CN113444263B - 一种可被mmp9蛋白酶特异性识别降解的水凝胶及其制备方法和应用 - Google Patents
一种可被mmp9蛋白酶特异性识别降解的水凝胶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种可被MMP9蛋白酶特异性识别降解的水凝胶及其制备方法和应用,本发明首先蛋白构建方面,利用基因编辑技术,构建出ELP‑GPLGLWAR‑ELP蛋白序列;其次引入两个额外的暴露在溶剂中的半胱氨酸残基让它们能够与含有聚乙二醇的降冰片烯反应生成水凝胶;最后在宏观调控上,通过控制溶液中MMP9的的浓度来影响水凝胶被降解的速率。本发明中基于可被MMP9蛋白酶特异性识别降解的水凝胶可以广泛用于伤口敷料、药物释放、MMP9含量检测。
Description
技术领域
本发明涉及酶降解水凝胶技术领域,具体涉及可被MMP9蛋白酶特异性识别降解的水凝胶及其制备方法和应用。
背景技术
水凝胶是一类极为亲水的三维网络结构凝胶,具有良好的生物相容性、柔软性和可降解性等诸多优点,可以提供类似于天然组织的环境,这种发展迅速的新型材料现已广泛应用于多种领域,如药物递送、生物工程、再生医学等。
同时近些年来出现了很多可以响应外界条件变化的水凝胶,这其中具备受酶促降解能力的水凝胶因其降解效率高、过程可靠、适用范围广而备受关注。
目前,缺乏一种可被MMP9蛋白酶特异性识别降解的水凝胶及其制备方法和应用。
发明内容
本发明的目的是提供一种可被MMP9蛋白酶特异性识别降解的水凝胶及其制备方法和应用。
为了解决现有技术的问题,本发明提供了如下技术方案:本发明的一种可被MMP9蛋白酶特异性识别降解的水凝胶的制备方法,包括如下步骤:(1)质粒的构建:在多肽序列GPLGLWAR两端插入ELP蛋白序列,将蛋白ELP-GPLGLWAR-ELP的基因片段通过限制性内切酶处理克隆到pQE80L载体中;
(2)蛋白的表达纯化:将步骤(1)所得质粒转入大肠杆菌BL21感受态细胞表达蛋白并将所得蛋白纯化;
(3)可降解凝胶的制备:将步骤(2)所得蛋白与多臂亲水高分子4臂-降冰片烯-聚乙二醇溶液混合,制得基于可被MMP9蛋白酶特异性识别降解的蛋白的水凝胶。
进一步地,在步骤(1)中,ELP-GPLGLWAR-ELP的基因片段通过限制性内切酶BamHI和KpnI处理,pQE80L载体通过限制性内切酶BglII和KpnI处理,蛋白基因序列带有pQE80L载体的N-末端6×His标记。
进一步地,在步骤(2)中,蛋白表达条件为16-37℃,4-20小时,用Co2+-NTA蛋白树脂纯化,透析至1×磷酸盐缓冲液,并在使用前保存在4℃下。
更进一步地,在步骤(2)中,所述的1×磷酸盐缓冲液的摩尔浓度10mM,pH为6.21-8.21,含137mM NaCl和2.7mM KCl。
进一步地,在步骤(3)中,蛋白ELP-GPLGLWAR-ELP与4臂-降冰片烯-聚乙二醇以摩尔比2:1混合,随后加入1mg/ml的苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐lap,紫外灯照射1.5小时成胶,如图2所示。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的蛋白的水凝胶在检测MMP9含量中的应用。
进一步地,当浸泡于MMP9蛋白酶水溶液中时,水凝胶会发生降解。
进一步地,可被降解的蛋白,其中间含有多肽序列GPLGLWAR,可被MMP9蛋白酶特异性识别。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶在伤口敷料药物中的应用。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶在药物释放中的应用。
有益效果:本发明的当浸泡于MMP9蛋白酶水溶液中时,水凝胶会发生降解,本发明可被降解的蛋白,其中间含有多肽序列GPLGLWAR,可被MMP9蛋白酶特异性识别。本发明的基于可被MMP9蛋白酶特异性识别降解的蛋白的水凝胶能够被外界的MMP9蛋白酶降解;无细胞毒害性。
与现有技术相比,本发明具有如下优点:
(1)该种水凝胶可被酶降解,在功能上,仅需微量MMP9,约10nM即可使水凝胶发生明显降解。
附图说明
图1为本发明的可被MMP9蛋白酶特异性识别降解的蛋白水凝胶的降解原理示意图。
图2为本发明的基于可被MMP9蛋白酶特异性识别降解的水凝胶设计原理示意图。
图3为本发明的基于可被MMP9蛋白酶特异性识别降解的水凝胶在MMP9蛋白酶溶液中进行降解实验图。
具体实施方式
下面结合附图对本发明内容作进一步详细说明。
实施例1
如图1所示,水凝胶网络中,ELP-GPLGLWAR-ELP蛋白中间的GPLGLWAR多肽序列可以直接被MMP9识别,并从中间切断。
本发明的一种可被MMP9蛋白酶特异性识别降解的水凝胶,制备方法包括如下步骤:(1)质粒的构建:在多肽序列GPLGLWAR两端插入ELP蛋白序列,将蛋白ELP-GPLGLWAR-ELP的基因片段通过限制性内切酶处理克隆到pQE80L载体中;ELP-GPLGLWAR-ELP的基因片段通过限制性内切酶BamHI和KpnI处理,pQE80L载体通过限制性内切酶BglII和KpnI处理,蛋白基因序列带有pQE80L载体的N-末端6×His标记。
(2)蛋白的表达纯化:将步骤(1)所得质粒转入大肠杆菌BL21感受态细胞表达蛋白并将所得蛋白纯化;蛋白表达条件为25℃,16小时,用Co2+-NTA蛋白树脂纯化,透析至1×磷酸盐缓冲液,并在使用前保存在4℃下。所述的1×磷酸盐缓冲液的摩尔浓度10mM,pH为7.21,含137mM NaCl和2.7mM KCl。
(3)可降解凝胶的制备:将步骤(2)所得蛋白与多臂亲水高分子4臂-降冰片烯-聚乙二醇溶液混合,制得基于可被MMP9蛋白酶特异性识别降解的蛋白的水凝胶。蛋白ELP-GPLGLWAR-ELP与4臂-降冰片烯-聚乙二醇以摩尔比2:1混合,随后加入1mg/ml的苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐lap,紫外灯照射1.5小时成胶。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的蛋白的水凝胶在检测MMP9含量中的应用。
当浸泡于MMP9蛋白酶水溶液中时,水凝胶会发生降解。
可被降解的蛋白,其中间含有多肽序列GPLGLWAR,可被MMP9蛋白酶特异性识别。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶在伤口敷料药物中的应用。
本发明所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶在药物释放中的应用。
受到某些特定多肽序列可被MMP9蛋白酶特异性识别、降解现象的启发,本发明提出一种可被MMP9蛋白酶特异性识别降解的蛋白的水凝胶。其主体思路在于,设计一种浸泡在MMP9水溶液中时会被MMP9降解的水凝胶。首先蛋白构建方面,利用基因编辑技术,构建出ELP-GPLGLWAR-ELP蛋白序列;其次在宏观调控上,当水凝胶浸泡在含有MMP9的溶液中时,其蛋白将会被MMP9分解,从而使整块水凝胶降解。
本发明的设计的原理,首先,通过突变以及氨基酸序列的添加为蛋白ELP-GPLGLWAR-ELP引入两个额外的暴露在溶剂中的半胱氨酸残基让它们能够与含有聚乙二醇的降冰片烯反应生成水凝胶。其次,当加入MMP9蛋白酶,它会特异性识别暴露在外的GPLGLWAR序列,并将其在中间切断。最后,可以通过控制MMP9的浓度及外界温度来控制水凝胶降解的速度。
使用这样的蛋白水凝胶设计,所用材料无细胞毒害性,可以通过MMP9蛋白酶来控制水凝胶的降解。MMP9酶本就存在于生物体内,不会造成机体损伤,可以将其制作成伤口敷料,在MMP9的作用下降解而释放出内含的药物。也可以利用MMP9对其特异性识别的特点,用来检测溶液中是否含有MMP9蛋白酶及其浓度。
实施例2
实施例2与实施例1的区别在于:在步骤(2)中,蛋白的表达纯化:将步骤(1)所得质粒转入大肠杆菌BL21感受态细胞表达蛋白并将所得蛋白纯化;蛋白表达条件为16℃,20小时,用Co2+-NTA蛋白树脂纯化,透析至1×磷酸盐缓冲液,并在使用前保存在4℃下。所述的1×磷酸盐缓冲液的摩尔浓度10mM,pH为6.21,含137mM NaCl和2.7mM KCl。
实施例3
实施例3与实施例1的区别在于:在步骤(2)中,蛋白的表达纯化:将步骤(1)所得质粒转入大肠杆菌BL21感受态细胞表达蛋白并将所得蛋白纯化;蛋白表达条件为37℃,4小时,用Co2+-NTA蛋白树脂纯化,透析至1×磷酸盐缓冲液,并在使用前保存在4℃下。所述的1×磷酸盐缓冲液的摩尔浓度10mM,pH为8.21,含137mM NaCl和2.7mM KCl。
试验例1
下面对本发明各项性能进行实施例测试:
本发明在MMP9蛋白酶水溶液中的降解实验
如图3所示,水凝胶在MMP9作用下,24小时内已经降解近半,36小时时已经几乎完全降解。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述试验例的限制,上述试验例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。
Claims (10)
1.一种可被MMP9蛋白酶特异性识别降解的水凝胶的制备方法,其特征在于包括如下步骤:
(1)质粒的构建:在多肽序列GPLGLWAR两端插入ELP蛋白序列,将蛋白ELP-GPLGLWAR-ELP的基因片段通过限制性内切酶处理克隆到pQE80L载体中;
(2)蛋白的表达纯化:将步骤(1)所得质粒转入大肠杆菌BL21感受态细胞表达蛋白并将所得蛋白纯化;
(3)可降解凝胶的制备:将步骤(2)所得蛋白与多臂亲水高分子4臂-降冰片烯-聚乙二醇溶液混合,制得可被MMP9蛋白酶特异性识别降解的水凝胶。
2.根据权利要求1所述的可被MMP9蛋白酶特异性识别降解的水凝胶的制备方法,其特征在于:在步骤(1)中,ELP-GPLGLWAR-ELP的基因片段通过限制性内切酶BamHI 和 KpnI处理,pQE80L载体通过限制性内切酶BglII 和 KpnI处理,蛋白基因序列带有pQE80L载体的N-末端6×His标记。
3.根据权利要求1所述的可被MMP9蛋白酶特异性识别降解的水凝胶的制备方法,其特征在于:在步骤(2)中,蛋白表达条件为16 - 37℃,4 - 20小时,用Co2+-NTA蛋白树脂纯化,透析至1×磷酸盐缓冲液,并在使用前保存在4℃下。
4.根据权利要求3所述的可被MMP9蛋白酶特异性识别降解的水凝胶的制备方法,其特征在于:在步骤(2)中,所述的1×磷酸盐缓冲液的摩尔浓度10 mM,pH为6.21 - 8.21,含137mM NaCl和2.7 mM KCl。
5.根据权利要求4所述的可被MMP9蛋白酶特异性识别降解的水凝胶的制备方法,其特征在于:在步骤(3)中,蛋白ELP-GPLGLWAR-ELP与4臂-降冰片烯-聚乙二醇以摩尔比2:1混合,随后加入1mg/ml的苯基(2,4,6-三甲基苯甲酰基)磷酸锂盐lap,紫外灯照射1.5小时成胶。
6.权利要求1至5任一项所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶。
7.权利要求1至5任一项所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶在检测MMP9含量中的应用。
8.根据权利要求7所述的应用,其特征在于:当浸泡于MMP9蛋白酶水溶液中时,水凝胶会发生降解。
9.根据权利要求7所述的应用,其特征在于:含有可被降解的蛋白,其中间含有多肽序列GPLGLWAR,可被MMP9蛋白酶特异性识别。
10.权利要求1至5任一项所述的方法制备的可被MMP9蛋白酶特异性识别降解的水凝胶在伤口敷料药物中的应用。
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