CN107501395B - 泽陆蛙抗氧化肽及其基因和应用 - Google Patents
泽陆蛙抗氧化肽及其基因和应用 Download PDFInfo
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- CN107501395B CN107501395B CN201710975835.2A CN201710975835A CN107501395B CN 107501395 B CN107501395 B CN 107501395B CN 201710975835 A CN201710975835 A CN 201710975835A CN 107501395 B CN107501395 B CN 107501395B
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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,所述的泽陆蛙抗氧化肽是由9个氨基酸组成的环肽,分子量1122.62道尔顿,等电点8.01,其氨基酸序列为:Arg Cys Phe Tyr Phe Gly Gln Cys Thr(RCFYFGQCT)(SEQ ID NO.1),上述多肽的第二位半胱氨酸和第八位半胱氨酸形成分子内二硫键,第九位苏氨酸带有酰胺化修饰。所述泽陆蛙抗氧化肽的基因序列由SEQ ID NO.4组成,其中编码有功能的成熟泽陆蛙抗氧化肽是第196‑222位核苷酸。该泽陆蛙抗氧化肽具有结构简单、人工合成方便的特点,可以作为制备抗氧化功效的药物和食品添加剂中的应用。
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以及在生物制药中的用途。
背景技术
生物分子的氧化是一个自由基介导的过程,它对食品和生物系统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系统,抗氧化多肽是其中的一类重要组分。目前,从R.pleuraden、R.catesbeiana、O.livida等物种中鉴定了是十几中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。此外,来自两栖动物的皮肤抗氧化肽通常还兼有免疫调节、组胺释放和免疫调节等功能,如,Pipinin、XO-4、pLR、pYR和HR-II等有显著的促肥大细胞脱粒和组胺释放功能,pleurain-D4、pleurain-E1、pleurain-G1、pleurain-J1、pleurain-K1、pleurain-M1、pleurain-N1、pleurain-N1、pleurain-P1和antioxidin-RP1能调节细胞因子的合成(Chem Rev.2015,115(4):1760-1846);因此两栖动物皮肤也是免疫调节肽的重要来源。中国博大的多物种生物资源是结构多样性小分子有机化合物的重要来源,其中一些生物活性物质早已被中医记载和采用。很多两栖类动物作为中国传统中药和民族药物而广泛应用。泽陆蛙(Fejervarya limoncharis)是传统中药材,其全体有清热解毒,健脾消积功效。可用于治疗痈肿、疔疖、乳痈、小儿疳积、热痢等多种病症。但是由于泽陆蛙个体小和捕捉困难,目前对其皮肤药理活性物质的结构与功能研究在世界范围内还未见报道,其作为中药疗效的物质基础仍不为人们所知。
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;本发明利用基因组学方法和药理学研究获得泽陆蛙抗氧化肽编码基因,发明人将本发明的泽陆蛙抗氧化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。发明人将本发明的泽陆蛙抗氧化肽全序列结构经蛋白数据库进行搜索比较未发现有任何相同的多肽。
发明内容
本发明的目的是基于上述技术背景,针对天然抗氧化剂的不足和人工合成抗氧化剂的安全性问题,提供一种新的具有抗氧化和免疫调节活性的泽陆蛙抗氧化肽及其基因和它在制备抗氧化功效的药物和食品添加剂中的应用。
为了解决上述技术问题,本发明提供了一种泽陆蛙抗氧化肽,所述泽陆蛙抗氧化肽的序列如SEQ ID No.1所示。
RCFYFGQCT SEQ ID NO.1
一种泽陆蛙抗氧化肽,所述的泽陆蛙抗氧化肽是由9个氨基酸组成的多肽,分子量1122.62道尔顿,等电点8.01,其氨基酸序列为:Arg Cys Phe Tyr Phe Gly Gln Cys Thr(RCFYFGQCT)(SEQ ID NO.1),上述多肽的第二位半胱氨酸和第八位半胱氨酸形成分子内二硫键,第九位苏氨酸带有酰胺化修饰。
所述泽陆蛙抗氧化肽的编码基因由225个核苷酸组成,自5’端至3’端序列为其序列为(SEQ ID NO.4):
SEQ ID NO.4
序列中第196-222位核苷酸编码具有功能的成熟泽陆蛙抗氧化肽。
一种编码上述的泽陆蛙抗氧化肽的核苷酸。
所述泽陆蛙抗氧化肽可在制备抗氧化功效的药物和食品添加剂中的应用。
所述泽陆蛙抗氧化肽在制备清除自由基药物或美容用品中的用途。
所述泽陆蛙在制备治疗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的药物的用途,优选神经退行性疾病为治疗帕金森病和类风湿性关节炎。
本发明的有益效果在于:
由泽陆蛙抗氧化肽编码基因推导其氨基酸结构,合成的泽陆蛙抗氧化肽具有显著的免疫调节和抗氧化作用。该泽陆蛙抗氧化肽具有结构简单、人工合成方便、毒性低的有益特点。
附图说明
图1为本发明泽陆蛙抗氧化肽HPLC纯化鉴定结果;
图2为本发明泽陆蛙抗氧化肽质谱鉴定结果;
图3为本发明泽陆蛙抗氧化肽清除ABTS(A)和DPPH(B)自由基的“量-效”动力学曲线;
图4为本发明泽陆蛙抗氧化肽对RBL-2H3细胞的毒性测试结果(A)和对RBL-2H3细胞脱颗粒(B)和组胺释放(C)的影响;
图5为本发明泽陆蛙抗氧化肽对RAW 264.7细胞分泌对分泌IL-10的促进作用(A)和TNF-α的抑制作用(B)的结果。
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:
本发明的泽陆蛙抗氧化肽,所述的泽陆蛙抗氧化肽是由9个氨基酸组成的环肽,分子量1122.62道尔顿,等电点8.01,其氨基酸序列为Arg Cys Phe Tyr Phe Gly Gln CysThr(RCFYFGQCT)(SEQ ID NO.1),上述多肽的第二位半胱氨酸和第八位半胱氨酸形成分子内二硫键,第九位苏氨酸带有酰胺化修饰。
所述泽陆蛙抗氧化肽的基因序列SEQ ID NO.4的第196-222位核苷酸编码。本发明的泽陆蛙抗氧化肽及其基因的制备过程包括如下步骤:
实施例1,泽陆蛙抗氧化肽基因克隆:
I、泽陆蛙皮肤总RNA提取:活体泽陆蛙用水清洗干净,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,管底的沉淀物即为泽陆蛙皮肤总RNA。
II、泽陆蛙皮肤mRNA的纯化:泽陆蛙皮肤mRNA分离纯化采用美国PROMEGA公司的mRNA Isolation Systems试剂盒。具体如下:取泽陆蛙皮肤总RNA500μg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,则上清中为纯化的泽陆蛙皮肤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水中得到泽陆蛙皮肤mRNA。
III、泽陆蛙皮肤cDNA文库构建:采用CLONTECH公司CreatorTM SMARTTM 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泽陆蛙皮肤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、1.0μl10mM dNTP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泽陆蛙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、泽陆蛙抗氧化肽基因克隆筛选:扩增引物长度为25个核苷酸,其序列为5’ATGTTCACCTTGAAGAAATCCCT 3’(SEQ ID NO.2),PCR另一扩增引物为CLONTECH公司SMARTTMcDNALibrary 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、泽陆蛙抗氧化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’CGCCAGGGTTTTCCCAGTCACGAC 3’(SEQ IDNO.6)。基因测序结果自5’端至3’端序列为(SEQ ID NO.4):
泽陆蛙抗氧化肽基因核苷酸的序列表为:序列长度为225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:泽陆蛙皮肤。
根据泽陆蛙抗氧化肽的基因推断编码由功能的成熟活分泌肽为第196-222位核苷酸,氨基酸序列为:RCFYFGQCT(见序列SEQ ID NO.1)
实施例2,泽陆蛙抗氧化肽的制备:
I、泽陆蛙抗氧化肽的制备方法:根据泽陆蛙抗氧化肽的基因推断编码由功能的成熟活分泌肽氨基酸序列后用自动多肽合成仪合成多肽。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液梯度为22min内15-37%,检测波长为220nm,多肽出现在15.662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、纯化的泽陆蛙抗氧化肽用高效液相色谱(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。
泽陆蛙抗氧化肽是中国两栖类动物泽陆蛙抗氧化肽基因编码的含9个氨基酸的环肽,分子量1122.62道尔顿,等电点8.01,其氨基酸序列为:Arg Cys Phe Tyr Phe Gly GlnCys Thr(RCFYFGQCT)(SEQ ID NO.1),上述多肽的第二位半胱氨酸和第八位半胱氨酸形成分子内二硫键,第九位苏氨酸带有酰胺化修饰。
实施例3,泽陆蛙抗氧化肽的活性实验
I、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。
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。泽陆蛙抗氧化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,泽陆蛙抗氧化肽也可以应用于美容护肤品中。
Ⅱ、免疫调节作用:
A、对RBL-2H3细胞毒性和脱粒肽活性、组胺释放的影响
RBL-2H3细胞培养于37℃,5%CO2培养箱中,RBL-2H3细胞汇合度约80-90%时消化细胞,离心后收集,弃上清,加入2ml培养基使其混匀,调整细胞悬液密度为1x105个/mL,并以90μL/well接种于96孔平底板,每孔加入10μL不同浓度的样品,阳性对照和阴性对照分别为同体积的0.1%Triton X-100和PBS。CO2恒温培养箱中培养细胞1h后,MTT发测定细胞活力。
B、肥大细胞脱粒肽活性检测(MCDT)
RBL-2H3细胞汇合度约80-90%时消化细胞,调整细胞悬液密度为2x104个/well接种于96孔平底板,CO2恒温培养箱中培养24小时后,加入一定浓度的样品与贴壁细胞孵育1小时,随后4℃,1000rpm离心10min。吸取50μL上清,加入50μL底物继续孵育6h后加入100μLpH 10.0的Na2CO3终止反应,于405nm下测光吸收。其中阴性对照为PBS,样品阳性对照为0.1%(v/v)的Triton X-100。该实验重复三次。
β-氨基己糖苷酶释放率=(上清样品测定OD值-调零OD值)/(总酶组孔OD值-调零OD值)×100%
C、组胺释放实验
RBL-2H3细胞汇合度约80-90%时消化细胞,调整细胞悬液密度为1x105个/mL,并以90μL到1.5mL EP管,加入一定浓度的样品与悬浮细胞37℃恒温培养箱中培养1小时后,加1mL提前预冷的生理盐水溶液并置于冰上操作,3000rpm离心5min吸出上清。沉淀用2μL台氏液悬浮,95℃变性10min。上清中加入0.5g NaCl,2.5mL正丁醇和0.2mL2.5mol/L NaOH;沉淀加入0.3g NaCl,2.5mL正丁醇和0.2mL 2.5mol/L NaOH,立即混匀,振荡后离心1500rpm,5min;取2mL有机相加到已加0.6mL 0.1mol/L HCl和2.5mL正庚烷的试管中,振荡后低速离心5min,弃有机相;取0.5mL HCl相用ddH2O稀释1倍后,加入0.25mL 0.4mol/L NaOH和0.05mL 1mg/ml OPT甲醇溶液,在20℃条件下准确反应10min后,加入0.25mL 0.5mol/LHC1终止反应。其中阴性对照为生理盐水,样品阳性对照为40μg/mL的C48/80。利用多功能微孔检测仪于激发波360nm,荧光波长450nm的条件下测定荧光强度读数(T)。该实验重复三次。
组胺释放率=[(样本的上清S/沉淀P荧光强度Tx-对照组上清S/沉淀P荧光强度T0)/样本的上清S+沉淀P荧光强度Tx-(对照组上清S+沉淀P荧光强度T0)]×100%。
D、对细胞因子分泌的影响
1ml浓度为1×106/ml RAW 264.7细胞用含100U/ml青霉素、0.1mg/ml链霉素、10%胎牛血清的DMEM(Gibco公司产品)37℃5%CO2在24孔细胞培养板中培养。终浓度是0、20、40、80和160nM的多肽样品分别加入到含0或1μg/ml LPS(from E.coli 055:B5,Sigma-Aldrich,USA)刺激的细胞中孵育24h。细胞上清被收集,1000rpm离心10min,上清液中TNF-α和IL-10的含量用上海酶联公司生产的ELISA检测试剂盒按照说明书检测。该试验重复三次。
肥大细胞脱粒肽和组胺能调节哺乳动物中免疫应答模型,特别是迟发性超敏反应和T与B细胞功能的模型。结果如图4所示,泽陆蛙抗氧化肽对肥大细胞没有毒性,却能够显著调节肥大细胞的细胞脱粒肽和组胺释放活性,因此,该肽能够调节肥大细胞的功能,抑制多肽作为药物使用的时候的迟发性超敏反应,以及T和B细胞的功能。从结果图5所示,泽陆蛙抗氧化肽能够显著促进IL-10的释放和抑制TNF-α的释放,这两个细胞因子与炎症相关,而肥大细胞也是炎性细胞,因此,泽陆蛙抗氧化肽具有很好的抗炎性作用,抑制炎症对机体造成的损害。神经退行性疾病如,帕金森病、老年痴呆症、糖尿病、类风湿性关节炎的疾病进展中炎症有重要作用,因此泽陆蛙抗氧化肽可以用于这些疾病的治疗。
SEQUENCE LISTING
<110> 南方医科大学
<120> 泽陆蛙抗氧化肽及其基因和应用
<160> 6
<170> PatentIn version 3.3
<210> 1
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<212> PRT
<213> 泽陆蛙抗氧化肽
<400> 1
Arg Cys Phe Tyr Phe Gly Gln Cys Thr
1 5
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<400> 2
atgttcacct tgaagaaatc cct 23
<210> 3
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<212> DNA
<213> 人工序列
<400> 3
attctagagg ccgaggcggc cgacatg 27
<210> 4
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<212> DNA
<213> 人工序列
<400> 4
atgttcacct tgaagaaatc cctcgtattg gtggtctttc ttgggatggt ctccttttct 60
ctctgccagg aagagagcca tgatggaaat gaaacaacca cagttgttgc agagggagaa 120
aatacggcca aaaatgtgga aaaagaagag gaagtgaaac tggaaaagga agtttctgat 180
tacgctaacc tcgagagatg tttttatttt gggcagtgca cgtga 225
<210> 5
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<212> DNA
<213> 人工序列
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gagcggataa caatttcaca cagg 24
<210> 6
<211> 24
<212> DNA
<213> 人工序列
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cgccagggtt ttcccagtca cgac 24
Claims (6)
1.一种泽陆蛙抗氧化肽,所述泽陆蛙抗氧化肽的氨基酸序列如SEQ ID No.1所示。
2.一种泽陆蛙抗氧化肽,其特征在于,所述的泽陆蛙抗氧化肽是由9个氨基酸组成的多肽,分子量1122.62道尔顿,等电点8.01,其氨基酸序列为:Arg Cys Phe Tyr Phe Gly GlnCys Thr(RCFYFGQCT)(SEQ ID NO. 1),上述多肽的第二位半胱氨酸和第八位半胱氨酸形成分子内二硫键,第九位苏氨酸带有酰胺化修饰。
3.泽陆蛙抗氧化肽基因,其特征在于,其核苷酸序列如SEQ ID NO. 4所示。
4.一种编码权利要求1或2所述的泽陆蛙抗氧化肽的核酸。
5.权利要求1或2所述的泽陆蛙抗氧化肽在制备抗氧化的药物、食品添加剂、美容用品或防腐剂中的应用。
6.权利要求1或2所述的泽陆蛙抗氧化肽在制备清除自由基的药物、食品添加剂、防腐剂或美容用品中的用途。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998025961A1 (en) * | 1996-12-13 | 1998-06-18 | Sbl Vaccin Ab | Antimicrobially active polypeptides |
CN106399319A (zh) * | 2016-10-18 | 2017-02-15 | 南方医科大学 | 一种泽陆蛙基因及其编码的多肽和该多肽的用途 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998025961A1 (en) * | 1996-12-13 | 1998-06-18 | Sbl Vaccin Ab | Antimicrobially active polypeptides |
CN106399319A (zh) * | 2016-10-18 | 2017-02-15 | 南方医科大学 | 一种泽陆蛙基因及其编码的多肽和该多肽的用途 |
Non-Patent Citations (2)
Title |
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Purification, characterization and cloning of two novel tigerinin-like peptides from skin secretions of Fejervarya cancrivora;Yuzhu Song, et al.;《Peptides》;20090409;第30卷;第1228–1232页 * |
抗菌肽抗氧化作用机制及其应用前景;肖明珠等;《食品科学》;20101231;第31卷(第11期);摘要 * |
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