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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| CN110616255B (zh) * | 2019-10-14 | 2023-01-24 | 康妍葆(北京)干细胞科技有限公司 | Rna水平鉴别牙髓干细胞与牙龈干细胞的引物组、试剂盒及鉴定方法 |
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