CN106399320B - 一种花狭口蛙的基因及其编码的多肽和该多肽的用途 - Google Patents
一种花狭口蛙的基因及其编码的多肽和该多肽的用途 Download PDFInfo
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
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Abstract
本发明涉及一种源于成熟花狭口蛙皮肤的基因，该基因的核酸序列如SEQ ID NO.5所示。所述的基因可编码氨基酸序列如SEQ ID NO.1所示的多肽，该多肽及其突变体具有抗菌、抗肿瘤以及心血管系统疾病和促肠道收缩的活性，可用于制备相关的药物。
Description
技术领域：
本发明涉及来自动物的抵抗物质，具有涉及源于花狭口蛙的基因及其编码的多于20个氨基酸的肽。
背景技术：
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。它们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，很难诱导菌株产生耐药性，同时兼有免疫调节、促进回肠肌收缩、抗氧化、促胰岛素分泌、抗肿瘤和抗病毒等多种功能(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。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、对心血管系统的作用等(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。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)。
肿瘤是危害人类健康的一类主要疾病，统计资料表明，我国从上世纪80年代起，恶性肿瘤的发病率呈明显上升趋势，常见肿瘤的发病率的增长在5-10％之间，年恶性肿瘤发病人数为160万，每年死于恶性肿瘤的患者为130万人，目前肿瘤病人的数目在540万。大中城市中肺癌、乳腺癌发病率最高；农村地区则以胃癌、食管癌发病率最高。可见，我国抗肿瘤药的市场潜力巨大。治疗恶性肿瘤的相关研究工作正在不断取得进展，据了解，目前疗效较好的常用抗肿瘤药物约有50种，WHO公布的常用抗肿瘤药物为49种，我国能够生产其中的40种抗肿瘤药物。由于抗肿瘤药物的研发周期长，我国生产企业大部分只能再单纯依靠仿制国外上市的新型抗肿瘤药物来维持本企业的生存。另外在化疗中所使用的药物，如阿霉素、环磷酰胺、肿瘤坏死因子等均具有较大的人体毒性，副作用非常大，因而肿瘤治疗药物的开发是药物研制的热点。目前从两栖动物皮肤中发现一些带有抗肿瘤活性的多肽，这些多肽大部分是抗菌肽，如，magainins、aureins、citropins、brevinin-1、ranatuerin-2、temporin和dermaseptin等(Chem Rev.2015,115(4):1760-1846)。这些多肽是潜在的肿瘤治疗药物。此外，目前也从两栖动物皮肤中鉴定带抗菌活性的bradykinin、bombesin、tachykinin等亲肌肉肽。这些多肽能够促进回肠肌或平滑肌收缩，在动物体内参与了许多病理反应，如血管舒张、血管渗透、血压控制、疼痛产生和炎症反应等。其中的bradykinin及其受体已被证实是血管紧张素转化酶抑制剂和血管紧张素AT受体拮抗剂在高血压、心肌梗死、心力衰竭治疗中的作用靶点。因此，从两栖动物皮肤中鉴定的亲肌肉肽可以用作心血管系统药物和促肠道收缩药物应用(Chem Rev.2015,115(4):1760-1846)。
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。花狭口蛙(Kaloulapulchra)主要分布于我国分布于福建、广东、广西、海南、云南等地，是国家保护的有益的或者有重要经济、科学研究价值的陆生野生动物。由于其个体小，难于捕捉，对其药理活性物质的研究很少。
发明内容：
本发明所要解决的技术问题是提供一种源于花狭口蛙皮肤的基因，该基因编码的多肽具有具有多种生物活性。
本发明解决上述技术问题的方案如下：
一种源于成熟花狭口蛙皮肤的基因，该基因的核酸序列如SEQ ID NO.5所示。
上述基因中第127-216位核苷酸编码的多肽的氨基酸序列如SEQ ID NO.1所示。
上述多肽为由30个氨基酸组成的环肽，其第二十四位半胱氨酸和第三十位半胱氨酸形成分子内二硫键，分子量为3054.97道尔顿，等电点为9.061。
一种SEQ ID NO.1所示多肽的突变体，该突变体的氨基酸序列如SEQ ID NO.2所示。
上述SEQ ID NO.2所示突变体由SEQ ID NO.1所示序列第5位的丙氨酸和第20位的赖氨酸分别被苏氨酸和精氨酸替代得到，其分子量为3140.22道尔顿，等电点为9.116。
上述多肽及其突变体具有抗菌、抗肿瘤以及心血管系统疾病和促肠道收缩的活性，可用于制备相关的药物。
本发明所述基因中第127-216位核苷酸编码的多肽及其突变体具有结构简单、人工合成方便、活性强等优点。
附图说明：
图1为本发明所述多肽的HPLC色谱图。
图2为本发明所述突变体的HPLC色谱图。
图3为本发明所述多肽的质谱图。
图4为本发明所述突变体的质谱图。
图5本发明所述多肽和突变体促进豚鼠离体回肠肌收缩的波形图。
图6为本发明所述多肽及其突变体对高脂模型SD大鼠体重影响结果的条形图。
图7为本发明所述多肽及其突变体对对高脂模型SD大鼠血清TC、TG、HDL-C影响结果的条形图。
具体实施方式：
为了便于理解，先将下述实施例中所涉及的基因、肽和突变体名称作如下说明：
下述的花狭口蛙多功能抗菌肽即为SEQ ID NO.5所示序列第127-216位核苷酸编码的多肽，其氨基酸序列如SEQ ID NO.1所示；下述花狭口蛙多功能抗菌肽的编码基因即为SEQ ID NO.5所示的源于成熟花狭口蛙皮肤的基因；下述花狭口蛙多功能抗菌肽2即为所述的突变体，其氨基酸序列如SEQ ID NO.2所示。
实施例1(基因克隆)
I、花狭口蛙皮肤总RNA提取：取300mg花狭口蛙的皮肤组织，加入10m1美国GIBCOBRL公司产品生产的Trizol溶液，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狭口蛙皮肤总RNA。
II、花狭口蛙皮肤mRNA的纯化：花狭口蛙皮肤mRNA分离纯化采用美国PROMEGA公司的mRNA Isolation Systems试剂盒。具体如下：取花狭口蛙皮肤总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0.15m1 DEPC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狭口蛙皮肤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水中得到花狭口蛙皮肤mRNA。
III、花狭口蛙皮肤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花狭口蛙皮肤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花狭口蛙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、花狭口蛙多功能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’ATGTTCACCATGAAGAAATCCCTG3’(SEQ ID NO.3)，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.4)。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、花狭口蛙多功能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.7)。基因测序结果自5’端至3’端序列为如SEQ ID NO.5所示。
花狭口蛙多功能抗菌肽基因核苷酸的序列表为：序列长度为219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：花狭口蛙皮肤。
根据花狭口蛙多功能抗菌肽的基因推断编码由功能的成熟活分泌肽为第127-216位核苷酸，氨基酸序列为：GVITDALKGAAKTVAAELLKKAHCKLTNSC(见序列SEQ ID NO.1)
实施例2(所述多肽及其突变体的制备)
Ⅰ、花狭口蛙多功能抗菌肽及其突变体的制备方法：根据花狭口蛙分泌肽的基因推断编码有功能的成熟分泌肽氨基酸序列，利用蛋白组学方法设计狭口蛙多功能抗菌肽的突变体，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。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25min内25-50％，检测波长为220nm。花狭口蛙多功能抗菌肽1出现在16.876分钟，花狭口蛙多功能抗菌肽2出现在18.036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、纯化的花狭口蛙多功能抗菌肽及其突变体用高效液相色谱(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。
花狭口蛙多功能抗菌肽是中国两栖类动物花狭口蛙多功能抗菌肽基因编码的一种环状多肽，分子量3054.97道尔顿，等电点9.061，其氨基酸序列为：Gly Val Ile Thr AspAla Leu Lys Gly Ala Ala Lys Thr Val Ala Ala Glu Leu Leu Lys Lys Ala His CysLys Leu Thr Asn Ser Cys(GVITDALKGAAKTVAAELLKKAHCKLTNSC)(SEQ ID NO.1)，上述多肽的其第二十四位半胱氨酸和第三十位半胱氨酸形成分子内二硫键。
花狭口蛙多功能抗菌肽突变体是通过蛋白组学的方法设计的一组具有改良的抗菌、抗肿瘤和促回肠肌收缩活性的环状多肽.无酶活性，它的全序列如下：
Gly Val Ile Thr Asp Thr Leu Lys Gly Val Ala Lys Thr Val Ala Ala GluLeu Leu Arg Lys Ala His Cys Lys Leu Thr Asn Ser Cys(GVITDTLKGVAKTVAAELLRKAHCKLTNSC)
实施例3(所述多肽及其突变体的活性实验)
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，合成的花狭口蛙多功能抗菌肽及其突变体都能显著的抑制细菌生长。
表1.花狭口蛙多功能抗菌肽及其突变体的抗菌活性
Ⅱ、抑制肿瘤生长的作用
在96孔细胞培养板上，将待测样品用完全培养基进行5倍或2倍倍比稀释，共六个稀释度，每个稀释度设3个重复孔，每孔100μl，同时设置正常细胞对照。每孔滴加3×105个/ml的NCI-h460、MCF-7和MDA-MB-231细胞100μl。置37℃，5％CO2培养箱内培养。48小时后用MTT法测定待测化合物对细胞的毒性作用。EC50是对50％宿主细胞产生细胞毒性作用时的浓度。
表2花狭口蛙多功能抗菌肽及其突变体的抑制肿瘤细胞生长的作用
由表1可见，花狭口蛙多功能抗菌肽能显著抑制肺癌细胞系(NCI-h460)和两种乳腺癌细胞系(MCF-7和MDA-MB-231)的生长，这表明花狭口蛙多功能抗菌肽及其突变体具有很强的抑制肿瘤细胞生长作用，同时还具有序列简单、合成方便等优点。可作为制备治疗肿瘤、胃炎、胰腺炎药物的应用。
Ⅲ、促回肠肌收缩活性
A、对离体回肠肌的作用
豚鼠断颈处死，取其离体回肠肌标本测定生物活性，大鼠离体回肠肌标本2cm长于37℃台氏液中保温，通氧气，带肌肉收缩舒张稳定后加入不同浓度的样品，图像的采集与处理采用成都泰盟科技有限公司生产的BL-420生物机能实验系统。实验过程以没有加样品的正常收缩为对照。图5显示花狭口蛙多功能抗菌肽及其突变体能显著增加豚鼠离体回肠收缩幅度。
B、对高脂模型SD大鼠的作用
选用清洁级的健康成年SD雄性大鼠，50只体重200克左右，按照体重随机分成3组，每组10只，空白组给予维持饲料，实验组给予高脂肪饲料(在维持饲料中添加20％蔗糖、1.2％胆固醇、0.2％胆酸钠、1.5％猪油)，实验组每天经口给予花狭口蛙多功能抗菌肽及其突变体(200mg/kg)，空白组和模型对照组给予等体积的生理盐水，大鼠自由进食和饮水，连续观察30，记录每周体重变化，第31天不禁食采血，采血后分离血清，测定血清TC、TG、HDL-C水平才有GLAMOUR3000全自动生化记录仪进行测定。数据处理采用统计学分析，结果图6显示开始时实验组与对照组相比均无统计学差异(P>0.05)，图7显示饲喂30天后，实验组大鼠血清TG和HDL-C水平显著低于高脂肪对照组(P<0.01)。
Claims (1)
1.SEQ ID NO：1所示的多肽在制备抗肿瘤的药物中的应用，其中，SEQ ID NO：1所示的多肽是由SEQ ID NO.5所示的成熟花狭口蛙皮肤的基因中第127-216位核苷酸编码的；所述的肿瘤为肺癌或乳腺癌。
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