CN107501394B - 高特异性mmp-14底物肽及其制备方法和应用 - Google Patents
高特异性mmp-14底物肽及其制备方法和应用 Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
本发明属于生物酶检测领域,具体涉及一种高特异性MMP‑14底物肽及其制备方法和应用。该底物肽具有如式I所示的结构:其中,n为4‑30的整数,F1和F2分别为荧光基团及其对应的淬灭基团。本发明的底物具有高特异性,该底物以多肽及PEG结构为基础,在基质金属蛋白酶检测及癌症检测治疗应用方面具有广阔的前景。
Description
技术领域
本发明涉及生物酶检测领域,更具体地,涉及一种高特异性MMP-14底物肽及其制备方法和应用。
背景技术
基质金属蛋白酶(matrix metalloprotease,MMPs)是一类自然进化中高度保守的,活性依赖于锌离子和钙离子的蛋白水解酶,其广泛分布于植物、脊椎动物、无脊椎动物中。目前该家族已分离鉴别出28个成员,编号分别为MMP1-28。该家族成员具有相似的结构,一般由5个功能不同的结构域组成,包括疏水信号肽序列、前肽区(保持酶原的稳定)、催化活性区(Zn2+结合位点)、富含脯氨酸的铰链区和羧基末端区(与酶的底物特异性有关)。MMPs成员在上述共同结构的基础上各有特点,不同MMPs结构中底物结合区内侧残基侧链大小不同直接导致底物结合的差异性。根据作用底物的种类以及片断同源性,MMPs可分为6类:胶原酶(MMP-1、MMP-8、MMP-13、MMP-18);明胶酶(MMP-2、MMP-9);间质水解酶(MMP-3、MMP-10、MMP-11);基质水解酶(MMP-7、MMP-26);膜型MMP(MMP-14、MMP-15、MMP-16、MMP-17、MMP-24、MMP-25)和其他分泌型MMP(MMP-19、MMP-21、MMP-23、MMP-27、MMP-28)。
MMPs的主要功能是降解细胞外基质的有效成分,在细胞迁移、血管生成、胚胎发育、动脉粥样硬化、肝肾纤维化疾病、恶性肿瘤的浸润与转移等病理生理过程中发挥重要的作用。通过对基质金属蛋白酶的检测,可以深度了解其性质及致病机理,并进一步了解病症的恶化程度,从而进行及时检测治疗。鉴于MMPs在生理和病理进程中起着关键性的作用,对于基质金属蛋白酶检测技术的研究开发是十分必要的。
MMP-14作为MMPs家中的重要一员膜蛋白,发挥着降解基底膜与细胞外基质以及调节细胞运动、生长、分化、凋亡等多种生命功能。研究表明,其在结肠癌、胃癌、乳腺癌和***的转移与侵袭中都扮演着重要的角色。研究还发现,MMP-14参与另两个重要的MMPs家族成员MMP-2、MMP-13酶原的激活过程,而这两个蛋白酶也已被实验证实与肿瘤的侵袭和转移紧密相关,换言之,MMP-14是研究肿瘤侵袭和转移机制中非常关键的一环,对其性质的深入研究将有望揭示致病机理,实现对疾病的有效诊断和治疗。
目前常见的MMPs酶活检测方法有:荧光明胶酶检测法、明胶酶谱法、DQ明胶原位酶谱分析、高效液相色谱法、肽链修饰的荧光探针技术以及荧光分子断层成像技术。其中荧光探针技术相比其它技术具有可实现“实时、可视、定量”的高灵敏度检测的潜力,具有一定的技术优势。目前报道的肽链修饰的荧光探针主要可以分为3类:酶活性荧光成像探针、靶向探针和交联成像探针。其中基于多肽的荧光探针是研究最广的酶活性成像探针。此类成像探针的底物是基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)的原理设计。FRET作为一种方便、快捷、灵敏的光学手段被广泛应用于化学分析、生物结构研究等领域,特别是在蛋白酶活性的测定中效果显著。
然而由于MMPs家族蛋白酶之间具有较明显的底物交叉活性,如何提高底物肽的特异性成为荧光类检测探针开发中的关键问题。
发明内容
本发明的目的是提供一种提高MMPs乃至酶底物特异性的方法。该法通过在底物中引入适当的linker和具有特异性结合的非底物结合肽,极大提高了酶对底物催化的特异性。
为了实现上述目的,本发明提供一种高特异性MMP-14底物肽,该底物肽具有如式I所示的结构:
其中,n为4-30的整数,F1和F2分别为荧光基团及其对应的淬灭基团优选地,n为6-24的整数,进一步优选地,n为10-15的整数。
该底物肽的序列通式可表达为:F1-PLGL-F2-AR-(PEG)n-HWKHLHNTKTFL,其中,PLGL如SEQ ID NO:1所示,HWKHLHNTKTFL如SEQ ID NO:2所示。
本发明中,F1和F2可以为常规的荧光-淬灭基团对。优选地,所述底物肽具有式II所示结构(序列通式为:(MCA)-PLGL-(Dpa)-AR-(PEG)n-HWKHLHNTKTFL),或具有式III所示结构。
其中,p和q均为为4-30的整数,优选地,p和q均为6-24的整数,进一步优选地,p和q均为10-15的整数。具有优选长度聚乙二醇linker的底物肽具有更高的催化特异性。
本发明还提供上述高特异性MMP-14底物肽的制备方法,该制备方法包括如下步骤:
合成顺序:从C端到N端;
(1)将2-氯三苯甲基氯树脂进行溶胀;
(2)完成第一个Fmoc保护的氨基酸的连接,随后重复脱保护、检测、洗涤、加入Fmoc保护的氨基酸进行缩合、检测、洗涤的步骤,按所设计序列连入相应的氨基酸,最后接MCA;
(3)抽干并洗涤树脂;
(4)用切割液将多肽从树脂上切割下来;
(5)将裂解液用氮气吹干,多肽用***洗涤,然后常温干燥;
(6)使用HPLC对粗品进行分析提纯。
上述方法的具体步骤为本领域技术人员公知。
例如,步骤(1)中所述溶胀可通过加入DCM实现。步骤(2)可以为常规的氨基酸合成步骤。步骤(3)中洗涤可依次利用DMF、乙醇、DMF、DCM进行。步骤(4)中切割液例如为TFA95%;水1%;EDT 2%;TIS 2%的混合液。
为便于保存,该制备方法还包括将目标多肽进行冻干。
此外,本发明还提供上述高特异性MMP-14底物肽作为基质金属蛋白酶-14检测试剂的应用。
本发明通过一段聚乙二醇(PEG)作为linker在原有的广谱性MMPs多肽底物上连入一段MMP-14的特异性结合多肽,利用多肽的锚定性以及特异性把MMP-14对原有多肽的催化特异性大大提高了。本发明的底物具有高特异性,该底物以多肽及PEG结构为基础,在基质金属蛋白酶检测及癌症检测治疗应用方面具有广阔的前景。
此外,所用的聚乙二醇linker通过了美国FDA认证,符合美国药典(USP),国家处方集(NF),食品化学法典(FCC)标准,结构简单且体内安全性高,具有临床应用开发的巨大潜力。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
具体实施方式
通过以下实施例更详细地描述本发明。
所用材料及仪器:
1、树脂:取代度为1.03mmol/g的2-Chlorotrityl Chloride Resin(天津市南开合成科技有限公司)
2、氨基酸:
Fmoc-Leu-OH(成都诚诺,>99%),Fmoc-Phe-OH(成都诚诺,>99%),Fmoc-Thr(But)-OH(成都诚诺,>99%)等
3、原料:
Fmoc-NH-PEG24-CH2CH2COOH(博美生物,99%),Fmoc-Dap(Dnp)-OH(博美生物,99%),7-甲氧基香豆素-4-乙酸(MCA,百灵威科技,>99.0%)
4、合成试剂:
DMF(原产地韩国),DCM(原产地韩国),MEOH(原产地日本),
DIEA(新德化工,99%),HBTU(昊帆生物科技,99%)
5、脱保护试剂:
哌啶(国药集团上海化学试剂公司,99%)
6、检测试剂:
苯酚试剂(自配),吡啶试剂(自配),茚三酮试剂(自配)
7、裂解试剂:
95%切割液:TFA(J.T.Baker,99%),TIS(上海达瑞精细化工,98%),EDT(上海达瑞精细化工,98%),无水***(上海实验,实测99.7%)
8、氮气:(新联气体)
9、仪器:
十二通道半自动多肽合成仪:上海强耀生物科技有限公司
SHIMADZU高效液相色谱仪(型号:制备型,分析型,软件:Class-VP.SevialSystem,厂商:SHIMADZU)
LABCONCO冻干机(型号:Freezone.Plus.6,厂商:LABCONCO)
离心机(上海安亭科学仪器厂型号:TDL-40B)
实施例1-3
合成顺序:从C端到N端。合成步骤如下:
(1)树脂溶胀
将2-Chlorotrityl Chloride Resin放入反应管中,加DCM(15mL/g),振荡30min。
(2)接第一个氨基酸
通过沙芯抽滤掉溶剂,加入3倍摩尔过量的Fmoc-Leu-OH氨基酸,加入DMF溶解,再加入10倍摩尔过量的DIEA,振荡60min。用甲醇封闭。
(3)脱保护
去掉DMF,加20%哌啶DMF溶液(15mL/g),5min,去掉再加20%哌啶DMF溶液(15mL/g),15min。
(4)检测
抽掉哌啶溶液,取十几粒树脂,用乙醇洗三次,加入检测试剂检测,105℃-110℃加热5min,变深蓝色为阳性反应。
(5)洗涤:DMF(10mL/g)两次,DCM(10mL/g)两次,DMF(10mL/g)两次。
(6)缩合
保护氨基酸三倍过量,HBTU三倍过量,均用尽量少DMF溶解,加入反应管,立刻加入十倍过量的DIEA,反应30min。
(7)检测
取十几粒树脂,用乙醇洗三次,加入检测试剂检测,105℃-110℃加热5min,无色为阴性反应。
(8)洗涤
DMF(10mL/g)一次,DCM(10mL/g)两次,DMF(10mL/g)两次。
(9)重复步骤(3)至步骤(6),从右到左依次连接序列中的氨基酸,最后接MCA。
(10)抽干,按照下列方法洗树脂:
DMF(10mL/g)两次,甲醇(10mL/g)两次,DMF(10mL/g)两次,DCM(10mL/g)两次,抽干10min。
(11)从树脂上切割多肽
配制切割液(10mL/g):TFA 95%;水1%;EDT 2%;TIS 2%。
切割时间:120min。
(12)吹干洗涤
将裂解液用氮气尽量吹干,用***洗六次,然后常温挥干。
(13)分析提纯:用高效液相色谱将粗品提纯。
(14)冻干
收集目标多肽溶液放入冻干机中进行浓缩,冻干成白色粉末。
通过质谱检验,证明分别合成产物肽B-肽D,序列如表1所示。
表1
多肽名称 | 多肽序列 |
肽A | (MCA)-PLGL-(Dpa)-AR |
肽B | (MCA)-PLGL-(Dpa)-AR-(PEG)<sub>24</sub>-HWKHLHNTKTFL |
肽C | (MCA)-PLGL-(Dpa)-AR-(PEG)<sub>12</sub>-HWKHLHNTKTFL |
肽D | (MCA)-PLGL-(Dpa)-AR-(PEG)<sub>6</sub>-HWKHLHNTKTFL |
测试例1
对肽B-肽D以及肽A(通过常规方法合成)进行酶活性检测。检测方法如下:
酶促反应动力学法,具体操作为:在96孔酶标板中依次加入底物肽(1μM)、MMPAssay Buffer(20mM Tris-HCl pH 8.0,150mM NaCl,5mM CaCl2,100μM ZnCl2,0.05%Brij35)与不同浓度的MMP-14,同时以加入MMP-14相同体积的缓冲溶液作为对照组,在反应温度为37℃,激发波长328nm、发射波长393nm的条件下测定单位时间内荧光的变化。
结果如表2所示。
表2
由表2检测结果可见,本发明的底物肽对于MMP-14具有高特异性,特别是具有优选范围聚乙二醇linker的底物肽,催化特异性更为显著。
以上已经描述了本发明的各实施例,上述说明是示例性的,并非穷尽性的,并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下,对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。
SEQUENCE LISTING
<110> 天津医科大学
<120> 高特异性MMP-14底物肽及其制备方法和应用
<130> 1700201
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 4
<212> PRT
<213> 人工序列
<400> 1
Pro Leu Gly Leu
1
<210> 2
<211> 12
<212> PRT
<213> 人工序列
<400> 2
His Trp Lys His Leu His Asn Thr Lys Thr Phe Leu
1 5 10
Claims (5)
3.权利要求1-2中任意一项所述的高特异性MMP-14底物肽的制备方法,其特征在于,该制备方法包括如下步骤:
合成顺序:从C端到N端;
(1)将2-氯三苯甲基氯树脂进行溶胀;
(2)完成第一个Fmoc保护的氨基酸的连接,随后重复脱保护、检测、洗涤、加入Fmoc保护的氨基酸进行缩合、检测、洗涤的步骤,按所设计序列连入相应的氨基酸,最后接7-甲氧基香豆素-4-乙酸;
(3)抽干并洗涤树脂;
(4)用切割液将多肽从树脂上切割下来;
(5)将裂解液用氮气吹干,多肽用***洗涤,然后常温干燥;
(6)使用HPLC对粗品进行分析提纯。
4.根据权利要求3所述的方法,其中,该制备方法还包括:(7)将目标多肽进行冻干。
5.权利要求1-2中任意一项所述的高特异性MMP-14底物肽在制备基质金属蛋白酶-14检测试剂中的应用。
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