CN106399338B - A kind of bioprobe detecting living cell membrane surface tension variations - Google Patents

A kind of bioprobe detecting living cell membrane surface tension variations 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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cell membrane
cell
surface tension
membrane surface
bioprobe
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CN106399338A (en
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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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

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Abstract

The invention belongs to cell biology and technical field of molecular biology, it is a kind of bioprobe based on FRET technology, it can dynamically detect the variation of living cell membrane surface tension, specifically disclose a kind of bioprobe for detecting living cell membrane surface tension variations.The probe includes FRET fluorescin to ECFP and five YPet, elastomeric sequences Linker, Lyn sequence and K-ras sequence parts.After the probe and the Transfected Recombinant Plasmid to living cells of pcDNA3.1 (+), spontaneous in cell fluorescin fusion probe can be given expression to, qualitatively detect the variation of cell layer tension, and do not influence on cell original function.The probe realizes in active somatic cell to the dynamic detection of cell membrane surface tension variation, have the characteristics that it is at low cost, to cytotoxic side effect, provide a kind of visualization tool for research cell membrane tension variation.

Description

A kind of bioprobe detecting living cell membrane surface tension variations
Technical field
It is based on fluorescence resonance energy transfer the invention belongs to cell biology and technical field of molecular biology (fluorescence resonance energy transfer, FRET) technology and the biology of Subcloned technology design preparation are visited Needle.It can voluntarily be expressed in living cells, and the variation of cell membrane surface tension is qualitatively reflected by the variation of fluorescence signal.
Background technique
Shear stress causes the change of cell membrane surface tension in cell membrane upper surface first, this physically changed It may be the basis of tumour cell directional migration.The method of current detecting cell film tension has deformable polymer substrate Method, microtrabeculae method etc., but they there are spatial resolutions it is low, high to base material requirement the defects of, there is no a kind of has at present conscientiously The method of effect can measure the variation of living cell membrane surface tension.The present invention proposes a kind of based on FRET skill as a result, Art, the bioprobe for detecting cell membrane surface tension variation, it has temporal resolution height, low manufacture cost, to cell The advantages that nonhazardous acts on.
Summary of the invention
The present invention provides a kind of new bio probe of detectable living cell membrane surface tension variations, will in design FRET technology is in conjunction with Subcloned technology.
Technical solution of the present invention:
A kind of bioprobe detecting living cell membrane surface tension variations, which includes FRET fluorescin To five ECFP (1-2), YPet (1-4), elastomeric sequences Linker (1-3), Lyn sequence (1-1) and K-ras sequence (1-5) portions Point, DNA sequence dna is sheared by Subcloned technology and is spliced, and forms recombinant plasmid with vector plasmid pcDNA3.1 (+).
Wherein, the amino acid sequence of Lyn are as follows:
AAATMGCIKSKRKDNLNDDGVDMKT
Its corresponding DNA sequence dna are as follows:
GCGGCCGCCACCATGGGCTGCATCAAGAGCAAGCGCAAGGACAACCTGAACGACGACGGCGTGGACAT GAAGACC；
The amino acid sequence of K-ras are as follows:
KKKKKKSKTKCVIM
Corresponding DNA sequence dna are as follows:
AAGAAGAAGAAGAAGAAGAGCAAGACCAAGTGCGTGATCATG；
The amino acid sequence of elastomeric sequences Linker are as follows:
GPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGA
Corresponding DNA sequence dna are as follows:
GGTCCAGGAGGCGCAGGACCTGGCGGGGCTGGACCGGGTGGCGCGGGACCCGGCGGAGCCGGCCCAGG TGGGGCGGGCCCTGGTGGTGCTGGTCCGGGAGGGGCAGGGCCCGGAGGTGCC。
Beneficial effects of the present invention: the probe, which is realized, examines the dynamic of cell membrane surface tension variation in active somatic cell Survey, have the characteristics that it is at low cost, to cytotoxic side effect, provide a kind of visual chemical industry to study cell membrane tension variation Tool.
Detailed description of the invention
Fig. 1 is the schematic diagram of bioprobe MSS for detecting living cell membrane surface tension variations a kind of.
Fig. 2 is the working principle diagram for detecting the bioprobe MSS of living cell membrane surface tension variations.
In figure: 1-1Lyn sequence；1-2FRET fluorescin is to ECFP；1-3 elastomeric sequences Linker；1-4YPet；
1-5K-ras sequence.
Specific embodiment
Below in conjunction with attached drawing and technical solution, a specific embodiment of the invention is further illustrated.
After this probe is transfected into living cell body using transfection reagent, it can voluntarily give expression to fluorescin fusion and visit The Lyn sequence 1-1 and K-ras sequence 1-5 of needle construction, both ends is connected with cell membrane respectively, when external force makes cell membrane surface When power changes, elastomeric sequences Linker1-3 can be stretched or compress, cause FRET fluorescin to ECFP 1-2 and The change of distance between YPet1-4, so as to cause the change of FRET signal.Swashing for 436nm wavelength is given to the cell after transfection Hair is acquired the fluorescent image on 474nm and 530nm wavelength simultaneously using FRET microscope, then passes through 474nm/530nm fluorescence The efficiency of intensity analysing energy transfer, to obtain the information of cell membrane surface tension variation.
A kind of bioprobe that can detect living cell membrane surface tension variations, nucleotide sequence:
A kind of bioprobe that can detect living cell membrane surface tension variations, amino acid sequence:

Claims (1)

1. a kind of bioprobe for detecting living cell membrane surface tension variations, which is characterized in that the bioprobe includes FRET fluorescin is to ECFP (1-2), YPet (1-4), elastomeric sequences Linker (1-3), Lyn sequence (1-1) and K-ras sequence Five parts (1-5) are sheared DNA sequence dna by Subcloned technology and are spliced, and form recombination matter with vector plasmid pcDNA3.1 (+) Grain；
Wherein, the amino acid sequence of Lyn are as follows:
AAATMGCIKSKRKDNLNDDGVDMKT
Its corresponding DNA sequence dna are as follows:
GCGGCCGCCACCATGGGCTGCATCAAGAGCAAGCGCAAGGACAACCTGAACGACGACGGCGTGGACATGAAG ACC；
The amino acid sequence of K-ras are as follows:
KKKKKKSKTKCVIM
Corresponding DNA sequence dna are as follows:
AAGAAGAAGAAGAAGAAGAGCAAGACCAAGTGCGTGATCATG；
The amino acid sequence of elastomeric sequences Linker are as follows:
GPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGA
Corresponding DNA sequence dna are as follows:
GGTCCAGGAGGCGCAGGACCTGGCGGGGCTGGACCGGGTGGCGCGGGACCCGGCGGAGCCGGCCCAGGTGGG GCGGGCCCTGGTGGTGCTGGTCCGGGAGGGGCAGGGCCCGGAGGTGCC。
CN201610752206.9A 2016-08-29 2016-08-29 A kind of bioprobe detecting living cell membrane surface tension variations Active CN106399338B (en)

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CN108120836B (en) * 2017-12-06 2020-08-14 大连理工大学 Fluorescent biological probe for detecting force transfer of Paxillin protein in living cells
CN111378050B (en) * 2018-12-29 2023-04-07 深圳先进技术研究院 Fusion protein and application thereof
CN110981943B (en) * 2019-12-02 2021-08-03 清华大学 Polypeptide, application thereof in preparation of medicine and medicine
CN114672503B (en) * 2022-03-21 2023-10-13 大连理工大学 Biological stress sensor for connecting microfilaments and local adhesive spots in living cells
CN115947866B (en) * 2022-09-28 2024-04-19 大连理工大学 FRET-based biological probe for detecting Paxillin protein activity in living cells and recombinant plasmid thereof

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 (en) * 2010-09-27 2013-07-31 国立大学法人京都大学 Linker for unimolecular FRET biosensor based on principle of fluorescence resonance energy transfer
CN105784656A (en) * 2016-03-16 2016-07-20 大连理工大学 Biological probe for detecting activity of RhoGDIalpha (Rho GDP dissociation inhibitor alpha) protein in living cell

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 (en) * 2010-09-27 2013-07-31 国立大学法人京都大学 Linker for unimolecular FRET biosensor based on principle of fluorescence resonance energy transfer
CN105784656A (en) * 2016-03-16 2016-07-20 大连理工大学 Biological probe for detecting activity of RhoGDIalpha (Rho GDP dissociation inhibitor alpha) protein in living cell

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