CN105784656B - 一种检测活细胞内RhoGDIα蛋白活性的生物探针 - Google Patents
一种检测活细胞内RhoGDIα蛋白活性的生物探针 Download PDFInfo
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Abstract
本发明属于细胞生物学及分子生物学技术领域,涉及一种根据FRET基本原理设计制备的、可检测活细胞内RhoGDIα蛋白活性的生物探针。该探针为融合的蛋白质结构,包括FRET荧光蛋白对ECFP与Ypet,RhoGDIα全长序列,RhoGDIα的底物结构域switch II及中间的linker序列五个部分。本发明的融合质粒转入活细胞后,可由细胞自行表达出探针的融合蛋白结构,从而在细胞内发挥探针功能,具有特异性高、成本低、对细胞无副作用等特点。
Description
技术领域
本发明属于细胞生物学及分子生物学技术领域,是基于荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理和亚克隆技术进行设计制备,通过荧光蛋白颜色与强度变化检测活细胞内RhoGDIα(Rho GDP dissociation inhibitor α)蛋白活性变化的生物探针。
背景技术
RhoGDIα是Rho家族成员的负性调控蛋白,可通过抑制Rho家族不同成员的活性参与细胞迁移过程,对癌细胞侵袭具有至关重要的作用。目前一般采用基于蛋白抗原抗体结合的免疫组织化学反应、Western Blotting检测、或基于核酸水平的原位PCR反应等手段检测细胞内RhoGDIα蛋白,这些手段都存在一定的缺陷:1.必须对细胞进行破碎或固定等预处理,破坏了细胞的正常生理状态;2.不能在单细胞或亚细胞水平上动态观测;3.只能检测蛋白的量多少,不能检测蛋白的活性高低;4.成本高。因此,目前没有一种切实有效的方法能对活细胞内RhoGDIα活性进行检测。针对上述缺点,本发明提出一种基于FRET技术的生物探针,可以检测活细胞中RhoGDIα活性变化,同时具备以下优点:1.亚克隆技术大大降低了探针制备周期及成本;2.可在单细胞及亚细胞水平上进行观测;3.可以对同一细胞进行实时动态检测;4.在活细胞上观察,不伤害细胞,能反映细胞内的真实状态。
发明内容
本发明提供一种可检测活细胞中RhoGDIα活性的新型生物探针,设计中将FRET技术与亚克隆技术结合,利用RhoGDIα活性改变能与其底物结合而改变探针空间结构,从而引起FRET效率发生变化的特点,使其能实时检测出单一活细胞中RhoGDIα活性及空间分布的动态变化。
本发明的技术方案如下:
如附图所示,该RhoGDIα-FRET探针为蛋白质,结构共包括FRET荧光蛋白对ECFP(1-1)与Ypet(1-5),RhoGDIα全长蛋白(1-4),Rho家族共同底物序列switch II(1-2)及连接linker序列(1-3)。在设计及制备过程中,采用亚克隆技术对FRET荧光蛋白对ECFP(1-1)、RhoGDIα的底物结构域switch II(1-2)、linker序列(1-3)、RhoGDIα全长序列(1-4)和Ypet(1-5)五个部分在DNA水平上进行连接,并包装入商品化的pcDNA3.1质粒,从而构建出含RhoGDIα-FRET探针DNA序列的融合质粒,该质粒用常规方法转染活细胞,即可在细胞内自行表达出探针蛋白。通过FRET显微镜观察荧光,从而发挥检测功能。
其中,RhoGDIα全长氨基酸序列如下:
MAEQEPTAEQLAQIAAENEEDEHSVNYKPPAQKSIQEIQELDKDDESLRKYKEALLGRVAVSADPNVPNVIVTRLTLVCSTAPGPLELDLTGDLESFKKQSFVLKEGVEYRIKISFRVNREIVSGMKYIQHTYRKGVKIDKTDYMVGSYGPRAEEYEFLTPMEEAPKGMLARGSYNIKSRFTDDDKTDHLSWEWNLTIKKEWKD
其对应的DNA序列为:
ATGGCAGAACAGGAACCCACTGCTGAGCAGCTCGCTCAGATAGCTGCAGAGAATGAGGAAGACGAGCACTCTGTGAACTACAAGCCTCCAGCCCAGAAGAGCATCCAGGAGATCCAGGAACTGGACAAGGATGATGAAAGCCTTCGAAAGTACAAGGAGGCCCTGCTGGGCCGAGTAGCTGTCTCTGCAGACCCCAATGTCCCCAACGTCATTGTGACCCGCTTGACCTTGGTGTGCAGCACTGCCCCAGGCCCTCTGGAACTGGACCTGACAGGTGATCTGGAGAGCTTCAAGAAACAGTCATTTGTGTTGAAGGAAGGTGTGGAGTACCGGATAAAAATCTCTTTCCGGGTGAACAGAGAGATCGTGTCAGGCATGAAGTACATCCAGCACACATACAGGAAAGGGGTCAAAATTGACAAGACTGACTACATGGTCGGGAGCTATGGGCCAAGGGCTGAGGAGTATGAGTTCCTGACACCCATGGAAGAGGCCCCCAAAGGCATGCTTGCTCGGGGCAGTTACAACATCAAGTCCCGCTTCACAGATGATGACAAAACTGACCACCTGTCCTGGGAGTGGAATCTCACCATCAAAAAGGAATGGAAGGACTGA
Rho家族共有底物序列Switch II氨基酸序列全长:
DTAGQEDYDRLRPLS
对应的DNA序列为:
GATACTGCAGGGCAAGAGGATTATGACAGACTACGACCGTTATCC
Linker氨基酸序列为:GGSGGT
对应的DNA序列为:GGTGGCTCTGGCGGTACT。
该生物探针的融合质粒转入活细胞内,由细胞自行表达出探针的融合蛋白结构,在活细胞内通过FRET荧光显微镜动态检测RhoGDIα蛋白活性及分布情况变化。
本发明的有益效果:该探针具有特异性高、成本低、对细胞无副作用等特点,实现了在单一活细胞中对RhoGDIα活性变化及空间分布情况的实时观测,是一种廉价高效的FRET生物探针,为科学研究或者临床检测细胞内RhoGDIα活性状态提供一种工具。
附图说明
图1是本发明的RhoGDIα-FRET探针结构示意图。
图2是本发明的RhoGDIα-FRET探针工作原理图。
图中:1-1FRET荧光蛋白对ECFP;1-2RhoGDIα的底物结构域switch II;1-3linker序列;1-4RhoGDIα全长序列;1-5Ypet。
具体实施方式
以下结合附图和技术方案,进一步说明本发明的具体实施方式。
本探针可在真核细胞中自行表达,能准确反映RhoGDIα活性及空间分布的变化情况且不影响细胞自身功能。具体实施方法如下:对真核细胞转染RhoGDIα-FRET探针的融合质粒,细胞即自行表达RhoGDIα-FRET探针的融合蛋白。当该探针上的RhoGDIα蛋白(1-4)具备活性时,可与探针上的底物switchII(1-2)相互结合,整个探针发生折叠改变探针的空间结构,探针上的ECFP(1-1)与Ypet(1-5)之间的距离改变,就会影响两个荧光蛋白之间的能量转移效率。在FRET显微镜下对转入RhoGDIα-FRET探针的活细胞拍照,以波长420nm的激发光进行激发,同时在485nm、535nm波长上采集荧光图像,通过485nm/535nm荧光强度比例分析能量转移的效率变化,即可获得活细胞中RhoGDIα活性及空间分布情况。
Claims (2)
1.一种检测活细胞内RhoGDIα蛋白活性的生物探针,其特征在于,该生物探针为融合的蛋白质结构,包括FRET荧光蛋白对ECFP(1-1)与Ypet(1-5),RhoGDIα全长序列(1-4),RhoGDIα的底物结构域switch II(1-2)及linker序列(1-3)五个部分;FRET荧光蛋白对ECFP(1-1)、RhoGDIα的底物结构域switch II(1-2)、linker序列(1-3)、RhoGDIα全长序列(1-4)和Ypet(1-5)依次连接;
其中,RhoGDIα全长序列的氨基酸序列如下:
MAEQEPTAEQLAQIAAENEEDEHSVNYKPPAQKSIQEIQELDKDDESLRKYKEALLGRVAVSADPNVPNVIVTRLTLVCSTAPGPLELDLTGDLESFKKQSFVLKEGVEYRIKISFRVNREIVSGMKYIQHTYRKGVKIDKTDYMVGSYGPRAEEYEFLTPMEEAPKGMLARGSYNIKSRFTDDDKTDHLSWEWNLTIKKEWKD
其对应的DNA序列为:
ATGGCAGAACAGGAACCCACTGCTGAGCAGCTCGCTCAGATAGCTGCAGAGAATGAGGAAGACGAGCACTCTGTGAACTACAAGCCTCCAGCCCAGAAGAGCATCCAGGAGATCCAGGAACTGGACAAGGATGATGAAAGCCTTCGAAAGTACAAGGAGGCCCTGCTGGGCCGAGTAGCTGTCTCTGCAGACCCCAATGTCCCCAACGTCATTGTGACCCGCTTGACCTTGGTGTGCAGCACTGCCCCAGGCCCTCTGGAACTGGACCTGACAGGTGATCTGGAGAGCTTCAAGAAACAGTCATTTGTGTTGAAGGAAGGTGTGGAGTACCGGATAAAAATCTCTTTCCGGGTGAACAGAGAGATCGTGTCAGGCATGAAGTACATCCAGCACACATACAGGAAAGGGGTCAAAATTGACAAGACTGACTACATGGTCGGGAGCTATGGGCCAAGGGCTGAGGAGTATGAGTTCCTGACACCCATGGAAGAGGCCCCCAAAGGCATGCTTGCTCGGGGCAGTTACAACATCAAGTCCCGCTTCACAGATGATGACAAAACTGACCACCTGTCCTGGGAGTGGAATCTCACCATCAAAAAGGAATGGAAGGACTGA;
RhoGDIα的底物结构域switch II的氨基酸序列全长:
DTAGQEDYDRLRPLS
对应的DNA序列为:
GATACTGCAGGGCAAGAGGATTATGACAGACTACGACCGTTATCC;
Linker氨基酸序列为:GGSGGT
对应的DNA序列为:GGTGGCTCTGGCGGTACT。
2.根据权利要求1所述的生物探针的应用,其特征在于,该生物探针的融合质粒转入活细胞内,由细胞自行表达出探针的融合蛋白结构,在活细胞内通过FRET荧光显微镜动态检测RhoGDIα蛋白活性及分布情况变化。
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