CN116064464A - Fluorescence pair based on FRET effect, probe comprising fluorescence pair and application of fluorescence pair - Google Patents

Fluorescence pair based on FRET effect, probe comprising fluorescence pair and application of fluorescence pair Download PDF

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CN116064464A
CN116064464A CN202211203313.8A CN202211203313A CN116064464A CN 116064464 A CN116064464 A CN 116064464A CN 202211203313 A CN202211203313 A CN 202211203313A CN 116064464 A CN116064464 A CN 116064464A
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camkii
fluorescence
probe
pair
fluorescence pair
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郑秋凌
郝海平
吕志文
郑啸
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China Pharmaceutical University
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Abstract

The invention provides a fluorescence pair based on a FRET effect, a probe containing the same and application thereof, wherein the fluorescence pair is Cerulean3 and Venus, and the invention also provides a probe containing the fluorescence pair, which can be used for detecting the activity of CaMKII in living cells and researching the accurate space-time regulation of the activity of the CaMKII in the living cells.

Description

Fluorescence pair based on FRET effect, probe comprising fluorescence pair and application of fluorescence pair
Technical Field
The invention belongs to the fields of cell biology and molecular biology, and particularly relates to a fluorescence pair based on a FRET effect, a probe containing the fluorescence pair and application of the fluorescence pair.
Background
CaMKII is calmodulin (Ca 2+ CaM) dependent serine/threonine kinase by sensing Ca 2+ Signaling plays an important role in cellular signaling. Removal ofAbout 30 splice variants were expressed in humans, along with four CaMKII isozymes (a, b, c, and d). Subtypes a and b are brain-specific, and subtypes c and d are expressed in most tissues, with more expressed in heart tissue. The research shows that CaMKII plays an important role in learning and memory, synapse formation, cardiovascular diseases and the like, so that the research on the accurate space-time regulation of the activity of the CaMKII in living cells is significant. At present, CAMUI based on FRET principle is often used as a sensor for detecting CaMKII activity in living cells, and traditional CAMUI adopts classical fluorescent protein pairs, including cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) as fluorescent pairs. .
The traditional camul design has the following disadvantages: fluorescence quantum yields of CFP and YFP are not high and pH sensitivity of the CFP and YFP is high; CFP has strong fluorescence switching phenomenon, affecting FRET ratio; when calcium chloride is commonly used as a source of calcium ions, YFP is sensitive to and quenched by chloride ions (Cl-). The above drawbacks have made CFP/YFP unusable for accurate spatial-temporal regulation studies of CaMKII activity in living cells.
Disclosure of Invention
The invention aims to provide a Cerulean3/Venus as a fluorescence pair to replace CFP/YFP and design a new CAMUI to replace the traditional CAMUI. Venus, a variant of YFP, folds well, is relatively tolerant to acidic environments and chloride exposure, and reduces the effect of chloride on fluorescence when living cells are detected with a calcium chloride reagent. Cerulean3 as a variant of CFP is brighter, a protein with a single fluorescence index, cerulea3 has higher fluorescence quantum yield and significantly reduced fluorescence switching behavior, and light is more stable than CFP.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a fluorescence pair based on FRET effect, said fluorescence pair being Cerulean3 and Venus.
A probe comprising the above fluorescent pair has the structure mVenus-CaMKII delta-mCerulean 3, see FIG. 1. Compared with the traditional CAMUI structure, the CAMUI structure of the invention replaces CFP with mVenus and YFP with mCerulean3. The CAMUI structure is that mCerulean3 is connected to the C end of CaMKII delta, mVenus is connected to the N end of CaMKII delta, and the amino acid sequence of the mVenus is shown as SEQ ID NO. 1.
Use of the probe for detecting the activity of CaMKII in living cells. The detection mechanism is shown in fig. 2: calcium ion/calmodulin (Ca) 2+ CaM) binds to the regulatory region (regulatory) of CaMKII, and the kinase region (catabolic) is separated from the regulatory region such that the CaMKII conformation changes, increasing the distance between mcrulean and mVenus, resulting in a decrease in FRET between the two fluorescent proteins.
A preparation method of the probe is a gene recombination method mature in the industry, specifically a gene taking a human CaMKII delta gene as a target gene, adding enzyme cutting sites before and after CDS of the target gene, and then recombining the target gene between two fluorescent protein gene sequences of mCerulean3 (SEQ ID NO. 2) and mVenus (SEQ ID NO. 3) so as to generate a fusion of mVenus-CaMKII delta-mCerulean 3, wherein the nucleotide sequence of the fusion is shown as SEQ ID NO. 4. Cloning the fusion sequence fragment on a plasmid vector to form a recombinant plasmid, transferring the recombinant plasmid into competent bacteria for cloning and copying, purifying the obtained plasmid, and sequencing and analyzing the fusion gene; and (3) transfecting the purified plasmid into a host cell for expression to obtain the probe.
As a further improvement of the technical scheme, the plasmid vector is a pCMV-Blank vector.
Compared with the prior art, the fluorescence pair has outstanding substantive characteristics and remarkable progress, and in particular, compared with the prior classical protein pair CFP/YFP, the fluorescence pair has high fluorescence quantum yield, low pH sensitivity of the fluorescence pair and the fluorescence quantum yield, stable FRET ratio and reduced sensitivity to chloride ions (Cl-). Furthermore, the probe provided by the invention can be used for accurate space-time regulation detection and research of CaMKII activity in living cells. In addition, the probe of the invention is prepared by adopting a conventional gene recombination method, and has simple operation and low cost.
Drawings
Fig. 1 is a schematic diagram of a conventional and inventive CAMUI structure.
FIG. 2 is a schematic diagram of the detection of CaMKII activity in living cells by CAMUI according to the present invention.
FIG. 3 is a graph showing the emission spectrum of transfected cells at an excitation wavelength of 430 nm.
FIG. 4 is a chart showing the change of FRET patterns of transfected cells.
Detailed Description
The technical scheme of the invention is further described in detail through the following specific embodiments.
Experimental methods for which specific conditions are not noted in the examples in this disclosure are generally in accordance with conventional conditions, or in accordance with conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.
EXAMPLE 1 detection of changes in CaMKII Activity in living cells under drug stimulation
Cell culture: 10 wells per well in 96 well plate 4 HEK293T cells were cultured with DMEM containing 10% FBS by volume for 12 hours and replaced with serum-free DMEM for 1 hour before transfection.
Transfection step: two sets of 1.5ml centrifuge tubes were prepared, each tube was filled with 50. Mu.L of serum-free DMEM, one set was filled with constructed plasmid pmVenus-CaMKII delta-mCerulean 3, gently blown with a pipette gun for 5 uses, the other set was filled with Genjet ™ in vitro DNA transfection reagent, gently blown with a pipette gun for 5 uses, and left to stand for 5min at a desired plasmid to transfection reagent ratio of 1. Mu.g: 2.5. Mu.L. After 5min of standing, a group of DMEM mixed solution containing pmVenus-CaMKII delta-mCerulean 3 constructed plasmids was transferred to a group of DMEM mixed solution containing transfection reagents, gently blown under 5 using a pipette, mixed well and stood for 15min, 96-well cell culture plates were taken out of a cell incubator, 10. Mu.L of mixed solution was added to each well, 10 wells were added, and 100ng of constructed plasmids were contained in each well, and the above steps required complete light shielding. After the addition, the 96-well cell plates are placed in a cell incubator again for 18 hours, and the complete medium is replaced for further 18 hours for further culture. The recombinant plasmid pmVenus-CaMKII delta-mCerulean 3 adopts CMV-Blank vector of Biyun biotechnology Co., ltd, and the recombination step comprises preparing linearization vector (enzyme cutting or PCR); amplifying a target fragment with a homology arm; mixing the carrier, the fragment and 2× GenRec Master Mix (2×GenRec kit), and reacting at 50deg.C for 15-30 min; and (3) converting and coating a flat plate. The method mainly comprises the following steps of recombination reaction, transformation and clone identification, and comprises the following operation steps:
(1) 2X GenRec Master Mix thawing on ice, and mixing; adding the carrier and the fragments into a sterile tube, wherein the addition amount of the fragments (ng) =0.02×base pair number (bp) of the fragments (the corresponding concentration of the fragments is 0.03 pmol), supplementing to 10 μl with sterile water, adding 10 μl of 2× GenRec Master Mix, and gently sucking and beating with a pipette to avoid bubble generation; reacting at 50deg.C for 15-30min, and placing on ice for 3-5min. (2) conversion: chemically transformed competent cells were thawed on ice: 10. Mu.L of the reaction solution was added to competent cells and mixed well. Ice bath for 10min: placing the centrifuge tube in a water bath at 42 ℃ for heat shock for 90s, and then rapidly placing the centrifuge tube back on ice to cool the cells for 2-3 min: adding 600 mu L of preheated sterile LB (or SOC) culture solution into the centrifuge tube, and carrying out constant-temperature shaking culture at 37 ℃ for 45-60 min; the supernatant was discarded by brief centrifugation, 100. Mu.L of fresh LB medium was aspirated for resuspension, and the bacterial solution was spread evenly on plates containing the appropriate antibiotics. The plates were inverted and incubated overnight at 37 ℃. (3) clone identification, colony PCR. Single colonies were picked up to 20-50. Mu.L of LB medium with sterile tips or toothpicks and mixed well, 1. Mu.L was directly used as PCR template, and 2X Taq DNAPolymerase Master progressive colony PCR experiments were used.
Drug stimulation experiments:
experimental principle: ionomycin is an ionophore with affinity for calcium ions, which can be brought from outside the cell into the cytosol or released from a calcium reservoir within the cell into the cytosol. Under the action of ionomycin, the concentration of free calcium ions in cells can be increased, and calcium ions and calmodulin can be promoted to form more complexes, namely Ca 2+ /CaM。Ca 2+ CaM is an important factor for activating CaMKII delta, and various activation pathways of CaMKII delta are basically required to activate CaMKII delta by Ca 2+ Binding of/CaM is premised, thereby inducing Ca 2+ The CaM complex is combined with the CaMKII delta peptide chain in the novel CAMUI to promote the opening of the CAMUI peptide chain, the regulation region of the CaMKII delta is separated from the catalytic region, the catalytic region is exposed, the activity of the CaMKII delta is increased, the distance between two fluorescent proteins is increased, and the FRET phenomenon is reduced.
The experimental steps are as follows: the ionomycin solution is added into each hole, so that the concentration of the ionomycin in the hole is 4 mu mol/L, and the mixture is uniformly mixed and cultured in a cross way. FRET ratios were measured 1, 6, 12, 24 hours before dosing, respectively.
Instrument measurement: the medium in the wells was discarded before measurement and replaced with 100. Mu.L of extracellular fluid (130 mmol/LNaCl,20 mmol/LHEPES,2 mmol/LNaHCO) 3 ，25mmol/LD-Glucose，2.5mmol/LKCl，1.25mmol/LNaH 2 PO 4 ，0.8 mmol/LMgCl 2 ，1.8 mmol/LCaCl 2 ) Emission spectra of excitation light wavelengths 430 at 1, 6, 12, 24 hours prior to dosing were measured using a BioTek Synergy2 microplate reader, respectively, as shown in fig. 3.
Data processing and analysis: FRET ratios were detected and calculated using a microplate reader. FRET measurement method: FRET ratio = a/B, a is 430 excitation measurement 530 emission intensity, B is 430 excitation measurement 480 emission intensity. Higher FRET ratios indicate more CaMKII is inactive, lower CaMKII is active. The results show that the CAMUI has obvious FRET phenomenon, the activity of CaMKII in the CAMUI is activated after ionomycin is added, the FRET ratio is reduced, the novel CAMUI is successfully constructed, and the method can be used for observing the influence of different drugs on the activity of intracellular CaMKII at different time points.
Example 2 detection of the spatiotemporal State of real-time CaMKII activation in living cells
Cell culture: HEK293T cells are cultured by adopting a special culture dish D35-20-0-N (small dish diameter 35 mm-bottom surface hole diameter 20mm-0 slide thickness 0.085-0.115 mm) with the laser confocal, and 10 parts of each culture dish are added 5 Cells were incubated with DMEM containing 10% FBS by volume for 12 hours and replaced with serum-free DMEM for 1 hour prior to transfection.
Transfection step: 100 mu L of the plasmid-liposome mixture was added to the dish, and the mixture was gently shaken in a cross manner, placed in a cell incubator, and cultured for 18 hours, followed by replacing the complete medium and further culturing for 18 hours.
Instrument measurement: transfected cells were photographed in real time using an LSM700 laser confocal microscope, and three detection channels were set up for 405/488 laser light and 405-488 (donor), 488-530 (acceptor), 405-530 (FRET) and time series for 15min, 30s apartShooting, and adding CaCl 2 The extracellular solution was brought to a calcium ion concentration of 10mM, and the resulting map was photographed after 15 min.
Data processing and analysis: at the time of adding CaCl 2 After the extracellular liquid calcium ions reach a certain concentration, the bright areas in the FRET images of the transfected cells, which are observed within 15 minutes, are distributed in cytoplasm and obviously reduced in the FRET images of the transfected cells, which is shown that the FRET phenomenon is reduced. As shown in fig. 4, the bright areas are distributed in the cytoplasm, the bright areas decrease in brightness with time, and the bright areas decrease in size significantly. That is, the concentration of calcium ions in cells is increased, the concentration of calcium ions combined with calmodulin to form a complex is increased, and the interaction between the complex and CaMKII is promoted to induce the CaMKII to change from an inhibition state to an activation state. The method can observe the activity state and distribution of CaMKII in living cells in real time, and is helpful for understanding the pathological mechanism of CaMKII.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same; while the invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that the present invention may be modified and equivalents substituted for elements thereof; without departing from the spirit of the invention, it is intended to cover the scope of the invention as claimed.

Claims (5)

1. Fluorescence pair based on FRET effect, characterized in that said fluorescence pair is Cerulean3 and Venus.
2. A probe comprising the fluorescent pair of claim 1, which has the structure of Venus-CaMKII-Cerulean3, wherein Cerulean3 is attached to the C-terminus of CaMKII, and Venus is attached to the N-terminus of CaMKII, and has the amino acid sequence shown in SEQ ID No. 1.
3. Use of a probe according to claim 2 for detecting the activity of CaMKII in living cells.
4. A method of preparing a probe according to claim 2, wherein the human CaMKII delta gene is used as a target gene, which is cloned between two fluorescent protein genes of mveulan 3 and mveuns, thereby producing a fusion of mveuns-CaMKII delta-mveulan 3; cloning the construct on a plasmid vector to form a recombinant plasmid; transferring the recombinant plasmid into competent bacteria for cloning and copying, purifying the obtained plasmid, and sequencing and analyzing the fusion gene; and expressing the purified pelletization transfected host cells to obtain the probe.
5. The method of claim 4, wherein the granulation carrier is pCMV-Blank carrier.
CN202211203313.8A 2022-09-29 2022-09-29 Fluorescence pair based on FRET effect, probe comprising fluorescence pair and application of fluorescence pair Pending CN116064464A (en)

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