WO2003016339A1 - A frog skin antibacterial peptide derivative - Google Patents

A frog skin antibacterial peptide derivative Download PDF

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Publication number
WO2003016339A1
WO2003016339A1 PCT/CN2002/000317 CN0200317W WO03016339A1 WO 2003016339 A1 WO2003016339 A1 WO 2003016339A1 CN 0200317 W CN0200317 W CN 0200317W WO 03016339 A1 WO03016339 A1 WO 03016339A1
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WO
WIPO (PCT)
Prior art keywords
antibacterial peptide
frog skin
skin antibacterial
peptide derivative
lys
Prior art date
Application number
PCT/CN2002/000317
Other languages
English (en)
French (fr)
Inventor
Yukun Sun
Dengxi Wu
Zhiyong Zhu
Gang Yu
Chunjuan Shen
Shaoling Zhao
Jiaxiang Zhou
Original Assignee
Shanghai Huayi Bio Lab
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Publication date
Application filed by Shanghai Huayi Bio Lab filed Critical Shanghai Huayi Bio Lab
Priority to JP2003521261A priority Critical patent/JP4194941B2/ja
Priority to AU2002257498A priority patent/AU2002257498B2/en
Priority to CA2446848A priority patent/CA2446848C/en
Priority to KR1020037014458A priority patent/KR100902209B1/ko
Priority to BRPI0209684A priority patent/BRPI0209684B8/pt
Priority to EP02727168.3A priority patent/EP1386928B1/en
Publication of WO2003016339A1 publication Critical patent/WO2003016339A1/zh
Priority to US10/705,106 priority patent/US7232800B2/en
Priority to US11/686,190 priority patent/US20070166790A1/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
    • 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
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to antibacterial peptide derivatives, in particular to a class of frog skin antibacterial peptide derivatives having antibacterial action.
  • Antibacterial peptides have different amounts of positive charges.
  • the mechanism of their antibacterial effect is that the positive charges they combine with the negative charges of the phospholipids of the bacterial cell wall membrane form ion channels on the cell membrane to increase permeability. It can make bacterial cells lyse and die, so its antibacterial effect has nothing to do with the binding of specific receptors.
  • Antibacterial peptides have a wide range of antibacterial properties, and they have inhibitory effects on Gram-positive, Gram-negative bacteria, aerobic bacteria, and anaerobic bacteria. They are different from antibiotics and do not produce resistance, even if they are resistant to a variety of antibiotics The bacteria can also be suppressed. In addition, antibacterial peptides can also inhibit protozoa and viruses. The metabolites of antimicrobial peptides are amino acids, and therefore have low toxicity to host cells. In summary, the antibacterial peptides are a class of compounds with broad antibacterial and medicinal prospects.
  • Frog skin antibacterial peptide Magainin is a kind of natural antibacterial peptide extracted from frog skin with antibacterial effect and widely studied. It has the characteristics of easy synthesis, low cost, and low hemolysis.
  • US patent USP 5589364 discloses a method for preparing natural frog skin antibacterial peptide Magaininll (23 amino acids) using genetic engineering technology.
  • the frog skin antibacterial peptide Magainin II is a kind of natural frog skin antibacterial peptide Magainin. Its amino acid sequence is as follows ⁇
  • Gly is glycine, He is isoleucine, Lys is lysine, Phe is phenylalanine, Leu is leucine, His is histidine, Ser is serine, Ala is alanine, Val Is valine, Glu is glutamic acid, Met is methionine, and Asn is asparagine.
  • US patent USP 6183992 discloses a method for preparing frog skin antibacterial peptide derivative MSI-78 (22 amino acids) by using genetic engineering technology.
  • the amino acid sequence of MSI-78 is as follows:
  • the object of the present invention is to provide a class of frog skin antibacterial peptide derivatives, which expands the types of frog skin antibacterial peptide derivatives. They are easy to prepare by solid-phase chemistry, and especially easy to prepare by genetic engineering techniques, thereby providing the possibility of reducing production costs. . They have the same or stronger antibacterial effect as natural frog skin antibacterial peptides. Summary of invention
  • the present invention relates to a class of frog skin antibacterial peptide derivatives with the following structure and its use in medicine
  • X is an amino acid residue selected from Met, lie or Leu
  • Y is a combination of two amino acid residues selected from the group consisting of Ser, Ly S , Il e , Arg, and Leu.
  • amino acid sequence of the frog skin antibacterial peptide derivative of the present invention is as described in ⁇ 210> 1 in the sequence listing.
  • the frog skin antibacterial peptide derivative provided by the present invention is an amphoteric compound and can react with acidic or basic substances to form a salt.
  • the acids commonly used to form acid addition salts are: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, Citric acid, benzoic acid, acetic acid, etc.
  • salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, Hydrochloride, bromide, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate , Oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dianate, hexyne-1 , 6-Diacid, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phenylacetic acid Salt, phenylpropionate, phenylbutyrate, citrate, lactate, Y-hydroxybutyrate, glycolate, tartrate, mes
  • Basic substances can also react with frog skin antibacterial peptides to form salts.
  • These basic substances include ammonium, alkali metal or alkaline earth metal hydroxides, as well as carbonates and bicarbonates. Typical are sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc.
  • the amino acid of the frog skin antibacterial peptide derivative in the present invention is an L-type or D-type isomer.
  • the preparation method of the frog skin antibacterial peptide derivative in the present invention is an L-type or D-type isomer.
  • the preparation method is a genetic engineering method, and the steps include: synthesizing a gene fragment according to the amino acid sequence of a frog skin antibacterial peptide derivative, ligating the gene fragments, constructing a plasmid, cloning, fermenting, separating and purifying and lyophilizing a product.
  • the preparation method is a solid-phase chemical synthesis method, which includes the steps of using a HMP resin as a solid-phase support, the ⁇ -amino group of an amino acid is protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group, and the synthesis is performed by a solid-phase peptide synthesizer.
  • the product was isolated, purified and lyophilized.
  • the invention also provides the application of the frog skin antibacterial peptide derivative and a salt suitable for medicament in the preparation of an antibacterial medicine.
  • the invention provides a class of frog skin antibacterial peptide derivatives, which expands the types of frog skin antibacterial peptide derivatives.
  • the provided frog skin antibacterial peptides are easy to prepare by solid-phase chemical synthesis, and are especially easy to prepare by genetic engineering techniques. This provides the possibility to reduce production costs. They have the same or stronger antibacterial effect as natural frog skin antibacterial peptides.
  • bactericidal effect and sterilization time on frog skin antibacterial peptide derivatives of the structure of the present invention show that the frog skin of the present invention is antibacterial
  • the peptide derivative is equivalent to the natural frog skin antibacterial peptide Magaininll in bactericidal effect on Escherichia coli, and also has an inhibitory effect on Staphylococcus aureus.
  • Use dermaseptin derivative of the invention is to 50 ⁇ 1 bacteria (106 bacteria / ml) for bactericidal assay, an amount of 0.5 ⁇ , 3 hours to kill 90% of bacteria, in an amount of 1 ⁇ ⁇ , 3 hours to kill almost Dead all bacteria.
  • Hemolysis experiments show that the ratio of 50% effective concentration (HC 5Q ) to 50% effective concentration (IC 5Q ) of the frog skin antibacterial peptide derivative of the present invention is about 50, and the natural frog skin antibacterial peptide Magaininll's HC 5Q / IC 5 . It is about 24, which shows that the product of the present invention has better safety than the natural frog skin antibacterial peptide Magaininll.
  • Acute toxicity experiments show that the frog skin antibacterial peptide derivative of the present invention has low acute toxicity.
  • Fig. 1 is a time chart of sterilization of E. coli by the frog skin antibacterial peptide derivative obtained in Example 1 of the present invention. Summary of the Invention
  • Example 1 A frog skin antibacterial peptide derivative of the structure of the present invention was prepared by genetic engineering, where X is Leu and Y is Ser-Arg.
  • the plasmid contains a temperature-controlled promoter PL (or a lactose promoter Lac, a tryptophan-lactose mixed promoter Tac), which is digested with restriction endonucleases EcoR I and Hind III, followed by phenol / chloroform extraction. Then, it was washed three times with chloroform, and then precipitated with isopropanol, and the plasmid was obtained by centrifugation. The plasmid is ligated with the frog skin antibacterial peptide derivative gene fragment to obtain a plasmid containing the frog skin antibacterial peptide derivative gene fragment. E. coli JM103 or JM109 was transformed, and colonies were selected by plate culture.
  • a plasmid containing the frog skin antibacterial peptide derivative gene fragment was extracted from a single colony, digested with EcoR I + HindIII, and then subjected to 1% agarose electrophoresis, stained with ethidium bromide, and compared with a standard sample The cloned gene fragments were checked by comparison, and then cloned by DNA sequence analysis and detection.
  • Inoculate 1L culture solution (LB culture solution) shake flask (10 bottles) containing 300ml of peptone, 5g of yeast extract powder, 5g of yeast extract powder, and sodium chloride 5g / L, 0.2mM isopropylthiosulfate at 37 ° C
  • IPTG lactose
  • Example 2 A frog skin antibacterial peptide derivative of the structure of the present invention was prepared by a genetic engineering method, wherein X is Leu and Y is Ser-Lys.
  • Trt trityl
  • the reagents are: N-methylpyrrolidone, dichloromethane, hexahydropyridine, methanol, dimethylaminopyridine (Dimethylaminopyridine) I DMF N, N- diisopropylethylamine (N, N- diisopropylethylamine) I NMP, HBTU 100 mmole / 0.5 M HOBT in DMF, N, N- dicyclohexylcarbodiimide ( ⁇ , ⁇ -Dicyclohexylcarbodiimide) / NMP
  • NMP is a test solution of N-methylpyrrolidone
  • HOBT is 1-Hydroxybenzotriazole
  • HBTU 2- (1 hydrogen-benzotriazolyl) -tetramethyl urea hexafluorophosphate (2- (1 ⁇ -benzotriazole-yl- 1,1,3,3 -tetramethyl-Uronium hexafluorophosphate)
  • the 0.25mmol scale Take the 0.25mmol scale as an example, weigh 0.25g of HMP resin, put it into the reactor on the peptide synthesizer, weigh various protective amino acids into 1mmol bottles, and follow the amino acid sequence of the insulinotropic peptide derivative from C-terminal to N-terminal are arranged in the synthesizer. At room temperature of 25 ° C, the Fmoc protection, activation, and connection are automatically controlled by a computer program, and then the next cycle is performed to complete the synthesis. After the synthesis is completed, the obtained peptide resin with a side chain protecting group may be dried on a peptide synthesizer and then weighed.
  • the protected insulin-promoting peptide derivative polypeptide resin was placed in a conical Erlenmeyer flask, and the lysis reagent was added as shown in the following table:
  • the bacteriostatic experiment was performed by comparing the frog skin antibacterial peptide derivative obtained in Example 1 with the natural frog skin antibacterial peptide II.
  • the specific operation is as follows:
  • test bacteria E. coli JM103 and Staphylococos Anreus were cultured separately, diluted to IxlO 6 bacteria / ml, sterilized Tris-HCl buffer 20 mM pH 6.5 was added, and different concentrations of the derivative and natural frog were added.
  • the antibacterial peptide II was incubated at 37 ° C for different times, and then 50 ⁇ 1 was spread on an agarose gel culture dish and cultured overnight at 37 ° C. The number of remaining colonies was recorded, and the sterilization percentage was calculated.
  • Bactericidal time chart See Figure 1 for the antibacterial time chart of the frog skin antibacterial peptide derivative obtained in Example 1 against E. coli.
  • Acute toxicity test Two Kunming mice were used for the acute toxicity test.
  • the frog skin antibacterial peptide derivative obtained in Example 1 was intraperitoneally injected, and the survival of the mice was observed after 1 hour.

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Description

蛙皮抗菌肽衍生物 技术领域
本发明涉及抗菌肽衍生物， 具体地说涉及一类有抗菌作用的蛙皮抗菌肽 衍生物。 技术背景
许多生物， 如昆虫、 微生物、 两栖类动物和人类都会产生保卫机体的抗 菌肽物质。 这些抗菌肽能将细胞膜脂质体穿孔使之失活， 并且对原虫类、 精 子、 甚至病毒也有作用， 因而被称为超级抗生素。 抗菌肽类物质都带有不同 数量的正电荷， 其抗菌作用的机制在于， 它所带的正电荷与细菌细胞壁膜的 磷脂质的负电荷相结合， 在细胞膜上形成离子通道， 扩大通透性， 使细菌细 胞溶解、 死亡， 因此其抗菌作用与特异受体的结合无关。
抗菌肽的抗菌范围很广， 对革兰氏阳性、 革兰氏阴性菌、 好氧菌以及厌 氧菌都有抑制作用， 并且与抗菌素不同， 不产生抗药性， 即使对多种抗菌素 产生抗药性的细菌也可被抑制。 另外， 抗菌肽也可以抑制原虫和病毒。 抗菌 肽的代谢产物为氨基酸， 因此对宿主细胞的毒性低。 综上所述抗菌肽是一类 有广阔的抗菌药用前景的化合物。
蛙皮抗菌肽 Magainin是一类天然的从蛙皮中提取的有抗菌作用而又得到 广泛研究的抗菌肽。 它具有易合成， 成本低， 溶血性低等特点。
美国专利 USP 5589364公开了如何利用基因工程技术制备天然蛙皮抗菌 肽 Magaininll (23 个氨基酸） 的方法。 蛙皮抗菌肽 Magainin II是天然蛙皮 抗菌肽 Magainin的一种， 其氨基酸序列如下-
Gly-Glu-Ile-Met-Asn-Ser-OH
式中的 Gly为甘氨酸， He为异亮氨酸， Lys为赖氨酸， Phe为苯丙氨酸, Leu为亮氨酸， His为组氨酸， Ser为丝氨酸， Ala为丙氨酸， Val为缬氨酸， Glu为谷氨酸， Met为甲硫氨酸， Asn为天冬酰胺。
美国专利 USP 6183992公开了利用基因工程技术制备蛙皮抗菌肽衍生物 MSI-78 (22个氨基酸） 的方法， MSI-78的氨基酸序列如下：
Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val- Lys-Ile-Leu-Lys-Lys-NH2
美国 HarrietM. lamb等人在《ADIS NEW DRUG PROFILE》杂志上报导： 蛙皮抗菌肽衍生物 MSI-78 应用在治疗外伤感染及糖尿病下肢溃疡等方面有 明显疗效。 发明目的
本发明的目的是提供一类蛙皮抗菌肽衍生物， 扩大了蛙皮抗菌肽衍生物 的种类， 它们易于用固相化学制备， 尤其易于用基因工程技术制备， 从而为 降低生产成本提供了可能。 它们具有与天然蛙皮抗菌肽相同或者更强的抗菌 作用。 发明概要
本发明的内容涉及一类如下结构的蛙皮抗菌肽衍生物及其适用于药用的
Gly-Glu-Ile-X-Asn-Y-OH
其中： X为选自 Met, lie或 Leu的氨基酸残基，
Y为选自 Ser,LyS,Ile,Arg， Leu的两个氨基酸残基的组合。
本发明的蛙皮抗菌肽衍生物的氨基酸序列如序列表中 <210>1所述。
本发明提供的蛙皮抗菌肽衍生物是两性化合物， 可与酸性或碱性物质反 应形成盐。 通常采用的形成酸加成盐的酸为： 盐酸， 氢溴酸， 氢碘酸， 硫酸， 磷酸， 对 -甲苯磺酸， 甲磺酸， 草酸， 对溴苯基磺酸， 碳酸， 琥珀酸， 柠檬酸， 苯甲酸， 乙酸等。 这类盐的例子包括硫酸盐， 焦硫酸盐， 硫酸氢盐， 亚硫酸 盐， 亚硫酸氢盐， 磷酸盐， 磷酸氢盐， 磷酸二氢盐， 偏磷酸盐， 焦磷酸盐， 盐酸盐， 溴化物， 碘化物， 乙酸盐， 丙酸盐， 癸酸盐， 辛酸盐， 丙烯酸盐， 甲酸盐， 异丁酸盐， 己酸盐， 庚酸盐， 丙炔酸盐， 草酸盐， 丙二酸盐， 丁二 酸盐， 辛二酸盐， 癸二酸盐， 富马酸盐， 马来酸盐， 丁炔 -1,4-二酸盐， 己炔- 1,6-二酸盐， 苯甲酸盐， 氯苯甲酸盐， 甲基苯甲酸盐， 二硝基苯甲酸盐， 羟 基苯甲酸盐， 甲氧基苯甲酸盐， 苯乙酸盐， 苯丙酸盐， 苯丁酸盐， 柠檬酸盐， 乳酸盐， Y -羟基丁酸盐， 甘醇酸盐， 酒石酸盐， 甲磺酸盐， 丙磺酸盐， 萘 -1- 磺酸盐， 萘 -2-磺酸盐， 扁桃酸盐等， 优选的酸加成盐是蛙皮抗菌肽衍生物与 无机酸如盐酸， 氢溴酸， 尤其是盐酸形成的盐。
碱性物质也可以与蛙皮抗菌肽反应形成盐， 这些碱性物质包括铵， 碱金 属或碱土金属的氢氧化物， 以及碳酸盐， 碳酸氢盐等。 典型的有氢氧化钠， 氢氧化钾， 氢氧化铵， 碳酸钾等。
本发明中所述的蛙皮抗菌肽衍生物的氨基酸是 L型或者 D型异构体。 本发明中所述的蛙皮抗菌肽衍生物的制备方法。
该制备方法是基因工程方法， 其步骤包括： 按蛙皮抗菌肽衍生物的氨基 酸序列合成基因片段， 基因片段连接， 质粒构建， 克隆， 发酵， 分离纯化及 冻干得产品。
本发明中所述的蛙皮抗菌肽衍生物的制备方法。
该制备方法是固相化学合成法， 其步骤包括以 HMP树脂为固相载体， 氨基酸的 α -氨基用 9-芴基甲氧羰基（Fmoc)基团保护， 用固相多肽合成仪 合成， 经分离提纯及冻干得产品。
本发明还提供了该蛙皮抗菌肽衍生物及其适用于药用的盐在制备抗菌药 物中的应用。
本发明提供了一类蛙皮抗菌肽衍生物， 扩大了蛙皮抗菌肽衍生物的种 类， 所提供的该类蛙皮抗菌肽易于用固相化学合成法制备， 尤其易于用基因 工程技术制备， 从而为降低生产成本提供了可能。 它们具有与天然蛙皮抗菌 肽相同或者更强抗菌作用。 对本发明所述结构的蛙皮抗菌肽衍生物进行的杀 菌效果和杀菌时间实验， 以及溶血和急性毒性实验表明： 本发明的蛙皮抗菌 肽衍生物与天然蛙皮抗菌肽 Magaininll对大肠杆菌的杀菌效果相当， 对金黄 葡萄球菌亦有抑制作用。 使用本发明蛙皮抗菌肽衍生物对 50μ1 的菌液（106 菌 /ml) 进行杀菌实验， 用量为 0.5μ ， 3 小时可杀死 90%以上的细菌， 用量 为 1μ§， 3 小时几乎杀死全部细菌。 溶血实验表明本发明的蛙皮抗菌肽衍生 物溶血 50%有效浓度（HC5Q) 与抑制 50%有效浓度（IC5Q) 比值约为 50， 而 天然蛙皮抗菌肽 Magaininll的 HC5Q /IC5。约为 24， 这说明本发明的产品安全 性优于天然蛙皮抗菌肽 Magaininll。 急性毒性实验表明本发明的蛙皮抗菌肽 衍生物的急性毒性较低。 附图说明
现结合附图和实施例对本发明的内容作进一步说明。
附图 1是本发明实施例 1所得的蛙皮抗菌肽衍生物对大肠杆菌的杀菌时 间图。 发明内容
下面实施例是对本发明的进一步阐述而不是对本发明的限制。 实施例 1 基因工程法制备本发明结构的蛙皮抗菌肽衍生物， 其中 X为 Leu， Y为 Ser-Arg。
(1)按该蛙皮抗菌肽衍生物的氨基酸序列合成基因片段如下：
(a)5， AAT CC ATG GGT A C GGT AAA TIT CTG CAC AGC GCG AAA AAA
φ)5' TIT GGT AAA GCG TIT G G GGT GAA ATC CIG AAC AGC CGT TAG A
(c)5， AG CIT CTA AOG GCT GTT CAG GAT TIC AGC CAC AAA CGC TIT
(φ5， ACC AAA TIT ΠΤ GGC GCT GTG CAG AAA TIT AOC GAT ACC CAT GG
(2) DNA片段的连接
取光密度为 0.1 (A260mn ) 的上述四个片段 ， 二个试管中分别加片段 a 与 d和 b与 c， 再加聚核苷酸激酶缓冲液（Polynucleotide Kinase Buffer) 和 聚核苷酸激酶（Polynucleotide Kinase) 。 37°C保温 60分钟后， 在 95°C水浴 中保温 10分钟， 随后徐徐自然冷却至室温， 再加 T4连接酶缓冲液（ T4 Ligase Buffer)及三磷酸腺苷（ATP)溶液， 最后加 T4连接酶（T4 Ligase) ， 于 15°C 保温过夜， 使片段完成连接。
(3) 克隆
质粒含启动子为温控启动子 PL (或乳糖启动子 Lac， 色氨酸 -乳糖混合启 动子 Tac) ，用限制性内切酶 EcoR I和 Hind III双酶切之后， 经过酚 /氯仿 抽提， 再以氯仿洗涤 3次， 然后以异丙醇沉淀， 经离心得质粒。 将质粒与该 蛙皮抗菌肽衍生物基因片段连接， 即得含有该蛙皮抗菌肽衍生物基因片段的 质粒。 转化大肠杆菌 JM103或 JM109, 平板培养筛选菌落。
(4)验证
从单菌落中抽提含该蛙皮抗菌肽衍生物基因片段的质粒， 经过 EcoR I +HindIII双酶切之后， 进行 1%琼脂糖电泳， 以溴化乙锭（Ethidum Bromide) 染色， 与标准样品对照检査克隆的基因片段， 再经 DNA序列分析、 检测完 成克隆。
(5)发酵
在 1L含 300ml组成为蛋白胨 10g、 酵母抽提粉 5g、 氯化钠 5g/L的培养 液（LB培养液）摇瓶（10瓶） 中接种， 37°C0.2mM的异丙基硫代半乳糖苷 (IPTG)诱导， 振荡培养过夜， 离心收集菌体。 利用温控 PL启动子质粒时， 于 30°C培养 8小时， 然后将培养液升温至 42°C， 维持 4小时使基因表达。
(6)经溶菌酶 37°C、 1 小时破碎细胞壁后离心， 将沉淀加 6M盐酸胍抽 提， 离心透析， 再离心， 得蛋白质包涵体。 将包涵体洗涤 3次， 洗涤溶液为 1%氯化钠， 0.1%曲拉通 x-100 ( TrifoX-100)， Tris-HCl缓冲液（20mM， pH8)， 以十二垸基磺酸钠 （SDS) -12%聚丙烯酰凝胶（PAGE) 电泳对融合蛋白进 行鉴定。
(7)将包涵体溶于 8M尿素溶液中， 50mM HC1条件下， 加溴化氰裂解， 避光通氮气条件下，搅拌反应 2小时。反应完毕，用琼脂糖凝胶分子筛 G-25/ 快速蛋白分离系统（FPLC) 分离， 得该蛙皮抗菌肽衍生物粗品， 再经高效 液相层析仪 （HPLC， C18柱） ， 以乙氰 /0.1%三氟乙酸梯度洗脱。 结果得该 蛙皮抗菌肽衍生物产品， 该产品 HPLC图谱与化学合成品完全一致。 实施例 2 基因工程法制备本发明结构的蛙皮抗菌肽衍生物， 其中 X为 Leu， Y为 Ser-Lys。
(1)按该蛙皮抗菌肽衍生物的氨基酸序列合成基因片段如下：
(a) 5， AAT TCC A G GGT ATC GGT AAA TIT CTG CAC AGC GCG AAA AAA
(b) 5， TIT GGT AAA GCG TIT GTG GGT GAA ATC CTG AAC AGC AAG TAG A
(c) 5' AG CTT CTA CTT GCT GTT CAG GAT TIC ACC CAC AAA CGC TIT
(d) 5' ACC AAA TIT TIT CGC GCT GTG CAG AAA TIT AGC GAT AGC CAT GG 本实施例以下步骤（2)〜（7)均同实施例 1。 结果得该蛙皮抗菌肽衍生 物产品， 该产品 HPLC图谱与化学合成品完全一致。 实例例 3 固相化学合成法制备本发明结构的蛙皮抗菌肽衍生物， 其中 X为 Leu， Y为 Lys-Arg。
(1) 所采用的氨基酸单体：
Figure imgf000007_0001
上式中缩写表示：
Fmoc: 9-芴基甲氧羰基
BOC: 叔丁氧羰基 ( tert-butyloxycarbonyl )
Trt: 三苯甲基（trityl) ,
OtBu: 叔丁基酯
TBu: 叔丁基（tert-butyl)
(2)合成所采用的仪器设备及试剂：
仪器为： Applied Biosystem多肽合成仪， 433A型， 美国
试剂有： N-甲基吡咯垸酮， 二氯甲烷， 六氢吡啶， 甲醇， 二甲氨基吡啶 ( Dimethylaminopyridine ) I DMF N,N-二异丙基乙胺 ( Ν,Ν- diisopropylethylamine ) I NMP， HBTU 100 mmole / 0.5 M HOBT in DMF， N,N- 二环己基碳二亚胺（Ν,Ν-Dicyclohexylcarbodiimide) /NMP
其中： DMF为 Ν,Ν-二甲基甲酰胺 （Ν,Ν-Dimethylformamide)
NMP为试一水苯苯巯三 N-甲基吡咯烷酮 （N-methylpyrrolidone)
HOBT为 1酚基氟甲-羟基苯并三唑 （1-Hydroxybenzotriazole)
HBTU为 2- ( 1 氢-苯并三唑基） -四甲基脲六氟磷酸盐 （2-(1Η- benzotriazole-yl- 1,1,3 ,3 -tetramethyl-Uronium hexafluorophosphate)
(3)操作：
a. 合成
以 0.25mmol规模为例， 称取 HMP树脂 0.25g, 置入多肽合成仪上的反 应器中， 将各种带保护基氨基酸称取 lmmol装瓶， 按该促胰岛素分泌肽衍生 物的氨基酸序列从 C-端向 N-端排列在合成仪中， 于 25°C室温条件下， 由计 算机程序控制自动进行脱 Fmoc保护、 活化、 连结， 然后再进行下一轮循环， 如此完成合成。 合成结束后， 将得到的带侧链保护基的多肽树脂在多肽合成 仪上吹干后称重即可。
b. 脱保护基及切断树脂：
将带保护基的该促胰岛素分泌肽衍生物多肽树脂置于具塞三角烧瓶， 加 入裂解试剂如下表：
用量
0.50 ml
醚 0.50 ml
0.75 g
乙醇 0.20 ml
乙酸 10.0 ml
然后在恒温 30°C条件下， 电磁搅拌反应 6小时； 过滤、 收集滤液， 树脂 用少量三氟乙酸洗涤； 合并收集液与洗涤液， 加入乙醚产生沉淀， 过滤， 沉 淀用少量乙醚洗涤后放入干燥器中干燥， 得到粗品。 c. HPLC分离纯化、 冻干
将得到的粗品用制备型 HPLC进行分离、 提纯， 最后经冷冻干燥后得到 产品。 经色质联用， 该衍生物分子量实测值与理论值相符。 实施例 4 药效实验
以实施例 1所得蛙皮抗菌肽衍生物与天然蛙皮抗菌肽 II对照进行抑菌实 验， 具体操作如下：
待试菌大肠杆菌 E.coli JM103,金黄葡萄球菌 Staphylococos Anreus分别 培养， 稀释至 IxlO6菌 /ml， 加入灭菌的 Tris-HCl缓冲液 20mM pH6.5， 加入 不同浓度的该衍生物和天然蛙皮抗菌肽 II， 37°C保温不同时间， 然后取 50μ1 涂于琼脂糖凝胶培养皿中， 37°C培养过夜， 记录残存菌落数， 计算杀菌百分 率。
(1) 杀菌效果表；
实施例 1所得蛙皮抗菌肽衍生物与天然蛙皮抗菌肽 II对大肠杆菌的杀菌 效果对比表
P 残余菌落数 杀菌百分率％ 口 Λ7 浓度
口
g/ml Ohr 3hr 4hr 3hr 4hr
407 228
0
317
实施例 1
所得蛙皮 109 73 160 195
10 71.3 44.2 抗菌肽衍 91 177
生物
9 5 2 5
20 97.8 99.1
7 3
99 57 52 44
10 75.4 84.9 天然蛙皮 78 48
抗菌肽 II 12 4 6 4
20 97.5 99.2
8 5 实施例 1所得蛙皮抗菌肽衍生物对金黄葡萄球菌的杀菌效果表
Figure imgf000010_0001
(2) 杀菌时间图： 实施例 1 所得蛙皮抗菌肽衍生物对大肠杆菌的抗菌时 间图见附图 1
(3) 溶血实验表；
Figure imgf000010_0002
(4) 急性毒性实验： 以二只昆明小鼠进行急性毒性实验， 腹腔注射实施 例 1所得蛙皮抗菌肽衍生物， 观察 1小时后小鼠的存活情况。
腹腔注射量 存活率
lOO g 100%
200μ 100%
lOOO g 100%

Claims

权利要求
1. 一类蛙皮抗菌肽衍生物及其适用于药用的盐， 该蛙皮抗菌肽衍生物 的氨基酸序列如序列表中 <210>1所述。
2. 根据权利要求 1 所述的蛙皮抗菌肽衍生物的制备方法， 该制备方法 是基因工程方法， 其步骤包括： 按蛙皮抗菌肽衍生物的氨基酸序列合成基因 片段， 基因片段连接， 质粒构建， 克隆， 发酵， 分离纯化及冻干得产品。
3. 根据权利要求 1 所述的蛙皮抗菌肽衍生物的制备方法， 该制备方法 是固相化学合成法， 其步骤包括以 HMP树脂为固相载体， 氨基酸的 α -氨基 用 9-芴基甲氧羰基（Fmoc)基团保护， 用固相多肽合成仪合成， 经分离提纯 及冻干得产品。
4. 根据权利要求 1 所述的蛙皮抗菌肽衍生物及其适用于药用的盐在制 备抗菌药物中的应用。
PCT/CN2002/000317 2001-05-10 2002-05-08 A frog skin antibacterial peptide derivative WO2003016339A1 (en)

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