CN113480628B - 一种贵州疣螈多肽tk-cath及其编码基因和应用 - Google Patents
一种贵州疣螈多肽tk-cath及其编码基因和应用 Download PDFInfo
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Abstract
本发明涉及一种贵州疣螈来源抗炎和促进伤口修复双功能多肽TK‑CATH及其编码基因和应用，贵州疣螈多肽TK‑CATH由55个氨基酸残基组成，分子量6196.99Da，等电点4.65，氨基酸序列如SEQ ID：1，其中Cys45和Cys48之间形成一对分子内二硫键，贵州疣螈多肽TK‑CATH具有分子量小、人工合成简单、能促进皮肤创口修复、具有较强的抗炎活性且溶血活性极低的优点，具有广泛的应用前景。
Description
技术领域
本发明涉及生物医学技术领域，尤其涉及一种贵州疣螈来源多肽 TK-CATH及其编码基因和应用。
背景技术
多肽是一种由氨基酸组成的小分子蛋白质，具有多样的结构和活性。生物体内能够产生大量不同结构和活性的天然活性多肽分子，在生物体生存过程中发挥重要的作用。活性多肽具有分子量小、结构简单、活性强等优点，在不同领域具有很大的开发应用前景。
两栖动物皮肤裸露，没有鳞片、毛发等保护。其皮肤作为和外界接触的第一道屏障，通常会受到来自外界的侵袭，继而会使得皮肤遭受损伤，从而启动机体的自我修复显得至关重要。两栖动物皮肤富含大量天然活性多肽分子，到目前为止已从各种两栖类动物体内发现数百种不同的多肽分子，并且其数目还在不断增加。
发明内容
本发明提供一种从贵州疣螈(Tylototriton kweichowensis)皮肤中发现的具有良好的抗炎和促进皮肤修复活性的新型多肽 TK-CATH，以及编码这条多肽的核酸序列。还发现了该多肽在多个领域中的新应用。
本发明所述贵州疣螈多肽TK-CATH由55个氨基酸残基组成，分子量6196.99Da，等电点4.65，其氨基酸序列为：
Gly1Gly2Gln3Asp4Thr5Gly6Lys7Glu8Gly9Glu10Thr11Gly12Lys13Lys14Lys15L ys16Ser17
Asp18Asn19Trp20Phe21Met22Asn23Leu24Leu25Asn26Lys27Phe28Leu29Glu30Leu31Ile32Gly33
Leu34Lys35Glu36Ala37Gly38Asp39Asp40Ser41Glu42Pro43Phe44Cys45Phe46Thr 47Cys48Ile49Phe50Asp51Met52Phe53Ser54Gln55(SEQ ID：1)，其中其中Cy s45和Cys48之间形成一对分子内二硫键。
贵州疣螈多肽TK-CATH编码基因通过从贵州疣螈皮肤提取总R NA，反转录合成第一链cDNA，采用长末端聚合酶链式反应(LD-P CR)方法扩增第二链cDNA。
根据蝾螈科cathelicidins家族抗菌肽信号肽区的保守序列，人工设计合成正向引物5’-TKCATH:5’-ATGGAGGTCTGGCAGTGTG TGTTAT-3’，反向引物为CLONTECH公司In-Fusion SMARTer TM Directional cDNA Library Construction Kit中的3’-PCR引物，其序列为5’-CGGGGTACGATGAGACACCAT-3’。PCR反应扩增，扩增完成后回收目的片段，将回收的目的片段连接到质粒载体上，转化感受态细胞，筛选挑取阳性菌落，进行核苷酸测序。
该贵州疣螈多肽TK-CATH前体的编码基因由1011个核苷酸组成，其5’端至3’端序列为SEQ ID NO:2所示的核苷酸序列，编码贵州疣螈多肽TK-CATH的为第385-549位核苷酸。
贵州疣螈多肽TK-CATH在制备皮肤创口修复药物中的应用。
贵州疣螈多肽TK-CATH在制备抗炎药物中的应用。
贵州疣螈多肽TK-CATH在制备保健品中的应用。
贵州疣螈多肽TK-CATH在制备化妆品中的应用。
本发明中的贵州疣螈多肽TK-CATH具有分子量小、人工合成简单、能促进皮肤创口修复、具有较强的抗炎活性且溶血活性极低的优点，具有广泛的应用前景。
附图说明
图1 TK-CATH促进HaCAT细胞增殖；
图2 TK-CATH促进HaCAT细胞迁移；
图3 TK-CATH加速小鼠全层创面愈合；
图4 TK-CATH对于LPS诱导的促炎因子分泌的影响。
具体实施方式
下面用实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：
贵州疣螈多肽TK-CATH编码基因克隆：
1)贵州疣螈皮肤总RNA提取：
①取200mg贵州疣螈背部皮肤组织，放入研钵中加入液氮研磨成粉末，转移到EP管中，加入1ml总RNA提取缓冲液(Trizol，美国Life公司产品)，充分混匀，而后于4℃，12000rpm离心10min。
②离心取上清，加入0.2ml氯仿溶液，剧烈混匀，室温放置10 分钟，而后以4℃，12000rpm离心10分钟，弃除沉淀。
③上清加入等体积的异丙醇，室温放置10分钟，以4℃，12000rpm 离心10分钟，收集沉淀用75％(V/V)乙醇洗一次，晾干，管底沉淀物即为贵州疣螈皮肤总RNA。
2)贵州疣螈皮肤cDNA二链合成：采用CLONTECH公司 In-Fusion SMARTerTMDirectional cDNA Library Construction Kit合成。
(1)cDNA第一链合成(mRNA反转录)：
①RNase-free的PCR管中加入1μl贵州疣螈皮肤总RNA、1μl 3’端一链合成引物(3’In-Fusion SMARTer CDS Primer)和2.5μl RNase-free水使总体积达到4.5μl，混匀后短暂离心(2000rpm，30s)，离心后于72℃保温3分钟；保温后再将离心管在42℃孵育2分钟。
②在上述离心管中加入以下试剂(均为CLONTECH公司 In-Fusion SMARTerTMDirectional cDNA Library Construction Kit建库试剂盒中配备)，2.0μl 5×第一链缓冲液、0.25μl 100mM DTT、1.0μl 10mM dNTP Mix、1.0μl SMARTer V Oligonucleotide、0.25μl RNase Inhibitor和1.0μl SMARTScribe Reverse Transcriptase反转录酶，混合离心管中试剂并短暂离心(2000rpm，30s)，在42℃保温90min，然后68℃保温10min。保温处理后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。
(2)采用长末端聚合酶链式反应(LD-PCR)方法扩增第二链(所用试剂均为CLONTECH公司In-Fusion SMARTerTM Directional cDNA Library Construction Kit建库试剂盒中配备)
①将2μl cDNA第一链(mRNA反转录)、80μl去离子水、10μl 10×Advantage 2PCR缓冲液、2μl 50×dNTP混合物、2μl 5’PCR引物、2μl CDS III/3’PCR引物以及2μl 50×Advantage 2Polymerase Mix 在95℃预热的PCR管中进行混合。
②在PCR仪中按以下程序扩增：
95℃，1min；18个循环：95℃，15sec，65℃，30sec，68℃，6 min。循环结束后，将离心管中合成的cDNA双链－80℃保存。
(3)贵州疣螈多肽TK-CATH编码基因克隆：
根据蝾螈科cathelicidins家族抗菌肽信号肽区的保守序列，人工设计合成正向引物:5’-TK-CATH: 5’-ATGGAGGTCTGGCAGTGTGTGTTAT-3’，反向引物为 CLONTECH公司In-Fusion SMARTerTM Directional cDNA Library Construction Kit中的3’-PCR引物，其序列为 5’-CGGGGTACGATGAGACACCAT-3’。PCR反应在如下条件下进行： 95℃4min，95℃30sec，57℃30sec和72℃1min，30个循环。扩增完成后用胶回收试剂盒(天根生物)进行目的片段回收。将回收的目的片段连接到pMD19-T载体(Takara,大连)，转化DH5α感受态细胞。涂板并进行氨苄青霉素筛选，挑取单菌落用M13引物PCR检测***片段大小。挑取阳性菌落，摇菌提取质粒，使用Applied Biosystems DNA sequencer,model ABI PRISM 377进行核苷酸测序。
测定结果：
编码贵州疣螈多肽TK-CATH前体的基因自5’端至3’端序列为：
贵州疣螈多肽TK-CATH前体的编码基因核苷酸序列表为：序列长度为1011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：贵州疣螈皮肤。
实施例2
贵州疣螈多肽TK-CATH的制备：
(1)贵州疣螈多肽的化学合成方法：根据编码基因推导的氨基酸序列，用自动多肽合成仪(433A,Applied Biosystems)合成其全序列，通过HPLC反相柱层析脱盐纯化。
(2)分子量测定采用基质辅助激光解析电离飞行时间质谱 (MALDI-TOF)。
(3)纯化的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。
贵州疣螈多肽TK-CATH是基因编码的一种小分子多肽，由55 个氨基酸残基组成，分子量6196.99Da，等电点4.65。贵州疣螈多肽全序列为：
Gly1Gly2Gln3Asp4Thr5Gly6Lys7Glu8Gly9Glu10Thr11Gly12Lys13Lys14Lys15Lys16Ser17Asp18
Asn19Trp20Phe21Met22Asn23Leu24Leu25Asn26Lys27Phe28Leu29Glu30Leu31Ile32Gly33Leu34
Lys35Glu36Ala37Gly38Asp39Asp40Ser41Glu42Pro43Phe44Cys45Phe46Thr47Cys 48Ile49Phe50Asp51Met52Phe53Ser54Gln55，其中Cys45和Cys48之间形成一对分子内二硫键。
实施例3
贵州疣螈多肽TK-CATH细胞增殖活性检测：
细胞培养：HaCAT细胞从液氮中拿出，放入37℃的水浴锅中快速融化，加入适量培养基混匀后，1200rpm离心5min，弃去上清，用含有10％血清的无支原体的DMEM培养基重悬混匀后转至培养瓶中，放入37℃，5％CO2培养箱培养，待细胞生长至对数生长期后传代用于后续实验。
细胞给药：用0.25％胰酶对上述细胞进行消化，将离心管放置 1200rpm离心机中离心5min，弃去上清，用DMEM培养基将细胞团块轻轻的吹打混匀并用血球计数板计数，并稀释至1×105个/ml。取 96孔板，每孔加入100μl稀释好的细胞悬液，使得每孔中含有约10000个左右的HaCAT细胞，实验设定5个调零孔，调零孔加入100μl DMEM培养基，在37℃，5％CO2培养箱中培养16-24h。培养好后在样品孔加入不同浓度的TK-CATH或Ot-WHP(已发表的促伤口愈合肽作为阳性对照)。空白孔加入同样体积的PBS。37℃，5％CO2培养箱中培养过夜。
每孔加入20μl MTT溶液，继续放入37℃，5％CO2培养箱培养 4h后终止培养，每孔加入150μl DMSO(二甲基亚枫)置于摇床上， 110rpm低速振荡10min，使得结晶充分溶解，于570nm处测吸光值。
结果由图1可见，实验结果表明，表明TK-CATH可促进 HaCAT(人永生化角质细胞)的体外增殖。(阴性对照：PBS，阳性对照：Ot-WHP)
实施例4
贵州疣螈多肽TK-CATH细胞迁移活性测定：
按上述方法培养HaCAT细胞并并稀释至1×106个/ml，取6孔板，每孔加入1ml细胞悬液，放入37℃，5％CO2培养箱培养过夜。
待细胞长满孔后，弃去孔内液体，加入适当2％胎牛血清的 DMEM培养基，饥饿培养24h，PBS轻轻洗涤一次后，用200μl黄枪头垂直于培养孔，自上而下均匀力度划线，使得刮痕边缘完整，用PBS洗去被枪尖刮下来的细胞团，加入适当浓度的TK-CATH或阳性对照Ot-WHP，显微镜下拍照观察后，放入37℃，5％CO2培养箱培养，在培养过程中，取三个时间点进行拍照观察(0h、24h、48h)，使用Image-pro plus 6.0计算细胞刮痕的间距。细胞刮痕率＝(0h-24h) /0h×100。
实验结果如图2所示，TK-CATH在作用24，48h后，与阴性对照组(PBS)相比有明显的差别，甚至优于阳性对照组(Ot-WHP)，在 TK-CATH作用24h和48h的划痕修复率分别达到55％和82％。说明 TK-CATH具有促进角质细胞迁移的作用。
实施例5
贵州疣螈多肽TK-CATH促创口愈合功能
建立起小鼠全层皮肤伤口模型，实验分为三组，第一组在小鼠左边伤口滴加20μlPBS作为阴性对照，右侧滴加20μl TK-CATH(多肽浓度为200μg/ml)，第二组则在左边滴加20μl Ot-WHP作为阳性对照、右侧滴加20μl TK-CATH(多肽浓度为200μg/ml)，于0、2、 4、6、8天拍摄创面并用Image-pro plus 6.0计算不同时间伤口面积的变化，伤口愈合率为(0day-2day)/0day×100。
结果如图3所示，TK-CATH的创口修复效果比Control组的好，且与阳性对照效果相当，进一步证明了TK-CATH有明显的促进伤口修复的作用，为进一步了解创口面积的收缩变化，使用Image pro plus 软件计算创口面积，并求得创口修复率，计算公式为：(0d-2d)/0d*100。从图3 B中得知，当给药达到第八天时候，Control组对于小鼠创口的修复率达到61％，而TK-CATH组修复率达到了84％左右，差异比较明显，其愈合作用与阳性对照相接近。(空白对照：PBS、阳性对照： Ot-WHP促伤口愈合肽)
实施例6
贵州疣螈多肽TK-CATH抗炎活性测定：
将RAW264.7细胞培养至对数生长期，用0.25％胰酶对上述细胞进行消化，将离心管放置1200rpm离心机中离心5min，弃去上清，并用DMEM培养基将细胞团块轻轻的吹打混匀并用血球计数板计数，稀释至2×105/ml，孔加入稀释好的细胞悬液200μl，得每孔有 40000个细胞，放入37℃，5％CO2培养箱中培养过夜。次日加不同浓度的TK-CATH多肽(终浓度为5、10、20μg/ml)以及大肠杆菌 LPS(终浓度为100ng/ml)，放入37℃，5％CO2培养箱培养6h后取上清并用ELISA试剂盒检测炎症因子的表达。
结果如图4所示，TK-CATH能抑制LPS诱导的促炎因子的产生，且呈浓度依赖性。当多肽浓度达到20μg/ml时，TK-CATH对于TNF- α、IL-6的抑制率达到了33％、68％。
实施例7
贵州疣螈多肽TK-CATH溶血活性测定：
将采集的新鲜C57小鼠血与阿氏液混合抗凝，生理盐水洗涤2 次并重悬成107-108cell/ml的悬浮液。上述稀释好的红细胞悬液与溶解于生理盐水的贵州疣螈多肽样品混合，37℃保温30min，再于1000 rpm离心5min，上清液于540nm测吸收值。阴性对照使用生理盐水，阳性对照使用Triton X-100，溶血百分比按以下公式计算：溶血百分比H％＝A样品-A阴性对照/A阳性对照×100％。
结果表明浓度为200μg/ml时，溶血百分比为小于2％。说明 TK-CATH对哺乳动物红细胞具有极低的溶血活性。
通过上述实施例可以看出，本发明中的贵州疣螈多肽TK-CATH 可通过化学合成的方式获得，该多肽具有较强的促进皮肤创口修复活性和抗炎活性，其次该多肽具有分子量小、合成简单、溶血活性低的特点，可用作皮肤创口修复的药物、抗炎药物、术后医疗器械的消毒剂、保健品、化妆品等的生产原料或辅料。
将贵州疣螈多肽TK-CATH按照常规剂量与常规的辅料结合，通过现有的加工方法制备成对应的药物、抗炎药物、术后医疗器械的消毒剂、保健品、化妆品等产品。
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Claims (4)
1.一种贵州疣螈多肽TK-CATH，其特征在于：所述的多肽TK-CATH由55个氨基酸残基组成，其氨基酸序列如SEQ ID No：1所示，其中Cys45和Cys48之间形成一对分子内二硫键。
2.根据权利要求1所述的贵州疣螈多肽TK-CATH在制备皮肤创口修复药物中的应用。
3.根据权利要求1所述的贵州疣螈多肽TK-CATH在制备抗炎药物中的应用。
4.根据权利要求1所述的贵州疣螈多肽TK-CATH在制备化妆品中的应用。
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Citations (3)
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| CN1809364A (zh) * | 2003-06-23 | 2006-07-26 | 杰龙公司 | 增加端粒酶活性的组合物和方法 |
| CN104288750A (zh) * | 2014-10-23 | 2015-01-21 | 中国科学院昆明动物研究所 | 红瘰疣螈皮肤修复肽cathelicidin-TV2的应用 |
| WO2018157773A1 (zh) * | 2017-02-28 | 2018-09-07 | 暨南大学 | 一种促进创伤后组织修复与再生的修复肽及其应用 |
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