CN106399338B - 一种检测活细胞细胞膜表面张力变化的生物探针 - Google Patents

一种检测活细胞细胞膜表面张力变化的生物探针 Download PDF

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CN106399338B
CN106399338B CN201610752206.9A CN201610752206A CN106399338B CN 106399338 B CN106399338 B CN 106399338B CN 201610752206 A CN201610752206 A CN 201610752206A CN 106399338 B CN106399338 B CN 106399338B
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刘波
李旺
于新磊
张树利
孙坚原
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Dalian University of Technology
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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，是一种基于FRET技术的生物探针，它能够动态地检测活细胞细胞膜表面张力的变化，具体公开了一种检测活细胞细胞膜表面张力变化的生物探针。该探针包括FRET荧光蛋白对ECFP与YPet、弹性序列Linker、Lyn序列以及K‑ras序列五个部分。该探针与pcDNA3.1(+)的重组质粒转染到活细胞后，可以在细胞中自发表达出荧光蛋白融合探针，定性地检测细胞膜张力的变化，且对细胞原有功能没有影响。该探针实现了在活体细胞内对细胞膜表面张力变化的动态检测，具有成本低、对细胞无毒副作用等特点，为研究细胞膜张力变化提供了一种可视化工具。

Description

一种检测活细胞细胞膜表面张力变化的生物探针
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)技术和亚克隆技术设计制备的生物探针。它能够在活细胞内自行表达，通过荧光信号的变化定性地反映细胞膜表面张力的变化。
背景技术
剪切应力作用于细胞膜上表面，首先引起细胞膜表面张力的改变，这种物理改变可能是肿瘤细胞定向迁移的基础。目前检测细胞膜表面张力的方法有可变形聚合物基底法、微柱法等，但它们存在空间分辨率低、对基底材料要求高等缺陷，目前尚无一种切实有效的方法能对活细胞细胞膜表面张力的变化进行测量。由此，本发明提出一种基于FRET技术、用于检测细胞膜表面张力变化的生物探针，它具有时间分辨率高、制作成本低、对细胞无毒害作用等优点。
发明内容
本发明提供一种可检测活细胞细胞膜表面张力变化的新型生物探针，设计中将FRET技术与亚克隆技术结合。
本发明的技术方案：
一种检测活细胞细胞膜表面张力变化的生物探针，该生物探针包括FRET荧光蛋白对ECFP(1-2)、YPet(1-4)、弹性序列Linker(1-3)、Lyn序列(1-1)和K-ras序列(1-5)五个部分，由亚克隆技术对DNA序列剪切拼接，并与载体质粒pcDNA3.1(+)形成重组质粒。
其中，Lyn的氨基酸序列为：
AAATMGCIKSKRKDNLNDDGVDMKT
其对应的DNA序列为：
GCGGCCGCCACCATGGGCTGCATCAAGAGCAAGCGCAAGGACAACCTGAACGACGACGGCGTGGACATGAAGACC；
K-ras的氨基酸序列为：
KKKKKKSKTKCVIM
对应的DNA序列为：
AAGAAGAAGAAGAAGAAGAGCAAGACCAAGTGCGTGATCATG；
弹性序列Linker的氨基酸序列为：
GPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGA
对应的DNA序列为：
GGTCCAGGAGGCGCAGGACCTGGCGGGGCTGGACCGGGTGGCGCGGGACCCGGCGGAGCCGGCCCAGGTGGGGCGGGCCCTGGTGGTGCTGGTCCGGGAGGGGCAGGGCCCGGAGGTGCC。
本发明的有益效果：该探针实现了在活体细胞内对细胞膜表面张力变化的动态检测，具有成本低、对细胞无毒副作用等特点，为研究细胞膜张力变化提供了一种可视化工具。
附图说明
图1是一种检测活细胞细胞膜表面张力变化的生物探针MSS的示意图。
图2是检测活细胞细胞膜表面张力变化的生物探针MSS的工作原理图。
图中：1-1Lyn序列；1-2FRET荧光蛋白对ECFP；1-3弹性序列Linker；1-4YPet；
1-5K-ras序列。
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。
利用转染试剂将本探针转染到活细胞体内后，它能够自行表达出荧光蛋白融合探针结构，两端的Lyn序列1-1和K-ras序列1-5分别与细胞膜相连，当外界力使细胞膜表面张力发生变化时，弹性序列Linker1-3会被拉伸或者压缩，引起FRET荧光蛋白对ECFP 1-2和YPet1-4之间距离的改变，从而导致FRET信号的改变。对转染后的细胞给予436nm波长的激发，利用FRET显微镜同时采集474nm和530nm波长上的荧光图像，然后通过474nm/530nm荧光强度比例分析能量转移的效率，从而得到细胞膜表面张力变化的信息。
一种能检测活细胞细胞膜表面张力变化的生物探针，核苷酸序列：
一种能检测活细胞细胞膜表面张力变化的生物探针，氨基酸序列：

Claims (1)

1.一种检测活细胞细胞膜表面张力变化的生物探针，其特征在于，该生物探针包括FRET荧光蛋白对ECFP(1-2)、YPet(1-4)、弹性序列Linker(1-3)、Lyn序列(1-1)和K-ras序列(1-5)五个部分，由亚克隆技术对DNA序列剪切拼接，并与载体质粒pcDNA3.1(+)形成重组质粒；
其中，Lyn的氨基酸序列为：
AAATMGCIKSKRKDNLNDDGVDMKT
其对应的DNA序列为：
GCGGCCGCCACCATGGGCTGCATCAAGAGCAAGCGCAAGGACAACCTGAACGACGACGGCGTGGACATGAAGACC；
K-ras的氨基酸序列为：
KKKKKKSKTKCVIM
对应的DNA序列为：
AAGAAGAAGAAGAAGAAGAGCAAGACCAAGTGCGTGATCATG；
弹性序列Linker的氨基酸序列为：
GPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGA
对应的DNA序列为：
GGTCCAGGAGGCGCAGGACCTGGCGGGGCTGGACCGGGTGGCGCGGGACCCGGCGGAGCCGGCCCAGGTGGGGCGGGCCCTGGTGGTGCTGGTCCGGGAGGGGCAGGGCCCGGAGGTGCC。
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CN108120836B (zh) * 2017-12-06 2020-08-14 大连理工大学 一种检测活细胞内Paxillin蛋白力传递的荧光生物探针
CN111378050B (zh) * 2018-12-29 2023-04-07 深圳先进技术研究院 一种融合蛋白及其应用
CN110981943B (zh) * 2019-12-02 2021-08-03 清华大学 多肽及其在制备药物中的用途和药物
CN114672503B (zh) * 2022-03-21 2023-10-13 大连理工大学 一种活细胞内连接微丝与局部黏着斑的生物应力传感器
CN115947866B (zh) * 2022-09-28 2024-04-19 大连理工大学 一种基于FRET的活细胞内Paxillin蛋白活性检测生物探针及其重组质粒

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CN103228669A (zh) * 2010-09-27 2013-07-31 国立大学法人京都大学 基于荧光共振能量转移的原理的单分子型fret生物传感器接头
CN105784656A (zh) * 2016-03-16 2016-07-20 大连理工大学 一种检测活细胞内RhoGDIα蛋白活性的生物探针

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003043577A2 (en) * 2001-11-19 2003-05-30 Iconix Pharmaceuticals, Inc. Modulators of rho c activity
CN103228669A (zh) * 2010-09-27 2013-07-31 国立大学法人京都大学 基于荧光共振能量转移的原理的单分子型fret生物传感器接头
CN105784656A (zh) * 2016-03-16 2016-07-20 大连理工大学 一种检测活细胞内RhoGDIα蛋白活性的生物探针

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