WO2014005241A1 - 抑血管生成素的药物用途 - Google Patents
抑血管生成素的药物用途 Download PDFInfo
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- WO2014005241A1 WO2014005241A1 PCT/CN2012/000907 CN2012000907W WO2014005241A1 WO 2014005241 A1 WO2014005241 A1 WO 2014005241A1 CN 2012000907 W CN2012000907 W CN 2012000907W WO 2014005241 A1 WO2014005241 A1 WO 2014005241A1
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- angiopoietin
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Classifications
-
- 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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/484—Plasmin (3.4.21.7)
-
- 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]
Definitions
- the present invention relates to the field of biomedical technology, and in particular to a pharmaceutical use of a biological material, and more particularly, to the use of an angiopoietin as an effective component of an anti-inflammatory drug. Background technique
- Angiostatin (angios tatin), also known as angiostatin and angiostatin, is a natural hydrolysate of plasminogen in the body, which can effectively inhibit the proliferation, migration and tubular vascular endothelial cell proliferation.
- Angiogenesis refers to the process of growing new branched blood vessels from existing blood vessels, which plays an important role in the development of tissues and organs of embryonic development, but is not active in general adult tissues (except tissues such as the female reproductive system). However, under certain pathological conditions, such as wound healing, inflammation, tumor growth and malignant metastasis, and atherosclerosis, it is often accompanied by an active angiogenic process.
- a tissue is capable of angiogenesis at a particular time depends on the balance between pro-angiogenic factors and angiogenic inhibitory molecules in the local environment. To date, nearly 30 endogenous angiogenesis inhibitors have been discovered. The inhibitory effect of angiopoietin on angiogenesis suggests its potential as a specific drug for vascular endothelial cells to treat angiogenesis-related diseases.
- angiopoietin can also bind to certain extracellular molecules such as tissue-type plasminogen inhibitors, suggesting that angiopoietin can also participate in the angiogenesis process by regulating the proteolytic activity surrounding the cells.
- angiopoietin The process of tumor growth and metastasis is inseparable from angiogenesis.
- a large number of studies have shown that recombinant or purified angiopoietin protein can significantly inhibit the growth of a variety of primary tumors.
- the clinical application of angiopoietin has entered the first phase.
- Inflammation is a common and frequently-occurring disease that threatens human health.
- the inflammatory response is a complex process involving multiple mediators, inflammatory factors and the body constantly fighting until a balance is reached.
- anti-inflammatory drugs mainly include steroia ant i-inf lammatory drugs (SAIDs), non-steroia ant i-inf lammatory drugs (NSAIDs) and traditional Chinese medicines.
- SAIDs steroia ant i-inf lammatory drugs
- NSAIDs non-steroia ant i-inf lammatory drugs
- Chinese medicines traditional Chinese medicines.
- steroidal drugs have excellent curative effects, long-term use can cause water and salt metabolism and serious disorders of sugar, fat and protein metabolism.
- Non-body drugs are currently the most widely used drugs for clinical inflammation treatment, because NSAIDs are inhibited.
- prostaglandins therefore, also has an anti-inflammatory effect, but also causes side effects on the gastrointestinal tract, mainly manifested as upper gastrointestinal bleeding caused by stomach and duodenal ulcer; can also cause sodium water retention And edema, hyperemia, nephrotic syndrome complicated by interstitial nephritis, renal insufficiency, acute interstitial nephritis, renal papillary necrosis and other serious kidney damage; can also increase the use of people with thrombotic adverse events (myocardial infarction, no Stable angina, cardiac thrombosis, sudden death, etc., congestive heart failure, hypertension, coronary heart disease, etc.; liver damage, lethargy, convulsions, headache, head pull, tinnitus, optic neuritis, asthma, skin damage, etc. may also occur Other adverse reactions; In addition, it can be seen from the literature in recent years that research on anti-inflammatory Chinese medicine Quite superficial understanding of the process of the specific role
- Angiogenes is a complex biological process in which vascular endothelial cells differentiate and migrate from existing vasculature to form new microvessels.
- Adult vascular endothelial cells are basically at rest, and new blood vessels are formed under physiological stimulation such as wound healing, tissue repair, female fertility and menstrual period, fetal development, which is physiological angiogenesis.
- physiological stimulation such as wound healing, tissue repair, female fertility and menstrual period, fetal development, which is physiological angiogenesis.
- angiogenesis-dependent diseases such as rheumatoid arthritis, diabetic or macular degeneration retinopathy, infantile hemangioma and malignant tumor.
- angiogenesis-dependent diseases such as rheumatoid arthritis, diabetic or macular degeneration retinopathy, infantile hemangioma and malignant tumor.
- Folkman clarified the importance of tumor angiogenesis in tumor development, metastasis and spread, and suggested that inhibitors of angiogenesis may become a new and valuable treatment for cancer.
- disrupting angiogenesis plays an important role in cancer research. Endogenous and exogenous tumor angiogenesis inhibitors provide a new clinical direction for the treatment of such diseases.
- Patent application 201010252717. 7 discloses an angiopoietin structure extracted from Agkistrodon acutus venom and preparation method thereof, and an antitumor drug prepared by using the same, and the medicinal preparation of angiostatin of the structure of the present invention The value was deeply measured and found to have new anti-inflammatory properties, which provided a new way and direction for the development of anti-inflammatory drugs. Summary of the invention
- the invention aims to solve the stagnant state of the technical development in the existing anti-inflammatory drug research, and proposes a new research direction of anti-inflammatory drugs.
- a technical solution for achieving the above object of the invention is the use of angiopoietin for the pharmaceutical use, and the use of angiopoietin as an active ingredient for the preparation of an anti-inflammatory drug.
- angiopoietin which is an active ingredient of an anti-inflammatory drug
- a reducing SDS-PAGE method two bands, which are respectively called an ⁇ chain and an anthracene chain, and have molecular weights of 12,000 to 22,000 Daltons and 9000 to 19,000 Daltons, respectively.
- amino acid sequence of the ⁇ chain is as shown in SEQ ID NO. 1
- amino acid sequence of the ⁇ chain is shown in SEQ ID NO.
- angiopoietin which is an active ingredient of an anti-inflammatory drug, generates three isomers corresponding to the ⁇ chain and the & chain, respectively, after two-dimensional electrophoresis.
- the above preparation method of angiopoietin as an active ingredient of an anti-inflammatory drug comprises the following steps:
- step (d) Purifying the crude angiogenin solution obtained in the step (b) by using the obtained immunoaffinity chromatography column, eluting with the elution buffer, and collecting the angiopoietin, and the elution buffer used is pH 2.0 ⁇ 4.0, 0.01 ⁇ 0.03 mol / L Gly-HCl buffer.
- the anti-angiogenic agent discovered by the present invention is a novel natural protein anti-inflammatory drug, which has no obvious toxic and side effects on the body, and has broad application prospects, and is a development of traditional anti-inflammatory drugs on the other hand. . DRAWINGS
- Figure 1 is a flow chart showing the preparation process of angiopoietin according to the present invention
- FIG. 2 shows the results of measurement of RAW 264.7 macrophages by mouse angiopoietin. detailed description
- the technical scheme of the present invention mainly relates to the use of angiopoietin as an anti-inflammatory drug, and firstly determines the anti-inflammatory effect according to specific experimental measurements.
- Acute inflammation of the hind paw of rats was induced by subcutaneous injection of an inflammatory shield (including carrageenan) into the hind paw of rats.
- Angiopoietin 2.5 mg/kg
- indomethacin positive control, 10 mg/kg
- vehicle 0.5% sodium carboxymethylcellulose CMC
- the volume of each of the two hind legs of each animal was measured using a volumetric measurer at a time of 1 h before the induction of inflammation and at intervals of 0.5 to 6 h after the injection of the inflammatory shield.
- the percentage increase in the right hind paw volume of each rat at each time point (foot swelling) was calculated according to the following formula: % increase (%) -100* (AB) / B, A represents the foot volume at each time point after injection, B Represents the volume of the foot before injection.
- the experimental results showed that the rats were orally administered with 2.5 mg/kg of the angiopoietin at 1 h before the induction of inflammation, and the carrageenan-induced acute inflammation of the rat's foot produced a significant anti-inflammatory effect, with 10 mg/ The anti-inflammatory effect of indomethacin, a positive control drug of kg, increased slightly, but there was no statistical difference.
- RNA extraction was stored at -80 *C.
- Lipopolysaccharide induces an inflammatory response and will therefore express IL-6, IL- ⁇ and TNF-a genes and will produce mRNA. If the angiopoietin inhibits the inflammatory response, the mRNA concentration decreases.
- RNA is extracted from the cells and then constructed into complementary DNA (cDNA) by reverse transcriptase (RT), which allows for stable storage.
- cDNA complementary DNA
- RT reverse transcriptase
- cDNAs are amplified by polymerase chain reaction (PCR) for detecting gene expression.
- PCR polymerase chain reaction
- the real-time PCR technique was used to determine the concentration of the inflammatory cytokines IL-6, 11-1 and -01 after treatment of mouse RAW 264. 7 macrophages with LPS and the angiopoietin.
- RAW 264 for each experimental group with or without angiogenesis and/or LPS treatment.
- the difference in the concentration of inflammatory cytokines in 7 macrophages is shown in Fig. 2.
- LPS stimulates the release of the inflammatory cytokines IL-6, IL- ⁇ and TNF-a.
- the release of these cytokines was significantly reduced after the addition of ⁇ to the angiogenic agent. Therefore, the angiopoietin has a significant resistance to LPS-induced mouse RAW 264. 7 macrophage inflammation.
- Figure 1 is a flow chart showing the preparation process of angiopoietin according to the present invention.
- the crude venom of Agkistrodon acutus is dissolved in the buffer for a certain period of time, and then the supernatant is centrifuged to obtain a solution of the venom of the venomous snake venom; the obtained snake venom solution is subjected to anion exchange chromatography to obtain a crude solution of angiogenin, followed by taking An appropriate amount of crude solution is prepared by cation exchange chromatography and gel chromatography to prepare an initial product of angiopoietin; and then, the primary purified product is used as an antigen to prepare a specific monoclonal antibody against the same;
- the appropriate affinity chromatography carrier is coupled to prepare an antibody affinity chromatography column; finally, the previously prepared crude angiopoietin inhibitory solution having angiogenic activity is prepared by using the coupled
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Abstract
本发明公开了一种抑血管生成素作为活性成分制备抗炎药物的应用。作为抗炎药物活性成分的抑血管生成素采用还原性SDS-PAGE法测定显示为两条带，分别称α链和β链，分子量分别为12000-22000道尔顿、9000-19000道尔顿，其中，a链的氨基酸序列如SEQ ID NO.1所示，β链的氨基酸序列如SEQ ID NO.2所示，经双向电泳后会分别产生与所述α链和β链对应的异构体各三个。
Description
抑血管生成素的药物用途 技术领域
本发明属于生物医药技术领域， 具体是涉及一种生物材料的药物 用途， 更具体地说， 本发明研究了一种抑血管生成素作为抗炎药物有 效成份的应用。 背景技术
说
抑血管生成素（angios tat in )， 又称血管抑素、血管生成抑制素， 是纤溶酶原（plasminogen )在体内的天然水解产物， 能够有效抑制血 管内皮细胞的增殖、 迁移和管状结书构的形成而参与对血管生成 ( angiogenes i s ) 的调节。 血管生成是指从已经存在的血管生长出新 的分枝血管的过程， 该过程在胚胎发育组织器官形成中发挥重要作用， 而在一般成体组织 （除雌性生殖系统等组织） 并不活跃。 然而在某些 病理条件下， 如伤口愈合、 炎症、 肿瘤的生长和恶性转移、 动脉粥样 硬化时， 却常常伴随活跃的血管生成过程。 某一组织在某一特定时间 是否能够进行血管生成取决于在本地环境中促血管生成因子和血管生 成抑制分子之间的平衡。 目前为止， 已经发现近 30种内源性的血管生 成抑制因子。 抑血管生成素对血管生成的抑制作用， 提示其作为一种 特异性的针对血管内皮细胞的药物治疗与血管生成有关的疾病的可能 性。
1994年， 哈佛医学院的 Folkman实验室首次从肺癌转移模型小鼠 的血清和尿液中分离得到一种抑血管生成素。 由于纤溶酶原存在不同 的裂解位点， 因此在不同的细胞环境中会产生不同形式的抑血管生成 的抑制作用， 尚未见报道。 无论是纤溶酵原还是纤溶酶在细胞膜表面 都存在多种受体， 然而它们并没有抑制血管生成功能， 这说明抑血管 生成素可能通过不同的细胞表面受体发挥作用。 此外， 不同的抑血管
生成素以及不同的翻译后修饰过程都会导致抑血管生成素以不同的方 式结合并将信号传递到胞内。 除了细胞表面受体外， 抑血管生成素也 能结合胞外的某些分子如组织型纤溶酶原抑制因子， 说明抑血管生成 素也可以通过调节细胞周围的蛋白水解活性参与血管生成过程。
肿瘤的生长和转移过程离不开血管生成， 在过去几年里， 大量的 研究表明重组或纯化的抑血管生成素蛋白能显著抑制多种原发性肿瘤 的生长。 目前， 关于抑血管生成素的临床应用已进入一期阶段。
炎症是威胁人类健康的一种常见病和多发病。 炎性反应是由多种 介质参与、 炎性因子与机体不断斗争直至达到一个平衡的复杂过程。 目前抗炎药主要包括甾体类抗炎药 ( steroia ant i-inf lammatory drugs , SAIDs ) 、 非甾体类抗炎药 ( non-steroia ant i-inf lammatory drugs , NSAIDs )和中药。 甾体类药物虽然有极好的疗效， 但长期使用 可引起水盐代谢和糖、 脂肪、 蛋白质代谢的严重紊乱； 非 体类药物 是目前临床上炎症治疗应用最广泛的药物，由于 NSAIDs 抑制了前列腺 素（PGs ) 的合成， 因此在起抗炎作用的同时， 也造成了对胃肠道的副 作用， 主要表现为胃、 十二指肠溃疡引起的上消化道出血； 还可引起 钠水潴留和水肿、 高血症、 肾病综合征并发间质性腎炎、 肾功能不全、 急性间质性腎炎、 肾乳头坏死等严重的腎损害； 亦可增加使用人群发 生血栓性不良事件（心肌梗死、 不稳定心绞痛、 心脏血栓、 猝死等） 、 充血性心力衰竭、 高血压、 冠心病等的风险； 还可能发生肝损害、 嗜 睡、 神情恍惚、 头痛、 头牽、 耳鸣、 视神经炎、 哮喘、 皮肤损害等其 他不良反应； 另外， 从近几年的文献可以看出， 对抗炎中药的研究还 比较肤浅， 对中药具体作用过程的了解还不够系统。
肿瘤血管生成（angiogenes is)是指血管内皮细胞从现存的血管系 统中分化、 迁移而形成新的微血管的复杂生物学过程。 成人的血管内 皮细胞基本处于静止状态， 在伤口愈合、 组织修复、 女性生育和月经 期、 胎儿发育等生理刺激下会生成新的血管， 这属于生理性血管生成。
有限时间内的一种有序的生理过程， 增生的内皮细胞很快恢复为正常 的静止状态。 当血管生成调节机制失控和血管生成过度时， 则成为致 病因素， 导致风湿性关节炎、 糖尿病性或黄斑变性视网膜病变、 婴儿 血管瘤和恶性肿瘤等血管生成依赖性疾病的发生和发展。早在 1971年， Folkman阐明了肿瘤血管生成在肿瘤发展、 转移和扩散中的重要意义， 并提出血管发生的抑制剂可能会成为一种新型的、 有价值的肿瘤治疗 手段。 目前， 破坏血管生成在癌症研究中占据重要的地位。 内源性和 外源性的肿瘤血管生成抑制物为这类疾病的治疗提供新的临床用药方 向。
专利申请 201010252717. 7公开了一种从尖吻蝮蛇蛇毒中提取的抑 血管生成素结构及其制备方法， 以及应用其制备的抗肿瘤药物， 本发 明对该结构的抑血管生成素的药用价值进行深入实验测量， 发现其还 具备新的抗炎特性， 为抗炎药物的研制提供了一种新的途径和方向。 发明内容
本发明旨在解决现有抗炎药物研究中的技术发展停滞不前状态， 提出一种新的抗炎药物研究方向。
实现上述发明目的的技术方案为， 抑血管生成素的药物用途， 抑 血管生成素作为活性成份制备抗炎药物中的应用。
上述作为抗炎药物活性成份的抑血管生成素采用还原性 SDS-PAGE 法测定显示为两条带，分别称 α链和 Ρ链，分子量分别为 12000 ~ 22000 道尔顿、 9000 ~ 19000道尔顿，其中， α链的氨基酸序列如 SEQ ID NO. 1 所示， β链的氨基酸序列如 SEQ ID NO. 2所示。
上述作为抗炎药物活性成份的抑血管生成素经双向电泳后会分别 产生与所述 α链和 &链对应的异构体各三个。
上述作为抗炎药物活性成份的抑血管生成素的制备方法包括如下 步骤：
( a )将尖吻蝮蛇粗毒溶解于緩冲液中， 其中蛇毒与緩冲液的体积
比为 1~2 : 10， 离心得到上清液， 这里所用的緩冲液是 pH 7.0 ~ 9.0, 0.01 ~ 0. lmol/L的 Tris-HCl緩冲液， 离心次数 2次， 离心速度为 4000r/min, 每次离心 15min'，
(b)将所得上清液过二乙胺乙基阴离子交换层析柱进行层析， 得 到抑血管生成素粗溶液， 层析具体为： 层析 pH 6.0 ~ 9.0的 0.01 ~ 0.05mol/L的 Tris-HCl緩沖液和 0.05 ~ 0.6mol/L NaCl溶液作为洗脱 液进行线性梯度洗脱， 洗脱时间为 1440min, 洗脱流速 5ml/min。
(c)将杂交瘤细胞株分泌的单克隆抗体与亲和层析载体偶联， 制 备得免疫亲和层析柱， 这里的亲和层析柱载体为 4B琼脂糖凝胶、 二乙 氨基葡聚糖凝胶、 羧甲基葡聚糖凝胶中的任意一种；
(d) 用所得免疫亲和层析柱纯化步骤（b) 所得抑血管生成素粗 溶液， 以洗脱緩沖液洗脱， 收集得到抑血管生成素， 所用洗脱緩冲液 为 pH2.0 ~ 4.0、 0.01 ~ 0.03 mol/L的 Gly-HCl緩冲液。
本发明所研究发现的抑血管生成素作为一种新型的天然蛋白质类 抗炎药物， 其对身体不存在明显的毒副作用， 具有广阔的应用前景， 是传统抗炎药物研究在另一方面的开拓。 附图说明
图 1是根据本发明的抑血管生成素的制备工艺流程图；
图 2是抑血管生成素对小鼠 RAW 264.7巨噬细胞的测量结果。 具体实施方式
本发明的技术方案主要是涉及抑血管生成素作为抗炎药物的药物 用途， 先根据具体实验测量详述确定其抗炎效果。
( 1 )对大鼠后足急性炎症的治疗作用
采用向大鼠后足皮下注射炎性物盾 (包括角叉藻聚糖）的方法诱导 大鼠后足急性炎症。 在炎症诱导前的 lh 口服给予抑血管生成素 (2.5mg/kg) 、 吲哚美辛（ indomethacin, 阳性对照药， 10mg/kg)或 溶媒（0.5%羧甲基纤维素钠 CMC)。 炎症诱导时， 向大鼠的右后足跖肌
下组织注射溶于生理盐水中 （0. 9% w/v NaCl )新鲜制备的角叉藻聚糖 溶液（0. 1ml , l% w/v ) 。 未注射的左后足作为对照。 在炎症诱导前的 lh以及注射炎性物盾后 0. 5-6h所设的不同时间，使用容积测量器对每 只动物的两只后足进行容积测定。 按以下公式计算各大鼠在各时间点 右后足容积的增加百分数（足部肿胀）：增加百分数（％ ) -100* (A-B) /B, A代表注射后各时间点的足容积， B代表注射前的足容积。
与未给药的左足对照，注射角叉藻聚糖溶液后大鼠右后足产生了明 显的炎症。 该抑血管生成素对角叉藻聚糖诱导的大鼠急性足部肿胀效 应的主要实验结果如下：
实验结果表明，在炎症诱导前 lh对大鼠口服给予 2. 5mg/kg该抑血 管生成素， 对角叉藻聚糖诱导的大鼠足部急性炎症产生了明显的抗炎 作用， 与 10mg/kg的阳性对照药物吲哚美辛相比抗炎作用略微提高， 但没有统计学差异。
( 2 )抑血管生成素在小鼠 RAW 264. 7巨噬细胞中的抗炎效应 将小鼠 RAW 264. 7 巨噬细胞培养于 100mm培养皿中，培养基为 DMEM (达尔伯克改良伊格尔培养基） ， 培养基中添加了 10%的 FBS (在 55
°C下加热 30min )和 1%的青霉素和链審素。 细胞达到 70%的融合后进行 传代培养。 10000细胞 /孔， 测定 0. Ol-ΙμΜ该抑血管生成素在小鼠 RAW 264. 7巨噬细胞中的毒性， 确定 ΙμΜ该抑血管生成素对小鼠 RAW 264. 7 巨噬细胞没有毒性。 将 RAW 264. 7 巨噬细胞接种于 4个 60mm的培养皿 中。在 Oh移除培养基并加入 ΙμΜ该抑血管生成素， 3h时加入 l (^g的脂 多糖（ LPS )，自第一次处理后细胞培养 24h,随后培养亚使用 PBS洗涤， 若不立即进行 RNA萃取则贮存于 -80 *C。
脂多糖（ LPS )可诱导炎症反应，因此将会表达 IL-6、 IL- Ιβ和 TNF-a 基因并将产生 mRNA。若该抑血管生成素抑制炎症反应， mRNA浓度下降。 从细胞中萃取出 RNA，然后经逆转录酶（RT )构建成互补的 DNA ( cDNA ), 这样便可稳定贮存。 这些 cDNA通过聚合酶链反应 （PCR ) 进行扩增， 用于检测基因表达。 使用实时 PCR技术测定 LPS和该抑血管生成素处 理小鼠 RAW 264. 7巨噬细胞后的致炎性细胞因子 IL-6、 11-1 和了 -01 浓度。
一组样品的成分
有或无该抑血管生成素和 /或 LPS处理后， 各实验组 RAW 264。 7巨 噬细胞内致炎性细胞因子浓度的差异如图 2， 从图中可知， LPS刺激了 致炎性细胞因子 IL-6、 IL-Ιβ和 TNF-a的释放。 加入 ΙμΜ的该抑血管生 成素后这些细胞因子的释放显著减少。 故该抑血管生成素对 LPS导致 的小鼠 RAW 264. 7巨噬细胞炎症具有明显的抵抗作用。
图 1是根据本发明的抑血管生成素的制备工艺流程图。如图 1所示:
首先， 将尖吻蝮蛇粗毒溶于緩冲液一定时间后离心取上清， 得尖吻蝮 蛇毒溶液； 再将所得蛇毒溶液经阴离子交换层析得抑血管生成素的粗 溶液， 接着取适量粗溶液先后经阳离子交换层析和凝胶层析， 制备得 抑血管生成素的初纯品； 然后， 以该初纯品为抗原， 制备针对其的特 异性单克隆抗体； 随后， 再与适宜的亲和层析载体偶联， 制备成抗体 亲和层析柱； 最后， 使用偶联得到的抗体亲和层析柱， 对前面制备的 具有抑制血管生成活性的抑血管生成素粗溶液进行免疫亲和层析， 制 备得到高纯的抑血管生成素。
上述技术方案仅体现了本发明技术方案的优选技术方案，本技术领 域的技术人员对其中某些部分所可能做出的一些变动均体现了本发明 的原理， 属于本发明的保护范围之内。
Claims
1、 抑血管生成素的药物用途， 其特征在于， 抑血管生成素作为活 性成份制备抗炎药物的应用。
2、 如权利要求 1所述的抑血管生成素的药物用途， 其特征在于， 作为抗炎药物活性成份的抑血管生成素采用还原性 SDS-PAGE法测定显 示为两条带，分别称 α链和 β链，分子量分别为 12000 22000道尔顿、 9000 ~ 19000道尔顿， 其中， α链的氨基酸序列如 SEQ ID NO.1所示， P链的氨基酸序列如 SEQ ID NO.2所示。
3、 如权利要求 2所述的抑血管生成素的药物用途， 其特征在于， 作为抗炎药物活性成份的抑血管生成素经双向电泳后会分别产生与所 述 α链和 β链对应的异构体各三个。
4、 如权利要求 1至 3任一项抑血管生成素的药物用途， 其特征在 于， 作为抗炎药物活性成份的抑血管生成素的制备方法包括如下步骤：
(a)将尖吻蝮蛇粗毒溶解于緩沖液中， 其中蛇毒与緩冲液的体积 比为 1~2 : 10， 离心得到上清液， 这里所用的緩冲液是 pH 7.0 ~ 9.0, 0.01 ~ 0. lmol/L的 Tris-HCl緩沖液， 离心次数 2次， 离心速度为 4000r/min, 每次离心 15min;
(b)将所得上清液过二乙胺乙基阴离子交换层析柱进行层析， 得 到抑血管生成素粗溶液， 层析具体为： 层析 pH 6.0~ 9. G的 0.01 ~ 0, 05mol/L的 Tris-HCl緩沖液和 0.05 ~ 0.6mol/L NaCl溶液作为洗脱 液进行线性梯度洗脱， 洗脱时间为 1440min， 洗脱流速 5ml/min。
(c)将杂交瘤细胞株分泌的单克隆抗体与亲和层析载体偶联， 制 备得免疫亲和层析柱， 这里的亲和层析柱栽体为 4B琼脂糖凝胶、 二乙 氨基葡聚糖凝胶、 羧甲基葡聚糖凝胶中的任意一种；
(d) 用所得免疫亲和层析柱纯化步骤（b) 所得抑血管生成素粗 溶液， 以洗脱緩沖液洗脱， 收集得到抑血管生成素， 所用洗脱緩沖液
为 pH2.0-4.0, 0.01 ~ 0.03 mol/L的 Gly-HCl緩沖液 <
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