CN110606869B - 一种促皮肤愈合肽oa-gp11及其制备方法与应用 - Google Patents
一种促皮肤愈合肽oa-gp11及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种促皮肤愈合肽OA‑GP11及其制备方法与应用。所述的促皮肤愈合肽OA‑GP11的氨基酸序列为GPLSGINAECM。本发明纯化得到的促皮肤愈合肽OA‑GP11在体外抗氧化活性检测、创伤修复细胞以及动物模型实验中显示了较强的活性，揭示本发明促皮肤愈合肽OA‑GP11有潜在的应用前景。本发明的有益效果在于：促皮肤愈合肽OA‑GP11具有结构简单、促创伤修复活性强的有益特点。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种促皮肤愈合肽OA-GP11及其制备方法与应用。
背景技术
作为人体最大的器官，皮肤可以调节体温并感知周围环境。它能保护体内组织和器官免受物理和化学伤害，以及来自致病微生物的侵害。皮肤的伤口修复过程是复杂和费时的，且任何修复过程的干扰都可能加重病情，并延迟伤口愈合。难治性的伤口也会导致病原体的入侵，导致继发感染、水和电解质紊乱、传染性休克、器官衰竭甚至死亡。为了改善康复过程，并减少在伤口愈合过程中出现的并发症，有效且快速地促进伤口愈合是至关重要的。目前，已知的几种创伤修复药物都存在一定的缺陷，包括稳定性差、活性低、存储成本高、疤痕增生等。因此，功能多样、活性高、稳定性高、特性专一的生物活性肽具有很高的探索价值。
两栖动物作为一种皮肤裸露且栖息地复杂的物种，很容易受到外部伤害，因而，它们形成了一种保护性的皮肤防御系统。之前关于两栖动物分泌物的研究已经确定了多种功能多样的生物活性肽，包括抗菌肽、抗氧化肽、伤口愈合肽、细胞因子、神经调节肽和神经毒素等。值得注意的是，两栖动物的皮肤在受损后可以迅速愈合，而且很少留疤，这表明了两栖动物皮肤分泌物中存在某种可以缩短伤口愈合的过程物质。
我们从云南臭蛙的皮肤分泌物中分离出一种新型肽，OA-GL17，它具有较高的抗氧化活性，可以有效促进体内外伤口的愈合，且具有较高的稳定性及促创伤修复活性。
发明内容
本发明的第一目的在于提供一种促皮肤愈合肽OA-GP11；第二目的在于提供所述的促皮肤愈合肽OA-GP11的制备方法；第三目的在于提供所述的促皮肤愈合肽OA-GP11的应用。
本发明的第一目的是这样实现的，所述的促皮肤愈合肽OA-GP11的氨基酸序列为GPLSGINAECM。
本发明的第二目的是这样实现的，包括以下步骤：
A、用电刺激法取云南臭蛙皮肤分泌物，溶解于PBS，真空冷冻干燥后得到物料a于-80℃保存备用；
B、将物料a溶解于水中得到物料b，物料b用Tris-HCl缓冲液进行洗脱，收集洗脱液得到物料c；
C、将物料c进行第一次高效液相色谱反相层析得到物料d；
D、将物料d进行第二次高效液相色谱反相层析得到目标物促皮肤愈合肽OA-GP11。
本发明的第三目的是这样实现的，所述的促皮肤愈合肽OA-GP11在制备促皮肤创伤修复的保健食品、化妆品或药品中的应用。
本发明是从云南臭蛙的皮肤分泌物中分离出一种新型肽，OA-GL17，它具有较高的抗氧化活性，可以有效促进体内外伤口的愈合，且具有较高的稳定性及促创伤修复活性。所述的OA-GL17具有结构简单、促创伤修复活性特点，可通过云南臭蛙皮肤分泌分离纯化得到的有益特点。本发明提供的促皮肤愈合肽OA-GP11，其制备方法可以通过生物化学分离纯化方法，由云南臭蛙皮肤分泌物中得到。上述方法制备的促皮肤愈合肽OA-GP11，可通过质谱及促创伤修复活性进行鉴定，促皮肤愈合肽OA-GP11的制备也可通过本领域常规的方法，采用化学合成或基因表达的方式得到。本发明纯化得到的促皮肤愈合肽OA-GP11在体外抗氧化活性检测、创伤修复细胞以及动物模型实验中显示了较强的活性，揭示本发明促皮肤愈合肽OA-GP11有潜在的应用前景。本发明的有益效果在于：促皮肤愈合肽OA-GP11具有结构简单、促创伤修复活性强的有益特点。
附图说明
图1为本发明促皮肤愈合肽OA-GP11的HPLC反相C18柱层析图；
图2为本发明促皮肤愈合肽OA-GP11的抗氧化活性图；
图3位本发明分离纯化促皮肤愈合肽OA-GP11的细胞模型促划痕修复活性图；
图4为本发明分离纯化促皮肤愈合肽OA-GP11的动物模型促创伤修复活性图。
具体实施方式
下面结合实施例和附图对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或替换，均属于本发明的保护范围。
本发明所述的促皮肤愈合肽OA-GP11的氨基酸序列为GPLSGINAECM。
本发明所述的促皮肤愈合肽OA-GP11的制备方法，包括以下步骤：
A、用电刺激法取云南臭蛙皮肤分泌物，溶解于PBS，真空冷冻干燥后得到物料a于-80℃保存备用；
B、将物料a溶解于水中得到物料b，物料b用Tris-HCl缓冲液进行洗脱，收集洗脱液得到物料c；
C、将物料c进行第一次高效液相色谱反相层析得到物料d；
D、将物料d进行第二次高效液相色谱反相层析得到目标物促皮肤愈合肽OA-GP11。
B步骤中所述的水为去离子水。
B步骤中所述的洗脱是取蛋白含量为80~150mg的物料b上预先用20mM Tris-HCl缓冲液平衡24h的Sephadex G75柱子，然后用Tris-HCl缓冲液进行洗脱。
所述的洗脱的流速为1.5ml/10min。
所述的Tris-HCl缓冲液pH值为7.8，含0.1M NaCl。
所述的第一次高效液相色谱反相层析是将物料c上样于预先用含0.1%三氟乙酸的超纯水平衡好的Hypersil ODS2 5mm柱子，在流速为0.5~1.5ml/min的条件下，用含0.1%三氟乙酸的乙腈在线性梯度条件下进行洗脱，监测波长为215nm。
所述的第二次高效液相色谱反相层析是将物料d真空冷冻干燥后溶于去离子水，然后重复第一次高效液相色谱反相层析过程。
本发明所述的促皮肤愈合肽OA-GP11的应用为所述的促皮肤愈合肽OA-GP11在制备促皮肤创伤修复的保健食品、化妆品或药品中的应用。
下面以具体实施案例对本发明做进一步说明：
实施例1
促皮肤愈合肽OA-GP11分离纯化和鉴定
1、分离纯化
云南臭蛙活体采自云南保山，用电刺激法取其皮肤分泌物（溶解于PBS），真空冷冻干燥，-80°保存备用。
第一步：Sephadex G75分子筛：按照上述方法获得皮肤分泌物冻干粉，溶解于去离子水中，取1 ml（蛋白含量为100 mg）上预先用20 mM Tris-HCl缓冲液（pH=7.8，含0.1 MNaCl）平衡24小时的Sephadex G75 (GE Healthcare，超细)柱子（长度40 cm，内径宽度1.5cm），用同样的缓冲液进行洗脱，流速为1.5ml/10min，每10 min收集一次。将收集的样品用HaCaT细胞划痕实验测定促创伤修复活性，活性较强的样品使用HPLC进行进一步提纯。
第二步：第一次高效液相色谱反相层析：
第一步所得样品上样于预先用超纯水（含0.1%的三氟乙酸）平衡好的HypersilODS2 5mm柱子（伊利特产品，尺寸为4.6 mm ×300 mm），实验仪器为Waters 1525高压液相系统，在流速为1 ml/min的条件下，用乙腈（含0.1%的三氟乙酸）在线性梯度（0-100% , 100min）条件下进行洗脱，监测波长为215 nm，所得的分离纯化图谱如图1A所示，箭头所指为具有促创伤修复活性峰，OA-GL17存在所示峰。
第三步：第二次高效液相色谱反相层析：
收集第一次具有促创伤修复活性峰，真空冷冻干燥后溶于去离子水，然后重复第一次HPLC过程，所得的分离纯化图谱如图1B所示，箭头所指为纯化的OA-GP11所在峰。
2、分子鉴定
氨基酸序列的测定：
纯化得到的促皮肤愈合肽OA-GP11在全自动蛋白质序列测定仪（岛津PPSQ-31A）上经Edman 降解法，测定了促皮肤愈合肽OA-GP11氨基酸全序列，结果表明促皮肤愈合肽OA-GP11具有本发明所示的氨基酸序列：GPLSGINAECM。
实施例2
促皮肤愈合肽OA-GP11的活性鉴定
1、体外抗氧化活性鉴定
本发明利用体外自由基清除活性检测实验检测促皮肤愈合肽OA-GP11的抗氧化活性，结果表明促皮肤愈合肽OA-GP11具有较强的抗氧化活性。
通过 OA-GP11 的 2, 2‘-偶氮二 (3-乙基苯并噻唑啉-6-磺酸)(ABTS)清除活性检测其抗氧化活性。将 2.8 mM 过硫酸钾与 7 mM ABTS 在暗室环境条件下与水中孵育不少于 6 h，制备 ABTS+自由基原液，并立即使用。用双蒸馏水将原液稀释 50 倍。将ddH2O溶解后的OA-GP11加入稀释后的原液中，以相同体积的溶剂为空白对照，维生素 C (vc)为阳性对照。在黑暗中反应 30 分钟， 415 nm 处测吸光度，ABTS+自由基清除率 (%) = (Ablank-Asample)×100/Ablank，结果如图2所示。
2、体外促细胞划痕修复活性鉴定
采用HaCaT细胞划痕愈合实验检测 OA-GP11 的促细胞划痕修复能力。细胞培养于DMEM培养基以及10 %胎牛血清和 1 %双抗 (青霉素-链霉素, 100 U/ml) 中，并置于含 5%二氧化碳的培养 (37°C) 环境内培养。采用 24 孔板培养 HaCaT细胞 (2.5×105个/孔)，血清饥饿培养 12-24 h，在形成的细胞单层上采用无菌移液管的尖端制作细胞机械划伤。用 PBS 轻轻洗两次去除漂浮的细胞, 然后加入含有一定浓度的 OA-GP11 的无血清培养基 (500 μl/孔)，继续培养24 h。 同时以不含 OA-GP11 的相同体积的无血清培养基作为阴性对照。采用倒置显微镜以不同的时间间隔拍摄细胞划痕修复的图像。使用 Image J 软件计算细胞的修复情况。促皮肤创伤修复的抗氧化多肽OA-GP11的促细胞划痕修复活性如图3所示。
3、体内动物模型促创伤愈合活性鉴定
选体重22-25g的SPF级昆明种小白鼠进行实验，将小鼠随机分成两组，每组10只。首先给小鼠腹腔注射1%的戊巴比妥钠（0.1ml/20g）麻醉小鼠，然后将小鼠背部毛剃除干净，并用75%的酒精消毒，最后在小鼠背部两侧用凿孔器凿出两个大小对称的孔,约8mm×8mm大小。术后将小鼠放在加热器旁待其醒后放回饲养室正常饲养。每天两次上样，每孔每次上样约20μl,第一组小鼠背部左侧涂生理盐水，右侧OA-GP11；第二组小鼠背部左侧涂康复新修复液，右侧OA-GP11。每隔2天给小鼠背部创伤拍照，最后通过Image J 软件计算创伤修复率。促皮肤创伤修复的抗氧化多肽OA-GP11的促动物模型创伤修复活性如图4所示。
SEQUENCE LISTING
<110> 昆明医科大学
<120> 一种促皮肤愈合肽OA-GP11及其制备方法与应用
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 11
<212> PRT
<213> 促皮肤愈合肽OA-GP11的氨基酸序列
<400> 1
Gly Pro Leu Ser Gly Ile Asn Ala Glu Cys Met
1 5 10
Claims (2)
1.一种促皮肤愈合肽OA-GP11，其特征在于所述的促皮肤愈合肽OA-GP11的氨基酸序列为GPLSGINAECM。
2.一种权利要求1所述的促皮肤愈合肽OA-GP11的应用，其特征在于所述的促皮肤愈合肽OA-GP11在制备促皮肤创伤修复的化妆品或药品中的应用。
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