CN107513100A - 虎纹蛙抗菌肽及其基因和应用 - Google Patents
虎纹蛙抗菌肽及其基因和应用 Download PDFInfo
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- CN107513100A CN107513100A CN201710975830.XA CN201710975830A CN107513100A CN 107513100 A CN107513100 A CN 107513100A CN 201710975830 A CN201710975830 A CN 201710975830A CN 107513100 A CN107513100 A CN 107513100A
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- mercuric chloride
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Abstract
本发明涉及一种虎纹蛙抗菌肽及其基因和在制药中的应用，所述虎纹蛙抗菌肽是由12个氨基酸组成的一种环状多肽，其氨基酸序列如SEQ ID NO.1所示，其第三位半胱氨酸和第十一位半胱氨酸相成分子内二硫键，第十二位酪氨酸带有酰胺化修饰。所述虎纹蛙抗菌肽的基因序列由SEQ ID NO.4组成，其中编码有功能的成熟虎纹蛙抗菌肽是第196‑231位核苷酸。本发明由虎纹蛙抗菌肽的基因推导其成熟功能多肽氨基酸序列，合成的虎纹蛙抗菌肽具有很强的抗菌和抗氧化功能。该虎纹蛙抗菌肽具有结构简单、人工合成方便的特点，可以作为制备病原微生物感染性疾病和自由基氧化导致的治疗药物、美容护肤药物和食品添加剂中应用。
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：
青霉素的发现使得人们能够对付各类病原微生物感染所引发的相关疾病，由此衍生出的各类抗生素更是对保护人类健康做出了巨大贡献。但是由于人们对这些“传统抗生素”的长期使用和滥用，导致许多细菌对它们产生了耐药性。多重耐药的超级细菌的出现使开发新型抗微生物药物变得尤其紧迫。抗菌肽是一类分子量小于1万，由10-50个氨基酸组成的、具有杀灭或抑制微生物生长的活性多肽。最新研究表明：抗菌肽不但具有广谱的抗微生物活性，同时具有“传统抗生素”无法比拟的优越性：如，除了可以直接杀灭微生物和很难诱导菌株产生耐药性外，抗菌肽还可以调节宿主的免疫功能、抑制炎症反应，间接地在感染性疾病治疗中发挥作用(Nat Rev Microbiol,2012,10(4):243-254)。此外，在严重细菌感染时，抗菌肽还能够中和内毒素、减轻脓毒症，在快速杀灭病原微生物的同时迅速停止或限制感染扩散(Antimicrob Agents Chemother,2014,58(9):5363-5371)。因此，抗菌肽有希望成为新一代抗微生物药物。
生物分子的氧化是一个氧自由基介导的过程，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、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变有关(FoodChemistry,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作用，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。
目前，对多肽类抗微生物药物的研制受到了日益广泛的重视(Future Med Chem,2013，5(3)；315-337)。据国内外文献报道，截至目前，至少有20种从不同生物分离得到的活性多肽候选药物进入了临床试验阶段(Future Med Chem,2013,5(3):315-337)。如，Xoma公司研发的用于治疗脑膜炎球菌血症的孤儿药Neuprex和Cutanea life science公司研发的用于治疗红斑痤疮的Omiganan外用乳膏均进入三期临床研究(Curt Protein Pept Sci,2012,13(7):611-619)；另外，Ellceutix公司研发的用于治疗MRSA引起的皮肤感染药物PMX-30063已经进入开发临床前研究(Expert Rev Anti Infect Ther,2014,12(12):1477-1486)。
很多两栖类动物作为中国传统中药和民族药物被广泛应用。如中华蟾蜍(Bufogargarizans)，大蹼铃蟾(Bombina maxima)，黑斑蛙(Pelophylax nigromaculata)和泽蛙(Euphlyctis limnocharis)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：抗微生物、抗肿瘤、镇痛、局部麻醉、免疫调节、对心血管系统作用等(Dongwuxue Yanjiu,2015,36(4):183-222；Chem Rev.2015,115(4):1760-1846)。在国外，两栖类皮肤特定药理活性单体化合物的寻找已是新药发明的热点。如从爪蟾(Xenopuslaevis)皮肤分泌物获得的活性多肽Magainin具广谱抗微生物作用，同时具有抗肿瘤活性，在美国已获准作为广谱抗菌药物，其基因工程产品已经作为抗菌药物被microbiologicalbiotech公司用于治疗糖尿病人足部溃疡(Curr Protein Pept Sci,2012,13(8):723-738.)。
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)主要分布于我国中南部各省、台湾、海南岛和香港，是我国特色资源动物之一。
发明人将本发明的虎纹蛙抗菌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抗菌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，提供种新的具有广谱抗微生物(包括革兰氏阴、阳性细菌、真菌)的虎纹蛙抗菌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，本发明提供了一种虎纹蛙抗菌肽，所述的虎纹蛙抗菌肽是由12个氨基酸组成的环肽，分子量1368.34道尔顿，等电点8.01，其氨基酸序列为：ArgVal Cys Ala Ala Phe Pro Ile Pro Met Cys Tyr(RVCAAFPIPMCY)(SEQ ID NO.1)，上述多肽的其第三位半胱氨酸和第十一位半胱氨酸相成分子内二硫键，第十二位酪氨酸带有酰胺化修饰。
所述虎纹蛙抗菌肽的编码基因由234个核苷酸组成，自5’端至3’端序列为其序列为(SEQ ID NO.4)：
序列中第196-231位核苷酸编码具有功能的成熟虎纹蛙抗菌肽(SEQ ID NO.1)。
本发明另一个方面提供了所述虎纹蛙抗菌肽可在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。优选地，所述病原微生物选自细菌、真菌或病毒；更优选地，所述病原微生物感染疾病选自由大肠杆菌、金黄色葡萄球菌、耐药金黄色葡萄球菌、枯草杆菌、痤疮杆菌、绿脓杆菌、白色念珠菌、黄曲霉中的一种或多种引发的疾病。
本发明另一个方面提供了所述虎纹蛙抗菌肽在制备抗细菌或抗真菌药物、消毒剂、食品添加剂或防腐剂中的用途，优选地，所述细菌选自大肠杆菌、金黄色葡萄球菌、耐药金黄色葡萄球菌、枯草杆菌、痤疮杆菌、绿脓杆菌中的一种或多种；优选地，所述真菌选自白色念珠菌、黄曲霉中的一种或多种。
本发明另一个方面提供了所述的虎纹蛙抗菌肽在制备抗氧化功效的药物和、食品添加剂、消毒剂、美容用品或防腐剂中的应用。
本发明另一个方面提供了所述的虎纹蛙抗菌肽在制备清除自由基药物、食品添加剂、消毒剂、防腐剂或美容用品中的用途。
本发明另一个方面提供了所述的虎纹蛙抗菌肽在制备治疗神经退行性疾病的药物的用途，优选神经退行性疾病为帕金森病、老年痴呆症、糖尿病、类风湿性关节炎和肌萎缩性脊髓侧索硬化症。
本发明另一个方面提供了所述的虎纹蛙抗菌肽作为抗氧化剂或抗自由基剂中的用途。
有益效果
由虎纹蛙抗菌肽编码基因推导其氨基酸结构，合成的虎纹蛙抗菌肽具有显著的抑制细菌、真菌和抗氧化作用。该虎纹蛙抗菌肽具有结构简单、人工合成方便、抗菌谱系广的有益特点。
附图说明
图1为本发明虎纹蛙抗菌肽HPLC纯化鉴定结果；
图2为本发明虎纹蛙抗菌肽质谱鉴定结果；
图3为本发明虎纹蛙抗菌肽清除抗氧化肽清除ABTS(A)和DPPH(B)自由基的“量-效”动力学曲线.
具体实施方式
下面结合附图和具体实施方式对本发明做进一步详细说明：
本发明的虎纹蛙抗菌肽，所述的虎纹蛙抗菌肽由12个氨基酸组成的环肽，分子量136.84道尔顿，等电点8.01，其氨基酸序列为：Arg Val Cys Ala Ala Phe Pro Ile ProMet Cys Tyr(RVCAAFPIPMCY)(SEQ ID NO.1)，上述多肽的其第三位半胱氨酸和第十一位半胱氨酸相成分子内二硫键，第十二位酪氨酸带有酰胺化修饰。
所述虎纹蛙抗菌肽的基因序列SEQ ID NO.4的第196-234位核苷酸编码。本发明的虎纹蛙抗菌肽及其基因的制备过程包括如下步骤：
实施例1，虎纹蛙抗菌肽基因克隆：
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、虎纹蛙抗菌肽基因克隆筛选：扩增引物长度为24个核苷酸，其序列为5’ATGTTCACCTTGAGGAAATCCC 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、虎纹蛙抗菌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，BcaBESTTMSequencing Primer RV-M序列：5`GAGCGGATAACAATTTCACACAGG 3’(SEQ ID NO.5)，BcaBESTTM Sequencing Primer M13-47：5’CGCCAGGGTTTTCCCAGTCACGAC 3’(SEQ ID NO.6)。基因测序结果自5’端至3’端序列为(SEQ ID NO.4):
虎纹蛙抗菌肽基因核苷酸的序列表为：序列长度为234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。
根据虎纹蛙抗菌肽的基因推断编码由功能的成熟活抗菌肽为第196-231位核苷酸，氨基酸序列为：RVCAAFPIPMCY(见序列SEQ ID NO.1)
实施例2，虎纹蛙抗菌肽的制备：
Ⅰ、虎纹蛙抗菌肽的制备方法：根据虎纹蛙抗菌肽的基因推断编码由功能的成熟活抗菌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，多肽洗脱梯度为B液浓度在20min内为25-47％，检测波长为220nm，肽出现在11.90822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、纯化的虎纹蛙抗菌肽用高效液相色谱(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。
虎纹蛙抗菌肽是中国两栖类动物虎纹蛙抗菌肽基因编码的一种环状多肽，分子量136.84道尔顿，等电点8.01，其氨基酸序列为：Arg Val Cys Ala Ala Phe Pro Ile ProMet Cys Tyr(RVCAAFPIPMCY)(SEQ ID NO.1)，上述多肽的其第三位半胱氨酸和第十一位半胱氨酸相成分子内二硫键，第十二位酪氨酸带有酰胺化修饰。
实施例3，虎纹蛙抗菌肽的活性实验
Ⅰ、抑制细菌生长能力测定
抗菌活性检测采用杯碟法，培养基为普通琼脂培养基。分别注入加热融化的培养基20m1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5m1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0.1-0.3mg/ml浓度的待测化合物溶液0.l ml，其余钢杯采用二倍稀释法加入样品液，37℃培养，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)。细菌菌株来源于昆明医学院第一附属医院，此试验重复四次，取平均值，结果如表1所示，合成的虎纹蛙抗菌肽能显著的抑制细菌生长。
表1.虎纹蛙抗菌肽抑制细菌生长活性
Ⅱ、抑制真菌生长能力测定
抗真菌活性检测采用杯碟法，培养基为改良沙保氏(Sabousand)培养基。分别注入加热溶化的培养基20ml于平皿中作为底层，使其在皿底均匀摊布，凝固后另取培养基适量加热溶化，分别向每皿中加入5ml菌悬液，摇匀，使其在底层上均匀摊布，作为菌层。冷却后，在平皿中等距离放入已消毒的不锈钢杯5个。第一个钢杯加入0.3mg/ml浓度的待测化合物溶液0.1ml，其余钢杯采用二倍稀释法加入样品液，37℃培养，24-48h后测量抑菌圈大小。抑菌圈10mm以上作为最小抑菌浓度(minimal inhibitory concentration,MIC)。细菌菌株来源于云南大学微生物研究所，此实验做三个平行，取几何平均值，结果如表2所示，合成的虎纹蛙抗菌肽能显著的抑制真菌生长。
表2.虎纹蛙抗菌肽抑制真菌生长活性
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，表明自由基清楚能力越强。
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3所示，虎纹蛙抗菌肽能显著清除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。虎纹蛙抗菌肽能很好地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，虎纹蛙抗菌肽也可以应用于美容护肤品中。
SEQUENCE LISTING
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Claims (10)
1.一种虎纹蛙抗菌肽，其特征在于，所述虎纹蛙抗菌肽的氨基酸序列如SEQ ID NO.1所示。
2.一种虎纹蛙抗菌肽，其特征在于，所述虎纹蛙抗菌肽是由12个氨基酸组成的多肽，其氨基酸序列如SEQ ID NO.1所示，其第三位半胱氨酸和第十一位半胱氨酸相成分子内二硫键，第十二位酪氨酸带有酰胺化修饰。
3.虎纹蛙抗菌肽基因的核苷酸序列，其特征在于：cDNA由234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或2所述的虎纹蛙抗菌肽的核苷酸。
5.权利要求1或2所述的虎纹蛙抗菌肽在制备病原微生物感染疾病的药物或美容护肤药物的应用；
优选地，所述病原微生物选自细菌、真菌或病毒；
更优选地，所述病原微生物感染疾病选自由大肠杆菌、金黄色葡萄球菌、耐药金黄色葡萄球菌、枯草杆菌、痤疮杆菌、绿脓杆菌、白色念珠菌、黄曲霉中的一种或多种引发的疾病。
6.权利要求1或2所述的虎纹蛙抗菌肽在制备抗细菌或抗真菌药物、消毒剂、食品添加剂或防腐剂中的用途，
优选地，所述细菌选自大肠杆菌、金黄色葡萄球菌、耐药金黄色葡萄球菌、枯草杆菌、痤疮杆菌、绿脓杆菌中的一种或多种；
优选地，所述真菌选自白色念珠菌、黄曲霉中的一种或多种。
7.权利要求1或2所述的虎纹蛙抗菌肽在制备抗氧化的药物、食品添加剂、消毒剂、美容用品或防腐剂中的应用。
8.权利要求1或2所述的虎纹蛙抗菌肽在制备清除自由基药物、食品添加剂、消毒剂、防腐剂或美容用品中的用途。
9.权利要求1或2所述的虎纹蛙抗菌肽在制备治疗神经退行性疾病的药物的用途，优选神经退行性疾病为帕金森病、老年痴呆症、糖尿病、类风湿性关节炎和肌萎缩性脊髓侧索硬化症。
10.权利要求1或2所述的虎纹蛙抗菌肽作为抗氧化剂或抗自由基剂中的用途。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108264539A (zh) * | 2017-12-28 | 2018-07-10 | 河南科技学院 | 一种抗菌肽rl-18及其应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106478811A (zh) * | 2016-10-20 | 2017-03-08 | 南方医科大学 | 虎纹蛙蛋白酶抑制肽及其基因和在制药中的应用 |
-
2017
- 2017-10-19 CN CN201710975830.XA patent/CN107513100A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106478811A (zh) * | 2016-10-20 | 2017-03-08 | 南方医科大学 | 虎纹蛙蛋白酶抑制肽及其基因和在制药中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| 王辉 等: "虎纹蛙皮肤组织cDNA文库的构建及抗菌肽基因tigerinin-HRs的克隆", 《华北农学报》 * |
| 肖明珠 等: "抗菌肽抗氧化作用机制及其应用前景", 《食品科学》 * |
| 金莉莉 等: "蛙科两栖动物皮肤抗菌肽的分子多样性及功能", 《遗传》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108264539A (zh) * | 2017-12-28 | 2018-07-10 | 河南科技学院 | 一种抗菌肽rl-18及其应用 |
| CN108264539B (zh) * | 2017-12-28 | 2020-12-25 | 河南科技学院 | 一种抗菌肽rl-18及其应用 |
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