CN107677655A - Rhodopsin fluorescence molecule complex and its preparation method and application - Google Patents
Rhodopsin fluorescence molecule complex and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of rhodopsin fluorescence molecule complex and its preparation method and application.Fluorescent small molecule is connected in N-terminal or the extracellular loop regions of insertion rhodopsin of rhodopsin by the present invention, provide a kind of spatial sensitivity height, speed is fast, brightness is high, there is the memebrane protein fluorescence molecule complex of spatial resolution and cell-specific simultaneously, and be applied to as film potential fluorescence probe in cell membrane potential and cell connecting detection.
Description
Technical field
The present invention relates to memebrane protein and fluorescence molecule complex, more particularly to a kind of rhodopsin-fluorescence molecule complex
And preparation method thereof, and its application in film potential and cell connecting detection.
Background technology
Film potential is prevalent in diversified cell, such as nerve cell, cardiac muscle cell, lymphocyte, cancer cell
Deng.Film potential is the important parameter and index of cell activities, and important Regulate signal, as neurotransmission, cell increase
Grow, cell differentiation and migration etc..By the detection of film potential, we can monitor the nervous activity of biology, heart state and thin
A variety of physiological activities such as born of the same parents' increment.Diagnosis for disease has very important significance.
The protein fluorescence that existing film potential detection method mainly has electro physiology, chemical dye probe and gene code is visited
Pin.Electro physiology directly records film potential by electrode, and chemical dye probe and protein fluorescence probe are read glimmering by microscope
The change of luminous intensity records film potential indirectly.But these three technologies have the shortcomings that respective.For example electro physiology detection does not have
Spatial resolution, chemical dye probe can not realize that cell-specific detects, and protein fluorescence probe has sensitivity, speed, bright
The various problems such as degree.
The content of the invention
The purpose of the present invention is to overcome existing technological deficiency, there is provided a kind of spatial sensitivity is high, speed is fast, brightness is high,
There is the memebrane protein of spatial resolution and cell-specific-fluorescence molecule complex simultaneously, and visited as film potential fluorescence
Pin is applied in cell membrane potential and cell connecting detection.
In memebrane protein provided by the invention-fluorescence molecule complex, the memebrane protein is rhodopsin (rhodopsin),
The albumen is seven transmembrane proteins, extracellular to have four regions, and the fluorescence molecule is connected to any in this four extracellular regions
On region.
As shown in figure 1, the parallel parallel bars of black represent cell membrane, parallel bars intermediate region represents intermembrane space, parallel bars top area
Domain is extracellular regions, and parallel bars lower zone is cytoplasmic region；Grey curves represent the rhodopsin of seven cross-films, black ball generation
Table is connected to rhodopsin N-terminal or the peptide sequence inserted in the extracellular loop regions of rhodopsin, and black asterisk represents glimmering
Optical molecule.
In some examples of the present invention, the peptide sequence is LAP peptide sequences, preferably LAP2 peptide sequences；Institute
It can be any in AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3 and Cy5 to state fluorescence molecule
One kind, added by the chemiluminescence small molecule with alkynyl and small molecule azido-methyl pyridine (pAz) initial ring containing azido
It is covalently attached into reaction on LAP peptide sequences, specific reaction principle can be found in Fig. 2.The first step, in the extracellular region of rhodopsin
The LAP peptide sequences of one section of 13 amino acid are added in the N-terminal in domain or extracellular loop；Second step, by LplA enzymes (LAP sequences
Special identification enzyme) and LplA enzymes substrate pAz and express rhodopsin-LAP cell be incubated, now, LplA meetings
PAz is connected in LAP sequences；3rd step, because pAz is the small molecule containing azido, it is possible to containing alkynyl
Cycloaddition reaction occurs for molecule, incubates the chemiluminescence small molecule with alkynyl and the reaction product of second step in this step
Educate, rhodopsin-LAP-pAz- fluorescent small molecules will be formed.
In other examples of the present invention, the peptide sequence is AP peptide sequences；The fluorescence molecule be selected from cy3,
Any one in Alex-488, Alex-568, Alex-594 and cy5, can pass through Streptavidin (streptavidin)
Combination between biotin (biotin) connects with AP peptide sequences.First in the extracellular regions (N of rhodopsin
In end or extracellular loop) the AP peptide sequences of one 15 amino acid of insertion, the sequence can identify by BirA enzymes, BirA meetings
Biotin is connected in AP sequences, then will be connected to Streptavidin and the cell expression of fluorescent small molecule
Rhodopsin-AP-biotin is incubated, and by the combination of Streptavidin and biotin, fluorescent small molecule just may be connected to
Above rhodopsin.
In other examples of the present invention, the peptide sequence is Halotag protein tags, and the fluorescence molecule is selected from
Any one in cy3, Alex-488, Alex-568, Alex-594 and cy5.Inserted first in the extracellular regions of rhodopsin
One Halotag label protein, then the rhodopsin-Halotag of fluorescent small molecule and cell expression is incubated, tied
Structure is the complex of rhodopsin-Halotag- fluorescent small molecules.
In other examples of the present invention, the peptide sequence is SNAPtag protein tags, and the fluorescence molecule is selected from
Any one in cy3, Alex-488, Alex-568, Alex-594 and cy5.Inserted first in the extracellular regions of rhodopsin
One SNAPtag label protein, then the rhodopsin-SNAPtag of fluorescent small molecule and cell expression is incubated, tied
Structure is the complex of rhodopsin-SNAPtag- fluorescent small molecules.
The preparation method of rhodopsin provided by the invention-fluorescence molecule complex comprises the following steps：
1) expression vector containing rhodopsin-peptide sequence antigen-4 fusion protein gene is built, polypeptide in the fusion protein
Sequence is connected to rhodopsin N-terminal or is inserted into the extracellular any loop regions of rhodopsin；
2) expression vector that step 1) is built is transferred in cell and expressed；
3) fluorescence molecule mark is carried out to the cell for expressing the rhodopsin-peptide sequence fusion protein, obtains visual purple
Red matter-fluorescence molecule complex.
In the preparation of " rhodopsin-LAP- fluorescent small molecules " complex, step 1) is by molecule clone technology by LAP
In polypeptide insertion rhodopsin extracellular regions, and by the rhodopsin-LAP antigen-4 fusion protein genes structure on expression vector；Step
Rapid 2) expression vector is transferred in cell is expressed；Step 3) regards LplA enzymes, substrate azido-methyl pyridine (pAz) with expression
The cell of rhodopsin-LAP fusion proteins is incubated, and picolyl azide is connected on LAP polypeptides, then washes away residual
Reagent, then into cell add the fluorescent small molecule AlexaFluor 488 containing alkynyl, AlexaFluor 568,
Any one in AlexaFluor 594, Cy3 and Cy5 is incubated, and makes fluorescence molecule that cycloaddition reaction, shape occur with pAz
Into " rhodopsin-LAP- fluorescent small molecules " complex.
In the preparation of " rhodopsin-AP- fluorescent small molecules " complex, step 1) is more by AP by molecule clone technology
In peptide insertion rhodopsin extracellular regions, and by the rhodopsin-AP antigen-4 fusion protein genes structure on expression vector；Step 2)
The BirA biotin ligase expression vectors that rhodopsin-AP fusion protein expression vectors and ER chambers position are transferred to cell jointly
In expressed, and biotin is added in cell culture medium；Step 3) adds to the cell of expression rhodopsin-AP fusion proteins
Add fluorescent small molecule AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, the Cy3 that streptavidin connects
With any one in Cy5, " rhodopsin-AP- fluorescent small molecules " complex is obtained.Wherein, step 2) rhodopsin-AP melts
When hop protein expression vector and BirA biotin ligase expression vector cotransfections, the mass ratio of two kinds of expression vectors is preferably 1:
1。
In the preparation of " rhodopsin-Halotag- fluorescent small molecules " complex, step 1) passes through molecule clone technology
Halotag is connected to the N-terminal of rhodopsin albumen, and the rhodopsin-Halotag antigen-4 fusion protein genes structure is being expressed
On carrier；Rhodopsin-Halotag fusion protein expression vectors are transferred in cell and expressed by step 2)；Step 3) is to table
Up to the cell addition fluorescent small molecule AlexaFluor 488 of rhodopsin-Halotag fusion proteins, AlexaFluor 568,
Any one in AlexaFluor 594, Cy3 and Cy5, obtains " rhodopsin-Halotag- fluorescent small molecules " complex.
In the preparation of " rhodopsin-SNAPtag- fluorescent small molecules " complex, step 1) passes through molecule clone technology
SNAPtag is connected to the N-terminal of rhodopsin albumen, and the rhodopsin-SNAPtag antigen-4 fusion protein genes structure is being expressed
On carrier；Rhodopsin-SNAPtag fusion protein expression vectors are transferred in cell and expressed by step 2)；Step 3) is to table
Up to the cell addition fluorescent small molecule AlexaFluor 488 of rhodopsin-SNAPtag fusion proteins, AlexaFluor 568,
Any one in AlexaFluor 594, Cy3 and Cy5, obtains " rhodopsin-SNAPtag- fluorescent small molecules " complex.
In above-mentioned preparation method, the cell is preferably 293T cells.Incubation time is not to be exceeded after 293T cell recoveries
60 days.The cell of longer time can reduce transfection efficiency, reduction flag effect.Density during 293T cell transfectings is 70-90%,
Transfection reagent can be calcium phosphate, Lipofectamine2000, PEI any one.Lower cell density can increase transfection
Toxicity, transfection efficiency is reduced, causes cell death.
The experiment of the present invention finds, after 293T cell transfectings 24 hours carry out fluorescence molecule marks, it can be seen that it is obvious
Marking signal.Less than 20 hours, or it is longer than 48 hours, marking signal can be weakened.
In above-mentioned preparation method, step 3) fluorescence molecule mark is initial, all fluorescence signals on cell membrane, with
The growth of time, the interior appearance fluorescence signal that can be different degrees of of kytoplasm, the signal is caused by kytoplasm endocytosis effect.Wherein " visual purple
Mark 30 minutes or so can in prepared by red matter-Halotag- fluorescent small molecules " and " rhodopsin-SNAPtag- fluorescent small molecules "
To see obvious kytoplasm endocytosis fluorescence signal, and " rhodopsin-LAP- fluorescent small molecules " and " rhodopsin-AP- fluorescence is small
In molecular labeling " preparation method, fluorescence signal is gulped down without obvious endoplasm in 120 minutes.
In above-mentioned preparation method, between different labeling methods, the intensity of fluorescence signal has bigger difference.Wherein, " depending on
Fluorescence signal in the preparation method of rhodopsin-Halotag- fluorescent small molecules " and " rhodopsin-SNAPtag- fluorescent small molecules "
Relatively strong, fluorescence signal is general in the preparation method of " rhodopsin-LAP- fluorescent small molecules ", " small point of rhodopsin-AP- fluorescence
Fluorescence signal is weaker in son " preparation method.
The basis in " rhodopsin-LAP- fluorescent small molecules " and " rhodopsin-AP- fluorescent small molecules " preparation method
The difference in the site of LAP or AP polypeptides insertion, different degrees of change occurs in the intensity and labeling effciency of fluorescence signal.
Rhodopsin provided by the invention-fluorescence molecule complex can be used for film potential detection, can be also used for cell
Connecting detection.
There is high sensitivity, speed in film potential record using rhodopsin provided by the invention-fluorescence molecule complex
Degree is fast, brightness is high has the advantages that spatial resolution and cell-specific simultaneously, can long-time tracking and monitoring cell film electricity
Position.
Brief description of the drawings
Fig. 1 is the structural representation of memebrane protein-fluorescence molecule complex of the present invention, is fluorescence molecule respectively from left to right
The situation in rhodopsin N-terminal and three extracellular Loop regions is connected to, wherein：The parallel parallel bars of black represent cell membrane, parallel bars
Intermediate region represents intermembrane space, and parallel bars upper area is extracellular regions, and parallel bars lower zone is cytoplasmic region；Grey curves represent
The rhodopsin of seven cross-films, black ball, which represents, to be connected to rhodopsin N-terminal or inserts in rhodopsin extracellular regions
Catenation sequence, black asterisk represents fluorescence molecule.
Fig. 2 is that the chemiluminescence small molecule with alkynyl is connected into LAP polypeptides by the small molecule pAz containing azido
Schematic diagram in sequence.
Fig. 3 is that 293T cells are prepared " rhodopsin-LAP2 amalgamation and expressions+fluorescent small molecule marks " using embodiment one
The fluorogram of fluorescence probe.
Fig. 4 is 293T cells using embodiment two, and to prepare " rhodopsin-AP amalgamation and expressions+fluorescent small molecule marks " glimmering
The fluorogram of light probe.
Fig. 5 is that 293T cells prepare " rhodopsin-Halotag amalgamation and expressions+fluorescent small molecule mark using embodiment three
The fluorogram of note " fluorescence probe.
Fig. 6 is that 293T cells prepare " rhodopsin-SNAPtag amalgamation and expressions+fluorescent small molecule mark using example IV
The fluorogram of note " fluorescence probe.
Fig. 7 is that 293T cells use " rhodopsin-LAP2 amalgamation and expressions+fluorescent small molecule marks " fluorescence in embodiment five
Probe application when film potential detects fluorescence with voltage change curve map.
Fig. 8 is that 293T cells use " rhodopsin-AP amalgamation and expressions+fluorescent small molecule marks " fluorescence to visit in embodiment six
Pin be applied to film potential detect when fluorescence with voltage change curve map.
Fig. 9 is that 293T cells use " rhodopsin-Halotag amalgamation and expressions+fluorescent small molecule marks " in embodiment seven
Fluorescence probe be applied to film potential detect when fluorescence with voltage change curve map.
Figure 10 is that 293T cells use " rhodopsin-SNAPtag amalgamation and expressions+fluorescent small molecule marks " in embodiment eight
Fluorescence probe be applied to film potential detect when fluorescence with voltage change curve map.
Figure 11 is that 293T cells use " rhodopsin-LAP2 amalgamation and expressions+fluorescent small molecule marks " glimmering in embodiment nine
Light probe is applied in cell connecting detection；Wherein, the fluorescent microscopic imaging figure after A. fluorescence probes mark；B. select in cell
Fluorescence with voltage change curve map；C and D corresponds respectively to A and B, but is connected with drugs block cells.
Embodiment
The present invention is described in further detail below by embodiment, but it should be appreciated by those skilled in the art these realities
Example is applied to be only illustrative of the invention and is not intended to limit the scope of the invention.The experiment of unreceipted actual conditions in the following example
Methods and techniques, generally carried out according to the normal condition of art or according to the condition proposed by manufacturer.
In following examples, the producer of reagent used is as shown in the table：
In following examples, the sequence of primer used is as shown in the table：
The preparation of embodiment one, " rhodopsin-LAP2 amalgamation and expressions+fluorescent small molecule mark ".
First, LAP2 polypeptides are inserted into rhodopsin albumin A ce.Pass through molecular cloning method using following primer pair
Build pcDNA3.1-pHluorin (Y65H)-Ace-mOrange2 (Y71A) mammalian cell expression vector：pcDNA3.1-
PHluorin-Ace junction F and pcDNA3.1-pHluorin-Ace junction R, pHluorin-Ace
Junction F and pHluorin-Ace junction R, pcDNA3.1-pHluorin-Ace-mOrange2 junction F
With pcDNA3.1-pHluorin-Ace-mOrange2 junction R, Ace-mOrange2 junction F and Ace-
MOrange2 junction R, pHluorin Dark Y65H-F and pHluorin Dark Y65H-R, mOrange2 Dark
Y71A-F and mOrange2 Dark Y71A-R.Then, using primer point mutation Ace-LAP2-loop1-F and
LAP2 polypeptides are inserted into regarding for above-mentioned carrier by point mutation Ace-LAP2-loop1-R by the method for direct mutagenesis
The extracellular first loop region of rhodopsin.Plasmid pcDNA3.1-pHluorin (the Y65H)-AceN-LAP2- that will be built
AceC-mOrange2 (Y71A) is transferred in 293T cells by way of PEI transfections.293T cells need to be layered in advance
On the sheet glass for a diameter of 12mm that matrigel was coated with, cell density during transfection is 80% or so, its culture medium be containing
There are 10%FBS DMEM culture mediums.PEI transfections concrete operations mode is as follows：By 0.5 μ g " rhodopsin-LAP2 fusions " plasmid
Successively it is added to the PEI that 1.5 μ L concentration are 1 μ g/ μ L inside 50 μ L opti-MEM, acutely mixes 10s, be incubated at room temperature
20min., it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free before PEI reagents are added to cell.By PEI
Transfection mixture is added dropwise in 293T cells.After 6 hours, the culture medium of cell is gained into normal culture medium.20 hours
Afterwards, the fluorescence labeling of click reaction mediations is carried out to cell.Mark is specifically divided into two steps.The first step, 300 μ L are contained 10 μM
LplA ligases, 200 μM of picolyl azide, 1mMATP and 5mM magnesium acetate normal cell culture medium
It is added in 293T cells, 37 DEG C of incubation 30min, is then washed three times using the DMEM culture mediums of serum-free.Second step, by 300 μ
L contains 40 μM of fluorophore-alkyne conjugate, 100 μM of CuSO4, 500 μM of BTTAA, 100 μM of TEMPOL and
2.5mM sodium ascorbateDPBS are added in 293T cells, are incubated at room temperature 5min, are then washed using DMEM culture mediums
Three times.At this point it is possible to obvious fluorescence signal is seen under the microscope, as shown in Figure 3.When the reaction reagent of second step is prepared,
Need 100 μM of CuSO first4, 500 μM of BTTAA, 100 μM of TEMPOL and 2.5mM sodium ascorbate are incubated 5-
10min, then add DPBS and 40 μM of fluorophore-alkyne conjugate.
The preparation of embodiment two, " rhodopsin-AP amalgamation and expressions+fluorescent small molecule mark ".
First, AP polypeptides are inserted into rhodopsin albumin A ce.Pass through molecular cloning method structure using following primer pair
Build pcDNA3.1-pHluorin (Y65H)-Ace-mOrange2 (Y71A) mammalian cell expression vector：pcDNA3.1-
PHluorin-Ace junction F and pcDNA3.1-pHluorin-Ace junction R, pHluorin-Ace
Junction F and pHluorin-Ace junction R, pcDNA3.1-pHluorin-Ace-mOrange2 junction F
With pcDNA3.1-pHluorin-Ace-mOrange2 junction R, Ace-mOrange2 junction F and Ace-
MOrange2 junction R, pHluorin Dark Y65H-F and pHluorin Dark Y65H-R, mOrange2 Dark
Y71A-F and mOrange2 Dark Y71A-R.Then, using primer point mutation Ace-AP-loop1-F and
AP polypeptides are inserted into the rhodopsin of above-mentioned carrier by the method for direct mutagenesis by point mutation Ace-AP-loop1-R
The extracellular first loop region of matter.Plasmid pcDNA3.1-pHluorin (the Y65H)-AceN-AP-AceC- that will be built
MOrange2 (Y71A) is transferred in 293T cells by way of PEI transfections.293T cells need to be layered on matrigel bags in advance
On a diameter of 12mm crossed sheet glass, cell density during transfection is 80% or so, and its culture medium is contains 10%FBS's
DMEM culture mediums.
PEI transfections concrete operations mode is as follows：By 0.25 μ g " rhodopsin-AP fusions " plasmids and 0.25 μ gBirA-ER
Plasmid and the PEI that 1.5 μ L concentration are 1 μ g/ μ L are successively added to inside 50 μ L opti-MEM, acutely mix 10s, room temperature is incubated
Educate 20min., it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free before PEI reagents are added to cell.Will
PEI transfection mixtures are added dropwise in 293T cells.After 6 hours, the culture medium of cell is gained and with the addition of 10 μM of biotins
With 10%FBS DMEM culture mediums.After 20 hours, AP polypeptide-biotin-streptavidins-fluorescence molecule is carried out to cell and is situated between
The fluorescence labeling led.Mark comprises the following steps that：300 μ L are contained into 5 μ g/mL streptavidin-conjugated
with Alexa568,1%BSA DPBS is added in 293T cells, is incubated at room temperature 10min, is then washed using DPBS
Three times.At this point it is possible to obvious fluorescence signal is seen under the microscope, as shown in Figure 4.
The preparation of embodiment three, " rhodopsin-Halotag amalgamation and expressions+fluorescent small molecule mark ".
First, Halotag polypeptides are merged with the N-terminal of Ace rhodopsins.Use primer pcDNA3.1-Halotag-Ace
Junction F and pcDNA3.1-Ace-Halotag junction R, Halotag-Ace junction F and Halotag-
Ace junction R build mammalian expression vector pcDNA3.1-Halotag-Ace by the method for molecular cloning.So
Afterwards, the plasmid is transferred in 293T cells by way of PEI transfections.293T cells, which need to be layered on matrigel in advance, to be coated with
A diameter of 12mm sheet glass on, cell density during transfection is 80% or so, and its culture medium is the DMEM containing 10%FBS
Culture medium.PEI transfections concrete operations mode is as follows：By 0.5 μ g " rhodopsin-Halotag fusions " plasmids and 1.5 μ L concentration
Successively it is added to for 1 μ g/ μ L PEI inside 50 μ L opti-MEM, acutely mixes 10s, is incubated at room temperature 20min.In PEI reagents
It is added to before cell, it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free.PEI transfection mixtures are added dropwise
Enter into 293T cells.After 6 hours, the culture medium of cell is gained to the DMEM culture mediums containing 10%FBS.It is right after 20 hours
Cell carries out Halotag- fluorescence molecule marks.Mark comprises the following steps that：300 μ L are contained into 5 μM of Halotag fluorescence to match somebody with somebody
The normal cell culture medium of body is added in 293T cells, is incubated at room temperature 10min, is then washed three times using DMEM.At this point it is possible to
Obvious fluorescence signal is seen under the microscope, as shown in Figure 5.
The preparation of example IV, " rhodopsin-SNAPtag amalgamation and expressions+fluorescent small molecule mark ".
First, SNAPtag polypeptides are merged with the N-terminal of Ace rhodopsins.Use primer pcDNA3.1-SNAPtag-Ace
Junction F and pcDNA3.1-Ace-SNAPtag junction R, SNAPtag-Ace junction F and SNAPtag-
Ace junction R build mammalian expression vector pcDNA3.1-SNAPtag-Ace by the method for molecular cloning.So
Afterwards, the plasmid is transferred in 293T cells by way of PEI transfections.293T cells, which need to be layered on matrigel in advance, to be coated with
A diameter of 12mm sheet glass on, cell density during transfection is 80% or so, and its culture medium is the DMEM containing 10%FBS
Culture medium.
PEI transfections concrete operations mode is as follows：0.5 μ g " rhodopsin-SNAPtag fusions " plasmids and 1.5 μ L is dense
Spend and be successively added to for 1 μ g/ μ L PEI inside 50 μ L opti-MEM, acutely mix 10s, be incubated at room temperature 20min.Tried in PEI
Agent is added to before cell, it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free.By PEI transfection mixtures dropwise
It is added in 293T cells.After 6 hours, the culture medium of cell is gained to the DMEM culture mediums containing 10%FBS.After 20 hours,
SNAPtag- fluorescence molecule marks are carried out to cell.Mark comprises the following steps that：300 μ L are contained into 5 μM of SNAPtag fluorescence
The normal cell culture medium of part is added in 293T cells, is incubated at room temperature 10min, is then washed three times using DMEM.Now, may be used
To see obvious fluorescence signal under the microscope, as shown in Figure 6.
Embodiment five, " rhodopsin-LAP2 amalgamation and expressions+fluorescent small molecule mark " fluorescence probe are in film potential detection
Application.
Plasmid pcDNA3.1-pHluorin (the Y65H)-AceN-LAP2-AceC-mOrange2 (Y71A) built is logical
The mode for crossing PEI transfections is transferred in 293T cells.293T cells need to be layered on a diameter of 12mm that matrigel was coated with advance
Sheet glass on, cell density during transfection is 80% or so, and its culture medium is the DMEM culture mediums containing 10%FBS.
PEI transfections concrete operations mode is as follows：With 1.5 μ L concentration it is 1 μ by 0.5 μ g " rhodopsin-LAP2 fusions " plasmids
G/ μ L PEI is successively added to inside 50 μ L opti-MEM, acutely mixes 10s, is incubated at room temperature 20min.Added in PEI reagents
, it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free before to cell.PEI transfection mixtures are added dropwise to
In 293T cells.After 6 hours, the culture medium of cell is gained into normal culture medium.After 8 hours, by cell according to 1: 5 ratio
On the sheet glass for passing on a diameter of 12mm that matrigel was coated with, after 20 hours, click reaction mediations are carried out to cell
Fluorescence labeling.
The film potential detection of electro physiology auxiliary is carried out after the completion of mark.Comprise the following steps that：Cell is placed on and buffered into XC
(125mMNaCl, 2.5mMKCl, 3mM CaCl in liquid2,1mM MgCl2,10mM HEPES,30mM glucose,pH 7.3,
305-310mOsm with sucrose), while add into XC final concentration of 50 μM of 2-aminoethoxydiphenyl
Borate, glass electrode is interior to add IC buffer solutions (125mM potassium gluconate, 8mMNaCl, 0.6mM MgCl2,
0.1mM CaCl2, 1mM EGTA, 10mM HEPES, 4mM Mg-ATP, 0.4mM Na-GTP (pH 7.3), 295mOsm
withsucrose).The tip resistance of glass electrode is 2.5-5M Ω.By glass electrode close to cell membrane, and further realize
High resistance seals (>500MΩ).Using voltage-clamp mode, the film potential of cell is controlled, while uses wide-field microscope record cell
Change in fluorescence.Data processing finally is carried out using MATLAB, obtains curve map of the fluorescence with voltage change, as shown in Figure 7.Can
To find out, fluorescence signal on cell membrane changes with voltage sensitive response, and sensitivity is up to 35%/100mV.
Embodiment six, " rhodopsin-AP amalgamation and expressions+fluorescent small molecule mark " fluorescence probe are in film potential detection
Using.
Plasmid pcDNA3.1-pHluorin (the Y65H)-AceN-AP-AceC-mOrange2 (Y71A) built is passed through
The mode of PEI transfections is transferred in 293T cells.293T cells need to be layered on a diameter of 12mm's that matrigel was coated with advance
On sheet glass, cell density during transfection is 80% or so, and its culture medium is the DMEM culture mediums containing 10%FBS.
PEI transfections concrete operations mode is as follows：By 0.25 μ g " rhodopsin-LAP2 fusions " plasmids and 0.25 μ gBirA matter
Grain and the PEI that 1.5 μ L concentration are 1 μ g/ μ L are successively added to inside 50 μ L opti-MEM, are acutely mixed 10s, are incubated at room temperature
20min., it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free before PEI reagents are added to cell.By PEI
Transfection mixture is added dropwise in 293T cells.After 6 hours, the culture medium of cell is gained with the addition of 10 μM of biotins and
10%FBS DMEM culture mediums.After 8 hours, cell is passed on according to 1: 5 ratio be coated with to matrigel it is a diameter of
On 12mm sheet glass.After 20 hours, the fluorescence of AP polypeptide-biotin-streptavidins-fluorescence molecule mediation is carried out to cell
Mark.
The film potential detection of electro physiology auxiliary is carried out after the completion of mark.Comprise the following steps that：Cell is placed on and buffered into XC
(125mMNaCl, 2.5mMKCl, 3mM CaCl in liquid2,1mM MgCl2,10mM HEPES,30mM glucose,pH 7.3,
305-310mOsm with sucrose), while add into XC final concentration of 50 μM of 2-aminoethoxydiphenyl
Borate, glass electrode is interior to add IC buffer solutions (125mM potassium gluconate, 8mMNaCl, 0.6mM MgCl2,
0.1mM CaCl2, 1mM EGTA, 10mM HEPES, 4mM Mg-ATP, 0.4mM Na-GTP (pH 7.3), 295mOsm
withsucrose).The tip resistance of glass electrode is 2.5-5M Ω.By glass electrode close to cell membrane, and further realize
High resistance seals (>500MΩ).Using voltage-clamp mode, the film potential of cell is controlled, while uses wide-field microscope record cell
Change in fluorescence.Data processing finally is carried out using MATLAB, curve map of the fluorescence with voltage change is obtained, as shown in figure 8, carefully
Fluorescence signal on after birth changes with voltage sensitive response, and sensitivity is up to 14%/100mV.
Embodiment seven ,] " rhodopsin-Halotag amalgamation and expressions+fluorescent small molecule mark " fluorescence probe examines in film potential
Application in survey.
The plasmid pcDNA3.1-Halotag-Ace built is transferred in 293T cells by way of PEI transfections.
293T cells need to be layered in advance on the sheet glass for a diameter of 12mm that matrigel was coated with, and cell density during transfection is
80% or so, its culture medium is the DMEM culture mediums containing 10%FBS.
PEI transfections concrete operations mode is as follows：0.5 μ g " rhodopsin-Halotag fusions " plasmids and 1.5 μ L is dense
Spend and be successively added to for 1 μ g/ μ L PEI inside 50 μ L opti-MEM, acutely mix 10s, be incubated at room temperature 20min.Tried in PEI
Agent is added to before cell, it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free.By PEI transfection mixtures dropwise
It is added in 293T cells.After 6 hours, the culture medium of cell is gained to the DMEM culture mediums containing 10%FBS., will after 8 hours
Cell is passed on onto the matrigel a diameter of 12mm being coated with sheet glass according to 1: 5 ratio.After 20 hours, cell is entered
Row Halotag- fluorescence molecules mark.
The film potential detection of electro physiology auxiliary is carried out after the completion of mark.Comprise the following steps that：Cell is placed on and buffered into XC
(125mMNaCl, 2.5mMKCl, 3mM CaCl in liquid2,1mM MgCl2,10mM HEPES,30mM glucose,pH 7.3,
305-310mOsm with sucrose), while add into XC final concentration of 50 μM of 2-aminoethoxydiphenyl
Borate, glass electrode is interior to add IC buffer solutions (125mM potassium gluconate, 8mMNaCl, 0.6mM MgCl2,
0.1mM CaCl2, 1mM EGTA, 10mM HEPES, 4mM Mg-ATP, 0.4mM Na-GTP (pH7.3), 295mOsm
withsucrose).The tip resistance of glass electrode is 2.5-5M Ω.By glass electrode close to cell membrane, and further realize
High resistance seals (>500MΩ).Using voltage-clamp mode, the film potential of cell is controlled, while uses wide-field microscope record cell
Change in fluorescence.Data processing finally is carried out using MATLAB, curve map of the fluorescence with voltage change is obtained, as shown in figure 9, carefully
Fluorescence signal on after birth changes with voltage sensitive response, and sensitivity is up to 4%/100mV.
Embodiment eight, " rhodopsin-SNAPtag amalgamation and expressions+fluorescent small molecule mark " fluorescence probe are examined in film potential
Application in survey.
The plasmid pcDNA3.1-SNAPtag-Ace built is transferred in 293T cells by way of PEI transfections.
293T cells need to be layered in advance on the sheet glass for a diameter of 12mm that matrigel was coated with, and cell density during transfection is
80% or so, its culture medium is the DMEM culture mediums containing 10%FBS.
PEI transfections concrete operations mode is as follows：0.5 μ g " rhodopsin-SNAPtag fusions " plasmids and 1.5 μ L is dense
Spend and be successively added to for 1 μ g/ μ L PEI inside 50 μ L opti-MEM, acutely mix 10s, be incubated at room temperature 20min.Tried in PEI
Agent is added to before cell, it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free.By PEI transfection mixtures dropwise
It is added in 293T cells.After 6 hours, the culture medium of cell is gained to the DMEM culture mediums containing 10%FBS., will after 8 hours
Cell is passed on onto the matrigel a diameter of 12mm being coated with sheet glass according to 1: 5 ratio.After 20 hours, cell is entered
Row Halotag- fluorescence molecules mark.
The film potential detection of electro physiology auxiliary is carried out after the completion of mark.Comprise the following steps that, cell is placed on and buffered into XC
(125mMNaCl, 2.5mMKCl, 3mM CaCl in liquid2,1mM MgCl2,10mM HEPES,30mM glucose,pH7.3,305–
310mOsm with sucrose), while add into XC final concentration of 50 μM of 2-aminoethoxydiphenyl
borate.IC buffer solutions (125mM potassium gluconate, 8mMNaCl, 0.6mM MgCl is added in glass electrode2,
0.1mM CaCl2, 1mM EGTA, 10mM HEPES, 4mM Mg-ATP, 0.4mM Na-GTP (pH7.3), 295mOsm
withsucrose).The tip resistance of glass electrode is 2.5-5M Ω.By glass electrode close to cell membrane, and further realize
High resistance seals (>500MΩ).Using voltage-clamp mode, the film potential of cell is controlled, while uses wide-field microscope record cell
Change in fluorescence.Data processing finally is carried out using MATLAB, obtains curve map of the fluorescence with voltage change, as shown in Figure 10,
Fluorescence signal on cell membrane changes with voltage sensitive response, and sensitivity is up to 16%/100mV.
Embodiment nine, " rhodopsin-LAP2 amalgamation and expressions+fluorescent small molecule mark " fluorescence probe are in cell connecting detection
In application.
Plasmid pcDNA3.1-pHluorin (the Y65H)-AceN-LAP2-AceC-mOrange2 (Y71A) built is logical
The mode for crossing PEI transfections is transferred in 293T cells.293T cells need to be layered on a diameter of 12mm that matrigel was coated with advance
Sheet glass on, cell density during transfection is 80% or so, and its culture medium is the DMEM culture mediums containing 10%FBS.
PEI transfections concrete operations mode is as follows：With 1.5 μ L concentration it is 1 μ by 0.5 μ g " rhodopsin-LAP2 fusions " plasmids
G/ μ L PEI is successively added to inside 50 μ L opti-MEM, acutely mixes 10s, is incubated at room temperature 20min.Added in PEI reagents
, it is necessary to which the culture medium of cell to be changed into the DMEM culture mediums of serum-free before to cell.PEI transfection mixtures are added dropwise to
In 293T cells.After 6 hours, the culture medium of cell is gained into normal culture medium.After 8 hours, by cell according to 1: 1 ratio
On the sheet glass for passing on a diameter of 12mm that matrigel was coated with, after 20 hours, click reaction mediations are carried out to cell
Fluorescence labeling.
The film potential detection of electro physiology auxiliary is carried out after the completion of mark.Comprise the following steps that：Cell is placed on and buffered into XC
(125mMNaCl, 2.5mMKCl, 3mM CaCl in liquid2,1mM MgCl2,10mM HEPES,30mM glucose,pH7.3,305–
310mOsm with sucrose), the interior addition IC buffer solutions of glass electrode (125mM potassium gluconate,
8mMNaCl,0.6mM MgCl2,0.1mM CaCl2,1mM EGTA,10mM HEPES,4mM Mg-ATP,0.4mM Na-GTP(pH
7.3), 295mOsmwithsucrose).The tip resistance of glass electrode is 2.5-5M Ω.By glass electrode close to cell membrane, and
And further realize high resistance seals (>500MΩ).Using voltage-clamp mode, the film potential of cell is controlled, while is shown using wide field
Micro mirror records the change in fluorescence of clamp cells and peripheral cell.Data processing finally is carried out using MATLAB, obtains pliers cell
And peripheral cell fluorescence is with the curve map of voltage change, and intercellular electrical connection is further analyzed, as shown in figure 11.Can be with
Find out, the fluorescence signal amplitude of variation of indicator cells film potential connects dependent on cell and with range attenuation, embodies the patent
The fluorescent film current potential probe of description can be used for the electrical activity of highly sensitive detection cell.
In summary, using " rhodopsin-fluorescence molecule complex " fluorescence probe provided by the invention have speed it is fast,
High sensitivity, brightness is high, can be with genetic coding the advantages of, and highly sensitive detection can be carried out to film potential, while can be with
Electrical connection to cell carries out highly sensitive detection.
SEQUENCE LISTING
<110>Peking University
<120>Rhodopsin-fluorescence molecule complex and its preparation method and application
<130> WX2017-03-136
<160> 36
<170> PatentIn version 3.3
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atgctggggc ccagccggcc agatctagta aaggagaaga acttttcact ggagttg 57
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<211> 56
<212> DNA
<213>Artificial sequence
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tcgctggtga ttctgctctt gctagctccg gacatcttct tgaagatagt ctcatg 56
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<212> DNA
<213>Artificial sequence
<400> 3
atggctgacg tggaaaccg 19
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tcggtttcca cgtcagccat ggtgctgtcc aggcccag 38
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atgctggggc ccagccggcc agatctagta aaggagaaga acttttcact ggagttg 57
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gtgattctgc tcttgctagc cttgtacagc tcgtccatgc cg 42
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<213>Artificial sequence
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tccggaaaca tggccatcat caaggagttc atg 33
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atgatggcca tgtttccgga cttgaagata gtctcatggg caatgagg 48
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cttgtcacta ctttaactca tggtgttcaa tgcttttcaa g 41
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cttgaaaagc attgaacacc atgagttaaa gtagtgacaa g 41
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cctgtcccct catttcaccg ccggctccaa ggcctacgtg aag 43
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cttcacgtag gccttggagc cggcggtgaa atgaggggac agg 43
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atcgacaagg tgtggtacga cctggacgcc accgagaccg gcatgattgc 50
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gtcgtaccac accttgtcga tctcgaagcc ttccacgtca gccatggtgc 50
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atcgacaagg tgtggtacga cctggacgcc aatggtgaaa ggcaggtggt ct 52
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gtcgtaccac accttgtcga tctcgaagcc cattgtcagg tcctggtagt tcacg 55
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cgacaaggtg tggtacgacc tggacgccga acacaagttc aaatgggtgt actttatcg 59
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gtcgtaccac accttgtcga tctcgaagcc atcctcgaag gcgcccag 48
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atcgacaagg tgtggtacga cctggacgcc ctgtccgtcg acaatgaggc ca 52
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gtcgtaccac accttgtcga tctcgaagcc cacgccacta ccagacccga 50
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atcttcgagg cccagaagat cgagtggcac gagaatggtg aaaggcaggt ggtctac 57
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gatcttctgg gcctcgaaga tgtcgttcag gcccattgtc aggtcctggt agttcacg 58
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atgctggggc ccagccggcc gaattcatgg acaaagattg cgaaatgaaa cgtac 55
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tcgctggtga ttctgctctt gctagctccg gacatcttct tgaagatagt ctcatg 56
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<212> DNA
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tgggtaaacc gggtctggga atggctgacg tggaaaccga 40
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tcccagaccc ggtttaccca 20
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atgctggggc ccagccggcc gaattcatgg cagaaatcgg tactggcttt c 51
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tcgctggtga ttctgctctt gctagctccg gacatcttct tgaagatagt ctcatg 56
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<213>Artificial sequence
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cgacgctcga gatttccggc atggctgacg tggaaaccg 39
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gccggaaatc tcgagcgtc 19
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cactgtgctg gatatctgca gaattcatgg ctgacgtgga aaccg 45
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gaaagcttgg taccgagctc ggatcctcat tacacctcgt tctcgtagca gaac 54
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gggcccagcc ggccagatct tccggaccag cgacacatg 39
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<213>Artificial sequence
<400> 34
tcgacctgca gccgcggccg cttgtacagc tcgtccatgc 40
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gtacaagcgg ccgcggctg 19
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gaagatctgg ccggctgg 18
Claims (10)
1. a kind of memebrane protein-fluorescence molecule complex, wherein：The memebrane protein is rhodopsin, and the albumen is seven cross-film eggs
In vain, extracellular to have four regions, the fluorescence molecule is connected on any region in this four extracellular regions.
2. memebrane protein as claimed in claim 1-fluorescence molecule complex, it is characterised in that N-terminal or born of the same parents in rhodopsin
A peptide sequence is connected with outer any loop regions, the fluorescence molecule is connected with the peptide sequence.
3. memebrane protein as claimed in claim 2-fluorescence molecule complex, it is characterised in that the peptide sequence is LAP polypeptides
Sequence, the fluorescence molecule is in AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3 and Cy5
Any one；The fluorescence molecule is mediated by small molecule azido-methyl pyridine, passes through alkynyl and the nitrine of azido-methyl pyridine
Base occurs cycloaddition reaction and is covalently attached on LAP peptide sequences.
4. memebrane protein as claimed in claim 2-fluorescence molecule complex, it is characterised in that the peptide sequence is AP polypeptides
Sequence, the fluorescence molecule is in AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3 and Cy5
Any one；AP peptide sequences connect biotin, and fluorescence molecule passes through the combination between Streptavidin and biotin
It is connected on AP peptide sequences.
5. memebrane protein as claimed in claim 2-fluorescence molecule complex, it is characterised in that the peptide sequence is Halotag
Protein tag or SNAPtag protein tags, the fluorescence molecule be selected from AlexaFluor 488, AlexaFluor 568,
Any one in AlexaFluor 594, Cy3 and Cy5.
6. the preparation method of memebrane protein described in claim 1-fluorescence molecule complex, comprises the following steps：
1) expression vector containing rhodopsin-peptide sequence antigen-4 fusion protein gene is built, peptide sequence in the fusion protein
It is connected to rhodopsin N-terminal or is inserted into the extracellular any loop regions of rhodopsin；
2) expression vector that step 1) is built is transferred in cell and expressed；
3) fluorescence molecule mark is carried out to the cell for expressing the rhodopsin-peptide sequence fusion protein, obtain rhodopsin-
Fluorescence molecule complex.
7. preparation method as claimed in claim 6, it is characterised in that LAP polypeptides are inserted the extracellular of rhodopsin by step 1)
In region, and by the rhodopsin-LAP antigen-4 fusion protein genes structure on expression vector；Expression vector is transferred to carefully by step 2)
Expressed in born of the same parents；Step 3) by LplA enzymes, substrate azido-methyl pyridine with expression rhodopsin-LAP fusion proteins cell
It is incubated, azido-methyl pyridine is connected on LAP polypeptides, then washes away remaining reagent, then added into cell and contain alkynes
It is any one in the fluorescent small molecule AlexaFluor 488 of base, AlexaFluor 568, AlexaFluor 594, Cy3 and Cy5
Kind is incubated, and is made fluorescence molecule that cycloaddition reaction occur with azido-methyl pyridine, is obtained rhodopsin-LAP- fluorescence molecules and answer
It is fit.
8. preparation method as claimed in claim 6, it is characterised in that step 1) inserts AP polypeptides the extracellular region of rhodopsin
In domain, and by the rhodopsin-AP antigen-4 fusion protein genes structure on expression vector；Rhodopsin-AP is merged egg by step 2)
The BirA biotin ligase expression vectors of white expression vector and ER chambers positioning are transferred in cell jointly is expressed, and in cell
Biotin is added in culture medium；Cell addition streptavidin connection of the step 3) to expression rhodopsin-AP fusion proteins
Any one in fluorescent small molecule AlexaFluor 488, AlexaFluor 568, AlexaFluor 594, Cy3 and Cy5,
Obtain rhodopsin-AP- fluorescence molecule complexs.
9. preparation method as claimed in claim 6, it is characterised in that Halotag or SNAPtag are connected to visual purple by step 1)
The N-terminal of red matter albumen, and by the rhodopsin-Halotag fusion proteins or the gene of rhodopsin-SNAPtag fusion proteins
Structure is on expression vector；Fusion protein expression vector is transferred in cell and expressed by step 2)；Step 3) to expression visual purple
The cell of red matter-Halotag fusion proteins or rhodopsin-SNAPtag fusion proteins addition fluorescence molecule AlexaFluor
488th, any one in AlexaFluor 568, AlexaFluor 594, Cy3 and Cy5, obtain rhodopsin-Halotag or
SNAPtag- fluorescence molecule complexs.
10. any described memebrane protein-fluorescence molecule complex of Claims 1 to 5 is in film potential detection and cell connecting detection
In application.
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Citations (3)
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|---|---|---|---|---|
| WO2009018379A1 (en) * | 2007-07-31 | 2009-02-05 | Purdue Pharma L.P. | Dielectric spectroscopy assays for screening of ion channel ligands |
| CN102119331A (en) * | 2008-06-05 | 2011-07-06 | 生命科技公司 | Activation and monitoring of cellular transmembrane potentials |
| CN103168236A (en) * | 2010-08-23 | 2013-06-19 | 哈佛大学管理委员会 | Optogenetic probes for measuring membrane potential |
-
2017
- 2017-09-20 CN CN201710854086.8A patent/CN107677655A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009018379A1 (en) * | 2007-07-31 | 2009-02-05 | Purdue Pharma L.P. | Dielectric spectroscopy assays for screening of ion channel ligands |
| CN102119331A (en) * | 2008-06-05 | 2011-07-06 | 生命科技公司 | Activation and monitoring of cellular transmembrane potentials |
| CN103168236A (en) * | 2010-08-23 | 2013-06-19 | 哈佛大学管理委员会 | Optogenetic probes for measuring membrane potential |
Non-Patent Citations (2)
| Title |
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| CHAYASITH UTTAMAPINANT,等: ""Site-specific protein labeling using PRIME and chelation-assisted click chemistry"", 《NATURE PROTOCOLS》 * |
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