CN106478811A - 虎纹蛙蛋白酶抑制肽及其基因和在制药中的应用 - Google Patents
虎纹蛙蛋白酶抑制肽及其基因和在制药中的应用 Download PDFInfo
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Abstract
本发明涉及一种活性多肽及其基因和在制药中的应用,所述的虎纹蛙蛋白酶抑制肽是由33个氨基酸组成的环肽,分子量3768.33道尔顿,等电点6.303,其氨基酸序列为SEQ ID NO.1,上述多肽的其第三半胱氨酸和第八位半胱氨酸形成分子内二硫键。所述虎纹蛙蛋白酶抑制肽的基因序列由SEQ ID NO.4组成,其中编码有功能的成熟虎纹蛙蛋白酶抑制肽是第379‑477位核苷酸。该虎纹蛙蛋白酶抑制肽具有结构简单、人工合成方便抗菌谱系广和活性强的特点,可以作为制备病原微生物感染性疾病的治疗药物和美容护肤药物应用。
Description
技术领域
本发明涉及生物医学领域,具体涉及一种从动物组织得到的蛋白。
背景技术
丝氨酸蛋白酶抑制剂(serine protease inhibitor,serpin)的最基本功能是防止蛋白质水解,调节丝氨酸蛋白酶的水解平衡。通过对丝氨酸蛋白酶的调节,丝氨酸蛋白酶抑制剂对生物体内的生理生化功能都有重要的影响。如,它们在血液凝固、补体形成、纤溶、蛋白质折叠、细胞迁移、细胞分化、细胞基质重建、激素形成及转运、细胞内蛋白质水解、血压调节、肿瘤抑制以及病毒或寄生虫致病性的形成等方面都有重要作用。丝氨酸蛋白酶抑制剂调节如此众多的生理过程导致它们有广泛的临床应用价值,如,抑酶肽aprotinin除在临床上广泛用于胃炎、胰腺炎等疾病的治疗外,也在胸外科手术中用于抗纤溶、抑制接触性激活、抗炎症等(Br J Anaesth.2013,110(5):675-8)。丝氨酸蛋白酶类似物如Kallikrein、tryptase在风湿性关节炎以及鼻炎、结膜炎、哮喘、胃肠炎、心血管***炎症等炎症发生中起着重要的作用(Biol Chem.2004,385(11):989-96)。因此,丝氨酸蛋白酶抑制剂已成为国际研究的热点,其研究与开发则蕴含着巨大的临床治疗药物的制备价值。
两栖动物一直以来都是传统药物的源泉。中华蟾蜍(Bufo gargarizans)、大蹼铃蟾(Bombina maxima)、黑斑蛙(pelophylax nigromaculata)、沼蛙(Hylarana guentheri)和泽蛙(Euphlyctis limnocharis)等两栖类动物被作为中国的传统中药材而被广泛的应用。现代研究表明:这些两栖动物的皮肤和内脏具有广泛的药理活性,如,广谱抗菌作用、抗肿瘤、局部麻醉、镇痛、免疫调节、对心血管***的作用等(Dongwuxue Yanjiu,2015,36(4):183-222)。传统中药药物成分的复杂性及其炮制方法的局限性造成药物活性成分不能更好发挥作用,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重要内容之一。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。目前有许多分子量小于10kDa的蛋白酶抑制剂多肽已经从两栖动物皮肤中被鉴定。这些两栖动物皮肤的蛋白酶抑制剂除在前体蛋白的加工和降解过程中有作用外,还在抗微生物感染中有功能,因此许多来自两栖动物的蛋白酶抑制剂同时具有抗菌和蛋白酶抑制剂活性,这些多肽包括ranacyclin-B、KPHTI、HV-BBI、HJTI、hylaserpin S2、OGTI、PYR、PSKP-1、PSKP-2、BOTI、BVTI、BMTI、BPTI、pLR和BSTI等(Chem Rev.2015,115(4):1760-1846)。
生物分子的氧化是一个自由基介导的过程,它对食品和生物***会造成许多不利的影响。在好氧器官内,与动脉硬化、癌症等多种疾病相关的游离自由基不可避免的随着氧代谢的过程而产生。另外,氧化应激被认为与多种疾病例如老年痴呆症、帕金森氏症,这此病理系引发于糖尿病、由糖尿病引起的并发症、类风湿性关节炎和肌萎缩性脊髓侧索硬化症引发的神经退行性变有关(Food Chemistry,2008,107,1485–1493)。在食品中,营养成分的氧化会产生过氧化物,其不仅会影响食品营养价值,造成食品品质的下降,严重的甚至还会导致摄入者的身体疾病(Journal of Food Science,1999,64,1000–1004)。因此寻找安全的抗氧化剂以抑制过氧化物产生一直是生化营养学的研究热点。由于BHT、BHA和没食子酸丙酯等化学合成抗氧化剂比天然抗氧化剂具有更好的效果和更便宜的价格,因此被广泛应用于食品行业中,但是,目前有研究发现合成抗氧化剂对人体肝、脾、肺等器官有积蓄性致癌左右,从而引起了人们对其安全性的担忧,并且开始慢慢限制起在食品中的使用,于是让人们把目光转向天然的抗氧化剂(Food Processing,1993,12,54–56)。α-生育酚是一种普遍适用的天然抗氧化剂,它能有效保持食品中油脂的稳定性,但却不利于食品保存。因此有必要寻找其他来源的天然抗氧化剂。由于两栖动物居住环境的特殊性,他们有时会受到自由氧等损害,这些活性自由氧能通过氧化脂肪、变性蛋白、破坏核酸,导致对代谢的严重后果和对组织和细胞的大量破坏。为了抵消这种氧化损害生存下来,两栖动物已经进化形成了抗氧化防御***,抗氧化多肽是其中的一类重要组分。目前,从R.pleuraden、R.catesbeiana、O.livida等物种中鉴定了十几种具有抗氧化活性的多肽(Chem Rev.2015,115(4):1760-1846)。因此,两栖动物皮肤是天然抗氧化多肽的重要来源。
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,对其皮肤活性肽类物质研究不多。虎纹蛙(Hoplobatrachus_ rugulosus)是被人工饲养重要的经济蛙类之一,国家Ⅱ级重点保护动物。目前对其皮肤药理活性物质的研究很少。
进入21世纪以来,基因组学的飞速发展及合成生物学的兴起大大促进了天然产物的结构与功能研究;本发明利用转录组学方法和药理学研究获得虎纹蛙蛋白酶抑制肽的全序列氨基酸结构经蛋白质数据库进行搜寻比较,未发现有任何相同多肽。发明人将本发明的虎纹蛙蛋白酶抑制肽编码基因经基因数据库进行搜寻比较,未发现有任何相同基因。
发明内容
本发明的目的是基于上述技术背景,提供一种具有广谱抗微生物(包括革兰氏阴、阳性细菌、真菌和病毒)、抗氧化和胰酶抑制剂活性的虎纹蛙蛋白酶抑制肽及其基因和它作为制备病原微生物感染性疾病的治疗药物和美容护肤药物的应用。
为了解决上述技术问题,本发明采用的技术方案是:
本发明一个方面提供了一种虎纹蛙蛋白酶抑制肽,所述多肽的序列如SEQ IDNo.1所示。
KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ SEQ ID NO.1
本发明另一个方面提供了一种虎纹蛙蛋白酶抑制肽,其特征在于,所述的虎纹蛙蛋白酶抑制肽是由33个氨基酸组成,分子量3768.33道尔顿,等电点6.303,其氨基酸序列为:Lys Glu Cys Lys Asp Tyr Tyr Cys Arg Leu Leu Met Lys Val Gly Ser Thr SerHis Ile Asp Ser Leu Asp Leu Gly Leu Ser His Glu Ala Gln Gln(KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ)(SEQ ID NO.1),上述多肽的其第三半胱氨酸和第八位半胱氨酸形成分子内二硫键。
本发明再一个方面提供了虎纹蛙蛋白酶抑制肽基因的核苷酸序列,其特征在于:cDNA由480个核苷酸组成,其自5’端至3’端序列如SEQ ID NO.4所示。
序列中第379-477位核苷酸编码具有功能的成熟虎纹蛙蛋白酶抑制肽。
本发明再一个方面提供了一种编码权利要求1所述的虎纹蛙蛋白酶抑制肽的核苷酸。
本发明再一个方面提供了前述的虎纹蛙蛋白酶抑制肽在制备病原微生物感染疾病的治疗药物和美容护肤药物的应用。
本发明再一个方面提供了前述的虎纹蛙蛋白酶抑制肽在制备清除自由基药物或美容用品中的用途。
本发明再一个方面提供了前述的虎纹蛙蛋白酶抑制肽在制备抗氧化药物或美容用品中的用途。
本发明再一个方面提供了前述的虎纹蛙蛋白酶抑制肽在制备预防或治疗与胃炎或胰腺炎的药物中的用途,优选,所述胃炎或胰腺炎为炎症相关的胃炎或炎症相关的胰腺炎。
本发明再一个方面提供了前述的虎纹蛙蛋白酶抑制肽在非诊断或治疗目的的抑制细菌中的用途,优选所述细菌为大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌和白色念球菌。
本发明再一个方面提供了前述的虎纹蛙蛋白酶抑制肽在制备抑制细菌的药物或美容用品中的用途,优选所述细菌为大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌和白色念球菌。
本发明的有益效果在于:
由虎纹蛙蛋白酶抑制肽编码基因推导其氨基酸结构,合成的虎纹蛙蛋白酶抑制肽具有显著的抑制细菌、真菌生长、抑制胰酶活性和抗氧化作用。该虎纹蛙蛋白酶抑制肽具有结构简单、人工合成方便、抗菌谱系广和活性强的有益特点。
附图说明
图1为本发明虎纹蛙蛋白酶抑制肽HPLC纯化鉴定结果;
图2为本发明虎纹蛙蛋白酶抑制肽质谱鉴定结果;
图3为本发明虎纹蛙蛋白酶抑制肽丝氨酸蛋白酶抑制剂活性结果;
图4为本发明虎纹蛙蛋白酶抑制肽清除DPPH和ABTS自由基“量-效”关系曲线。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明:
本发明的虎纹蛙蛋白酶抑制肽,所述的虎纹蛙蛋白酶抑制肽是由33个氨基酸组成多肽,分子量3768.33道尔顿,等电点6.303,其氨基酸序列为:Lys Glu Cys Lys Asp TyrTyr Cys Arg Leu Leu Met Lys Val Gly Ser Thr Ser His Ile Asp Ser Leu Asp LeuGly Leu Ser His Glu Ala Gln Gln(KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ)(SEQ IDNO.1),上述多肽的其第三位半胱氨酸和第八位半胱氨酸形成分子内二硫键从而成环。
所述虎纹蛙蛋白酶抑制肽的基因序列SEQ ID NO.4的第379-477位核苷酸编码。本发明的虎纹蛙蛋白酶抑制肽及其基因的制备过程包括如下步骤:
实施例1,虎纹蛙蛋白酶抑制肽基因克隆:
I、虎纹蛙皮肤总RNA提取:活体虎纹蛙用水清洗干净,放入液氮中速冻4h,取皮肤组织,称重,取300mg皮肤组织,加入10m1总RNA提取缓冲液(Trizol溶液,美国GIBCOBRL公司产品),于20m1玻璃匀浆器中匀浆30min。加入等体积酚/氯仿溶液,剧烈混匀,室温放置10min,4℃,12000rpm离心10min,弃除沉淀。向上清中加入等体积的异丙醇,室温放置10min,4℃,12000rpm离心10min,沉淀用75%乙醇洗一次,晾干,管底的沉淀物即为虎纹蛙皮肤总RNA。
II、虎纹蛙皮肤mRNA的纯化:虎纹蛙皮肤mRNA分离纯化采用美国PROMEGA公司的mRNA Isolation Systems试剂盒。具体如下:取虎纹蛙皮肤总RNA 500μg溶于500μl DEPC水中,放入65℃水浴10min,加人3μl Oligo(dT)探针和13μl 20×SSC溶液,混匀,放置室温冷却,称为A液。将磁珠轻弹混匀,至磁力架吸附30S,弃上清,加0.5×SSC0.3m1,至磁力架吸附30S,最后加0.1ml 0.5×SSC悬浮,称之为B液。将A液加入B液中,室温放置10分钟,至磁力架吸附30sec,弃上清,用0.1×SSC洗涤4次,最后弃上清,加0.L mlDEPC水悬浮,至磁力架上吸附30sec,将上清移至新的试管,再加入0.15m1DEPC水重新悬浮,至磁力架吸附30S,移上清至上述试管,则上清中为纯化的虎纹蛙皮肤mRNA。加入1/10体积3M乙酸钠,pH5.2,等体积异丙醇,于-70℃放置30分钟,4℃,12000rpm离心10min,弃上清,沉淀溶解于10μl DEPC水中得到虎纹蛙皮肤mRNA。
III、虎纹蛙皮肤cDNA文库构建:采用CLONTECH公司CreatorTM SMART TM cDNALibrary Construction Kit质粒cDNA文库构建试剂盒。
A.cDNA第一链合成(mRNA反转录):在0.5ml无菌的离心管加入1μl虎纹蛙皮肤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。混匀离心管中的试剂并以12000rpm离心15sec,72℃保温2min。将离心管在冰上孵育2min。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、1.0μl10mMdNTP混合物、1.0μl PowerScript反转录酶。混合离心管中试剂并以12000rpm离心15sec,在42℃保温1h。将离心管置于冰上中止第一链的合成。从离心管取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:95℃预热PCR仪。将2μlcDNA第一链(mRNA反转录)、80μl去离子水、10μl 10×Advantage 2PCR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。在PCR仪中按以下程序扩增:95℃20sec,95℃5sec,68℃6min,22个循环。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SV Gel and PCR Clean-Up System试剂盒进行抽提回收,步骤如下: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。以12000rpm离心30sec,倒掉收集管中的废液。加入700μl的洗脱液(含乙醇)于离心纯化柱中,以12000rpm离心30sec,倒掉收集管中的废液。重复步骤上述步骤。12000rpm离心5min。将离心纯化柱置于新的离心管中。加入30μl超纯水,在室温下静置5min。以12000rpm离心30sec,管底溶液即为所纯化过的cDNA双链。
D.酶切、连接以及连接产物的转化:在微量离心管中加入1μl Takara pMD18-T载体、4μl虎纹蛙cDNA双链溶液,全量为5μl。加入5μl的连接酶缓冲混合物。16℃反应2h。全量(10μl)加入至100μl DH5α感受态细胞中,冰中放置30min。42℃加热90Sec后,再在冰中放置1分钟。加入37℃孵育过的LB培养基890μl,37℃缓慢振荡培养60min。取200μl涂布于含有X-Gal、IPTG、Amp的LB培养基上37℃培养16h,形成单菌落。每个LB平皿用5m1LB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、虎纹蛙蛋白酶抑制肽基因克隆筛选:扩增引物长度为25个核苷酸,其序列为5’ATGAAGGTCTGGCAGTGCGCGCTC 3’(SEQ ID NO.2),PCR另一扩增引物为CLONTECH公司SMARTTMcDNA Library Construction Kit中的3’PCR Primer引物,其序列为5’ATTCTAGAGGCCGAGGCGGCCGACATG 3’(SEQ ID NO.3)。PCR反应在如下条件下进行:94℃30sec,50℃45sec和72℃2.5min,35个循环。
首先滴定构建的细菌cDNA文库,然后用含100μg/ml氨苄青霉素的LB培养基稀释至适当的细菌浓度(大约5000个细菌/毫升和30个细菌/毫升分别用于首轮筛选第二轮筛选),在96孔培养板上按8×8矩阵铺板(共64孔,每孔100μ1),37℃过夜培养。按行、列分别合并细菌培养液,有16个样品进行PCR鉴定,交叉阳性孔细菌样品进入第二轮筛选。
Ⅴ、虎纹蛙蛋白酶抑制肽基因序列测定和结果:提取质粒DNA用双脱氧法测定核苷酸序列,使用仪器为美国Applied Biosystems 373A全自动核苷酸序列测定仪,测序引物为BcaBESTTM Sequencing Primer RV-M和BcaBESTTM Sequencing Primer M13-47,BcaBESTTM Sequencing Primer RV-M序列:5`GAGCGGATAACAATTTCACACAGG 3’(SEQ IDNO.5),BcaBESTTM Sequencing Primer M13-47:5’CGCCAGGGTTTTCCCAGTCACGAC3’(SEQ IDNO.6)。基因测序结果自5’端至3’端序列为(SEQ ID NO.4):
虎纹蛙蛋白酶抑制肽基因核苷酸的序列表为:序列长度为480个碱基;序列类型:核酸;链数:单链;拓扑学:直链状;序列种类:cDNA;来源:虎纹蛙皮肤。
根据虎纹蛙蛋白酶抑制肽的基因推断编码由功能的成熟活多功能蛋白酶抑制肽为第379-477位核苷酸,氨基酸序列为:KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ(见序列SEQID NO.1)
实施例2,虎纹蛙蛋白酶抑制肽的制备:
Ⅰ、虎纹蛙蛋白酶抑制肽的制备方法:根据虎纹蛙蛋白酶抑制肽的基因推断编码有功能的成熟多功能蛋白酶抑制肽氨基酸序列后用自动多肽合成仪合成多肽。通过HPLC反相C18柱层析脱盐、纯化。二硫键的形成采用空气氧化法,具体为在烧瓶中将多肽溶解按照0.1mg/ml于0.1%醋酸溶液中后用氢氧化铵滴定成pH 7.8,然后室温搅拌过夜。通过HPLC反相C18柱层析脱盐、纯化。纯化时A液体为0.05%TFA+2%CH3CN,B液为0.05%TFA+90%CH3CN,B液浓度梯度为15min内25-40%,检测波长为220nm,多肽出现在9.248分钟。
Ⅱ、分子量测定采用快原子轰击质谱法(Fast atom bombardment massspectrometry,FAB-MS),以甘油:间硝基苄醇:二甲亚砜(1:1:l,V:V:V,体积比)为底物,Cs+作为轰击粒子,电流为1μA,发射电压为25Kv。
Ⅲ、纯化的虎纹蛙蛋白酶抑制肽用高效液相色谱(HPLC)方法鉴定其纯度,等电聚焦电泳测定等电点,用自动氨基酸测序仪测定氨基酸序列结构。
虎纹蛙蛋白酶抑制肽是中国两栖类动物虎纹蛙蛋白酶抑制肽基因编码的一种多肽,分子量3768.33道尔顿,等电点6.303,其氨基酸序列为:Lys Glu Cys Lys Asp Tyr TyrCys Arg Leu Leu Met Lys Val Gly Ser Thr Ser His Ile Asp Ser Leu Asp Leu GlyLeu Ser His Glu Ala Gln Gln(KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ)(SEQ ID NO.1),上述多肽的其第三半胱氨酸和第八位半胱氨酸形成分子内二硫键。
实施例3,虎纹蛙蛋白酶抑制肽的活性实验
Ⅰ、抗菌能力测定
抗菌活性检测采用杯碟法,细菌培养采用普通琼脂培养基,真菌培养采用培养基为改良沙保氏(Sabousand)培养基。分别注入加热融化的培养基20ml于平皿中作为底层,使其在皿底内均匀摊布,凝固后,另取培养基适量加热融化后,分别在每皿中加入5ml菌悬液,摇匀,使其在底层上均匀摊布,作为菌层。冷却后,在平皿中等距离均匀放入已消毒的不锈钢杯6个。第一个钢杯加入0.3mg/ml浓度的待测样品溶液0.l ml,其余钢杯采用二倍稀释法加入样品液,37℃培养,24h-48h后观察抑菌圈大小。抑菌圈l0mm以上的作为最小抑菌浓度(Minimal inhibitory concentration,MIC)。所有测试菌株均来源于广东微生物研究所,试验重复四次,取平均值,结果如表1所示,虎纹蛙蛋白酶抑制肽对于大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌和白色念球菌都具有显著的抑制作用,说明虎纹蛙蛋白酶抑制具有良好的广谱抑制细菌生长的作用。
表1.虎纹蛙蛋白酶抑制肽的抗菌活性
Ⅱ、丝氨酸蛋白酶抑制剂活性测定
不同量的虎纹蛙蛋白酶抑制肽溶解于0.05M Tris-HCl缓冲液与一定量的胰蛋白酶(最终浓度为40μg/ml)于0.05M Tris-HCl缓冲液于室温下保温2min,最后加入发色底物S-2238(终浓度为40μg/ml)启动反应,应用PERKIN ELMER(美国)公司生产的分光光度计于处监测吸收值变化2min,加入同样体积的0.05M Tris-HCl缓冲液与空白对照,抑制常数Ki=[I]/(V0/VI+1)计算。[I]为虎纹蛙蛋白酶抑制肽的摩尔浓度,V0为空白对照是胰蛋白酶与发色底物的反应速度,V1为加入虎纹蛙蛋白酶抑制肽后胰蛋白酶与发色底物的反应速度。此试验重复六次,取平均值。
结果如图3所示,虎纹蛙蛋白酶抑制肽能很有效地抑制胰蛋白酶的活性,在上述试验条件下抑制半数胰蛋白酶活性所需要的虎纹蛙蛋白酶抑制肽浓度为0.368μM,对胰蛋白酶的抑制常数Ki是3.2×10-7M。
丝氨酸蛋白酶抑制剂对生物体内的生理生化功能都有重要的影响。如,它们在血液凝固、补体形成、纤溶、蛋白质折叠、细胞迁移、细胞分化、细胞基质重建、激素形成及转运、细胞内蛋白质水解、血压调节、肿瘤抑制以及病毒或寄生虫致病性的形成等方面都有重要作用。虎纹蛙蛋白酶抑制肽能很有效地抑制胰蛋白酶,证实其能在体内调节众多的生理过程和有广泛的临床应用价值。胃炎、胰腺炎的发病过程涉及许多丝氨酸蛋白水解作用,因此抑制这些酶的水解能防止疾病的发生和减低随后的炎症因子对机体造成的损害。具有丝氨酸蛋白酶抑制剂活性的虎纹蛙蛋白酶抑制肽能应用与炎症相关的胃炎、胰腺炎中。
Ⅲ、抗氧化能力测定
1)DPPH自由基清除能力的测定
利用DPPH(1,1-diphenyl-2-picryl-hydrazyl)自由基清除率测定法研究抗氧化多肽。配制浓度为1×10-5mol/L的DPPH乙醇溶液,避光保存。将2ml,0.1mM的DPPH无水乙醇溶液加入到含有2ml不同酶解样品的洁净试管中,混匀。室温下放置30min后,于517nm处测定吸光度,吸光值越小,表明自由基清除能力越强。
清除率(%)=【1-(Ai-Aj)/A0】*100%
式中,A0为2ml,0.1mM的DPPH无水乙醇溶液+2ml的样品试剂,空白对照,Ai为2ml,0.1mM的DPPH无水乙醇溶液+2ml的样品,Aj为2ml的无水乙醇+2ml的样品。
2)ABTS自由基清除活性的测定
以去离子水将ABTS溶解,使ABTS浓度达到7mmol/L,加入过硫酸钾,使过硫酸钾的浓度为2.45nmol/L。之后将该溶液在室温下置于暗处过夜12~16h。将生成的ABTS自由基溶液用磷酸缓冲液(PBS,0.2mol/L,pH 7.4)稀释,使其在734nm下的吸光值为0.70。取0.1ml酶解液与2.9ml ABTS自由基液混合,摇匀30秒钟,暗处反应10分钟,然后在734nm下测定反应液的吸光值。以蒸馏水代替水解液作空白。
清除率(%)=(Ai-Aj)/A0*100%
式中,A0为2.9ml ABTS试剂与0.1ml蒸馏水混合液的吸光值,Aj为2.9ml ABTS+0.1ml的酶解液混合液的吸光值。
如图4所示,虎纹蛙蛋白酶抑制肽能显著清除DPPH和ABTS自由基。自由基氧化在老年痴呆症、帕金森氏症、糖尿病、类风湿性关节炎和肌萎缩性脊髓侧索硬化症引发的神经退行性疾病中有重要作用。虎纹蛙蛋白酶抑制肽能很好的清除自由基能说明其能应用于自由基氧化导致的相关疾病的治疗。另外,为了防止自由基对皮肤造成的损害,在美容护肤品中添加自由基清除剂必不可少。因此,虎纹蛙蛋白酶抑制肽也可以应用于美容护肤品中。
Claims (10)
1.一种虎纹蛙蛋白酶抑制肽,所述多肽的序列如SEQ ID No.1所示。
2.一种虎纹蛙蛋白酶抑制肽,其特征在于,所述的虎纹蛙蛋白酶抑制肽是由33个氨基酸组成,分子量3768.33道尔顿,等电点6.303,其氨基酸序列为:Lys Glu Cys Lys Asp TyrTyr Cys Arg Leu Leu Met Lys Val Gly Ser Thr Ser His Ile Asp Ser Leu Asp LeuGly Leu Ser His Glu Ala Gln Gln(KECKDYYCRLLMKVGSTSHIDSLDLGLSHEAQQ)(SEQ IDNO.1),上述多肽的其第三半胱氨酸和第八位半胱氨酸形成分子内二硫键。
3.虎纹蛙蛋白酶抑制肽基因的核苷酸序列,其特征在于:cDNA由480个核苷酸组成,其自5’端至3’端序列如SEQ ID NO.4所示。
4.一种编码权利要求1所述的虎纹蛙蛋白酶抑制肽的核苷酸。
5.权利要求1所述的虎纹蛙蛋白酶抑制肽在制备病原微生物感染疾病的治疗药物和美容护肤药物的应用。
6.权利要求1所述的虎纹蛙蛋白酶抑制肽在制备清除自由基药物或美容用品中的用途。
7.权利要求1所述的虎纹蛙蛋白酶抑制肽在制备抗氧化药物或美容用品中的用途。
8.权利要求1所述的虎纹蛙蛋白酶抑制肽在制备预防或治疗与胃炎或胰腺炎的药物中的用途,优选,所述胃炎或胰腺炎为炎症相关的胃炎或炎症相关的胰腺炎。
9.权利要求1所述的虎纹蛙蛋白酶抑制肽在非诊断或治疗目的的抑制细菌中的用途,优选所述细菌为大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌和白色念球菌。
10.权利要求1所述的虎纹蛙蛋白酶抑制肽在制备抑制细菌的药物或美容用品中的用途,优选所述细菌为大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌和白色念球菌。
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