CN106432459A - 花姬蛙抗菌肽及其基因和在制药中的应用 - Google Patents
花姬蛙抗菌肽及其基因和在制药中的应用 Download PDFInfo
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- CN106432459A CN106432459A CN201610917932.1A CN201610917932A CN106432459A CN 106432459 A CN106432459 A CN 106432459A CN 201610917932 A CN201610917932 A CN 201610917932A CN 106432459 A CN106432459 A CN 106432459A
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Abstract
本发明涉及一种花姬蛙抗菌肽及其基因和在制药中的应用,所述花姬蛙抗菌肽由30个氨基酸组成的一种环状多肽,分子量3440.53道尔顿,等电点10.726,其氨基酸序列如SEQ ID NO.1所示,上述多肽的其第三半胱氨酸和第七位半胱氨酸相成分子内二硫键。所述花姬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花姬蛙抗菌肽是第370‑459位核苷酸。本发明由花姬蛙抗菌肽的基因推导其成熟功能多肽氨基酸序列,合成的花姬蛙抗菌肽具有很强的抗菌和免疫调节功能。该花姬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的治疗药物应用。
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以及在生物制药中的用途。
背景技术:
两栖动物一直以来都是传统药物的源泉。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(Bufo gargarizans),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、促伤口愈合等(Chem Rev.2015,115(4):1760-1846)。因此,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。从爪蟾(Xenopus laevis)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Microbiological biotech公司用于治疗糖尿病人足部溃疡(Curr Protein Pept Sci,2012,13(8):723-738)。
抗菌肽是蛙类皮肤众多活性物质中的一种。与传统的抗生素,例如青霉素、红霉素和链霉素相比,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、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)。此外,部分抗菌肽除有抗菌功能外,还兼有免疫调节、组胺释放和促伤口愈合等功能,如,esculentin-2Cha、frenatin 2D、dersonin-D1、B2RP-Era能调节细胞因子的合成;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(Chem Rev.2015,115(4):1760-1846);因此,抗菌肽有希望成为新一代抗微生物药物。近年来,随着“传统抗生素”的滥用,微生物抗药性已成为临床微生物感染疾病治疗的重大问题,以至于某些病原细菌已没有临床治疗的一线药物。万古霉素抗性的葡萄球菌、肠球菌以及其他革兰氏阴性感染疾病目前均是世界范围内的临床难题,三类主要引起脑膜炎的细菌在临床上也出现了强的抗药性,抗青霉素、氯霉素脑膜炎双球菌、肺炎球菌,对抗新头孢菌素抗生素的肺炎球菌也广泛出现。因此新一类抗生素的研制已成为当务之急和国际上的热点。正是由于此,国际上许多大公司都大规模开展抗菌肽的发现与应用研究。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 MedChem,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 RevAnti Infect Ther,2014,12(12):1477-1486)。
作为新药发现与创新的源泉,新结构天然产物的发现和复杂化合物的高效制备一直是天然产物药物研究的核心。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生物结构与功能研究(Future Med Chem,2013,5(3);315-337);花姬蛙(Microhyla pulchra)是传统中药材和受国家保护的有重要经济和科学研究价值的陆生野生动物,其成体被白酒内浸泡或加酒捣敷后能用于治疗骨折、腰痛、风湿痹痛、体弱无力、跌打损伤、疮痈溃后化脓和久不封口等多种病症。但是由于花姬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。
本发明利用基因组学方法和药理学研究获得花姬蛙抗菌肽编码基因,发明人将本发明的花姬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。发明人将本发明的花姬蛙抗菌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,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)和免疫调节活性的花姬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应用。
为了解决上述技术问题,本发明采用的技术方案是:
一种花姬蛙抗菌肽,其特征在于,所述多肽的序列如SEQ ID No.1所示。
GRCNLLCKAKKKLRAVGNKIKEIKNVVFNR SEQ ID NO.1
一种编码上述的花姬蛙抗菌肽的核苷酸。
一种花姬蛙抗菌肽,所述的花姬蛙抗菌肽由30个氨基酸组成的多肽,分子量3440.53道尔顿,等电点10.726,其氨基酸序列为:Gly Arg Cys Asn Leu Leu Cys Lys AlaLys Lys Lys Leu Arg Ala Val Gly Asn Lys Ile Lys Glu Ile Lys Asn Val Val PheAsn Arg(GRCNLLCKAKKKLRAVGNKIKEIKNVVFNR)(SEQ ID NO.1),上述多肽的其第三半胱氨酸和第七位半胱氨酸相成分子内二硫键。
所述花姬蛙抗菌肽的编码基因由462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370-459位核苷酸编码具有功能的成熟花姬蛙抗菌肽。
所述花姬蛙抗菌肽在制备病原微生物感染性疾病的治疗药物应用。
所述花姬蛙抗菌肽在制备抗细菌的药物中的用途,优选地,所述细菌选自大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌中的一种或多种。
所述花姬蛙抗菌肽在制备抗真菌的药物中的用途,优选地,所述真菌选自白色念珠菌、黄曲霉中的一种或多种。
本发明的有益效果在于:
由花姬蛙抗菌肽编码基因推导其氨基酸结构,合成的花姬蛙抗菌肽具有显著的抑制细菌、真菌、病毒生长和免疫调节作用。该花姬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为本发明花姬蛙抗菌肽HPLC纯化鉴定结果;
图2为本发明花姬蛙抗菌肽质谱鉴定结果;
图3为本发明花姬蛙抗菌肽对肥大细胞组胺释放的影响;
图4为本发明花姬蛙抗菌肽对RAW 264.7细胞分泌INF-α的抑制作用;
图5为本发明花姬蛙抗菌肽对RAW 264.7细胞分泌IL-10的促进作用。
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:
本发明的花姬蛙抗菌肽,所述的花姬蛙抗菌肽由30个氨基酸组成的环肽,分子量3440.53道尔顿,等电点10.726,其氨基酸序列为:Gly Arg Cys Asn Leu Leu Cys Lys AlaLys Lys Lys Leu Arg Ala Val Gly Asn Lys Ile Lys Glu Ile Lys Asn Val Val PheAsn Arg(GRCNLLCKAKKKLRAVGNKIKEIKNVVFNR)(SEQ ID NO.1),上述多肽的其第三半胱氨酸和第七位半胱氨酸相成分子内二硫键。
所述花姬蛙抗菌肽的基因序列SEQ ID NO.4的第370-459位核苷酸编码。本发明的花姬蛙抗菌肽及其基因的制备过程包括如下步骤:
实施例1,花姬蛙抗菌肽基因克隆:
I、花姬蛙皮肤总RNA提取:活体花姬蛙用水清洗干净,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,管底的沉淀物即为花姬蛙皮肤总RNA。
II、花姬蛙皮肤mRNA的纯化:花姬蛙皮肤mRNA分离纯化采用美国PROMEGA公司的mRNA Isolation Systems试剂盒。具体如下:取花姬蛙皮肤总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,则上清中为纯化的花姬蛙皮肤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水中得到花姬蛙皮肤mRNA。
III、花姬蛙皮肤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花姬蛙皮肤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花姬蛙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、花姬蛙抗菌肽基因克隆筛选: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,48℃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、花姬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,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):
花姬蛙抗菌肽基因核苷酸的序列表为:序列长度为359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:花姬蛙皮肤。
根据花姬蛙抗菌肽的基因推断编码由功能的成熟活抗菌肽为第133-261位核苷酸,氨基酸序列为:GRCNLLCKAKKKLRAVGNKIKEIKNVVFNR(见序列SEQ ID NO.1)
实施例2,花姬蛙抗菌肽的制备:
Ⅰ、花姬蛙抗菌肽的制备方法:根据花姬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。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,梯度方法是B液体在15min从25%升到40%,检测波长为220nm,多肽出现在9.257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、纯化的花姬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。
花姬蛙抗菌肽是中国两栖类动物花姬蛙抗菌肽基因编码的一种环状多肽,分子量3440.53道尔顿,等电点10.726,多肽全序列一级结构为:GRCNLLCKAKKKLRAVGNKIKEIKNVVFNR,其第三位半胱氨酸和第七位半胱氨酸相成分子内二硫键。
实施例3,花姬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,合成的花姬蛙抗菌肽能显著的抑制细菌生长。
表1.花姬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(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,合成的花姬蛙抗菌肽能显著的抑制真菌生长。表2.花姬蛙抗菌肽抑制真菌生长活性
Ⅲ、免疫调节作用:
A.对肥大细胞组胺释放的影响
健康Wistar大鼠断颈处死,腹腔内注射10ml台氏液,腹部按摩10min,打开腹腔吸出含有肥大细胞的台氏液,1000rpm离心5min,重复用台氏液洗一次,再用1-2ml的台氏液悬浮肥大细胞。取10μl样品和90μl细胞悬液在37℃孵育10min,37℃孵育10min,然后加1.9ml冷的台氏液,3000rpm离心5min,吸出上清,沉淀用2ml台氏液悬浮,并在开水上煮10min。上清或者沉淀分别加入0.6g NaCl、2.5mL正丁醇和0.2ml 2.5mol/LNaOH,立即混匀,振荡后低速离心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,振荡后低速离心5min,弃有机相;取0.5mL HCl相用双蒸水稀释1倍后,加入0.25mL 0.4mol/L NaOH和0.05mL 1mg/L OPT甲醇溶液,在20℃条件下准确反应10min后,加入0.25mL 0.5mol/L HC1终止反应。空白管为先加入0.25mol/L HC1后,再加入邻苯二甲醛。利用PE公司的自动荧光分光光度计于激发波360nm,荧光波长450nm,狭缝10nm的条件下测定荧光强度读数(T)。组胺释放率按下式计算:
组胺释放率=[(样本的组胺释放量-自发组胺释放量)/组胺总含量]×100%
该试验重复三次。结果如图3所示,花姬蛙抗菌肽能显著促进肥大细胞释放组胺,并且呈现剂量依赖性。
B.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、5、10和25μg/ml的多肽样品分别加入到含0或100ng/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。结果如图4和5所示,花姬蛙抗菌肽能显著调节RAW 264.7细胞分泌细胞因子。
Claims (7)
1.一种花姬蛙抗菌肽,其特征在于,所述多肽的序列如SEQ ID No.1所示。
2.一种花姬蛙抗菌肽,其特征在于,所述花姬蛙抗菌肽是由30个氨基酸组成的一种多肽,分子量3440.53道尔顿,等电点10.726,其氨基酸序列为:Gly Arg Cys Asn Leu LeuCys Lys Ala Lys Lys Lys Leu Arg Ala Val Gly Asn Lys Ile Lys Glu Ile Lys AsnVal Val Phe Asn Arg (GRCNLLCKAKKKLRAVGNKIKEIKNVVFNR)(SEQ ID NO.1),其第三位半胱氨酸和第七位半胱氨酸相成分子内二硫键。
3.一种花姬蛙抗菌肽基因的核苷酸序列,其特征在于:cDNA由462个核苷酸组成,其自5’端至3’端序列为如SEQ ID NO.4所示。
4.一种编码权利要求1所述的花姬蛙抗菌肽的核苷酸。
5.权利要求1所述的花姬蛙抗菌肽在制备病原微生物感染疾病的治疗药物应用。
6.权利要求1所述的花姬蛙抗菌肽在制备抗细菌的药物中的用途,优选地,所述细菌选自大肠杆菌、金黄色葡萄球菌、枯草杆菌、绿脓杆菌中的一种或多种。
7.权利要求1所述的花姬蛙抗菌肽在制备抗真菌的药物中的用途,优选地,所述真菌选自白色念珠菌、黄曲霉中的一种或多种。
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