocin及其应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "New breakthrough in marine animal antimicrobial peptide research at Xiamen University", 17 March 2017 (2017-03-17), pages 1 - 2, XP055826249, Retrieved from the Internet <URL:https://std.xmu.edu.cn/_t353/2017/0317/c4739a269190/page.htm> *
DATABASE Protein 12 September 2019 (2019-09-12), ANONYMOUS: "hypothetical protein E2C01_023661 [Portunus trituberculatus]", XP055826244, retrieved from NCBI Database accession no. MPC30398 *
HE YIBIN, PENG HUI, LIU JIE, CHEN FANGYI, ZHOU YING, MA XIAOWAN, CHEN HUIYUN, WANG KEJIAN: "Chlorella Sp. Transgenic with Scy-Hepc Enhancing the Survival of Sparus Macrocephalus and Hybrid Grouper Challenged with Aeromonas Hydrophila.", FISH & SHELLFISH IMMUNOL, vol. 73, 1 February 2018 (2018-02-01), pages 22 - 29, XP055826245, ISSN: 1050-4648, DOI: 10.1016/j.fsi.2017.11.051 *
PENG, HUI: "The Optimized Expression Technology of Marine Animal-Sourced Antimicrobial Peptides in Pichia Pastoris and the Development of Antimicrobial Peptide as Feedstuff Additive (Abstract)", POSTDOCTORAL RESEARCH REPORT, 8 May 2019 (2019-05-08), CN, pages 1 - 17, XP009528982 *
QIAO KUN; XU WAN-FANG; CHEN HUI-YUN; PENG HUI; ZHANG YA-QUN; HUANG WEN-SHU; WANG SHU-PING; AN ZHE; SHAN ZHONG-GUO; CHEN FANG-YI; W: "A New Antimicrobial Peptide SCY2 Identified in Scylla Paramamosain Exerting a Potential Role of Reproductive Immunity", FISH & SHELLFISH IMMUNOLOGY, vol. 51, 18 February 2016 (2016-02-18), pages 251 - 262, XP029481863, ISSN: 1050-4648, DOI: 10.1016/j.fsi.2016.02.022 *
SHAN ZHONG-GUO; ZHU KE-XIN; CHEN FANG-YI; LIU JIE; CHEN BEI; QIAO KUN; PENG HUI; WANG KE-JIAN: "In Vivo Activity and the Transcriptional Regulatory Mechanism of the Antimicrobial Peptide SpHyastatin in Scylla Paramamosain.", FISH & SHELLFISH IMMUNOLOGY, vol. 59, 15 October 2016 (2016-10-15), pages 155 - 165, XP029821502, ISSN: 1050-4648, DOI: 10.1016/j.fsi.2016.10.023 *
WANG, XIAOFEI: "Investigation into the Expression and Antimicrobial Mechanisms of Crustins Fromscylla Paramamosain", CHINA MASTER'S THESES FULL-TEXT DATABASE, 8 May 2019 (2019-05-08), pages 1 - 67, XP055826815 *
ZHONGGUO SHAN, ZHU KEXIN, PENG HUI, CHEN BEI, LIU JIE, CHEN FANGYI, MA XIAOWAN, WANG SHUPING, QIAO KUN, WANG KEJIAN: "The New Antimicrobial Peptide SpHyastatin from the Mud Crab Scylla Paramamosain with Multiple Antimicrobial Mechanisms and High Effect on Bacterial Infection", FRONTIERS IN MICROBIOLOGY, vol. 7, 1140, 21 July 2016 (2016-07-21), pages 1 - 10, XP055709631, DOI: 10.3389/fmicb.2016.01140 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114304021A (zh) * 2022-01-12 2022-04-12 宁波大学 一种通过调控环境因子促进拟穴青蟹幼蟹生长及蜕壳的方法

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