WO2014198095A1 - 镇痛肽fi及其基因和应用 - Google Patents

镇痛肽fi及其基因和应用 Download PDF

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WO2014198095A1
WO2014198095A1 PCT/CN2013/084555 CN2013084555W WO2014198095A1 WO 2014198095 A1 WO2014198095 A1 WO 2014198095A1 CN 2013084555 W CN2013084555 W CN 2013084555W WO 2014198095 A1 WO2014198095 A1 WO 2014198095A1
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analgesic
analgesic peptide
peptide
pain
mice
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PCT/CN2013/084555
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French (fr)
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杜彦军
刘音
赖仞
容明强
朱玉琴
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四川科伦新光生物科技开发有限公司
中国科学院昆明动物研究所
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Publication of WO2014198095A1 publication Critical patent/WO2014198095A1/zh

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    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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, and in particular relates to an analgesic peptide FI and a gene and application thereof.
  • Analgesic drugs are a class of substances that can alleviate the pain of the disease. They can not only selectively reduce or alleviate the pain sensation, but also alleviate the unpleasant emotions such as fear, tension and anxiety caused by severe pain.
  • the current analgesic drugs can be divided into two types: antipyretic analgesics and anesthetic analgesics.
  • the former is represented by aspirin, and the sensitivity of nerve endings to pain-causing substances is alleviated by inhibiting the prostaglandin synthesis process in tissues. It is mainly used to relieve inflammatory pain and other dull pain; the latter is represented by morphine, which acts mainly in the thalamus and cortex and acts on the ⁇ -type opioid receptor in the central nervous system to relieve sharp pain, dull pain and Visceral visceral pain, resulting in analgesic effect, mainly used for pain caused by various diseases and severe pain of patients after surgery, especially advanced cancer pain.
  • anesthetic analgesic drugs represented by morphine are highly addictive and have serious withdrawal symptoms. Patients must continue to increase the dose to maintain the analgesic effect, and eventually lead to the failure of such drugs and the nerve damage. The main disadvantages of pain and no effect.
  • the northeast rain frog is an amphibious animal of the genus Hymenoptera, mainly distributed in Hokkaido and Yakushima, Japan, as well as Heilongjiang, Liaoning, Jilin, Inner Mongolia and other places. It mainly lives on trees, in order to escape the invasion and adaptation of various biological enemies.
  • the skin of the tree frog develops into a multi-functional organ, such as: secretion of a large number of antimicrobial peptides against microbial invasion; secretion of neurotoxin against various natural enemies; secretion growth factor promotion Wound healing, etc.
  • the object of the present invention is to overcome the shortcomings of the prior art, and provide an analgesic peptide FI;
  • Another object of the present invention is to provide an amino acid sequence and a gene sequence encoding an analgesic peptide FI;
  • an analgesic peptide FI comprising 6 amino acid residues and having a molecular weight of 717.87 Da, the isoelectric point is 5.52, and the amino acid sequence thereof is shown in SEQ ID: 1, which is: phenylalanine-tryptophan-valine-valine-isoleucine-glycine.
  • analgesic peptide FI of the present invention for the preparation of an analgesic drug.
  • the beneficial effects of the present invention are: experiments have proved that the analgesic peptide FI of the present invention has a good analgesic effect and can be used as an application for preparing an analgesic drug.
  • the analgesic peptide FI has the advantages of simple structure, convenient artificial synthesis, low production cost, etc., and utilizes industrial mass production, and has important application prospects and practical significance.
  • Figure 1 is a schematic diagram showing the analgesic effect of analgesic peptide FI on a mouse tail-tailing model.
  • Figure 2 is a schematic diagram showing the analgesic effect of analgesic peptide FI on a mouse hot plate model.
  • Figure 3 is a schematic diagram showing the analgesic effect of analgesic peptide FI on a mouse writhing analgesia model.
  • Figure 4 is a schematic diagram showing the analgesic effect of analgesic peptide FI on a mouse formalin analgesia model.
  • Example 1 Northeast Asian tree frog skin analgesic peptide FI gene cloning
  • the living northeast rain frog was cleaned with water, frozen in liquid nitrogen for 4 h, 300 mg of skin tissue was taken, 10 ml of Trizol extraction buffer (product of GIBCOBRL, USA) was added, and homogenized in a 20 ml glass homogenizer for 30 min;
  • the obtained total RNA of the skin was dissolved in an appropriate amount of DEPC water, and the concentration and purity of the extracted total RNA were measured at a wavelength of 280 nm at 260 nm.
  • the cDNA obtained by PCR The double strand was added to an equal volume of membrane binding buffer and mixed by inversion, and then the mixture was transferred to a centrifugal purification column, and allowed to stand at room temperature for 5 minutes to allow the DNA to sufficiently bind to the silica membrane. 16,000 g Centrifuge for 1 minute and drain the waste from the collection tube.
  • the bacteria were transferred to a sterile, single-use, ice-cold 50 ml polypropylene tube and placed on ice for 10 min to cool the culture to 0 °C.
  • the amplification primer is 17 nucleotides in length and its sequence is 5'TACCGAAAGAACTCAATTTA 3', another amplification primer for PCR is 3'PCR in CLONTECH's SMART cDNA Library Construction Kit Primer primer with a sequence of 5'ATTCTACAGGCCGAGGCGGCCGACATG 3'.
  • the PCR reaction was carried out under the following conditions: 94 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C. 45 seconds, 30 cycles.
  • the amplified product was detected by 1% agarose gel electrophoresis, and the desired band was excised and purified using a DNA purification kit.
  • the purified fragment of interest was ligated into the pMD19-T vector to obtain the ligated product.
  • the ligation product was transformed into pre-prepared E. coli DH5 ⁇ competent cells. Finally, an appropriate amount of the transformed product was applied to an Amp-containing LB plate and cultured at 37 ° C.
  • the single colony formed by h is the positive clone containing the fragment of interest.
  • the northeast rain frog skin FI contains 6 amino acid residues with a molecular weight of 717.87 Da and an isoelectric point of 5.52. Its amino acid sequence is SEQ. ID: 1 is: phenylalanine-tryptophan-valine-valine-isoleucine-glycine.
  • Example 3 Analgesic experiment of analgesic peptide FI on mouse tail-tail model
  • the Kunming mice weighing 18-22 g were used to screen 50 mice with a basic pain threshold of 3-7s for testing.
  • the method was as follows: the infrared beam of the rat tail pain meter was small. At 1/3 of the root of the rat tail, the pain threshold time is the time interval between when the mouse receives infrared beam irradiation and the tail motion of the mouse, and the basic pain threshold of the mouse is recorded.
  • the mice were divided into 5 groups, 10 in each group, which were negative control group and positive control group.
  • Treatment group 1, treatment group 2, treatment group 3, the analgesic peptide FI lyophilized powder was dissolved in sterile physiological saline to prepare 1.25mg/kg, 2.5mg/kg, 5.0mg/kg analgesic peptide FI, Dosing by intraperitoneal injection, positive control group injection 100 ⁇ L 5 mg/kg morphine, treatment group 1 was injected with 100 ⁇ L of 1.25 mg/kg analgesic peptide FI, treatment group 2 was injected with 100 ⁇ L of 2.5 mg/kg analgesic peptide FI, and treatment group 3 was injected with 100 ⁇ L.
  • analgesic peptide FI 5.0 mg/kg analgesic peptide FI, negative control group was injected with sterile physiological saline, the volume was 100 ⁇ L, and the pain threshold of the mice was determined at 10 min, 30 min, 60 min, 180 min, 360 min after the injection. In order to avoid tissue damage in mice, if the pain threshold is more than 10 s after administration, the pain threshold of the mouse is recorded as 10 s. The results are shown in Fig. 1.
  • Example 4 Analgesic experiment of analgesic peptide FI on mouse hot plate model
  • mice Female mice, Kunming, weighing 18-22 g, were used to screen 50 mice with a basic pain threshold of 5-30 s using a rat tail pain tester.
  • the test method was as follows: the mice were placed on a hot plate. The hot plate temperature is maintained at 55 ⁇ 0.5°C.
  • the reaction time of the mouse on the hot plate refers to the time interval from when the mouse is placed on the hot plate to the time when the mouse has a hind foot behavior or jumps directly out of the hot plate.
  • the basic pain threshold for mice The mice were divided into 5 groups, 10 in each group, which were negative control group and positive control group.
  • Treatment group 1, treatment group 2, treatment group 3, the analgesic peptide FI lyophilized powder was dissolved in sterile physiological saline to prepare 1.25mg/kg, 2.5mg/kg, 5.0mg/kg analgesic peptide FI, Dosing by intraperitoneal injection, positive control group injection 100 ⁇ L 5mg/kg morphine, treatment group 1 was injected with 100 ⁇ L 1.25mg/kg analgesic peptide FI, treatment group 2 was injected with 100 ⁇ L 2.5mg/kg analgesic peptide FI, and treatment group 3 was injected with 100 ⁇ L 5.0mg/kg analgesic peptide FI, negative control group was injected with sterile saline, the volume was 100 ⁇ L, and the pain threshold of mice was determined at 10min, 30min, 60min, 180min, 360min after injection, in order to avoid the tissue of mice. Injury, if the pain threshold is greater than 60 s after administration, the pain threshold of the mouse is recorded as
  • Example 5 Analgesic experiment of analgesic peptide FI on mouse writhing analgesia model
  • mice weighing 18-22 g were divided into 5 groups, 10 in each group, which were negative control group and positive control group.
  • Treatment group 1 treatment group 2
  • the analgesic peptide FI lyophilized powder was dissolved in sterile physiological saline to prepare 1.25 mg/kg, 2.5 mg/kg, 5.0 mg/kg of analgesic peptide FI, which was administered by intraperitoneal injection.
  • treatment group 1 was injected with 100 ⁇ L 1.25 mg/kg analgesic peptide FI
  • treatment group 2 was injected with 100 ⁇ L.
  • mice were injected with normal saline, 5 mg/kg morphine, 1.25 mg/kg FI, 2.5 mg/kg.
  • FI 5.0mg/kg FI, the number of writhing in mice were: 62, 27.9, 48.1, 32.5 and 26.9 times.
  • the experiment showed that the analgesic peptide FI can significantly alleviate the pain caused by acetic acid.
  • Example 6 Analgesic experiment of analgesic peptide FI on mouse formalin analgesia model
  • mice weighing 18-22 g were divided into 5 groups, 10 in each group, which were negative control group and positive control group.
  • Treatment group 1 treatment group 2, treatment group 3, the analgesic peptide FI lyophilized powder was dissolved in sterile physiological saline to prepare 1.25mg/kg, 2.5mg/kg, 5.0mg/kg analgesic peptide FI, Dosing by intraperitoneal injection, positive control group injection 100 ⁇ L 5 mg/kg morphine, treatment group 1 was injected with 100 ⁇ L of 1.25 mg/kg analgesic peptide FI, treatment group 2 was injected with 100 ⁇ L of 2.5 mg/kg analgesic peptide FI, and treatment group 3 was injected with 100 ⁇ L.
  • mice were injected with normal saline, 5 mg/kg morphine, 1.25 mg/kg FI, 2.5 mg/kg.
  • FI, 5.0 mg/kg FI, 0-5 min mice's toe time were: 142.2, 57.6, 108.4, 83.1 And 71.9 times; 15 ⁇ 30min mice's toe time was: 302.1, 258.7, 287.6, 247.7 and 216.6 times, the experiment showed that the analgesic peptide FI can significantly reduce the pain caused by formalin.

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Abstract

本发明公开了一种皮肤镇痛肽FI及其基因，它包含6个氨基酸残基，分子量为717.87Da，等电点为5.52，其氨基酸序列如SEQ ID:1所示，编码镇痛肽FI的DNA序列如SEQ ID:2所示。本发明还公开了所述镇痛肽在制备镇痛药物中的应用。

Description

[根据细则37.2由ISA制定的发明名称]　镇痛肽FI及其基因和应用 技术领域
本发明属于生物医学技术领域，具体涉及一种镇痛肽FI及其基因和应用。
 背景技术
疼痛是人们一生中经常遇到的不愉快的感觉，它提供躯体受到威胁的警报信号，是生命不可缺少的一种特殊保护功能；另一方面，它又是各种疾病最常见的症状，也是当今困扰人类健康最严重的问题之一。从当今世界范围来看，疼痛已经成为危害人类健康的主要“杀手”之一，也是造成人们劳动能力降低和出勤日减少的最普通、最直接的因素。镇痛药物是一类能够减轻疾病痛苦的物质，不仅能够选择地减轻或缓解疼痛感觉，也可以缓解因剧烈疼痛而引起的恐惧、紧张及焦虑等不愉快的情绪。目前的镇痛药物可以分为解热镇痛药和麻醉镇痛药物两类，前者以阿司匹林为代表，通过对组织中前列腺素合成过程的抑制作用，减轻神经末梢对致痛物质的敏感性，主要用于解除炎症性疼痛和其他钝疼；后者以吗啡为代表，其作用部位主要在丘脑和皮层，作用于中枢神经系统中的µ-型阿片受体，以缓解锐痛、钝痛和内脏绞痛，从而产生镇痛作用，主要用于各种疾病所引起的疼痛和手术后病人的严重疼痛，特别是晚期癌症疼痛。但是目前这种以吗啡为代表的麻醉性镇痛药物存在成瘾性强，戒断症状严重，病人必须不断增加剂量才能维持镇痛效果，而且最终导致对这类药物失效，同时对神经损伤性疼痛无疗效等主要缺点。
在中国的传统中药和民族医药中，许多两栖类动物被作为药材而被广泛的应用，如中华蟾蜍Bufo gargarizans，大蹼铃蟾Bombina maxima，黑斑蛙Pelophylax nigromaculata，沼蛙hyla ranaguentheri和泽蛙Euphlyctislimnocharis等，这些两栖类动物的皮肤和内脏具有广泛的药理活性和临床疗效，已报道药理活性有：光谱抗菌作用、抗肿瘤、局部麻醉、镇痛、免疫调节、对心血管系统的作用等；另一方面，传统中药药物成分的复杂性及其炮制方法的局限性也是造成药物活性成分不能更好发挥作用的中药原因，因而从这些传统药物中寻找特定的活性单体化合物是中药现代化的中药内容之一。在国外，两栖类皮肤特定药理活性单体化合物的寻找已经成为新药发明的热点。近十几年对170多种两栖类皮肤活性肽和类似物进行了筛选，证明有40多种活性肽有较好的药物开发前景。
我国对两栖类药物的应用有悠久的历史，但对其活性成分和药理性质的研究主要集中于生物碱等有机小分子，对其皮肤活性蛋白肽类物质的研究还较少。东北雨蛙为雨蛙科雨蛙属的两栖动物，主要分布于日本北海道和屋久岛，以及黑龙江、辽宁、吉林、内蒙等地，主要栖息于树上生活，为了逃避多种生物天敌的侵袭和适应多变的自然环境以及避免受伤机体损伤，在长期的进化过程中，雨蛙的皮肤发展成为具有多种功能的器官，如：分泌大量抗菌肽抵御微生物侵袭；分泌神经毒素对抗各类天敌；分泌生长因子促进伤口愈合等。
 技术问题
本发明的目的在于克服现有技术的缺点，提供一种镇痛肽FI；
本发明的另一个目的在于提供编码镇痛肽FI的氨基酸序列和基因序列；
本发明的又一目的在于提供一种镇痛肽FI在制备镇痛药物中的应用。
 技术解决方案
本发明的目的通过以下技术方案来实现，镇痛肽FI，它包含6个氨基酸残基，分子量为717.87 Da，等电点为5.52，其氨基酸序列如SEQ ID:1所示，为: 苯丙氨酸-色氨酸-脯氨酸-缬氨酸-异亮氨酸-甘氨酸。
编码一种镇痛肽FI的DNA序列，其核苷酸序列如SEQ ID:2所示，为：
Atggctttct tgaggaaatc gcttttcctt gtactatttc ttggattagt ctcactgtca 60
ttctgtgaag aagagaaaag agtggaagaa gaagagaaga aaagagatag tagtgtggag 120
gaagaaaaga gattttggcc agtgattggt cgtcgtgatg aagaaaagag attttggcca 180
gtgattggtc gtcgtgatga agaaaagaga ttctggccag tgattggtcg tcgtgatgaa 240
gaaaagagat tttggccagt gattggtcgt cgtgatgaag aaaagagatt ttggccagtg 300
attggtcgtc gtgatgaaga aaagagattt tggccagtga ttggtcgtcg tgatgaagaa 360
aagagatttt ggccagtgat tggtcgtcgt gatgaagaaa agagattttg gccagtgatt 420
ggtcgtcgtg atgaagaaaa gagattctgg ccagtgattg gtcgtcgtga tgaagaaaag 480
agattttggc cagtgactgg tcgtcgtgat gaataaaaga gattatggcc gcctggccgt 540
cattaataaa atgtaatgtt tcatccctaa ggaacacaat tactattagt tattccagat 600
ctatattaaa gcttatcaaa ctgataaaaa aaaaaaaaaa aaaaaaaaa 649
本发明的镇痛肽FI在制备镇痛药物中的应用。
 有益效果
本发明的有益效果是：实验证明，本发明中的镇痛肽FI具有良好的镇痛效果，能够作为制备镇痛药物中的应用。且该镇痛肽FI具有结构简单、人工合成方便，生产成本低等优点，利用工业大量生产，具有重要的应用前景和实际意义。
 附图说明
图1 为镇痛肽FI对小鼠摆尾模型的镇痛效果示意图。
图2 为镇痛肽FI对小鼠热板模型的镇痛效果示意图。
图3 为镇痛肽FI对小鼠扭体镇痛模型的镇痛效果示意图。
图4 为镇痛肽FI对小鼠福尔马林镇痛模型的镇痛效果示意图。
 本发明的实施方式
下面结合附图对本发明做进一步的描述，本发明的保护范围不局限于以下所述：
实施例1：东北雨蛙皮肤镇痛肽FI基因克隆
I. 东北雨蛙皮肤总RNA的提取
A. 活体东北雨蛙用水清洗干净，液氮中速冻4h，取皮肤组织300mg，加入10mlTrizol提取缓冲液(美国GIBCOBRL公司产品)，置于20ml玻璃匀浆器中匀浆30min；
B. 加入等体积酚/氯仿溶液，剧烈振荡混匀，室温放置10min后，于4℃，12000rpm离心机中离心10min，取上清液；
C. 上清液中加入等体积异丙醇，室温放置10min后，于4℃，12000rpm离心机中离心10min，沉淀用75%乙醇洗一次，晾干，即为东北雨蛙皮肤总RNA。
D. 将所获皮肤总RNA用适量DEPC水溶解，于28OIun、260nm波长处检测所提取的总RNA的浓度及纯度。
II. 东北雨蛙皮肤mRNA的纯化
东北雨蛙皮肤mRNA分离纯化采用美国PROMEGA公司的PolyATtract mRNAIsolation Systems试剂盒。
A. 取东北雨蛙皮肤总RNA500µg溶于500µl DEPC水中，65℃水浴10min，加入3µl的Oligo(dT)探针和13µl 20×SSC溶液，混合均匀，室温冷却，称为A液。
B. 磁珠(SA-PMP)的洗涤：将磁珠轻弹混匀，至磁力架吸附30s，弃上清，加0.5×SSC 0.3ml，至磁力架吸附30s，再加0.1ml 0.5×SSC 悬液，称为B液。
C. 将A液加入B液中，置室温10min，至磁力架吸附30s，弃上清，用0.1×SSC洗涤4次，再弃上清，加0.1ml DEPC水悬浮，至磁力架上吸附30s，将上清移至新的试管，再加入0.15ml DEPC水重新悬浮，至磁力架吸附30s，移上清至上述试管，上清液为纯化的东北雨蛙皮肤mRNA。
D. 加入1/10体积3M乙酸钠，pH 5.2，等体积异丙醇，于-70℃放置30min，4℃，12000rpm离心10min，弃上清，沉淀溶解于10µl DEPC水中。
III. 东北雨蛙皮肤cDNA文库构建
采用CLONTECH公司Creator SMARTTM cDNA Library Construction Kit 质粒cDNA文库构建试剂盒。
A. cDNA第一链合成（mRNA 反转录）：
1. 在0.5ml无菌的离心管中加入1µl华南雨蛙皮肤mRNA、1µl SMART IV寡聚核苷酸、1µl CDS III/3’ PCR 引物，加2µl去离子水使总体积达到5µl。
2. 混匀离心管中的试剂并短暂离心，72℃保温2min。
3. 将离心管在冰上孵育2min。
4. 在离心管中加入以下试剂：2.0µl 5×第一链缓冲、1.0µl 20mM二硫苏糖醇、1.0µl 10mM dNTP 混合物、1.0µl PowerScript 反转录酶。
5. 混合离心管中试剂并短暂离心，在42℃保温1h。
6. 将离心管置于冰上中止第一链的合成。
7. 从离心管去2µl所合成的cDNA第一链备用。
B. 采用长末端聚合酶链式反应(LD-PCR)方法扩增第二链
1. 95℃预热PCR仪。
2. 将2µl cDNA第一链（mRNA 反转录）、80µl去离子水、10µl 10×Asvantage2PCR缓冲、2µl 50×dNTP 混合物、2µl 5’PCR引物、2µl CDS III/3’ PCR 引物以及2µl大肠杆菌聚合酶离心管进行反应。
3. 在PCR仪中按以下程序扩增：
① 95℃ 20s
② 22个循环
95℃ 5s； 68℃ 6s
4. 循环结束后，将离心管中合成的cDNA双链进行抽提。
C. PCR产物用 PROMEGA 公司的 Wizard SV Gel and PCR Clean-Up System 试剂盒进行抽提回收，步骤如下：
1. 将通过PCR得到的cDNA 双链加入等体积的膜结合缓冲颠倒混匀，然后将混和液转入离心纯化柱，室温静置5分钟，使DNA充分与硅胶膜结合。16,000 g 离心1分钟，倒掉收集管中的废液。
2. 加入700µl的洗脱液（含乙醇）于离心纯化柱中，16,000g 离心1分钟，倒掉收集管中的废液。
3. 重复步骤2。
4. 16,000g 离心5分钟。
5. 将离心纯化柱置于新的离心管中。
6. 加入30µl超纯水，在室温下静置5分钟。
7. 16,000g 离心1分钟，管底溶液即为所纯化过的cDNA双链。
D. 大肠杆菌DH5α感受态细胞的制备：
1. 挑取单个DH5α菌落，接种于3m1不含氨苄青霉素的LB培养基中，37℃培养过夜，次日取上述菌液按比例1:100再接种于50m1 LB培养液中，37℃振荡2小时。当OD600值达到0.35时，收获细菌培养物。
2. 将细菌转移到一个无菌、一次性使用的、用冰预冷的50m1聚丙烯管中，在冰上方置10min，使培养物冷却至0℃。
3. 于4℃以4100r/min离心10min，以回收细胞。
4. 倒出培养液，将管倒置lmin以使最后的痕量培养液流尽。
5. 每50ml初始培养液且30 ml预冷的0.1mol/L CaCl2-MgCl2溶液(80mmol/L MgCl2，20mmol/L CaCl2)重悬每份细胞沉淀。
6. 于4℃以4100r/min离心10min，以回收细胞。
7. 倒出培养液，将管倒置lmin以使最后的痕量培养液流尽。
8. 每50m1初始培养物用2m1用冰预冷的0.1mol/L CaCl2重悬每份细胞沉淀，分装后备用。
E. 酶切、连接以及连接产物的转化：
1. 在微量离心管中加入1µl Takara pMD19-T 载体、4µl东北雨蛙 cDNA双链溶液，全量为5µl。
2. 加入5µl（等量）的连接酶缓冲混合物。
3. 16℃反应2 小时。
4. 全量（10µl）加入至100µl DH5α感受态细胞中，冰中放置30分钟。
5. 42℃加热90秒钟后，再在冰中放置1分钟。
6. 加入37℃温浴过的LB培养基890µl，37℃缓慢振荡培养60分钟。
7. 取200µl涂布于含有X-Gal、IPTG、Amp的LB培养基上37℃培养16小时，形成单菌落。
8. 每个LB平皿用5m1 LB液体培养基洗涤菌落，加30%甘油冻存，构建的cDNA大约含1 × 106个单独克隆。
IV. 东北雨蛙皮肤FI基因克隆筛选
扩增引物长度为17个核苷酸其序列为5’TACCGAAAGAACTCAATTTA 3’，PCR另一扩增引物为CLONTECH 公司SMART cDNA Library Construction Kit中的3’PCR Primer引物，其序列为5＇ATTCTACAGGCCGAGGCGGCCGACATG 3＇。
PCR反应在如下条件下进行：94 ℃ 30秒钟，56℃ 30秒钟和72 ℃ 45秒钟，30个循环。
将扩增产物用1%琼脂糖凝胶电泳检测，切取目的条带，用DNA纯化试剂盒进行纯化。将纯化的目的片段连接到pMD19-T载体中，即得连接产物。将连接产物转化预先制备好的大肠杆菌DH5α感受态细胞。最后，取适量转化产物涂布至含Amp的LB平板上，经37℃培养16 h形成的单菌落即为含目的片段的阳性克隆。
V. 东北雨蛙皮肤FI基因测定
挑取单菌落用M13引物检测插入片段的大小。挑选含目的片段的阳性克隆送DNA测序公司测序。
东北雨蛙皮肤FI包含6个氨基酸残基，分子量为717.87 Da，等电点为5.52，其氨基酸序列如SEQ ID:1所示，为: 苯丙氨酸-色氨酸-脯氨酸-缬氨酸-异亮氨酸-甘氨酸。
编码东北雨蛙皮肤镇痛肽FI的DNA序列，其核苷酸序列如SEQ ID:2所示，为：
atggctttct tgaggaaatc gcttttcctt gtactatttc ttggattagt ctcactgtca 60
ttctgtgaag aagagaaaag agtggaagaa gaagagaaga aaagagatag tagtgtggag 120
gaagaaaaga gattttggcc agtgattggt cgtcgtgatg aagaaaagag attttggcca 180
gtgattggtc gtcgtgatga agaaaagaga ttctggccag tgattggtcg tcgtgatgaa 240
gaaaagagat tttggccagt gattggtcgt cgtgatgaag aaaagagatt ttggccagtg 300
attggtcgtc gtgatgaaga aaagagattt tggccagtga ttggtcgtcg tgatgaagaa 360
aagagatttt ggccagtgat tggtcgtcgt gatgaagaaa agagattttg gccagtgatt 420
ggtcgtcgtg atgaagaaaa gagattctgg ccagtgattg gtcgtcgtga tgaagaaaag 480
agattttggc cagtgactgg tcgtcgtgat gaataaaaga gattatggcc gcctggccgt 540
cattaataaa atgtaatgtt tcatccctaa ggaacacaat tactattagt tattccagat 600
ctatattaaa gcttatcaaa ctgataaaaa aaaaaaaaaa aaaaaaaaa 649
实施例2：制备镇痛肽FI
A. 称取树脂0.2g放置于干燥洁净的反应管中，加入适量的N,N-二甲基酰胺（DMF）,活化30分钟作用，将称取第一氨基酸残基1mmol，DMAP150mg加入到反应管中，DMF做为溶剂反应3小时。反应完毕后用DMF洗3-6次，加入适当的吡啶和乙酸酐，体积比为1:1，反应30 min。反应完毕后DMF洗3-6次。然后用哌啶洗脱氨基酸的保护基团Fmoc，洗脱两次每次15min，再用DMF洗4次，甲醇洗2次。
B. 称第二个氨基酸3mmol，HBTU3mmol于反应管中，加入DIEA0.5ml，反应40分钟，用DMF 洗3-6次，加入哌啶溶液两次洗脱氨基酸的保护基团Fmoc，每次10分钟，再用DMF洗4次，甲醇洗2次。
C. 重复第二个步骤直到最后一个氨基酸残基。
D. 最后一个氨基酸反应完毕后，用三氟乙酸切割2小时，反应抽滤，得到多肽的三氟乙酸溶液，用乙醚沉淀，离心，再用乙醚洗3-5遍，得到白色固体，经过HPLC脱盐冻干得到多肽样本。
实施例3：镇痛肽FI对小鼠摆尾模型的镇痛实验
取重量为18～22g昆明小鼠，用鼠尾测痛仪筛选出基础痛阈在3～7s的小鼠50只用于试验，测定方法为：鼠尾测痛仪的红外光束照在距离小鼠尾巴根部的1/3处，痛阈时间是指从小鼠接受红外光束照射到小鼠发生摆尾动作之间的时间间隔，并记录小鼠的基础痛阈值。将小鼠分为5组，每组10只，分别为阴性对照组、阳性对照组组、 治疗组1、治疗组2、治疗组3，将镇痛肽FI冻干粉溶解于无菌生理盐水中，制备1.25mg/kg、2.5mg/kg、5.0mg/kg的镇痛肽FI，采用腹腔注射方式给药，阳性对照组注射100µL 5mg/kg吗啡， 治疗组1注射100µL 1.25mg/kg镇痛肽FI，治疗组2注射100µL 2.5mg/kg镇痛肽FI，治疗组3注射100µL 5.0mg/kg镇痛肽FI，阴性对照组注射无菌生理盐水，体积为100µL，在注射给药后10min、30min、60min、180min、360min测定小鼠的痛阈值。为了避免造成小鼠的组织伤害，如果小鼠给药后痛阈值大于10s，该小鼠的痛阈值就记为10s，结果见图1。
如图1所示：与对照组相比，小鼠注射镇痛肽FI后，能够明显减轻小鼠的疼痛。
实施例4：镇痛肽FI对小鼠热板模型的镇痛实验
取雌性小鼠，昆明种，重量为18～22g，用鼠尾测痛仪筛选出基础痛阈在5～30s的小鼠50只用于试验，测试方法为：将小鼠置于热板上，热板温度保持在55±0.5℃，小鼠在热板上的反应时间是指从小鼠放到热板上到小鼠发生添后足行为或者直接跳出热板之间的时间间隔，并记录小鼠的基础痛阈值。将小鼠分为5组，每组10只，分别为阴性对照组、阳性对照组、 治疗组1、治疗组2、治疗组3，将镇痛肽FI冻干粉溶解于无菌生理盐水中，制备1.25mg/kg、2.5mg/kg、5.0mg/kg的镇痛肽FI，采用腹腔注射方式给药，阳性对照组注射100 µL 5mg/kg吗啡， 治疗组1注射100µL 1.25mg/kg镇痛肽FI，治疗组2注射100µL 2.5mg/kg镇痛肽FI，治疗组3注射100µL 5.0mg/kg镇痛肽FI，阴性对照组注射无菌生理盐水，体积为100µL，在注射给药后10min、30min、60min、180min、360min测定小鼠的痛阈值，为了避免造成小鼠的组织伤害，如果小鼠给药后痛阈值大于60s，该小鼠的痛阈值就记为60s，结果见图2。
如图2所示：与对照组相比，小鼠注射镇痛肽FI后，能够明显减轻热板造成的疼痛。
实施例5：镇痛肽FI对小鼠扭体镇痛模型的镇痛实验
取重量为18～22g昆明小鼠50只，分为5组，每组10只，分别为阴性对照组、阳性对照组、 治疗组1、治疗组2、 治疗组3，将镇痛肽FI冻干粉溶解于无菌生理盐水中，制备1.25mg/kg、2.5mg/kg、5.0mg/kg的镇痛肽FI，采用腹腔注射方式给药，阳性对照组注射 100 µL 5mg/kg吗啡， 治疗组1注射100 µL 1.25mg/kg镇痛肽FI，治疗组2注射100 µL 2.5mg/kg镇痛肽FI，治疗组3注射100 µL 5.0mg/kg镇痛肽FI，阴性对照组注射无菌生理盐水，体积为100µL，30min后，腹腔注射0.6%的乙酸（10ml/kg体重），计数小鼠注射乙酸后30min的扭体次数，结果见图3。
如图3所示：小鼠注射生理盐水、5mg/kg吗啡、1.25mg/kg FI、2.5mg/kg FI、5.0mg/kg FI，小鼠扭体次数分别为：62、27.9、48.1、32.5 和26.9次，实验表明：镇痛肽FI能够明显减轻乙酸造成的疼痛。
实施例6：镇痛肽FI对小鼠福尔马林镇痛模型的镇痛实验
取重量为18～22g昆明小鼠50只，分为5组，每组10只，分别为阴性对照组、阳性对照组、 治疗组1、治疗组2、治疗组3，将镇痛肽FI冻干粉溶解于无菌生理盐水中，制备1.25mg/kg、2.5mg/kg、5.0mg/kg的镇痛肽FI，采用腹腔注射方式给药，阳性对照组注射100µL 5mg/kg吗啡， 治疗组1注射100µL 1.25mg/kg镇痛肽FI，治疗组2注射100µL 2.5mg/kg镇痛肽FI，治疗组3注射100µL 5.0mg/kg镇痛肽FI，阴性对照组注射无菌生理盐水，体积为100µL，30min后，在小鼠的脚掌处注射20mL浓度为0.92%福尔马林，采用摄像记录小鼠总舔脚时间，结果见图4。
如图4所示：小鼠注射生理盐水、5mg/kg吗啡、1.25mg/kg FI、2.5mg/kg FI、5.0mg/kg FI，0～5min小鼠舔趾时间分别为：142.2、57.6、108.4、83.1 和71.9次；15～30min小鼠舔趾时间分别为：372.1、258.7、287.6、247.7和216.6次，实验表明：镇痛肽FI能够明显减轻福尔马林造成的疼痛。

Claims (3)

  1. 一种镇痛肽FI，其特征在于，它包含6个氨基酸残基，分子量为717.87 Da，等电点为5.52，其氨基酸序列如SEQ ID:1所示。
  2. 编码权利要求1所述一种镇痛肽FI的DNA序列，其核苷酸序列如SEQ ID:2所示。
  3. 如权利要求1所述一种镇痛肽FI在制备镇痛药物中的应用。
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