WO2017012132A1 - 姚虻天然抗炎症多肽cecropin-TY1及其应用 - Google Patents

姚虻天然抗炎症多肽cecropin-TY1及其应用 Download PDF

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WO2017012132A1
WO2017012132A1 PCT/CN2015/085252 CN2015085252W WO2017012132A1 WO 2017012132 A1 WO2017012132 A1 WO 2017012132A1 CN 2015085252 W CN2015085252 W CN 2015085252W WO 2017012132 A1 WO2017012132 A1 WO 2017012132A1
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cecropin
inflammatory
natural anti
yao
inflammatory polypeptide
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卫林
徐薇
李敏
黄春景
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苏州大学张家港工业技术研究院
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    • A—HUMAN NECESSITIES
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention relates to a natural anti-inflammatory polypeptide cecropin-TY1 extracted from the salivary gland of Yao Yao and its new application in anti-inflammation, and belongs to the field of biomedical technology.
  • an object of the present invention is to provide a natural anti-inflammatory polypeptide cecropin-TY1 and its use.
  • Cecropin antibacterial peptide is a kind of small molecule polypeptide with antibacterial function. Some of the cecropin antibacterial peptides also have anti-inflammatory functions and can neutralize endotoxin, that is, neutralize bacterial infection while being released. Toxins and exert anti-inflammatory properties. According to It is reported that researchers have studied the successful blood-sucking mechanism of the blood-sucking insect Yaoban (Tabanus yao) based on the host-external parasite interaction. The cecropin-like peptide cecropin-TY1 was found in the salivary glands of Yao.
  • the natural anti-inflammatory polypeptide cecropin-TY1 of the present invention is extracted from the salivary gland of Yaojing, consisting of 39 amino acids, amidated at the carbon end, molecular weight of 3970.22 Dalton, isoelectric point of 11.17, and its amino acid sequence is SEQ. ID: 1, ie:
  • the natural anti-inflammatory polypeptide cecropin-TY1 of the present invention is used for preparing anti-inflammatory drugs, sepsis caused by bacterial infection, endotoxin shock drugs, veterinary drugs, animal feed and cosmetics.
  • the present invention has at least the following advantages: the natural anti-inflammatory polypeptide cecropin-TY1 extracted from the salivary gland of Yao Yao in the present invention has the characteristics of small molecular weight, simple synthesis, obvious anti-inflammatory effect, and low cytotoxicity.
  • the fields of medicine, cosmetics and aquaculture have broad application prospects.
  • Figure 1 is a graph showing the transcriptional inhibition of inducible nitric oxide synthase by Cecropin-TY1 of the present invention
  • Figure 2 is a graph showing the results of inhibition of nitrite production by Cecropin-TY1 of the present invention.
  • Figure 3 is a graph showing the results of inhibition of the production of inflammatory cytokines by Cecropin-TY1 of the present invention.
  • Figure 4 is a graph showing the results of inhibition of activation of MAPKs by Cecropin-TY1 of the present invention.
  • Figure 5 is a graph showing the results of inhibition of NF- ⁇ B activation by cecropin-TY1 of the present invention.
  • Figure 6 is a graph showing the toxicity of cecropin-TY1 of the present invention on mouse macrophages
  • Figure 7 is an endotoxin neutralizing activity of cecropin-TY1 of the present invention.
  • Figure 8 is a circular dichroic chromatogram of the cecropin-TY1 of the present invention in different solutions
  • Fig. 9 is a schematic view showing the simulation of the solution structure of the cecropin-TY1 of the present invention.
  • Inducible nitric oxide synthase is a synthetic enzyme essential for the production of nitric oxide (NO).
  • iNOS Inducible nitric oxide synthase
  • LPS lipopolysaccharide
  • cecropin-TY inhibited the transcription of iNOS in a dose-dependent manner.
  • the transcription level of iNOS was only 100% in cells treated with LPS, and the levels of iNOS were only 41.2%, 31.3%, and 15.8%, respectively, after treatment with cecropin-TY1 at 5, 10, and 20 ⁇ g/mL, cecropin- TY1 reduced transcription of iNOS by 58.8%, 68.7%, and 84.2%.
  • nitrite in the cell culture medium can indirectly reflect the production of NO, and then the effect of cecropin-TY1 on nitrite production is examined.
  • C57BL/6 mouse peritoneal macrophages were plated in 24-well cell culture plates (2.5 ⁇ 10 5 cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 ⁇ g/mL streptomycin sulfate.
  • RMPI-1640 medium purchased from Gbico, USA was cultured. After the cells were attached, as shown in Fig. 2, 100 ng/mL lipopolysaccharide (LPS from Escherichia coli 0111:B4) was added to the cells.
  • cecropin-TY inhibited the production of nitrite in a dose-dependent manner.
  • the cumulative level of nitrite in the LPS-only group was 20.4 ⁇ M.
  • the cumulative levels of nitrite were only 12.0, 7.3, and 5.0 ⁇ M, respectively, and cecropin-TY1 was reduced. Production of 40.0, 63.7 and 75.0% nitrite.
  • C57BL/6 mouse peritoneal macrophages were plated in 24-well cell culture plates (2.5 ⁇ 10 5 cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 ⁇ g/mL streptomycin sulfate.
  • RMPI-1640 medium purchased from Gbico, USA was cultured. After the cells were attached, as shown in Fig. 3, 100 ng/mL lipopolysaccharide (LPS from Escherichia coli 0111:B4) was added to the cells.
  • cecropin-TY1 inhibited the transcription and production of LPS-induced inflammatory cytokines TNF- ⁇ , IL-1 ⁇ and IL-6 in a dose-dependent manner.
  • cecropin-TY1 inhibited the transcription of 60.3% of TNF-a, 67.5% of IL-1 ⁇ and 94.1% of IL-6, while the 20 mg/mL of cecropin-TY1 decreased by 72.1%.
  • C57BL/6 mouse peritoneal macrophages were plated in 6-well cell culture plates (1 ⁇ 10 6 cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 ⁇ g/mL streptomycin sulfate.
  • RMPI-1640 medium purchased from Gbico, USA was cultured. After the cells were attached, as shown in Fig. 4 and Fig.
  • LPS lipopolysaccharide
  • B4 purchased from Sigma
  • cecropin-TY1 at 5, 10 and 20 ⁇ g/mL, respectively
  • various control groups including: only LPS group, only peptide group (10 ⁇ g/mL) and both No LPS and no cecropin-TY1 group.
  • RIPA lysate purchased from Biyuntian
  • BCA protein quantification kit Purchased from Biyuntian for protein quantification.
  • the lysed protein was electrophoresed on a 10% polyacrylamide gel at a voltage of 100 volts for 90 minutes according to a loading of 40 ⁇ g, and then the protein was electroporated (300 mA, 90 minutes) onto the hybridization membrane, and then 5 at room temperature. % bovine serum albumin was blocked for 2 hours.
  • cecropin-TY1 inhibited phosphorylation of ERK, JNK, and p38 in a dose-dependent manner, inhibiting LPS-induced activation of the MAPKs signaling pathway.
  • cecropin-TY1 inhibits the activation of nuclear transcription factor kappa B (NF- ⁇ B)
  • cecropin-TY1 inhibited phosphorylation of I ⁇ B ⁇ and p65 in a dose-dependent manner, inhibiting LPS-induced activation of the NF- ⁇ B signaling pathway.
  • C57BL/6 mouse peritoneal macrophages and RAW264.7 cells were plated in 96-well culture plates (2 ⁇ 10 4 cells/well) with 2% fetal bovine serum, 100 U/mL ampicillin and 100 ⁇ g/ MLPI-1640 medium (100 ⁇ L/well, purchased from Gbico, USA) was cultured in mL of streptomycin sulfate. After the cells were attached, a series of 2-fold gradient dilutions of cecropin-TY1 as shown in Figure 6 were added to co-culture. At 24 hours, the cell proliferation and toxicity test reagent CCK-8 (10 ⁇ L/well, purchased from Beijing Wobison Technology Co., Ltd.) was added to each well. After 4 hours of incubation, the light absorption at 450 nm was measured, and the cell viability without the added polypeptide was defined as 100%, and calculated cell viability after treatment with different concentrations of cecropin-TY1.
  • cecropin-TY1 did not detect cytotoxicity against mouse macrophages at concentrations up to 100 ⁇ g/mL. It is indicated that its anti-inflammatory activity is not achieved by affecting macrophage activity.
  • the polypeptide was dissolved in 10 mM phosphate buffer of pH 6.0, and different concentrations of cecropin-TY1 (0, 12.5, 25, 50, 100 ⁇ g/mL) were mixed with LPS (1 ⁇ g/mL, purchased from Sigma).
  • cecropin-TY1 neutralized the endotoxin activity of LPS in a dose-dependent manner. At concentrations of 12.5, 25, 50 and 100 mg/mL, cecropin-TY1 neutralized 28.8, 41.3, 47.9 and 65.1% endotoxin activity.
  • the circular dichroism chromatogram of the sample in different solution environments was measured by a circular dichroism instrument JASCO J-810 (JASCO, Tokyo, Japan) to analyze the composition of the secondary structure of cecropin-TY1 in different solutions.
  • the cecropin-TY1 sample was dissolved in water, membrane simulated environment (TFE/H 2 O, LPS/H 2 O, SDS/H 2 O solution) at a dissolved concentration of 0.2 mg/mL.
  • the test temperature was set to 25 ° C, the scanning wavelength range was 190-260 nm, the wavelength interval was 0.2 nm, the sample cell length was 0.1 cm, and the bandwidth was 1 nm. Each sample was continuously scanned 3 times. Circular dichroism was used to estimate the secondary structure of cecropin-TY1 in different solution environments according to the Yang formula.
  • the three-dimensional solution structure of cecropin-TY1 was calculated by Easymodeller version 2.0, and the surface charge distribution was analyzed.
  • the structure was optimized and visualized using MODELL and PYMOL software (http://www.pymol.org) using papiliocin (similarity 59%, PDB entry code 2LA2) derived from butterfly as a template for homology modeling.
  • cecropin-TY1 exhibits a helical-hinge-helical conformation, ie two alpha-helical domains (red regions, Leu 3 -Thr 24 and Ile 27 -Leu 38 ) consist of a hinge domain (green) Area, Ile 25 - Pro 26 ) connection (Figure 9A).
  • Surface charge analysis showed that the surface area of cecropin-TY1 was positively charged (Fig. 9B, blue region).

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Abstract

本发明公开了一种提取自姚虻唾液腺的天然抗炎症多肽cecropin-TYl，其由39个氨基酸组成，碳末端酰胺化。该多肽具有分子量小、抗炎效果明显、细胞毒性低的特点，可用于医药、动物饲料及化妆品领域。

Description

姚虻天然抗炎症多肽cecropin-TY1及其应用
本申请要求了申请日为2015年07月20日，申请号为201510424164.1，发明名称为“姚虻天然抗炎症多肽cecropin-TY1及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种提取自姚虻唾液腺的天然抗炎症多肽cecropin-TY1及其在抗炎症中的新应用，属于生物医学技术领域。
背景技术
近年来，随着传统抗生素的滥用，微生物对传统抗生素产生了越来越强的耐受性，微生物感染已成为严重威胁人类健康的难题。这些微生物中特别是革兰氏阴性细菌感染时能释放脂多糖(LPS)，会引发脓毒血症和内毒素休克。一直以来对微生物感染引发的脓毒血症的主要治疗方法为输液、改善机体供氧、抗感染治疗和免疫治疗等。同时这些治疗方法又存在着诸多不足，比如抗感染治疗会导致大量细菌被杀死而释放出大量内毒素，加剧脓毒血症。因此，这就需要持续开发新的治疗脓毒血症的制剂。
发明内容
为解决上述技术问题，本发明的目的是提供一种姚虻天然抗炎症多肽cecropin-TY1及其应用。
本发明的技术方案是：
Cecropin抗菌肽是一类具有抗菌功能的一种小分子多肽，其中一部分cecropin抗菌肽还具有抗炎的功能，而且能中和内毒素，即在抗菌的同时能中和细菌感染是所释放的内毒素，并发挥抗炎功能。根据 报道，有研究者基于宿主-体外寄生虫相互作用的关系，对吸血昆虫姚虻(Tabanus yao)成功吸血机制进行研究，从姚虻唾液腺中发现了cecropin类多肽cecropin-TY1。
本发明所述的姚虻天然抗炎症多肽cecropin-TY1，提取自姚虻唾液腺，由39个氨基酸组成，碳末端酰胺化，分子量为3970.22道尔顿，等电点为11.17，其氨基酸序列为SEQ ID:1，即：
Gly1Try2Leu3Lys4Lys5Ile6Gly7Lys8Lys9Ile10Glu11Arg12Val13Gly14Gln15Asn16Val17Arg18Asn19Ala20Ala21Ile22Ser23Thr24Ile25Pro26Ile27Ala28Gln29Gly3 0Ala31Ala32Gly33Val34Ala35Gly36Ala37Leu38Asn39-NH2，且其中所有氨基酸均为L-型。
本发明所述的姚虻天然抗炎症多肽cecropin-TY1在制备抗炎药物、细菌感染引起的脓毒血症和内毒素休克药物、兽药、动物饲料及化妆品中的应用。
其具体的为：
本发明所述的姚虻天然抗炎症多肽cecropin-TY1在抑制脂多糖诱导的巨噬细胞产生一氧化氮中应用；在抑制脂多糖诱导的巨噬细胞产生炎症细胞因子中的应用；在抑制脂多糖诱导的炎症信号通路中的应用，包括在抑制脂多糖诱导的丝裂原活化蛋白激酶信号通路的激活中的应用，和在抑制脂多糖诱导的核转录因子NF-κB的激活中的应用；在中和内毒素功能中的应用。
借由上述方案，本发明至少具有以下优点：本发明中所述提取自姚虻唾液腺中的天然抗炎症多肽cecropin-TY1具有分子量小、合成简单、抗炎效果明显、细胞毒性低的特点，在医药、化妆品和养殖业等领域具有广泛的应用前景。
上述说明仅是本实用新型技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是本发明Cecropin-TY1抑制诱导型一氧化氮合酶的转录结果图；
图2是本发明Cecropin-TY1抑制亚硝酸盐的产生的结果图；
图3是本发明Cecropin-TY1抑制炎症细胞因子的产生的结果图；
图4是本发明Cecropin-TY1抑制MAPKs的激活的结果图；
图5是本发明cecropin-TY1抑制NF-κB的激活的结果图；
图6是本发明cecropin-TY1对小鼠巨噬细胞的毒性；
图7是本发明cecropin-TY1的内毒素中和活性；
图8是本发明cecropin-TY1在不同溶液中的圆二色谱图；
图9是本发明cecropin-TY1的溶液结构模拟示意图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
一、姚虻唾液免抗炎症多肽cecropin-TY1对脂多糖(LPS)诱导的巨噬细胞一氧化氮(NO)产生的影响。
(1)、抑制诱导型一氧化氮合酶(iNOS)的转录：
诱导型一氧化氮合酶(iNOS)是一氧化氮(NO)产生所必须的合成酶，首先检测cecropin-TY1对一氧化氮合酶转录水平的影响。C57BL/6小鼠腹腔巨噬细胞铺于24孔细胞培养板中(2.5×105细胞/孔)，用加有2％胎牛血清、100U/mL氨苄青霉素和100μg/mL硫酸链霉素的RMPI-1640培养基(购买自美国Gbico公司)培养，待细胞贴壁后，如图1标注所示，在细胞中加入100ng/mL脂多糖(LPS，来自于大肠杆菌Escherichia coli 0111:B4，购买自Sigma)，同时分别加入5、10和20μg/mL的cecropin-TY1，并分别设置多种对照组，包括：只加 LPS组、只加多肽组(10μg/mL)和既不加LPS又不加cecropin-TY1组。共孵育6小时后，收集细胞，用Trizol(购买自Takara)裂解细胞，提取RNA，用逆转录试剂盒(购买自Takara)合成cDNA，并用荧光定量PCR检测不同处理的细胞中iNOS的转录水平。
由图1可见，cecropin-TY以剂量依赖的方式抑制了iNOS的转录。只加LPS处理的细胞的iNOS的转录水平设定为100％，而加入5、10和20μg/mL的cecropin-TY1处理后，iNOS的转录水平分别只有41.2％、31.3％和15.8％，cecropin-TY1减少了58.8％、68.7％和84.2％的iNOS的转录。
(2)、抑制亚硝酸盐的产生：
细胞培养基中亚硝酸盐(nitrite)的累积水平可以间接反映NO的产生，接着检测了cecropin-TY1对亚硝酸盐产生的影响。将C57BL/6小鼠腹腔巨噬细胞铺于24孔细胞培养板中(2.5×105细胞/孔)，用加有2％胎牛血清、100U/mL氨苄青霉素和100μg/mL硫酸链霉素的RMPI-1640培养基(购买自美国Gbico公司)培养，待细胞贴壁后，如图2标注所示，在细胞中加入100ng/mL脂多糖(LPS，来自于大肠杆菌Escherichia coli 0111:B4，购买自Sigma)，同时分别加入5、10和20μg/mL的cecropin-TY1，并分别设置多种对照组，包括：只加LPS组、只加多肽组(10μg/mL)和既不加LPS又不加cecropin-TY1组。共孵育24小时后，收集培养上清，用格里斯试剂(购买自碧云天)检测不同处理的细胞后细胞培养上清亚硝酸盐的累积水平。
由图2可见，cecropin-TY以剂量依赖的方式抑制了亚硝酸盐的产生。只加LPS处理组亚硝酸盐的累积水平为20.4μM，加入5、10和20μg/mL的cecropin-TY1处理后，亚硝酸盐的累积水平分别只有12.0、7.3和5.0μM，cecropin-TY1减少了40.0、63.7和75.0％的亚硝酸盐的产生。
二、姚虻唾液抗炎症肽cecropin-TY1对脂多糖(LPS)诱导的巨噬细胞炎症细胞因子产生的影响。
将C57BL/6小鼠腹腔巨噬细胞铺于24孔细胞培养板中(2.5×105细胞/孔)，用加有2％胎牛血清、100U/mL氨苄青霉素和100μg/mL硫酸链霉素的RMPI-1640培养基(购买自美国Gbico公司)培养，待细胞贴壁后，如图3标注所示，在细胞中加入100ng/mL脂多糖(LPS，来自于大肠杆菌Escherichia coli 0111:B4，购买自Sigma)，同时分别加入5、10和20μg/mL的cecropin-TY1，并分别设置多种对照组，包括：只加LPS组、只加多肽组(10μg/mL)和既不加LPS又不加cecropin-TY1组。共孵育6小时后，收集细胞培养上清，用酶联免疫试剂盒(购买自达科为)检测细胞因子肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)和白介素-6(IL-6)的累积水平。同时收集细胞，用Trizol(购买自Takara)裂解细胞，提取RNA，用逆转录试剂盒(购买自Takara)合成cDNA，并用荧光定量PCR检测不同处理的细胞中炎症细胞因子TNF-α、IL-1β和IL-6的转录水平。
如图3所示，cecropin-TY1以剂量依赖的方式抑制了LPS诱导的炎症细胞因子TNF-α、IL-1β和IL-6的转录和产生。在浓度为20mg/mL时,cecropin-TY1抑制了60.3％的TNF-a、67.5％的IL-1β和94.1％的IL-6的转录,同时，20mg/mL的cecropin-TY1减少了72.1％的TNF-a、63.4％的IL-1β和76.2％的IL-6的产生。
三、Cecropin-TY1对炎症信号通路的影响。
将C57BL/6小鼠腹腔巨噬细胞铺于6孔细胞培养板中(1×106细胞/孔)，用加有2％胎牛血清、100U/mL氨苄青霉素和100μg/mL硫酸链霉素的RMPI-1640培养基(购买自美国Gbico公司)培养，待细胞贴壁后，如图4和图5标注所示，在细胞中加入100ng/mL脂多糖(LPS，来自于大肠杆菌Escherichia coli 0111:B4，购买自Sigma)，同时分别加入5、10和20仩g/mL的cecropin-TY1，并分别设置多种对照组，包括：只加LPS组、只加多肽组(10μg/mL)和既不加LPS又不加cecropin-TY1组。孵育30分钟后，收集细胞，用RIPA裂解液(购买自碧云天)裂解细胞，收取细胞蛋白，再用BCA蛋白定量试剂盒(购 买自碧云天)进行蛋白定量。将裂解好的蛋白按照40微克的上样量用10％聚丙酰胺凝胶在100伏的电压下电泳90分钟，再将蛋白电转(300毫安，90分钟)到杂交膜上，然后室温用5％牛血清白蛋白封闭2小时。
(1)、抑制丝裂原活化蛋白激酶(MAPKs)信号通路的激活
将MAPKs信号通路相关的一抗：P-ERK、ERK、P-JNK、JNK、P-p38和p38(购买自美国CST公司)，以及内参GAPDH(购买自上海鼎国生物技术有限公司)用5％的牛血清白蛋白按照1:2000的方法稀释，把封闭好的杂交膜与稀释的一抗4℃孵育过夜，用含0.1％吐温-20的Tris缓冲液(TBST,2.42g/LTrisbase,8g/L NaCl,0.1％Tween 20,pH7.6)洗涤3次，每次5分钟，再用5％的牛血清白蛋白稀释后的辣根过氧化物酶标记的二抗(1:5000，购买自美国CST公司)在室温下孵育1小时后，用TBST洗涤3次，每次10分钟，再用发光底物(购买自中国天根生化科技有限公司)在暗示显影曝光。
如图4所示，cecropin-TY1以剂量依赖的方式抑制了ERK、JNK和p38的磷酸化，抑制了LPS诱导的MAPKs信号通路的激活。
(2)、cecropin-TY1抑制核转录因子kappa B(NF-κB)的激活
将NF-κB信号通路相关的一抗：P-IκBα、IκBα、P-p65、和p65(购买自美国CST公司)，以及内参β-actin(购买自上海鼎国生物技术有限公司)用5％的牛血清白蛋白按照1:2000的方法稀释，把封闭好的杂交膜与稀释的一抗4℃孵育过夜，用含0.1％吐温-20的Tris缓冲液(TBST,2.42g/L Trisbase,8g/L NaCl,0.1％Tween 20,pH 7.6)洗涤3次，每次5分钟，再用5％的牛血清白蛋白稀释后的辣根过氧化物酶标记的二抗(1:5000，购买自美国CST公司)在室温下孵育1小时后，用TBST洗涤3次，每次10分钟，再用发光底物(购买自中国天根生化科技有限公司)在暗示显影曝光。
如图5所示，cecropin-TY1以剂量依赖的方式抑制了IκBα和p65的磷酸化，抑制了LPS诱导的NF-κB信号通路的激活。
四、cecropin-TY1细胞毒性的测定。
分别将C57BL/6小鼠腹腔巨噬细胞和RAW264.7细胞铺于96孔培养板中(2×104细胞/孔)，用加有2％胎牛血清、100U/mL氨苄青霉素和100μg/mL硫酸链霉素的RMPI-1640培养基(100μL/孔，购买自美国Gbico公司)培养，待细胞贴壁后，加入如图6所示的一系列2倍梯度稀释的cecropin-TY1，共培养24小时候，每孔加入细胞增殖与毒性检测试剂CCK-8(10μL/孔，购买自北京沃比森科技有限公司)，孵育4小时候，检测450nm处光吸收，将未加入多肽的细胞活力定义为100％，并计算不同浓度cecropin-TY1处理后的细胞活力。
如图6所示，cecropin-TY1在浓度高达100μg/mL时，未检测出对小鼠巨噬细胞的细胞毒性。表明其抗炎活性不是通过影响巨噬细胞活性来实现的。
五、cecropin-TY1中和内毒素功能的测定。
将多肽溶解于10mM、PH6.0的磷酸盐缓冲中，将不同浓度的cecropin-TY1(0、12.5、25、50、100μg/mL)分别与LPS(1μg/mL，购买自Sigma)混合在一个无热源的试管中，37℃共孵育30分钟后，每100仩L的多肽-LPS混合物中加入100仩L的鲎试剂(购买自厦门鲎试剂有限公司)，37℃共孵育10分钟，然后加入试剂盒提供的底物，37℃孵育6分钟后，于酶标仪上测定545纳米处的光吸收，然后计算不同浓度的cecropin-TY1的中和能力，其中不加多肽的LPS定义为0中和管。
如图7所示，cecropin-TY1以剂量依赖的方式中和了LPS的内毒素活性，在浓度为12.5、25、50和100mg/mL时，cecropin-TY1分别中和了28.8、41.3、47.9和65.1％的内毒素活性。
六、cecropin-TY1溶液二级结构的测定。
用圆二色谱仪JASCO J-810(JASCO，Tokyo，Japan)检测样品在不同溶液环境中的圆二色谱谱图，来分析cecropin-TY1在不同溶液中二级结构的组成。cecropin-TY1样品溶解于水、膜模拟环境(TFE/H2O、LPS/H2O、SDS/H2O溶液)，溶解浓度为0.2mg/mL。设置试验温度为 25℃，扫描波长范围190-260nm，波长间隔为0.2nm，样品池长0.1cm，带宽1nm每个样品连续扫描3次。圆二色谱根据杨公式(Yang formula)估算cecropin-TY1在不同溶液环境的二级结构。
如图8所示，在H2O中，cecropin-TY1在199nm处呈现最大负吸收，表明此时主要的构象为无规则卷曲。当在膜模拟环境(TFE/H2O、LPS/H2O、SDS/H2O溶液)，cecropin-TY1在208和222nm处呈现双重负吸收，表明此时主要的二级构象为α-螺旋。结果表明，在膜模拟环境中，有一部分无规则卷曲构象转换成了α-螺旋构象。
七、cecropin-TY1溶液三级结构的测定。
用Easymodeller version 2.0来计算cecropin-TY1的三维溶液结构，并分析其表面电荷分布情况。用来源于蝴蝶的papiliocin(相似性为59％,PDB entry code 2LA2)作为同源模拟的模板，用MODELLER和PYMOL软件(http://www.pymol.org)来对结构进行优化和可视化。
如图9所示，cecropin-TY1呈现出螺旋-铰链-螺旋的构象，即两个α-螺旋结构域(红色区域，Leu3-Thr24和Ile27-Leu38)由一个铰链结构域(绿色区域，Ile25-Pro26)连接(图9A)。表面电荷分析显示，cecropin-TY1部分表面区域带有正电荷(图9B，蓝色区域)。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2015085252-appb-000001

Claims (10)

  1. 一种姚虻天然抗炎症多肽cecropin-TY1，其特征在于：提取自姚虻唾液腺，由39个氨基酸组成，碳末端酰胺化，分子量为3970.22道尔顿，等电点为11.17，其氨基酸序列为SEQ ID:1，且其中所有氨基酸均为L-型。
  2. 根据权利要求1所述的姚虻天然抗炎症多肽cecropin-TY1在制备抗炎药物中的应用。
  3. 根据权利要求1所述的姚虻天然抗炎症多肽cecropin-TY1在制备细菌感染引起的脓毒血症和内毒素休克药物中的应用。
  4. 根据权利要求1所述的姚虻天然抗炎症多肽cecropin-TY1在制备兽药、动物饲料及化妆品中的应用。
  5. 根据权利要求2-4中任一权利要求所述的应用，其特征在于：姚虻天然抗炎症多肽cecropin-TY1在抑制脂多糖诱导的巨噬细胞产生一氧化氮中应用。
  6. 根据权利要求2-4中任一权利要求所述的应用，其特征在于：姚虻天然抗炎症多肽cecropin-TY1在抑制脂多糖诱导的巨噬细胞产生炎症细胞因子中的应用。
  7. 根据权利要求2-4中任一权利要求所述的应用，其特征在于：姚虻天然抗炎症多肽cecropin-TY1在抑制脂多糖诱导的炎症信号通路中的应用。
  8. 根据权利要求7所述的应用，其特征在于：姚虻天然抗炎症多肽cecropin-TY1在抑制脂多糖诱导的丝裂原活化蛋白激酶信号通路的激活中的应用。
  9. 根据权利要求7所述的应用，其特征在于：姚虻天然抗炎症多肽cecropin-TY1在抑制脂多糖诱导的核转录因子NF-κB的激活中的应用。
  10. 根据权利要求2-4中任一权利要求所述的应用，其特征在于：姚虻天然抗炎症多肽cecropin-TY1在中和内毒素功能中的应用。
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