CN106478803B - 花姬蛙促胰岛素释放肽及其基因和在制药中的应用 - Google Patents
花姬蛙促胰岛素释放肽及其基因和在制药中的应用 Download PDFInfo
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- CN106478803B CN106478803B CN201610915763.8A CN201610915763A CN106478803B CN 106478803 B CN106478803 B CN 106478803B CN 201610915763 A CN201610915763 A CN 201610915763A CN 106478803 B CN106478803 B CN 106478803B
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- hypsizygus marmoreus
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- insulin
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Abstract
本发明涉及花姬蛙促胰岛素释放肽及其基因和在制药中的应用，所述花姬蛙促胰岛素释放肽由33个氨基酸组成的环肽，分子量3535.14道尔顿，等电点10.3，其氨基酸序列如SEQ ID NO.1所示，上述多肽的第四位半胱氨酸和第九位半胱氨酸相成分子内二硫键。所述花姬蛙促胰岛素释放肽的基因序列由SEQ ID NO.4组成，其中编码有功能的成熟花姬蛙促胰岛素释放肽是第379‑477位核苷酸。本发明由花姬蛙促胰岛素释放肽的基因推导其成熟功能多肽氨基酸序列，合成的花姬蛙促胰岛素释放肽具有很好的促进胰岛素释放作用，同时还具有细胞毒性、无血浆凝固活性等优点，可作为制备治疗糖尿病药物的应用。
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：
糖尿病是多遗传基因和环境因素引起的以糖、脂肪、蛋白质代谢紊乱，而导致多系统，多脏器功能损害的一种多发的内分泌、代谢性疾病。统计显示，我国80年代糖尿病患病率仅为0.67％，但2010年国家疾病监测地区数据显示，全国18周岁以上居民糖尿病患病率为9.7％，并且有1.5亿人处于糖尿病前期。糖尿病本身不可怕，可怕的是并发症，糖尿病并发症包括癌、睡眠呼吸暂停、抑郁、心肌梗死、外周血管病、高血压。中国Ⅱ型糖尿病的发病率在过去的30年里猛增(Int J Med Sci.,2014,11(11):1185-200)。Ⅱ型糖尿病又称“非胰岛素依赖型糖尿病”，主要病理表现为胰岛素抵抗和由胰岛细胞功能失调所致的胰岛素分泌相对不足，形成持久的高血糖，并可产生多种致命性并发症。其临床表现症状大多与机体产生胰岛素抗性、自身分泌的胰岛素少或滞后等有关。至今为止，该病尚无根治的办法，它不仅会加重患者经济负担，还可使患者致残、早亡。
目前，世界上治疗Ⅱ型糖尿病以磺酰脲类药物为主，辅以双胍类、a-糖苷酶抑制剂等药物。但是，由于磺酰脲类药物的副作用大，长期使用会因为胰岛素过度分泌而导致胰岛β细胞衰竭，服用此类药物的糖尿病人中，每年有10％的病人会因治疗失效而改用胰岛素等其他类药物(Int J Med Sci,2014,11(11):1185-200)。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。来自脊椎动物和无脊椎动物的活性肽能够利用肠促胰岛素降低血糖水平(Am J Med,2010,123:S2-S10)。肠促胰岛素是肠被收营养物质刺激后在外周组织分泌产生以促进胰岛素分泌和葡萄糖的吸收。内源性促胰岛素释放肽及其结构类似物因能延缓胃排空，抑制胰高血糖素产生，增加胰岛素的生物合成及β细胞数量等特点已成为治疗Ⅱ型糖尿病的新亮点(Best PractRes Clin Endocrinol Metab,2007,21:497-516)。人体内最强的肠促胰岛素GLP-1肽和其数种结构类似物目前已经被应用于临床上治疗糖尿病(Drugs,2009,69:1985-2004)。基于此，从自然界中寻找具有这些特点的多肽的研究已经在全球被广泛开展。从吉拉毒蜥(Heloderma suspectum)的毒液中发现的exendin-4是GLP-1受体的长效激动剂，以其为基础的商业化产品Byetta目前也被应用于临床上作治疗糖尿病(Expert OpinPharmacother,2007,8:2593-608)。
两栖动物一直以来都是传统药物的源泉。最近的研究表明许多蛙皮肤抗菌肽在对细胞没有毒性的浓度下在体外能够刺激大鼠BRIN-BD11β细胞释放胰岛素。这些促胰岛素释放肽已经从Hylarana güntheri、Lithobates catesbeianus、Lithobates palustris、Lithobates pipiens、Lithobates septentrionalis、Pelophylax saharicus、Hylomantislemur和Pseudis paradoxa的皮肤中被鉴定。并且其中的brevinin-2GUb、phylloseptin-L2、B2-RP及其相关类似物在体内能够增加胰岛素浓度，能够降低血浆血糖水平，改善小鼠的血糖耐受性Chem Rev.2015,115(4):1760-1846)。因此，这些化合物能够用于治疗糖尿病。
中国博大的多物种天然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。很多两栖类动物在中国属于传统中药和民族药物而广泛应用，如，花姬蛙(Microhyla pulchra)是传统中药材，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。
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花姬蛙胰岛素释放促进肽编码基因，发明人将本发明的花姬蛙胰岛素释放促进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胰岛素释放促进肽(Microhylain)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，提供一种新的具有促进胰岛素释放的花姬蛙促胰岛素释放肽及其基因在制药中的应用。
为了解决上述技术问题，本发明采用的技术方案是：
一种花姬蛙促胰岛素释放肽，其序列如SEQ ID No.1所示。
RLPCKGIFCKVGHGSPIGRHKNNNATLSPFSTV SEQ ID NO.1
一种花姬蛙促胰岛素释放肽，所述的花姬蛙促胰岛素释放肽是由33个氨基酸组成的环肽，分子量3535.14道尔顿，等电点10.3，其氨基酸序列为：Arg Leu Pro Cys Lys GlyIle Phe Cys Lys Val Gly His Gly Ser Pro Ile Gly Arg His Lys Asn Asn Asn AlaThr Leu Ser Pro Phe Ser Thr Val(RLPCKGIFCKVGHGSPIGRHKNNNATLSPFSTV)(SEQ IDNO.1)，上述多肽的第四位半胱氨酸和第九位半胱氨酸形成分子内二硫键。
所述花姬蛙促胰岛素释放肽的编码基因由480个核苷酸组成，其序列为(SEQ IDNO.4)：
序列中第379-477位核苷酸编码具有功能的成熟花姬蛙促胰岛素释放肽(SEQ IDNO.1)。所述的花姬蛙促胰岛素释放肽可作为制备治疗糖尿病药物应用。
所述花姬蛙促胰岛素释放肽在促进胰岛素释放中的应用。
所述花姬蛙促胰岛素释放肽在制备治疗血糖升高的药物中的应用。
本发明的有益效果在于:
由花姬蛙促胰岛素释放肽编码基因推导其氨基酸结构，合成的花姬蛙促胰岛素释放肽具有很好的促进胰岛素释放作用，同时还具有细胞毒性、无血浆凝固活性等优点，可作为制备治疗糖尿病药物的应用。
附图说明：
图1为本发明花姬蛙促胰岛素释放肽HPLC纯化鉴定结果；
图2为本发明花姬蛙促胰岛素释放肽质谱鉴定结果；
图3为本发明花姬蛙促胰岛素释放肽促进INS-1细胞分泌胰岛素的“量-效关系”结果；
图4为本发明花姬蛙促胰岛素释放肽降低小鼠体内血清血糖效果结果；
图5为本发明花姬蛙促胰岛素释放肽降低小鼠体内血糖AUC水平结果。
具体实施方式：
下面结合附图和具体实施方式对本发明做进一步详细说明：
本发明的花姬蛙促胰岛素释放肽由33个氨基酸组成的环肽，分子量3535.14道尔顿，等电点10.3，其氨基酸序列为：Arg Leu Pro Cys Lys Gly Ile Phe Cys Lys Val GlyHis Gly Ser Pro Ile Gly Arg His Lys Asn Asn Asn Ala Thr Leu Ser Pro Phe SerThr Val (RLPCKGIFCKVGHGSPIGRHKNNNATLSPFSTV)(SEQ ID NO.1)，上述多肽的其第四位半胱氨酸和第九位半胱氨酸相成分子内二硫键。
所述花姬蛙促胰岛素释放肽的基因序列由SEQ ID NO.4序列组成，序列中第379-477位核苷酸编码具有功能的成熟花姬蛙促胰岛素释放肽。本发明的花姬蛙促胰岛素释放肽及其基因的制备过程包括如下步骤：
实施例1，花姬蛙促胰岛素释放肽基因克隆
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 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、花姬蛙促胰岛素释放肽基因克隆筛选：扩增引物长度为27个核苷酸，其序列为5’ATGAAGGTCTGGCAGTGTGTGCTCTGG 3’(SEQ ID NO.2)，PCR另一扩增引物为CLONTECH公司SMARTTM cDNA 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℃50sec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、花姬蛙促胰岛素释放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):
花姬蛙促胰岛素释放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：花姬蛙皮肤。
根据花姬蛙促胰岛素释放肽的基因推断编码由功能的成熟活胰岛素释放促进肽为第379-477位核苷酸，氨基酸序列为SEQ ID NO.1。
实施例2，花姬蛙促胰岛素释放肽的制备
Ⅰ、花姬蛙促胰岛素释放肽的制备方法：根据花姬蛙促胰岛素释放肽的基因推断编码其有功能的成熟活性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0.1％TFA+100％acetonitrile，B液为0.1％TFA+100％water，多肽出现在13.5分钟，流速1.0ml/min，检测波长为220nm。
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、纯化的花姬蛙促胰岛素释放肽用高效液相色谱(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。
花姬蛙促胰岛素释放肽是中国两栖类动物花姬蛙促胰岛素释放肽基因编码的一种环状多肽，3535.14道尔顿，等电点10.3，多肽全序列一级结构为：其氨基酸序列为：ArgLeu Pro Cys Lys Gly Ile Phe Cys Lys Val Gly His Gly Ser Pro Ile Gly Arg HisLys Asn Asn Asn Ala Thr Leu Ser Pro Phe Ser Thr Val(RLPCKGIFCKVGHGSPIGRHKNNNATLSPFSTV)(SEQ ID NO.1)，上述多肽第四位半胱氨酸和第九位半胱氨酸形成分子内二硫键使多肽成环。
实施例3，花姬蛙促胰岛素释放肽的活性实验
Ⅰ、花姬蛙促胰岛素释放肽的体外促进胰岛素释放作用
花姬蛙促胰岛素释放肽的促进胰岛素释放活性采用葡萄糖诱导的胰岛素释放活性检测方法(GSIS，Glucose-stimulated insulin secretion)测定。具体步骤如下：大鼠胰岛细胞瘤INS-1细胞用含100U/ml青霉素、0.1mg/ml链霉素、10％胎牛血清和11.1mM葡萄糖的RPMI-164037℃5％CO2在24孔细胞培养板中培养，细胞形成单层后用pH 7.4，含5.6或16.8mM葡萄糖、0.1％牛血清白蛋白的Krebs–Ringer bicarbonate缓冲液中37℃预孵育40min，吸去上清，细胞同含样品的同样缓冲液37℃预孵育20min，吸去上清，1000rpm离心10min，上清液用于胰岛素含量检测，每个样品4个重复。胰岛素含量用上海酶研公司生产的胰岛素ELISA检测试剂盒按照说明书检测。结果见说明书附图3。由说明书附图3可见，花姬蛙促胰岛素释放肽具有显著的促进胰岛素释放的作用，INS-1细胞释放出的胰岛素随着花姬蛙促胰岛素释放肽浓度的增加呈现上升的趋势。
Ⅱ、花姬蛙促胰岛素释放肽的体内促胰岛素释放作用
初始血糖差异不显著的30只8周龄C57BL/6J雄性小鼠按照单因素完全随机试验设计随机分为3组：100nmol/kg的花姬蛙促胰岛素释放肽、25nmol/kg利拉鲁肽阳性药组和PBS阴性对照组。口服葡萄糖耐量试验(oral glucose tolerance test，OGTT)前小鼠适应1周，试验前空腹14h，并在葡萄糖(1.5g/kg)灌胃前30min腹腔给药处理。灌胃后的0、10、20、30、60、90min取小鼠尾尖血测血糖，并观察期间小鼠的状态。血糖浓度测定采用酶联公司的葡萄糖氧化酶活性测定试剂盒按照说明测定。结果见说明书附图4和5。由说明书附图4和5可见对C57BL/6J小鼠进行OGTT后，花姬蛙促胰岛素释放肽同阳性药利拉鲁肽一样均显示出显著的降血糖作用，花姬蛙促胰岛素释放肽和阳性药利拉鲁肽在120min的OGTT过程中的血糖曲线下面积均显著小于PBS组，因此，花姬蛙促胰岛素释放肽具有降低血糖的生物活性，可作为制备治疗糖尿病药物的应用。
Claims (5)
1.一种花姬蛙促胰岛素释放肽，其特征在于，所述花姬蛙促胰岛素释放肽是 由33个氨基酸组成的多肽，分子量3535.14道尔顿，等电点10.3，其氨基酸序列为：Arg Leu Pro CysLys Gly Ile Phe Cys Lys Val Gly His Gly Ser Pro Ile Gly Arg His Lys Asn AsnAsn Ala Thr Leu Ser Pro Phe Ser Thr Val(RLPCKGIFCKVGHGSPIGRHKNNNATLSPFSTV)(SEQ ID NO.1)，上述多肽的第四位半胱氨酸和第九位半胱氨酸形成分子内二硫键。
2.一种编码权利要求1所述的花姬蛙促胰岛素释放肽基因的核苷酸。
3.根据权利要求1所述的花姬蛙促胰岛素释放肽在制备治疗糖尿病的药物中的应用。
4.根据权利要求1所述的花姬蛙促胰岛素释放肽在制备促进胰岛素释放的药物中的应用。
5.根据权利要求1所述的花姬蛙促胰岛素释放肽在制备治疗血糖升高的药物中的应用。
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| CN1900109A (zh) * | 2006-05-30 | 2007-01-24 | 中国科学院昆明动物研究所 | 棕点湍蛙胰岛素释放促进肽和在制药中的应用 |
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| CN1900109A (zh) * | 2006-05-30 | 2007-01-24 | 中国科学院昆明动物研究所 | 棕点湍蛙胰岛素释放促进肽和在制药中的应用 |
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| Brevinin-1 and multiple insulin-releasing peptides in the skin of the frog Rana palustris;Marenah 等;《Journal of Endocrinology》;20040531;第181卷(第2期);第347-354页 * |
| 蛙皮抗菌肽促胰岛素分泌功能研究进展;赵茜 等;《生命的化学》;20140415;第34卷(第2期);第249-256页 * |
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