CN108120836B - Fluorescent biological probe for detecting force transfer of Paxillin protein in living cells - Google Patents
Fluorescent biological probe for detecting force transfer of Paxillin protein in living cells Download PDFInfo
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Abstract
The invention belongs to the technical field of cell biology and molecular biology, and particularly relates to a fluorescent biological probe for detecting the force transmission of a Paxillin protein in a living cell, which is a biological probe based on FRET technology and can dynamically detect the force transmission change of the Paxillin protein in the living cell. The probe comprises five parts of a FRET fluorescent protein pair ECFP and YPet, an elastic sequence Linker, an Ld12 sequence and a Lim1234 sequence. After the probe and the recombinant plasmid of pcDNA3.1(+) are transfected into a living cell, a fluorescent protein reconstitution fusion probe can be spontaneously expressed in the cell and automatically integrated into a local adhesion spot region where the Paxillin protein is located, the force transmission change of the Paxillin protein in the living cell can be qualitatively detected, and no toxic or side effect is caused on the cell.
Description
Technical Field
The invention belongs to the technical field of cell biology and molecular biology, and particularly relates to a fluorescent biological probe for detecting the force transfer of Paxillin protein in living cells, which is designed and prepared based on Fluorescence Resonance Energy Transfer (FRET) technology and conventional biological engineering subcloning technology. The probe structure of the kit can be expressed in living cells by self, and qualitatively reflects the change of the force transmission of the Paxillin protein in the living cells through the change of a fluorescent signal.
Background
Abnormal responses of cells after exposure to external forces are associated with certain diseases. The external force acts on the cell membrane of the living cell and then is physically transmitted in the cell through structures such as cytoskeletal proteins connected with the cell membrane. The Paxillin protein is an important structural protein on local focal adhesion spots in cells, and the transmission of force is directly related to the dynamic change of the local focal adhesion spots of the cells. At present, no effective method for detecting force transfer on a Paxillin protein in a living cell can be realized. In view of this, the invention provides a fluorescence biological probe which is based on FRET technology and is applied to detecting the force transmission of the Paxillin protein in living cells, and the fluorescence biological probe has the advantages of no toxicity to cells, dynamic detection, high time resolution, low cost and the like.
Disclosure of Invention
The invention provides a novel fluorescent biological probe capable of detecting the force transmission of Paxillin protein in living cells, wherein a FRET technology is combined with a conventional biological engineering subcloning technology in design, and the specific technical scheme is as follows:
a fluorescent biological probe for detecting force transmission of a Paxillin protein in a living cell comprises five parts of a FRET fluorescent protein pair ECFP (1-2) and YPet (1-4), an elastic sequence Linker (1-3), a structural domain Ld12 sequence (1-1) of the Paxillin protein and another structural domain Lim1234 sequence (1-5) of the Paxillin protein, wherein the DNA sequences of the five parts are sheared, spliced and reconstructed by using a subcloning technology, and form a recombinant plasmid together with pcDNA3.1 (+).
Wherein the Ld12 amino acid sequence is:
DLDALLADLESTTELDRLLLELNAVQ
the corresponding DNA sequence is:
GCGGCCGCCACCATGGGCTGCATCAAGAGCAAGCGCAAGGACAACCTGAACGACGACGGCGTGGACATGAAGACC
the Lim1234 amino acid sequence is:
CGACKKPIAGQVVTAMGKTWHPEHFVCTHCQEEIGSRNFFERDGQPYCEKDYHCYYCNGPILDKVVTALDRTWHPEHFFCAQCGAFFGPEGFHEKDGKAYCRKDYCGGCARAILENYISALNTLWHPECFVCRECFTPFVNGSFFEHDGQPYCEVHYHCSGCQKPITGRCITAMAKKFHPEHFVCAFCLKQLNKGTFKEQNDKPYCQNCF
the corresponding DNA sequences were:
TGCGGCGCCTGCAAGAAGCCCATCGCCGGCCAGGTGGTGACCGCCATGGGCAAGACCTGGCACCCCGAGCACTTCGTGTGCACCCACTGCCAGGAGGAGATCGGCAGCAGGAACTTCTTCGAGAGGGACGGCCAGCCCTACTGCGAGAAGGACTACCACTGCTACTACTGCAACGGCCCCATCCTGGACAAGGTGGTGACCGCCCTGGACAGGACCTGGCACCCCGAGCACTTCTTCTGCGCCCAGTGCGGCGCCTTCTTCGGCCCCGAGGGCTTCCACGAGAAGGACGGCAAGGCCTACTGCAGGAAGGACTACTGCGGCGGCTGCGCCAGGGCCATCCTGGAGAACTACATCAGCGCCCTGAACACCCTGTGGCACCCCGAGTGCTTCGTGTGCAGGGAGTGCTTCACCCCCTTCGTGAACGGCAGCTTCTTCGAGCACGACGGCCAGCCCTACTGCGAGGTGCACTACCACTGCAGCGGCTGCCAGAAGCCCATCACCGGCAGGTGCATCACCGCCATGGCCAAGAAGTTCCACCCCGAGCACTTCGTGTGCGCCTTCTGCCTGAAGCAGCTGAACAAGGGCACCTTCAAGGAGCAGAACGACAAGCCCTACTGCCAGAACTGCTTC
the Linker amino acid sequence is as follows:
GPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGAGPGGA
the corresponding DNA sequences were:
GGTCCAGGAGGCGCAGGACCTGGCGGGGCTGGACCGGGTGGCGCGGGACCCGGCGGAGCCGGCCCAGGTGGGGCGGGCCCTGGTGGTGCTGGTCCGGGAGGGGCAGGGCCCGGAGGTGCC
the application of the fluorescent biological probe for detecting the force transfer of the Paxillin protein in the living cell comprises the steps of transfecting the probe into the living cell, automatically expressing a fluorescent protein reconstructed fusion probe structure, automatically integrating the fluorescent protein reconstructed fusion probe structure on a local adhesion spot where the Paxillin protein is located, and dynamically detecting and analyzing the dynamic efficiency change of energy transfer in the living cell by using a FRET (fluorescence resonance energy transfer) fluorescence microscope, so that the change of the force transfer of the Paxillin protein in the living cell is detected.
The invention has the beneficial effects that:
the probe realizes dynamic detection of force transfer on intracellular Paxillin protein in living cells, has the characteristics of low cost, no toxic or side effect on cells, dynamic detection and the like, and provides a visual tool for researching intracellular force transfer.
Drawings
FIG. 1(a) is a schematic view of a Paxillin protein force transmission detection probe a.
FIG. 1(b) is a schematic diagram b of a Paxillin protein force transmission detection probe.
Detailed Description
The following further describes a specific embodiment of the present invention with reference to the drawings and technical solutions.
After the probe is transfected into a living cell body by using a transfection reagent, the cell can express a reconstructed fusion fluorescent protein, and Ld12(1-1) and Lim1234(1-5) sequences at two ends are respectively connected with a cytoskeletal protein and a signal protein so as to be automatically integrated to the position of the original Paxillin protein on a local focal adhesion. When external force is applied and the force is transmitted to the Paxillin protein part replaced by the probe in the local adhesive spot, the Linker (1-3) contained in the probe is stressed, deformed, contracted or stretched, and the fluorescent protein changes the distance between ECFP (1-2) and YPet (1-4), so that the FRET efficiency is changed. Giving excitation light with 415nm wavelength to the transfected cells, simultaneously acquiring fluorescence images with 485nm and 535nm wavelength by using a FRET microscope, and analyzing dynamic efficiency change of energy transfer by using a 485nm/535nm fluorescence intensity ratio so as to obtain information of intracellular Paxillin protein force transfer.
Sequence listing
<110> university of Large Community
<120> fluorescent bioprobe for detecting force transfer of Paxillin protein in living cells
<130>2017
<141>2017-12-06
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>901
<212>PRT
<213> Artificial sequence (human)
<400>1
Met Asp Asp Leu Asp Ala Leu Leu Ala Asp Leu Glu Ser Thr Thr Ser
1 5 10 15
His Ile Ser Lys Arg Pro Val Phe Leu Ser Glu Glu Thr Pro Tyr Ser
20 25 30
Tyr Pro Thr Gly Asn His Thr Tyr Gln Glu Ile Ala Val Pro Pro Pro
35 40 45
Val Pro Pro Pro Pro Ser Ser Glu Ala Leu Asn Gly Thr Ile Leu Asp
50 55 60
Pro Leu Asp Gln Trp Gln Pro Ser Ser Ser Arg Phe Ile His Gln Gln
65 70 75 80
Pro Gln Ser Ser Ser Pro Val Tyr Gly Ser Ser Ala Lys Thr Ser Ser
85 90 95
Val Ser Asn Pro Gln Asp Ser Val Gly Ser Pro Cys Ser Arg Val Gly
100 105 110
Glu Glu Glu His Val Tyr Ser Phe Pro Asn Lys Gln Lys Ser Ala Glu
115 120 125
Pro Ser Pro Thr Val Met Ser Thr Ser Leu Gly Ser Asn Leu Ser Glu
130 135 140
Leu Asp Arg Leu Leu Leu Glu Leu Gly Ser Met Val Ser Lys Gly Glu
145 150 155 160
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
165 170 175
Val Asn Gly His Arg Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
180 185 190
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu
195 200 205
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Trp Gly Val Gln
210 215 220
Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
225 230 235 240
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
245 250 255
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
260 265 270
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
275 280 285
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Ile Ser His Asn
290 295 300
Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala His Phe
305 310 315 320
Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
325 330 335
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
340 345 350
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
355 360 365
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Glu Leu
370 375 380
Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly
385 390 395 400
Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro
405 410 415
Gly Gly Ala Gly Pro Gly Gly Ala Met Ser Lys Gly Glu Glu Leu Phe
420 425 430
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
435 440 445
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
450 455 460
Lys Leu Thr Leu Lys Leu Leu Cys Thr Thr Gly Lys Leu Pro Val Pro
465 470 475 480
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Val Gln Cys Phe Ala
485 490 495
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
500 505 510
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
515 520 525
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
530 535 540
Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile
545 550 555 560
Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile
565 570 575
Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
580 585 590
His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr Gln Gln
595 600 605
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
610 615 620
Leu Ser Tyr Gln Ser Ala Leu Phe Lys Asp Pro Asn Glu Lys Arg Asp
625 630 635 640
His Met Val Leu Leu Glu Phe Leu Thr Ala Ala Gly Ile Thr Glu Gly
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Met Asn Glu Leu Tyr Lys Glu Phe Lys Gly Val Cys Gly Ala Cys Lys
660 665 670
Lys Pro Ile Ala Gly Gln Val Val Thr Ala Met Gly Lys Thr Trp His
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Pro Glu His Phe Val Cys Thr His Cys Gln Glu Glu Ile Gly Ser Arg
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Asn Phe Phe Glu Arg Asp Gly Gln Pro Tyr Cys Glu Lys Asp Tyr His
705 710 715 720
Asn Leu Phe Ser Pro Arg Cys Tyr Tyr Cys Asn Gly Pro Ile Leu Asp
725 730 735
Lys Val Val Thr Ala Leu Asp Arg Thr Trp His Pro Glu His Phe Phe
740 745 750
Cys Ala Gln Cys Gly Ala Phe Phe Gly Pro Glu Gly Phe His Glu Lys
755 760 765
Asp Gly Lys Ala Tyr Cys Arg Lys Asp Tyr Phe Asp Met Phe Ala Pro
770 775 780
Lys Cys Gly Gly Cys Ala Arg Ala Ile Leu Glu Asn Tyr Ile Ser Ala
785 790 795 800
Leu Asn Thr Leu Trp His Pro Glu Cys Phe Val Cys Arg Glu Cys Phe
805 810 815
Thr Pro Phe Val Asn Gly Ser Phe Phe Glu His Asp Gly Gln Pro Tyr
820 825 830
Cys Glu Val His Tyr His Glu Arg Arg Gly Ser Leu Cys Ser Gly Cys
835 840 845
Gln Lys Pro Ile Thr Gly Arg Cys Ile Thr Ala Met Ala Lys Lys Phe
850 855 860
His Pro Glu His Phe Val Cys Ala Phe Cys Leu Lys Gln Leu Asn Lys
865870 875 880
Gly Thr Phe Lys Glu Gln Asn Asp Lys Pro Tyr Cys Gln Asn Cys Phe
885 890 895
Leu Lys Leu Phe Cys
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Claims (2)
1. A fluorescent biological probe for detecting the force transmission of a Paxillin protein in a living cell is characterized in that the probe comprises five parts of a FRET fluorescent protein pair ECFP (1-2) and YPet (1-4), an elastic sequence Linker (1-3), a structural domain Ld12 sequence (1-1) of the Paxillin protein and another structural domain Lim1234 sequence (1-5) of the Paxillin protein, the DNA sequences of the five parts are sheared, spliced and reconstructed by using a subcloning technology to obtain a DNA sequence coding an amino acid sequence shown as SEQ ID NO. 1, and the DNA sequence and pcDNA3.1(+) form a recombinant plasmid.
2. The use of the fluorescent bioprobe for detecting the force transmission of Paxillin protein in living cells as claimed in claim 1, wherein the probe is transfected into living cells, self-expresses a fluorescent protein reconstituted fusion probe structure, automatically integrates the fluorescent protein reconstituted fusion probe structure on local focal adhesion where Paxillin protein is located, and dynamically detects and analyzes the dynamic efficiency change of energy transfer in living cells by using FRET fluorescence microscope, thereby detecting the change of the force transmission of Paxillin protein in living cells.
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| CN113292660B (en) * | 2021-05-25 | 2023-03-24 | 大连理工大学 | Biological probe for detecting directional differentiation state of mesenchymal stem cells |
| CN113321741B (en) * | 2021-05-27 | 2023-01-06 | 大连理工大学 | Biological probe for detecting stress transfer between cell membrane and skeleton of living cell |
| CN113234719B (en) * | 2021-06-08 | 2022-04-29 | 重庆医科大学 | Heritable fluorescent probe for detecting mitochondrial membrane potential and application thereof |
| 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 |
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