CN116903724A - 一种兼具镇痛和抗血栓功能的菲牛蛭源多肽及其前体蛋白和应用 - Google Patents
一种兼具镇痛和抗血栓功能的菲牛蛭源多肽及其前体蛋白和应用 Download PDFInfo
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Abstract
本发明提供了一种兼具镇痛和抗血栓功能的菲牛蛭源多肽及其前体蛋白和应用,属于功能肽技术领域。本发明提供的菲牛蛭源多肽,氨基酸序列如SEQ ID NO:1所示。本发明提供的菲牛蛭源多肽能够抑制弹性蛋白酶、FXIIa、kallikrein等蛋白酶的活性,具有良好的抗凝血作用,同时具有良好的镇痛和抗血栓作用;所述多肽具有结构简单、镇痛和抗血栓活性强等特点,能作为镇痛和抗血栓药物应用。
Description
技术领域
本发明属于功能肽技术领域,具体涉及一种兼具镇痛和抗血栓功能的菲牛蛭源多肽及其前体蛋白和应用。
背景技术
弹性蛋白酶属于丝氨酸蛋白酶中的糜蛋白酶家族,近年来的研究表明,弹性蛋白酶参与各种类型疼痛作用的发生。引起伤害性疼痛的机械伤口处含有大量的弹性蛋白酶,这些弹性蛋白酶可能参与了伤害性疼痛的发生;弹性蛋白酶通过激活Protease-activatedreceptor 2(PAR2)和Transient receptor potential vanilloid4(TRPV4)来诱导机体的炎症性疼痛反应;小鼠的中性粒细胞弹性蛋白酶抑制剂基因SerpinA3N缺乏在神经损伤模型中会增强神经性疼痛的敏感性,同时中性粒细胞弹性蛋白酶的敲除或者弹性蛋白酶抑制剂的应用均可以显著抑制小鼠的神经痛反应;弹性蛋白酶通过水解弹性蛋白、激活PAR2等过程参与了功能障碍性疼痛反应的发生;综上,弹性蛋白酶是痛觉发生的关键分子,其专一性抑制剂可以用于缓解各种原因引起的疼痛反应。已知的弹性蛋白酶抑制剂包括西维来司钠,现已批准作为临床用药,尽管具有良好的神经保护作用和减轻炎症反应,然而该药物属于西药,长期服用可能会有毒副作用。
血栓也是威胁人类健康生命的一类疾病,由血栓导致的心脑血管疾病大大影响人类生活品质。恢复血管通畅的方法包括人工机械方法,具体采用球囊导管术和外科栓子切除术,这给患者的身体和心理均带来巨大的负担。抗血栓疗法还包括运用溶栓药物、抗血小板药物和抗凝剂。溶栓药物可去除一个已形成的血栓,具有对机体损伤小的特点,较易被患者接受。常见的抗血栓的药物主要分为两大类:第一类为抗血小板药物,代表性药物有阿司匹林、氯吡格雷、替格瑞洛。抗凝药物为第二类,代表性药物有华法林、达比加群、利伐沙班,这些药物统称为抗血栓的药物。这些药物具有溶栓快的特点,但对肠胃刺激性较大,同时服用2~4周后容易产生耐药性。
发明内容
有鉴于此,本发明的目的在于提供一种菲牛蛭源多肽,兼具镇痛和抗血栓功能。
本发明提供了一种兼具镇痛和抗血栓功能的菲牛蛭源多肽,氨基酸序列如SEQ IDNO:1所示。
本发明提供了一种所述菲牛蛭源多肽的前体蛋白,氨基酸序列如SEQ ID NO:2所示。
本发明提供了一种编码所述前体蛋白的基因,核苷酸序列如SEQ ID NO:3所示。
本发明提供了一种镇痛和/或抗凝血药物,包括所述菲牛蛭源多肽和辅料。
本发明提供了一种所述菲牛蛭源多肽在制备抗凝血和/或镇痛的药物或制剂中的应用。
优选的,所述抗凝血包括以下一种或几种:抗弹性蛋白酶活性、抗凝血因子FXIIa活性和抗激肽释放酶活性。
优选的,所述镇痛包括抑制炎症反应导致的疼痛。
优选的,所述炎症反应由弹性蛋白酶诱导。
优选的,所述药物中菲牛蛭源多肽的浓度不低于10μM。
本发明提供了所述菲牛蛭源多肽在制备抗血栓的药物中的应用。
本发明提供了一种兼具镇痛和抗血栓功能的菲牛蛭源多肽,氨基酸序列如SEQ IDNO:1所示。菲牛蛭源多肽的前体肽含有76个氨基酸,去除信号肽后的成熟肽含有57个氨基酸,分子量为6061.6,等电点为4.07。本发明提供的菲牛蛭源多肽Poeciguamerin能够抑制弹性蛋白酶、FXIIa、kallikrein等蛋白酶的活性,具有良好的抗凝血作用,同时菲牛蛭源多肽能够有效发挥抑制弹性蛋白酶诱导的疼痛的作用,具有良好的镇痛作用,此外,以血栓小鼠模型为对象进行实验,发现菲牛蛭源多肽能够有效预防和治疗血栓的形成。可见,本发明提供的菲牛蛭源多肽具有结构简单、镇痛和抗血栓活性强等特点,能在制备镇痛和抗血栓药物中应用。
附图说明
图1为菲牛蛭中的弹性蛋白酶抑制剂Poeciguamerin的分离纯化结果,其中A为菲牛蛭唾液经Sephadex G-50葡聚糖凝胶过滤层析分离结果,B为Sephadex G-50葡聚糖凝胶过滤层析分离组分测定弹性蛋白酶活性结果,C为反向高压液相色谱分离结果,D为筛选弹性蛋白酶抑制剂Poeciguamerin的弹性蛋白酶活性抑制结果;
图2为菲牛蛭中的弹性蛋白酶抑制剂Poeciguamerin的抗凝血活性测定结果,其中A为弹性蛋白酶;B为凝血因子FXIIa;C为激肽释放酶;D为凝血因子FXa,E为胰蛋白酶和F为凝血酶;
图3为菲牛蛭Poeciguamerin对弹性蛋白酶、凝血因子FXIIa及激肽释放酶的抑制常数测定结果,其中A为弹性蛋白酶,B为凝血因子FXIIa,C为激肽释放酶;
图4为菲牛蛭Poeciguamerin对弹性蛋白酶诱导的疼痛的抑制作用结果;
图5为菲牛蛭Poeciguamerin对FeCl3诱导的颈动脉血栓的抑制结果,A为颈动脉血栓小鼠模型的抗血栓效果的活体成像结果,B为抗血栓活性柱形图。
具体实施方式
本发明提供了一种兼具镇痛和抗血栓功能的菲牛蛭源多肽,氨基酸序列如SEQ IDNO:1(VDEKAEVTDDLCGDKTCSGAQVCQNDACVCSPVRCMI MCPNGFKLDENGCEYPCSCA)所示。
在本发明中,所述菲牛蛭源多肽的来源通过菲牛蛭唾液中分离或者通过人工合成方法获得。所述菲牛蛭唾液中分离的方法,优选包括收集菲牛蛭的唾液,将所述唾液经分子排阻层析分离,筛选具有抑制弹性蛋白酶活性的组分进行反相高效液相色谱再次分离,将抑制弹性蛋白酶活性较高的组分进行质谱鉴定,得到菲牛蛭源多肽。所述分子排阻层析分离的条件优选为将冻干唾液样品溶解于0.1M PB缓冲液中上样于平衡好的Sephadex G-50凝胶过滤柱中;用PB进行洗脱,连接全自动部分收集器进行样品收集,设置流速为0.3mL/min,每10min收集一管;使用分光光度计每隔一管测定280nm和215nm吸光度值。所述PB缓冲液优选为Na2HPO4-NaH2PO4溶液,pH值6.0。上样前优选进行0.22μm滤膜过滤。所述SephadexG-50凝胶过滤柱的规格优选为100×2.6cm,购自GE Health公司。所述反相高效液相色谱再次分离的条件优选为:色谱柱为RP-HPLC C8(Sepax C8,30×0.46cm),每次蛋白上样量约为0.2mg,洗脱系统由A液和B液组成,流速为0.7mL/min,B液每分钟流量增加1%进行梯度洗脱,可见/紫外检测器检测样品280/215nm光吸收值,每一个吸收峰样品收集为一个单位。A液为含0.1%三氟乙酸的双蒸水。B液为含0.1%三氟乙酸的乙腈。
本发明提供了一种所述菲牛蛭源多肽的前体蛋白,氨基酸序列如SEQ ID NO:2(MKIAILLSLFLATLLVVRAVDEKAEVTDDLCGDKTCSGAQVCQ NDACVCSPVRCMIMCPNGFKLDENGCEYPCSCA)所示,N端包含信号肽MKIAILLSLFLATLLVVRA(SEQ ID NO:4)。
本发明提供了一种编码所述前体蛋白的基因,核苷酸序列如SEQ ID NO:3(ATGAAGATTGCAATCCTTTTGAGCCTTTTCCTCGCAACACTTCT TGTTGTCCGGGCAGTCGATGAAAAAGCAGAAGTAACCGATGATCTTTGCGGGGACAAGACATGTTCAGGAGCGCAAGTTTGTCAAAACGACGCATGCGTTTGCAGTCCGGTGAGATGCATGATCATGTGTCCGAATGGATTCAAGTTGGATGAAAATGGATGTGAATATCCTTGTTCCTGTGCTTAG)所示。
本发明提供了一种镇痛和/或抗凝血药物,包括所述菲牛蛭源多肽和辅料。本发明对所述辅料的种类没有特殊限制,采用本领域所熟知的辅料种类即可。本发明对所述药物的制备方法没有特殊限制,采用本领域所熟知的蛋白或多肽类药物的制备方法即可。
本发明提供了一种所述菲牛蛭源多肽在制备抗凝血和/或镇痛的药物或制剂中的应用。
在本发明中,所述抗凝血优选包括以下一种或几种:抗弹性蛋白酶活性、抗凝血因子FXIIa活性和抗激肽释放酶活性。实验证明,菲牛蛭源多肽对弹性蛋白酶、凝血因子FXIIa、激肽释放酶均具有明显的抑制作用,而对凝血因子FXa、胰蛋白酶及凝血酶没有明显的抑制作用。抑制常数测定结果表明,所述菲牛蛭源多肽对弹性蛋白酶、凝血因子FXIIa、激肽释放酶的抑制常数分别为:262.4nM、10.72μM、18.97μM。所述药物中菲牛蛭源多肽的浓度优选不低于10μM。
在本发明中,所述镇痛优选包括抑制炎症反应导致的疼痛。所述炎症反应优选由弹性蛋白酶诱导。实施例开展了以弹性蛋白酶诱导的小鼠后肢足底疼痛,以舔足时间的长短作为疼痛的表征指标,结果表明,处理后弹性蛋白酶诱导的舔足时间有显著降低,说明菲牛蛭源多肽可以明显抑制由弹性蛋白酶诱导的小鼠疼痛反应,为镇痛药物的制备提供了依据。
本发明提供了所述菲牛蛭源多肽在制备抗血栓的药物中的应用。
在本发明实施例中,以氯化铁诱导的颈动脉血栓模型检测了菲牛蛭源多肽的抗血栓作用,结果表明菲牛蛭源多肽在1.25和2.5mg/kg的浓度时显著抑制了氯化铁诱导的颈动脉血栓的形成。由此可知,所述菲牛蛭源多肽具有抗血栓的作用。
下面结合实施例对本发明提供的一种兼具镇痛和抗血栓功能的菲牛蛭源多肽及其前体蛋白和应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
菲牛蛭功能肽Poeciguamerin的分离纯化方法
用多功能电磁按摩器紧贴于菲牛蛭两侧嘴角进行5~10s刺激,刺激电压为6V,功率为2.5~345Hz,刺激后可见无色粘稠液体从口腔中溢出,用唾液刺激液(150mM NaCl,1mML-Arginine)反复冲洗,收集冲洗液,离心(4℃,12000g,1h),低压冷冻干燥,于-80℃低温保存备用。
上述冲洗液冻干成粉状物后,按照下述程序分离纯化弹性蛋白酶抑制剂,每步纯化后,进行弹性蛋白酶发色底物试验以确定目的蛋白所在的纯化峰。
(1)Sephadex G-50葡聚糖凝胶过滤层析
将冻干后的样品溶解于0.1M PB(Na2HPO4-NaH2PO4,pH 6.0)缓冲液中,经0.22μm滤器过滤后取5mL(蛋白量约为200mg)按照使用说明上样于已用相同缓冲液平衡好的Sephadex G-50凝胶过滤柱(100×2.6cm,GE Health)中。用相同PB进行洗脱,连接全自动部分收集器进行样品收集,设置流速为0.3mL/min,每10min收集一管。使用分光光度计每隔一管测定280nm和215nm吸光度值。根据吸收值收集整合各峰组分,采用弹性蛋白酶发色底物法跟踪样品进行活性检测,在96孔板中,取3μL样品与5μL浓度为0.1mg/mL的elastase混合于52μL缓冲液(100mM NaCl、50mM pH8.0的Tris-HCl、5mM CaCl2)中。室温放置5分钟后,加入35μL缓冲液与5μL浓度为0.1mg/mL的elastase底物混合液,终体积为100μL。检测OD405,间隔47s,连续测量20min。活性检测后低压冻干备用。
结果发现,图1中A中的2号峰具有弹性蛋白酶活性(图1中B),而1号峰和3号峰有微弱弹性的蛋白酶活性的作用,但与2号峰组分相比,活力较弱。
(2)反向高压液相色谱
将上述获得的具有抑制弹性蛋白酶活性的蛋白峰冻干样品溶解于双蒸水中,离心(12000g,20min)后取上清,经0.22μm滤器过滤后于液相色谱仪上进行分离纯化。使用色谱柱为RP-HPLC C8(Sepax C8,30×0.46cm),每次蛋白上样量约为0.2mg,洗脱系统由A液(含0.1%三氟乙酸的双蒸水)和B液(含0.1%三氟乙酸的乙腈)组成,流速为0.7mL/min,B液每分钟流量增加1%进行梯度洗脱,可见/紫外检测器检测样品280/215nm光吸收值,每一个吸收峰样品收集为一个单位。冻干重新溶解后按照上述方法测定各个组分对弹性蛋白酶的抑制作用。
结果发现图1中C中的6号峰具有弹性蛋白酶抑制作用,而4、5和7号峰与对照组均未显示出良好的抑制活性。
实施例2
菲牛蛭Poeciguamerin的功能分析
通过发色底物法测定菲牛蛭Poeciguamerin对弹性蛋白酶、凝血因子FXa、激肽释放酶、胰蛋白酶、凝血因子FXIIa及凝血酶的活性进行研究,发色底物法的具体检测过程为:在96孔板中,取3μL样品分别与酶混合于缓冲液(100mM NaCl、50mM Tris-HCl pH 8.0、5mMCaCl2)中,使总体积为60μL。室温放置5分钟后,加入酶底物的缓冲液,使总体积为100μL。然后测定酶反应的动力学。各种酶及底物使用浓度如表1所示:
表1
结果发现,Poeciguamerin对弹性蛋白酶(图2中A)、凝血因子FXIIa(图2中B)、激肽释放酶(图2中C)均具有明显的抑制作用,然而对凝血因子FXa(图2中D)、胰蛋白酶(图2中E)及凝血酶(图2中F)没有明显的抑制作用。
进一步测定了Poeciguamerin对弹性蛋白酶、凝血因子FXIIa、激肽释放酶的抑制常数。抑制常数测定的方法具体为:选用较常用的Dixonplot曲线来计算抑制常数Ki值,即抑制剂浓度为横坐标,纵坐标为反应速率的倒数,延长线交点的横坐标值为抑制剂对酶的抑制常数的负数。
结果如图3,Poeciguamerin对弹性蛋白酶、凝血因子FXIIa、激肽释放酶的抑制常数分别为:262.4nM、10.72μM、18.97μM。
实施例3
菲牛蛭Poeciguamerin的镇痛作用
通过体内实验检测了Poeciguamerin对弹性蛋白酶诱导的疼痛的抑制作用,具体的过程为:对照组小鼠右后肢足底注射弹性蛋白酶20μL,浓度约为6mg/kg,实验组小鼠注射弹性蛋白酶前10min尾静脉静脉注射Poeciguamerin(12mg/kg),然后通过记录30min内小鼠添足的总时间来评价疼痛的水平。
结果如图4,弹性蛋白酶注射组的的舔足时间比对照组显著延长,而经Poeciguamerin处理后弹性蛋白酶诱导的舔足时间有显著降低,由此说明Poeciguamerin可以明显抑制由弹性蛋白酶诱导的小鼠疼痛反应。
实施例4
菲牛蛭Poeciguamerin的抗血栓作用
通过构建氯化铁诱导的颈动脉血栓模型检测了菲牛蛭Poeciguamerin的抗血栓作用。氯化铁诱导的颈动脉血栓模型的构建过程具体如下:
取C57BL/6J小鼠经异氟烷麻醉后解剖分离小鼠的左颈动脉,然后在左颈动脉贴上预浸有10%(w/v)FeCl3溶液的滤纸(2×2mm)诱导血栓的形成,后通过激光多普勒血流仪连续记录颈动脉血流,记录血栓形成的时间。动物模型制备前10min分别给药Poeciguamerin(1.25、2.5mg/kg)或者肝素钠(20mg/kg),生理盐水组作为阴性对照。
结果如图5,Poeciguamerin在1.25和2.5mg/kg的浓度时显著抑制了氯化铁诱导的颈动脉血栓的形成,20mg/kg的肝素钠作为阳性对照。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种兼具镇痛和抗血栓功能的菲牛蛭源多肽,其特征在于,氨基酸序列如SEQ IDNO:1所示。
2.一种权利要求1所述菲牛蛭源多肽的前体蛋白,其特征在于,氨基酸序列如SEQ IDNO:2所示。
3.一种编码权利要求2所述前体蛋白的基因,其特征在于,核苷酸序列如SEQ ID NO:3所示。
4.一种镇痛和/或抗凝血药物,其特征在于,包括权利要求1所述菲牛蛭源多肽和辅料。
5.一种权利要求1所述菲牛蛭源多肽在制备抗凝血和/或镇痛的药物或制剂中的应用。
6.根据权利要求5所述应用,其特征在于,所述抗凝血包括以下一种或几种:抗弹性蛋白酶活性、抗凝血因子FXIIa活性和抗激肽释放酶活性。
7.根据权利要求5所述应用,其特征在于,所述镇痛包括抑制炎症反应导致的疼痛。
8.根据权利要求7所述应用,其特征在于,所述炎症反应由弹性蛋白酶诱导。
9.根据权利要求5~8中任意一项所述应用,其特征在于,所述药物中菲牛蛭源多肽的浓度不低于10μM。
10.权利要求1所述菲牛蛭源多肽在制备抗血栓的药物中的应用。
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