CN115197301A

CN115197301A - Calcium indication tool for intracellular calcium signal detection and related drug screening and application thereof

Info

Publication number: CN115197301A
Application number: CN202210657761.9A
Authority: CN
Inventors: 王友军; 章晓辉; 李佳
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2022-10-18

Abstract

The invention belongs to the field of biotechnology, and provides a calcium indicator protein for detecting calcium signals, wherein the amino acid sequence of the calcium indicator protein is shown as any one of SEQ ID NO 3-6. The calcium indicator protein has high fluorescence brightness and large dynamic range, and can directly indicate the calcium concentration.

Description

Calcium indication tool for intracellular calcium signal detection and related drug screening and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a calcium indication tool for detecting a calcium signal.

Background

Calcium ion is a second messenger commonly used by cells. Calcium signaling, the spatiotemporal variation of free calcium concentration within a cell, is essential for the maintenance of vital activities. In order to understand the molecular mechanisms of physiological and pathological activities of cells, especially neurons, in the research of bioscience, especially brain science, it is necessary to use fluorescent calcium indicators to detect the changes of intracellular calcium signals. In drug development, calcium ion channels (such as L-type calcium channel, TRP channel, pizeo channel, CRAC channel, IP) related to various serious diseases are screened on a large scale ₃ R and RyR, etc.), various high-throughput screening techniques based on fluorescent calcium indicators are also significantly superior to other approaches.

The GCaMP series calcium indicator prepared by utilizing green fluorescent protein (EGFP), calmodulin and target peptide thereof is a star-level calcium indicating tool, is widely applied in the field of neuroscience, and shows excellent performance in the process of detecting the single action potential of neurons, nerve subcellular signals, neuron discharge and other neuron activities. However, there are still some problems in the GCaMP series tool, for example, the inherent limitations of EGFP cause the following two problems in the improvement and application of GCaMP: 1) EGFP has a broad excitation spectrum and cross-color channels to CFP and YFP, resulting in a limited number of other fluorescence acquisitions that can be used simultaneously with GCaMP, which limits its application in multi-color fluorescence imaging of neurons. 2) The brightness of GCaMP is limited by the upper limit of EGFP fluorescence, which results in that the brightness of GCaMP has reached the optimized limit, and has the disadvantages of weak fluorescence at rest and maximum reaction time, small dynamic range, etc. This has led to slow progress in research relating to weak calcium signals at the sub-cellular level; when the drug screening aiming at the ion channel is carried out, an expensive high-sensitivity fluorescence detection platform is also required to be used, so that the application of the fluorescence detection platform in drug research and development is greatly limited. In conclusion, there is an urgent need to develop a green calcium indicator tool with narrower and brighter spectrum to detect subcellular calcium signals and better perform drug screening for ion channels.

Compared with EGFP, the excitation spectrum of mNeonGreen is very narrow and can be effectively separated from the spectrum of CFP, thereby avoiding the influence of the cross color problem; the mNeonGreen is the brightest known green fluorescent protein at present, the brightness of the mNeonGreen is 2.76 times of that of EGFP, and the signal-to-noise ratio of fluorescence imaging is greatly optimized; the light stability of the mNeonGreen is higher than that of the EGFP, so that the problem of light bleaching can be better solved. In conclusion, mNeon Green is a fluorescent protein with great development value, and can be transformed into a brighter, more sensitive and more stable green calcium indicator protein.

Some of the mNeon Green-engineered calc-indicator proteins have also appeared in the last five years. In 2016, barykina et al made the Calcium Indicator protein NTnC and recorded Calcium signals elicited by single action potentials in neurons (Barykina N V, subach O M, doronin D a, et al, a new design for the acquisition of Calcium signals, with a smaller dynamic range (< 3), i.e., ntc detected only a < 3-fold difference between the maximum and minimum fluorescence signals, with a small amplitude of the change in fluorescence intensity with Calcium concentration, not clearly indicating the change in Calcium signal, far without the GCaMP series sensitivity (the in vitro dynamic range of jGCaMP7c is 146), with a price value applied thereto, on this basis, subach et al made the current brightest green Calcium Indicator protein NTnC (GCaMP 7c, with a fast speed of activation, with a fast speed of gcaamp 7, about 2. The saturation speed of Calcium signal, about 2016. The saturation speed of Calcium signal, about 2. The saturation speed of Calcium signal under the test of phosphor-binding, while still showing the disadvantage of the high speed of the Calcium signal under the pdaxacuminal-binding kinetics of the test, with a fast speed of the voltage of the phosphor-binding protein, with a fast speed of the voltage of the probe, which was still applied to the test of the voltage of subzero.

Disclosure of Invention

The invention provides a set of ultra-sensitive green calcium indicator proteins, which are used for detecting cell calcium signals and screening drugs targeting calcium channels.

In a first aspect, the invention provides a calcium indicator protein which is one or more of:

(1) NEMOm: the amino acid sequence is shown as SEQ ID NO. 3;

(2) NEMOc: the amino acid sequence is shown as SEQ ID NO. 4;

(3) NEMOf: the amino acid sequence is shown as SEQ ID NO. 5;

(4) NEMOs: the amino acid sequence is shown in SEQ ID NO. 6.

In some embodiments, the invention also provides a biomaterial associated with the calcium indicator protein, comprising:

(1) A nucleic acid molecule encoding a calcemic indicator protein of the invention;

(2) Plasmids, cells, etc., comprising the nucleic acid molecules.

In a second aspect, the invention provides a product for detecting calcium signaling or screening for drugs targeting calcium channels, said product comprising a calcium indicator protein of the invention. Preferably, the product is a kit.

In a third aspect, the invention provides the use of a calcium indicator protein or product of the invention for detecting calcium signaling or screening for drugs targeting calcium channels.

In a fourth aspect, there is provided a method of detecting calcium signals, the method comprising the use of a calcium indicator protein or product of the invention.

In a fifth aspect, there is provided a method of screening for a drug targeting a calcium channel, the method comprising the use of a calcium indicator protein or product of the invention.

In some preferred embodiments, the methods and uses of the present invention are for living cells.

The calcium indicator proteins provided by the invention are modified based on NCaMP7, the calcium indicator proteins retain the advantage that the maximum fluorescence of NCaMP7 is far higher than that of GCaMP, and the dynamic range and the reaction speed are both remarkably improved compared with NCaMP 7. The excitation spectrum of the calcium indicator protein is narrower than that of GCaMP series, and the calcium indicator protein can be widely applied to multicolor fluorescence imaging; meanwhile, the dynamic range of the calcium indicator proteins under physiological conditions is improved by nearly 10 times, the calcium concentration can be directly indicated, and the resting fluorescence and the maximum fluorescence can be brighter, so that the tiny calcium signal difference in living cells can be more sensitively detected, and the calcium indicator proteins can be more conveniently applied to related drug screening.

Drawings

FIG. 1 shows the structure of NCaMP7 and the optimization scheme of the NEMO series;

FIG. 2 is a dynamic range test of NEMO, A is 100mM Ca detected using GCaMP6m, NEMOc ²⁺ Induced SOCE; b is dynamic range of in vitro calcium ion titration test and live cell calcium imaging detection NEMO, and GCaMP6m is subjected to normalization treatment.

FIG. 3 is a fluorescence value test of NEMO, A represents GCaMP6m, NCaMP7 indicates 100mM Ca ²⁺ Fluorescence change with time upon induced SOCE, B indicates NEMO series indicates 100mM Ca ²⁺ Fluorescence values over time for induced SOCE.

FIG. 4 is a kinetic assay of NEMO, A-C showing calcium oscillation and dynamic range using GCaMP6m, NCaMP7, NEMOf, D showing comparison of GCaMP6m, NCaMP7, and NEMOf measures the rate of single calcium oscillations, E shows the rate of TG-induced calcium release compared to NCaMP7 and NEMOf measures, and F shows K for GCaMP6m, NCaMP7, and NEMOf in an in vitro kinetic test _off 。

FIG. 5 shows the detection result of iPEEAQ method.

Detailed Description

The present disclosure will be described in detail below by way of specific embodiments, but the present disclosure is not limited to the following embodiments.

Unless otherwise specified, the reagents and apparatus used in the following embodiments are conventional in the art and are commercially available; the methods used are all conventional methods in the field, and the person skilled in the art can know how to implement the embodiments without any doubt according to the description of the embodiments and obtain the corresponding results.

Throughout this document, calcemic indicator proteins of the present invention include NEMOm, NEMOc, nemuf, NEMOs, collectively referred to as NEMO series. The application has disclosed the amino acid sequence of these proteins, and the skilled person can deduce the nucleotide sequence of these proteins based on the amino acid sequence and the common general knowledge and prior art in the field, and transform them into cells to express these proteins.

Experimental materials and methods:

1 in vivo assay

1.1 culture of cells

The medium used for the cells was DMEM complete medium supplemented with 10% FBS and 1% P/S, the culture environment was 37 ℃,5% CO ₂ The incubator of (1). The cells can be continuously expanded after being attached to the wall, when the confluence degree of the cells reaches about 80%, the cells need to be passaged, and the specific steps are as follows: the dishes on which the cells were aspirated were completely in DMEM with 1/2 of the volume of the medium without Ca ²⁺ 、Mg ²⁺ HBSS gently washed the cells twice and the residual medium was washed away. HBSS was aspirated and cells were digested with 0.25% trypsin containing EGTA in 1/10 volume of the medium. When the cell edge becomes bright and the shape becomes round, the new DMEM complete culture medium is used for blowing and beating the cells, and the cells are in a dispersed suspension state while the digestion is stoppedState. Appropriate amount of cells were removed for subsequent experiments, leaving appropriate amount of cells to continue culturing.

1.2 transfection of cells (before Living cell calcium imaging experiment)

Transfection refers to the transfer of foreign genes into eukaryotic cells and their heterologous expression. In this experiment, we used the electrotransfection method to perform gene transfection. The electrotransformation process comprises the following steps:

(1) Digesting the cells, taking out a proper amount of cells, and centrifuging at 800rpm for 5min;

(2) Resuspending the cells with OPTI-MEM at 4 ℃;

(3) Gently mixing the resuspended cells and plasmids, adding into an electric shock cup, and electrically shocking the cells (the volume of an electric transfer system and an electric transfer process used by different types of cells are different, the electric transfer process of the HEK293 cells in the experiment is 400 muL system, 180V,25ms and square wave pulse, and the electric transfer process of the HeLa cells is 500 muL system, 260V,525 muF and exponential decay pulse);

(4) And (3) paving the cells after the electric transformation on a circular glass sheet of a six-hole plate, culturing for about 1h to adhere the cells to the wall, supplementing 1-1.5mL of DMEM complete culture medium to each hole after the cells adhere to the wall, and continuously culturing for 24h to perform fluorescence detection.

1.3 live cell calcium imaging experiments

The composition of the living cell calcium imaging system comprises: light source, power supply for microscope and visible light source, zeiss inverted fluorescence microscope (Axio Observer Z1, ZEISS, germany), camera (ORCA-flash 4.0V 3 digital CMOS camera, C13440-20CU, HAMAMATSU, japan), filter wheel controller (LAMBDA 10-3, sutter instrument, USA), 40-fold objective oil-mirror lens (numerical aperture 1.3, ZEISS, germany), and Semrock

Filter system (filter information see table 1) and SlideBook 6 software operating system.

TABLE 1 Filter information

The materials used for the live cell fluorescence imaging experiments are shown in table 2:

table 2: information table of living cell fluorescence imaging experiment material

The solution preparation method comprises the following steps:

(1) Calcium imaging solution: 107mM NaCl, 7.2mM KCl, 1.2mM MgCl ₂ 20mM HEPES and 11.5mM glucose, adjusted to pH 7.20 with 2M NaOH solution. When simulating extracellular fluid, caCl with the final concentration of 1mM is added into the calcium imaging solution ₂ 。

(2) TG stock (1 mM): 1.536mL of DMSO was added to 5mg of TG powder, mixed well and dispensed into 1.5mL of EP tubes, and stored in a refrigerator at-20 ℃.

(3) Iono mother liquor (2.5 mM): 564.17. Mu.L of DMSO was added to 1mg of the iono powder, mixed and dissolved, and then dispensed into 1.5mL of EP tubes and stored in a refrigerator at-20 ℃.

(4) CCh mother liquor (100 mM): 54.75mL of ddH was added to 1g of CCh powder ₂ O, mixing and dissolving, and subpackaging into 1.5mL EP tubes for storage in a refrigerator at the temperature of-20 ℃.

(5)CaCl ₂ Mother liquor (1M): 11.1g of CaCl ₂ ddH2O is added into the powder to dissolve, the volume is determined to be 100mL, and the powder is stored at 4 ℃.

(6) EGTA mother liquor (0.5M): weighing 19.02g of EGTA, dissolving in ddH2O, adding a proper amount of NaOH powder to adjust the pH value to assist dissolution, metering the volume to 100mL after the dissolution, and storing at 4 ℃.

The live cell calcium imaging of this study indicated the calcium level and calcium signal of the cells by detecting the fluorescent changes of calcium indicator proteins, the specific experimental steps are as follows:

(1) Calcium indicator protein plasmids are transiently or stably transfected into HEK293 cells in advance, the transfected cells are laid on a round slide, and detection can be carried out after the cells are attached to the wall and the proteins are expressed.

(2) The cell-plated round slide was fixed in a special imaging chamber, and 400. Mu.L of 1mM CaCl was added ₂ The calcium imaging solution of (2) is incubated for 20min at rest and then the detection is started. The custom imaging chamber may hold a 24 x 24mm, 0.13-0.17 mm thick circular slide, and at least 400 μ L of imaging solution may be added to the imaging chamber to incubate the cells or induce the cells to produce a calcium signal.

(3) The same excitation light intensity, exposure time and Bin value were used for the experimental and control groups.

(4) Observing the state of the cells by using a bright field light source under an ocular lens, and then observing the transfection efficiency and the resting fluorescence value of the cells under the ocular lens to find the most suitable detection visual field.

(5) And collecting bright field and fluorescence images of the detection field by using SlideBook 6 software, and circling a blank background without cells and a cell region of interest.

(6) And (3) acquiring images at intervals of 2s after the experiment is started, pausing shooting when liquid needs to be changed, gently and quickly sucking out the solution in the imaging tank when the liquid needs to be changed, and adding a new solution to avoid the situation that the liquid is absorbed too hard or cells are dried up.

(7) And (4) after the experiment is finished, deriving a numerical value of the fluorescence value changing along with time, processing data, and repeating the experiment of each group for more than 3 times.

2 in vitro assay

2.1 inducible expression of Calcuim indicator protein

(1) Transetta (DE 3) cells transformed with pET28a-NEMO series, pET28a-NCaMP7 and pET28a-GCaMP6m plasmids were inoculated into approximately 5mL of LB liquid medium and subjected to scale-up culture at 37 ℃ and 200rpm for 12 hours. The 12 h-cultured broth was inoculated into 200mL of LB liquid medium at a volume ratio of 1.25.

(2) Then, IPTG was added to the above bacterial solution at a final concentration of 300. Mu.M to induce the expression of the protein, the culture was carried out at 20 ℃ and 150rpm for 12 hours, and after the induction, the bacterial solution was centrifuged at 9000rpm for 5min to collect the cells.

2.2 extraction and purification of Calcuim indicator protein

Preparing a solution: solution 1:20mM Tris,300mM NaCl,1mM imidazole, (pH = 7.20).

Solution 2:20mM Tris,500mM NaCl,10mM imidazole, (pH = 7.20).

Solution 3:20mM Tris,100mM NaCl,300mM imidazole, (pH = 7.20).

Extraction of total bacterial protein:

(1) The centrifuged cells stored at-20 ℃ were taken out and resuspended in 20mL of solution 1.

(2) And (3) adding lysozyme powder and a rotor into a small beaker, pouring the resuspended thalli, and magnetically stirring on ice for 5-10 min to dissolve cell walls.

(3) A further 800. Mu.L of 200mM PMSF inhibits the protease activity and TritonX-100 at a final concentration of 0.5% dissolves the bacterial cell membranes.

(4) And (3) carrying out ultrasonic disruption on the thalli, wherein the ultrasonic power is 20%, the work time is 3s, the buffering time is 9s, and the ultrasonic treatment is 100 times.

(5) The sonicated cells were centrifuged at 4 ℃ and 9000rpm for 20min.

(6) The precipitate and supernatant are retained for electrophoresis.

Purification of calndin (GECI):

(1) And (3) cleaning the column: about 1mL of nickel column material was added to an affinity chromatography column with a total volume of 6mL, and the column material was washed with 2mL of deionized water and solution 1 in that order.

(2) Binding protein: after mixing the centrifuged supernatant of total protein with the column, the mixed supernatant and column were poured into a 50mL centrifuge tube and mixed on a vertical mixer at 4 ℃ for 1h.

(3) Washing the hybrid protein: the column mixed with the protein was poured into a chromatography column, and after all the liquid was filtered off, the column was washed with 20mL of solution 1, and then with 10mL of solution 2 to wash away the impure protein that was not bound to the column.

(4) Elution GECI: the target protein was eluted with 5mL of solution 3, and the filtrate was collected in a volume of 1mL per wash. The collected purified protein is stored at 4 ℃, and the column material is soaked in the solution 1 and stored at 4 ℃. Before the column is used again, 2mL of 0.5M NaOH and the solution 1 are used for washing the column in sequence, and the column can be reused after washing.

Determination of protein concentration:

(1) Preparing a standard solution: solutions 1 were diluted with 5mg/mL BSA standard and graded to give 1.5, 1, 0.75, 0.5, 0.25 and 0.125mg/mL standards.

(2) Determination of the standard curve: mu.L of the standard solution was added to 750. Mu.L of G250 Coomassie brilliant blue, and the absorbance at 595nm was measured to draw a concentration-absorbance standard curve.

(3) And (3) determining the concentration of the target protein: mu.L of the target protein was added to 750. Mu.L of G250 Coomassie Brilliant blue, the absorbance at 595nm was measured, and the protein concentration was calculated from the standard curve. Identification of GECI after purification:

(1) SDS-PAGE: the purified GECI protein was electrophoresed, and the loading amount was 7. Mu.L. And (4) electrophoresis.

(2) Coomassie blue staining: the gel after electrophoresis was taken out in ddH ₂ Shake wash in O for a moment. Stationary liquid (25 mL ethanol, 20 mLH) ₂ O, 5mL acetic acid) for 1h. Coomassie brilliant blue R250 was stained for 4h. Washing with the decolorizing solution for 1-2 hr, and replacing the decolorizing solution for several times until the background of the glue is almost completely decolorized.

(3) And scanning and taking a picture. And identifying whether the size of the purified protein is in accordance with the expectation. Experiments were performed with proteins that fit the expectations.

2.3 calcium titration test

The solution used to test cytoplasmic GECI was formulated as follows: solution A:10mM EGTA,100mM KCl,30mM HEPES, pH =7.20. Solution K: solution A +10mM CaCl ₂ pH =7.20. Solutions B to J: solutions a and K were mixed according to the proportions in the table below to obtain solutions B to J, pH =7.20. The pH of the above solutions was adjusted with KOH solution, and the concentration of free calcium ion [ Ca ] in each solution ²⁺ ] _free Is calculated as follows (where K _d Represents Ca when the solution pH =7.20 and the temperature is 20 deg.C ²⁺ And dissociation constant between EGTA):

K _d ＝150.5nM

D＝[EGTA] _total -[Ca ²⁺ ] _total -K _d

[Ca ²⁺ ] _free ＝[-D+(4×K _d ×[Ca ²⁺ ] _total +D ² ) ^1/2 ]/2

TABLE 2 preparation method of cytosolic GECI calcium titration experimental solution and concentration of free calcium ions

The in vitro calcium titration test procedure is as follows:

(1) Adding 50 mu g/mL of protein to be detected into the cytoplasm A-K solution, uniformly mixing, and standing for 20min.

(2) The solution mixed with the protein to be tested was added to a full black 96-well plate at a volume of 200. Mu.L per well, with 4 parallel wells for each free calcium concentration. Background fluorescence was detected using a solution a without added protein.

(3) Determination of the different [ Ca ] s using the fluorescent end-point detection mode of the Flexstation 3 microplate reader ²⁺ ] _free Calcium in the solution indicates the fluorescence value of the protein. The excitation/emission light wavelength of GCaMP6m =485/510nm, the excitation/emission light wavelengths of ncamp7 and NEMO series =490/520nm, the pmtgain value was set at 300.

(4) Deriving data, removing background fluorescence value, fitting a curve with fluorescence value changing along with the concentration of free calcium by using Onesite-Specific binding with Hill slope in nonlinear fitting in Prism, and obtaining K after fitting _d And Hill value is the calcium affinity K of the calcium indicator protein _d And a Hill coefficient.

(5) If it is determined that the calindicator protein contains Mg ²⁺ When Kd and Hill values are determined, mgCl is added to a final concentration of 1mM when preparing the A-K solutions ₂ The solution, the rest steps are the same as (1) - (4)

2.4 kinetic testing

(1) Preparing a solution a:100mM KCl,30mM HEPES, 10. Mu.M CaCl ₂ ，pH＝7.20。

(2) Adding the protein to be detected into the solution a until the protein concentration is 50 mug/mL, uniformly mixing, and standing for 20min.

(3) The solution mixed with the protein to be detected was added to a full-black 96-well plate, the volume of the solution per well was 100. Mu.L, 4 parallel wells were provided for each calcuim-indicating protein, and the background fluorescence value was detected using the a solution to which no protein was added.

(4) The kinetic dissociation constant Koff of the calcuisine indicator protein was measured using the rapid kinetic test mode of the POLARstar Omega microplate reader from BMG Labtech. Mixing the raw materials in a ratio of 1:1 to the wells to be tested, solution A (10mM EGTA,100mM KCl,30mM HEPES, pH = 7.20) was added at a sampling interval of 0.06s, and the excitation/emission wavelength selected when fluorescence values were collected was 485/520nm.

(5) Deriving data, fitting a curve of the fluorescence value changing along with time by using onechase fix in nonlinear fitting in Prism after removing a background fluorescence value, wherein a K value obtained after fitting is a kinetic dissociation coefficient K of the calcium indicator protein _off 。

3Ca ²⁺ Intermittent quantitative measurement of concentration (iPEEQ)

Ca was performed using a Zeiss laser confocal imaging system (Zeiss LSM880 imaging system, 63 times objective oleoscope lens (numerical aperture 1.4), zen2.1 software system) ²⁺ Intermittent quantitative measurements of concentration. Intracellular assays were first performed using NEMOf-transfected HEK-293 cells. Before the start of the recording of the calcium signal, a cycle test of photoisomerization was first performed, the cells were continuously irradiated with 488nm excitation light for 25s at each cycle and with 405nm excitation light for 2.5s at the start of the cycle, the range of the emission light received throughout the process was 500-580 nm,5 cycles later the 10. Mu.M CCh-initiated intracellular calcium signal was indicated using NEMOf.

The in vitro calibration curve was then tested using exactly the same equipment and setup as for the cell assay, the nemuf purified protein was incubated with solutions a, E, G, H, I, J, K in table 2, and the nemuf protein was tested in vitro for 8 photoisomerization cycles (Δ F/F) at different free calcium concentrations ₀ ) _hv Along with Ca ²⁺ Exponentially fitting the curve of the concentration change to obtain K _d1 And hill value h ₁ (ii) a In vitro testing of F for NEMOf protein at different free calcium concentrations _end Associated with Ca ²⁺ Exponentially fitting the curve of the concentration change to obtain K _d2 And h ₂ . Will K _d1 、h ₁ And cellular calcium releaseMeasured at the last photoisomerization cycle before the end of the discharge test (Δ F/F) ₀ ) _hv Substituting into equation 1:

wherein F represents (Δ F/F) ₀ ) _hv ，F _diff ＝F _min -F _max (minimum and maximum (. DELTA.F/F) of in vitro testing ₀ ) _hv ). Obtaining free Ca in cells at rest ²⁺ Concentration [ Ca ] ²⁺ ] _rest . Will K _d2 、h ₂ And [ Ca ²⁺ ] _rest Into equation 1, where F represents the fluorescence value, F _diff ＝F _min -F _max (minimum and maximum fluorescence for in vitro tests). Calculating the fluorescence value F of NEMOf corresponding to the calcium concentration of the cells in the resting state _rest The actually measured resting fluorescence values F and F in the cell experiment _rest Dividing to obtain a calibration coefficient N, dividing all fluorescence values detected in a cell experiment by N for calibration, substituting all the calibrated fluorescence values into the formula, and obtaining [ Ca ] corresponding to each fluorescence value ²⁺ ]。

Preparation example:

according to the amino acid sequence (SEQ ID NO: 2) and structure of NCaMP7, the NEMO series calcium indicator proteins NEMOm, NEMOc, NEMOf and NEMOs are obtained by modifying the NCaMP 7. The specific modification is shown in fig. 1. Nucleotide sequences of NEMO series proteins (the nucleotide sequences of NEMOm, NEMOc, NEMOf and NEMOs are respectively shown in SEQ ID NO: 9-12) are designed according to the nucleotide sequence of NCaMP7, introduced into plasmids, and transformed into cells for expression. The expressed protein is completely consistent with the expectation through sequence analysis, and the amino acid sequences of NEMOm, NEMOc, NEMOf and NEMOs are respectively shown in SEQ ID NO. 3-6.

Example 1:

according to the method shown in the experimental materials and methods, the live cell calcium imaging experiment is carried out on each calcium indicator protein, and the specific method is as follows: expression of these Calcite proteins in HEK293 cells using a cell line containing 1. Mu. MTG + 2.5. Mu.M ioNo + 300. Mu.M EGTA free of CaCl ₂ The cells were treated with the calcium imaging solution for 10min to treat Ca in the endoplasmic reticulum calcium pool of the cells ²⁺ After the calcuim is completely emptied, the fluorescence intensity of the calcuim indicator protein at the moment is collected, namely the minimum fluorescence intensity (F) of the calcuim indicator protein _min ) (ii) a Then 1. Mu.M TG + 2.5. Mu.Miono +100mM Ca was used ²⁺ The calcium imaging solution of (2) was used to treat cells and detect high calcium (100 mM Ca) ²⁺ ) During induced SOCE (Store Operated Calcium Entry), calcium indicates the maximum fluorescence intensity of the protein, i.e., the maximum fluorescence intensity (F) _max )，F _max /F _min The ratio of (a) is the dynamic range of the calcium indicator in vivo test.

Then, according to the method shown in the experimental materials and methods, calcium ion titration test is carried out on each calcium indicator protein, and the dynamic range of each calcium indicator protein is detected in an in vitro experiment mode. The specific method comprises the following steps: in calcium titration experiments, 38870nM of free Ca ²⁺ The fluorescence intensity of the indicator measured under the conditions is recorded as the maximum F _max 0nM of free Ca ²⁺ The fluorescence intensity of the indicator measured under the conditions is recorded as the minimum value F _min ，F _max /F _min The ratio of (a) is the dynamic range of the in vitro test calcium indicator.

NEMOc is a calcium indicator with the greatest dynamic range in the NEMO series, and in vivo testing (live cell calcium imaging experiments) showed NEMOc to be high in calcium (100 mM Ca) ²⁺ ) During induced SOCE, the maximum calcium signal detected was 25-fold higher than NCaMP7 (FIG. 2A). At the same time, both in vivo (live cell calcium imaging experiments) and in vitro (calcium titration and kinetics) tests showed that the dynamic range of NEMO series calcium indicators was much higher than GCaMP6m and NCaMP7 (as shown in figure 2B).

Example 2: live cell calcium imaging experiments

At saturation of Ca ²⁺ In the case of (2), the NEMO series exhibited the advantage of having a maximum fluorescence higher than that of GCaMP6m (see FIG. 3), and in vivo testing (live cell calcium imaging experiments) showed that the NEMO series was at high calcium (100 mM Ca) ²⁺ ) The fluorescence value of the maximum calcium signal detected in the induced SOCE process is far higher than that of GCaMP6m, and the method is suitable for being usedCalcium signal detection in micro-areas such as organelles.

Example 3

Nemuf is the calcium indicator of the fastest response rate in the NEMO series, which is shorter than GCaMP6M and NCaMP7 in response to a single calcium oscillation detected in live cell calcium imaging experiments during 10 μ M CCh-induced calcium oscillations (see fig. 4A-4D); during 1 μ M TG-induced calcium release, the time taken to measure the entire calcium release process was also shorter (fig. 4E). At the same time, in vitro kinetic tests showed the dissociation rate constant K of NEMOf _off The results are much higher than those of GCaMP6m and NCaMP7 (as shown in FIG. 4F), and both indicate that NEMOf is the fastest version in the NEMO series and is more suitable for capturing the rapid dynamic change of calcium signals in neurons.

Example 4

Bierbuesse et al showed a relative change in fluorescence of GECI due to reversible photoisomerization of GECI (noted as (Δ F/F) ₀ ) _hv ) Independent of the amount of GECI expression, only Ca ²⁺ Is thus directly related to (Δ F/F) after calibration ₀ ) _hv Quantitative measurement of intracellular Ca ²⁺ Ca for realizing single fluorescence GECI by concentration and change thereof ²⁺ Quantitative measurement of concentration (PEAQ). On the basis, the method also develops an intermittent quantitative measurement method (Intermitent PEAQ, iPEEAQ) with slightly low time resolution and more convenient operation, does not continuously trigger the switch of photo-isomerism, and calculates the initial fluorescence value F after completing one PEAQ quantitative measurement ₀ Corresponding Ca ²⁺ Concentration, followed by Ca according to fluorescence ²⁺ The concentration of GECI is calibrated by a titration curve with the change of concentration, and Ca can be realized by only measuring the fluorescence value of GECI ²⁺ The quantitative measurement of the concentration greatly reduces phototoxicity and can be used for Ca in certain transient changes ²⁺ Rapid measurement of concentration.

The photoinduced isomerism characteristic of the NEMO series calcium indicator is detected by using an iPEEAQ method. As shown in FIG. 5A, when NEMOf is in a calcium-free environment, the irradiation of violet light (405 nm) results in an increase in its fluorescence value, (. DELTA.F/F) ₀ ) _hv Larger, with Ca ²⁺ Increase in concentration (. DELTA.F/F) ₀ ) _hv Gradually decrease in high calcium environment (39. Mu.M Ca) ²⁺ ) The change in fluorescence due to lower photoisomerization is minimal. Combining the photoisomerization characteristics of NEMO with the iPEEAQ method, NEMOf can achieve cytoplasmic Ca after calibration (see the left and right panels of FIG. 5B) ²⁺ Quantification of concentration indicates that when 10. Mu.M CCh is used to trigger calcium release, the cytosolic calcium level can rise from around 100nM to around 4. Mu.M at rest, as shown in FIG. 5C. The conventional GCaMP6 series calcium probes can only detect the relative change of calcium level before and after stimulation, cannot indicate accurate calcium ion concentration, and thus cannot be used for detecting the change of calcium level or calcium homeostasis. By using the iPEEAQ method, the NEMO series can directly indicate the absolute calcium concentration, so that the relative change of the calcium concentration can be detected, and the resting calcium level and the absolute level and the change of the calcium steady state can be indicated. Therefore, the NEMO tool has wider application range and is superior to GCaMP6 and derivative series thereof.

Sequence information:

GCaMP6m(SEQ ID NO:1)：

MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKTGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKGSYRDTEEEIREAFGVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK

NCaMP7(SEQ ID NO:2):

MVSKGEEENMASLPATHELHIFGSINGIDFDMVGQGTGNPNDGYEELNLKSTMGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVEGTGFPADGPVMTNSLTAEAHDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELRVMIIEVDADGDGTLDFPEFLAMMARKMKYRDTEEEIREAFGVFDKDGNGYIGAAELRHVMTNLGEKLTDEEVGELIREADIDGDGQVNYEEFVQMMTAKGGSGGGSSSRRKWNKAGHAVRAIGRLSSMYFADWCVSKKTCPNDKTIVSTFKWAFITDNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

NEMOm(SEQ ID NO:3)：

MLQNELALKLAGLDINKTGGGSHHHHHHVSKGEEENMASLPATHELHIFGSINGIDFDMVGQGTGNPNDGYEELNLKSTMGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVEGTGFPADGPVMTNSLTAEAHDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELRVMIIEVDADGDGTLDFPEFLAMMARKMKYRDTEEEIREAFGVFDKDGNGYIGAAELRHVMTNLGEKLTDEEVGELIREADIDGDGQVNYEEFVQMMTAKGGSGGSRRKWNKAGHAVRAIGRLSSIYFADWCVSKKTCPNDKTIVSTFKWAFITDNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

NEMOc(SEQ ID NO:4)：

MHHHHHHVSKGEEENMASLPATHELHIFGSINGIDFDMVGQGTGNPNDGYEELNLKSTMGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVEGTGFPADGPVMTNSLTAEAHDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELRVMIIEVDADGDGTLDFPEFLAMMARKMKYRDTEEEIREAFGVFDKDGNGYIGAAELRHVMTNLGEKLTDEEVGELIREADIDGDGQVNYEEFVQMMTAKGGGGSRRKWNKAGHAVRAIGRLSSIYFADWCVSKKTCPNDKTIVSTFKWAFITDNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

NEMOf(SEQ ID NO:5)：

MLQNELALKLAGLDINKTGGGSHHHHHHVSKGEEENMASLPATHELHIFGSINGIDFDMVGQGTGNPNDGYEELNLKSTMGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVEGTGFPADGPVMTNSLTAEAHDDLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVFRSLGQNPTEAELRVMIIEVDADGDGTLDFPEFLAMMARKMKYRDTEEEIREAFGVFDKDGNGYIGAAELRHVMTNLGEKLTDEEVGELIREADIDGDGQVNYEEFVQMMTAKGGGGSRRKWNKAGHAVRAIGRLSSIYFADWCVSKKTCPNDKTIVSTFKWAFITDNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

NEMOs(SEQ ID NO:6)：

MLQNELALKLAGLDINKTGGITLGMDELYKMHHHHHHVSKGEEENMASLPATHELHIFGSINGIDFDMVGQGTGNPNDGYEELNLKSTMGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVEGTGFPADGPVMTNSLTAEAHDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELRVMIIEVDADGDGTLDFPEFLAMMARKMKYRDTEEEIREAFGVFDKDGNGYIGAAELRHVMTNLGEKLTDEEVGELIREADIDGDGQVNYEEFVQMMTAKGGSGGGSSSRRKWNKAGHAVRAIGRLSSIYFADWCVSKKTCPNDKTIVSTFKWAFITDNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK

nucleotide sequence information:

GCaMP6m(SEQ ID NO:7)：

ATGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCTCGCCACCATGGTCGACTCATCACGTCGTAAGTGGAATAAGACAGGTCACGCAGTCAGAGCTATAGGTCGGCTGAGCTCACTCGAGAACGTCTATATCAAGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCGTGCAGTCCAAACTTTCGAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGTGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACCTGCCGGACCAACTGACTGAAGAGCAGATCGCAGAATTTAAAGAGGCTTTCTCCCTATTTGACAAGGACGGGGATGGGACAATAACAACCAAGGAGCTGGGGACGGTGATGCGGTCTCTGGGGCAGAACCCCACAGAAGCAGAGCTGCAGGACATGATCAATGAAGTAGATGCCGACGGTGACGGCACAATCGACTTCCCTGAGTTCCTGACAATGATGGCAAGAAAAGGGAGCTACAGGGACACGGAAGAAGAAATTAGAGAAGCGTTCGGTGTGTTTGATAAGGATGGCAATGGCTACATCAGTGCAGCAGAGCTTCGCCACGTGATGACAAACCTTGGAGAGAAGTTAACAGATGAAGAGGTTGATGAAATGATCAGGGAAGCAGACATCGATGGGGATGGTCAGGTAAACTACGAAGAGTTTGTACAAATGATGACAGCGAAG

NCaMP7(SEQ ID NO:8)：

ATGGTGAGCAAGGGCGAGGAGGAGAACATGGCCAGCCTGCCTGCTACACATGAACTGCACATTTTTGGAAGCATCAACGGCATCGACTTCGACATGGTGGGCCAGGGCACTGGAAATCCTAATGACGGATACGAGGAGCTGAATCTGAAGAGCACCATGGGCGACCTGCAGTTCAGCCCCTGGATACTGGTGCCTCATATTGGCTATGGCTTCCACCAATACCTGCCCTACCCCGACGGAATGAGCCCCTTTCAAGCTGCCATGGTGGATGGCAGCGGATACCAAGTGCATAGAACCATGCAGTTCGAGGACGGCGCCAGCCTTACAGTGAATTATAGATATACCTACGAGGGCAGCCACATCAAGGGCGAGGCACAAGTGGAAGGCACAGGATTTCCCGCTGATGGCCCCGTGATGACCAATAGCCTGACCGCTGAAGCCCACGACCAGCTGACAGAAGAACAGATCGCCGAATTCAAGGAGGCCTTTAGCCTGTTCGACAAGGACGGCGACGGCACCATTACCACAAAAGAGCTGGGCACAGTGATGAGAAGCCTGGGCCAAAATCCCACAGAAGCTGAGCTGAGAGTGATGATCATCGAGGTGGACGCCGACGGCGATGGAACACTGGATTTTCCTGAATTTCTGGCCATGATGGCCAGAAAGATGAAGTACAGAGACACCGAGGAGGAGATCAGAGAGGCCTTCGGCGTGTTTGACAAGGACGGCAATGGATACATTGGCGCCGCTGAACTGAGACACGTGATGACCAATCTGGGCGAGAAGCTGACCGACGAGGAGGTGGGAGAACTGATCAGAGAGGCTGACATTGACGGCGACGGCCAGGTGAATTATGAGGAGTTTGTGCAGATGATGACCGCCAAGGGCGGCAGCGGAGGAGGAAGCAGCAGCAGAAGAAAATGGAATAAGGCCGGCCACGCCGTGAGAGCCATTGGCAGACTTTCTAGCATGTACTTCGCCGACTGGTGCGTGAGCAAGAAGACCTGCCCTAACGACAAGACCATCGTGAGCACCTTCAAGTGGGCCTTCATCACCGACAACGGCAAGAGATACAGAAGCACCGCCAGAACCACCTACACCTTCGCCAAACCCATGGCCGCTAACTACCTGAAGAACCAGCCCATGTACGTGTTCAGAAAGACCGAGCTGAAGCACAGCAAGACCGAGCTGAACTTCAAGGAGTGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCTGTACAAG

NEMOm(SEQ ID NO:9)：

ATGCTGCAGAACGAGCTTGCTCTTAAGTTGGCTGGACTTGATATTAACAAGACTGGAGGAGGTTCTCATCATCATCATCATCATGTGAGCAAGGGCGAGGAGGAGAACATGGCCAGCCTGCCTGCTACACATGAACTGCACATTTTTGGAAGCATCAACGGCATCGACTTCGACATGGTGGGCCAGGGCACTGGAAATCCTAATGACGGATACGAGGAGCTGAATCTGAAGAGCACCATGGGCGACCTGCAGTTCAGCCCCTGGATACTGGTGCCTCATATTGGCTATGGCTTCCACCAATACCTGCCCTACCCCGACGGAATGAGCCCCTTTCAAGCTGCCATGGTGGATGGCAGCGGATACCAAGTGCATAGAACCATGCAGTTCGAGGACGGCGCCAGCCTTACAGTGAATTATAGATATACCTACGAGGGCAGCCACATCAAGGGCGAGGCACAAGTGGAAGGCACAGGATTTCCCGCTGATGGCCCCGTGATGACCAATAGCCTGACCGCTGAAGCCCACGACCAGCTGACAGAAGAACAGATCGCCGAATTCAAGGAGGCCTTTAGCCTGTTCGACAAGGACGGCGACGGCACCATTACCACAAAAGAGCTGGGCACAGTGATGAGAAGCCTGGGCCAAAATCCCACAGAAGCTGAGCTGAGAGTGATGATCATCGAGGTGGACGCCGACGGCGATGGAACACTGGATTTTCCTGAATTTCTGGCCATGATGGCCAGAAAGATGAAGTACAGAGACACCGAGGAGGAGATCAGAGAGGCCTTCGGCGTGTTTGACAAGGACGGCAATGGATACATTGGCGCCGCTGAACTGAGACACGTGATGACCAATCTGGGCGAGAAGCTGACCGACGAGGAGGTGGGAGAACTGATCAGAGAGGCTGACATTGACGGCGACGGCCAGGTGAATTATGAGGAGTTTGTGCAGATGATGACCGCCAAGGGCGGCAGCGGAGGAAGCAGAAGAAAATGGAATAAGGCCGGCCACGCCGTGAGAGCCATTGGCAGACTTTCTAGCATCTACTTCGCCGACTGGTGCGTGAGCAAGAAGACCTGCCCTAACGACAAGACCATCGTGAGCACCTTCAAGTGGGCCTTCATCACCGACAACGGCAAGAGATACAGAAGCACCGCCAGAACCACCTACACCTTCGCCAAACCCATGGCCGCTAACTACCTGAAGAACCAGCCCATGTACGTGTTCAGAAAGACCGAGCTGAAGCACAGCAAGACCGAGCTGAACTTCAAGGAGTGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCTGTACAAG

NEMOc(SEQ ID NO:10)：

ATGCATCATCATCATCATCATGTGAGCAAGGGCGAGGAGGAGAACATGGCCAGCCTGCCTGCTACACATGAACTGCACATTTTTGGAAGCATCAACGGCATCGACTTCGACATGGTGGGCCAGGGCACTGGAAATCCTAATGACGGATACGAGGAGCTGAATCTGAAGAGCACCATGGGCGACCTGCAGTTCAGCCCCTGGATACTGGTGCCTCATATTGGCTATGGCTTCCACCAATACCTGCCCTACCCCGACGGAATGAGCCCCTTTCAAGCTGCCATGGTGGATGGCAGCGGATACCAAGTGCATAGAACCATGCAGTTCGAGGACGGCGCCAGCCTTACAGTGAATTATAGATATACCTACGAGGGCAGCCACATCAAGGGCGAGGCACAAGTGGAAGGCACAGGATTTCCCGCTGATGGCCCCGTGATGACCAATAGCCTGACCGCTGAAGCCCACGACCAGCTGACAGAAGAACAGATCGCCGAATTCAAGGAGGCCTTTAGCCTGTTCGACAAGGACGGCGACGGCACCATTACCACAAAAGAGCTGGGCACAGTGATGAGAAGCCTGGGCCAAAATCCCACAGAAGCTGAGCTGAGAGTGATGATCATCGAGGTGGACGCCGACGGCGATGGAACACTGGATTTTCCTGAATTTCTGGCCATGATGGCCAGAAAGATGAAGTACAGAGACACCGAGGAGGAGATCAGAGAGGCCTTCGGCGTGTTTGACAAGGACGGCAATGGATACATTGGCGCCGCTGAACTGAGACACGTGATGACCAATCTGGGCGAGAAGCTGACCGACGAGGAGGTGGGAGAACTGATCAGAGAGGCTGACATTGACGGCGACGGCCAGGTGAATTATGAGGAGTTTGTGCAGATGATGACCGCCAAGGGCGGCGGAGGCAGCAGAAGAAAATGGAATAAGGCCGGCCACGCCGTGAGAGCCATTGGCAGACTTTCTAGCATCTACTTCGCCGACTGGTGCGTGAGCAAGAAGACCTGCCCTAACGACAAGACCATCGTGAGCACCTTCAAGTGGGCCTTCATCACCGACAACGGCAAGAGATACAGAAGCACCGCCAGAACCACCTACACCTTCGCCAAACCCATGGCCGCTAACTACCTGAAGAACCAGCCCATGTACGTGTTCAGAAAGACCGAGCTGAAGCACAGCAAGACCGAGCTGAACTTCAAGGAGTGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCTGTACAAG

NEMOf(SEQ ID NO:11)：

ATGCTGCAGAACGAGCTTGCTCTTAAGTTGGCTGGACTTGATATTAACAAGACTGGAGGAGGTTCTCATCATCATCATCATCATGTGAGCAAGGGCGAGGAGGAGAACATGGCCAGCCTGCCTGCTACACATGAACTGCACATTTTTGGAAGCATCAACGGCATCGACTTCGACATGGTGGGCCAGGGCACTGGAAATCCTAATGACGGATACGAGGAGCTGAATCTGAAGAGCACCATGGGCGACCTGCAGTTCAGCCCCTGGATACTGGTGCCTCATATTGGCTATGGCTTCCACCAATACCTGCCCTACCCCGACGGAATGAGCCCCTTTCAAGCTGCCATGGTGGATGGCAGCGGATACCAAGTGCATAGAACCATGCAGTTCGAGGACGGCGCCAGCCTTACAGTGAATTATAGATATACCTACGAGGGCAGCCACATCAAGGGCGAGGCACAAGTGGAAGGCACAGGATTTCCCGCTGATGGCCCCGTGATGACCAATAGCCTGACCGCTGAAGCCCACGACGATCTGACAGAAGAACAGATCGCCGAATTCAAGGAGGAGTTTAGCCTGTTCGACAAGGACGGCGACGGCACCATTACCACAAAAGAGCTGGGCACAGTGTTCAGAAGCCTGGGCCAAAATCCCACAGAAGCTGAGCTGAGAGTGATGATCATCGAGGTGGACGCCGACGGCGATGGAACACTGGATTTTCCTGAATTTCTGGCCATGATGGCCAGAAAGATGAAGTACAGAGACACCGAGGAGGAGATCAGAGAGGCCTTCGGCGTGTTTGACAAGGACGGCAATGGATACATTGGCGCCGCTGAACTGAGACACGTGATGACCAATCTGGGCGAGAAGCTGACCGACGAGGAGGTGGGAGAACTGATCAGAGAGGCTGACATTGACGGCGACGGCCAGGTGAATTATGAGGAGTTTGTGCAGATGATGACCGCCAAGGGCGGCGGAGGCAGCAGAAGAAAATGGAATAAGGCCGGCCACGCCGTGAGAGCCATTGGCAGACTTTCTAGCATCTACTTCGCCGACTGGTGCGTGAGCAAGAAGACCTGCCCTAACGACAAGACCATCGTGAGCACCTTCAAGTGGGCCTTCATCACCGACAACGGCAAGAGATACAGAAGCACCGCCAGAACCACCTACACCTTCGCCAAACCCATGGCCGCTAACTACCTGAAGAACCAGCCCATGTACGTGTTCAGAAAGACCGAGCTGAAGCACAGCAAGACCGAGCTGAACTTCAAGGAGTGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCTGTACAAG

NEMOs(SEQ ID NO:12)：

ATGCTGCAGAACGAGCTTGCTCTTAAGTTGGCTGGACTTGATATTAACAAGACTGGAGGGATCACTCTCGGCATGGACGAGCTTTACAAGATGCATCATCATCATCATCATGTGAGCAAGGGCGAGGAGGAGAACATGGCCAGCCTGCCTGCTACACATGAACTGCACATTTTTGGAAGCATCAACGGCATCGACTTCGACATGGTGGGCCAGGGCACTGGAAATCCTAATGACGGATACGAGGAGCTGAATCTGAAGAGCACCATGGGCGACCTGCAGTTCAGCCCCTGGATACTGGTGCCTCATATTGGCTATGGCTTCCACCAATACCTGCCCTACCCCGACGGAATGAGCCCCTTTCAAGCTGCCATGGTGGATGGCAGCGGATACCAAGTGCATAGAACCATGCAGTTCGAGGACGGCGCCAGCCTTACAGTGAATTATAGATATACCTACGAGGGCAGCCACATCAAGGGCGAGGCACAAGTGGAAGGCACAGGATTTCCCGCTGATGGCCCCGTGATGACCAATAGCCTGACCGCTGAAGCCCACGACCAGCTGACAGAAGAACAGATCGCCGAATTCAAGGAGGCCTTTAGCCTGTTCGACAAGGACGGCGACGGCACCATTACCACAAAAGAGCTGGGCACAGTGATGAGAAGCCTGGGCCAAAATCCCACAGAAGCTGAGCTGAGAGTGATGATCATCGAGGTGGACGCCGACGGCGATGGAACACTGGATTTTCCTGAATTTCTGGCCATGATGGCCAGAAAGATGAAGTACAGAGACACCGAGGAGGAGATCAGAGAGGCCTTCGGCGTGTTTGACAAGGACGGCAATGGATACATTGGCGCCGCTGAACTGAGACACGTGATGACCAATCTGGGCGAGAAGCTGACCGACGAGGAGGTGGGAGAACTGATCAGAGAGGCTGACATTGACGGCGACGGCCAGGTGAATTATGAGGAGTTTGTGCAGATGATGACCGCCAAGGGCGGCAGCGGAGGAGGAAGCAGCAGCAGAAGAAAATGGAATAAGGCCGGCCACGCCGTGAGAGCCATTGGCAGACTTTCTAGCATCTACTTCGCCGACTGGTGCGTGAGCAAGAAGACCTGCCCTAACGACAAGACCATCGTGAGCACCTTCAAGTGGGCCTTCATCACCGACAACGGCAAGAGATACAGAAGCACCGCCAGAACCACCTACACCTTCGCCAAACCCATGGCCGCTAACTACCTGAAGAACCAGCCCATGTACGTGTTCAGAAAGACCGAGCTGAAGCACAGCAAGACCGAGCTGAACTTCAAGGAGTGGCAGAAGGCCTTCACCGACGTGATGGGCATGGACGAGCTGTACAAG。

sequence listing

<110> university of Beijing teachers

<120> calcium indication tool for intracellular calcium signal detection and related drug screening and application thereof

<130> DSP1F213334ZJ

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly

1 5 10 15

Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp Leu

20 25 30

Ala Thr Met Val Asp Ser Ser Arg Arg Lys Trp Asn Lys Thr Gly His

35 40 45

Ala Val Arg Ala Ile Gly Arg Leu Ser Ser Leu Glu Asn Val Tyr Ile

50 55 60

Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg

65 70 75 80

His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Tyr His Tyr Gln Gln

85 90 95

Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr

100 105 110

Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp

115 120 125

His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly

130 135 140

Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val Ser Lys

145 150 155 160

Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp

165 170 175

Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly

180 185 190

Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly

195 200 205

Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly

210 215 220

Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe

225 230 235 240

Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile Phe

245 250 255

Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu

260 265 270

Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys

275 280 285

Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Leu Pro Asp

290 295 300

Gln Leu Thr Glu Glu Gln Ile Ala Glu Phe Lys Glu Ala Phe Ser Leu

305 310 315 320

Phe Asp Lys Asp Gly Asp Gly Thr Ile Thr Thr Lys Glu Leu Gly Thr

325 330 335

Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu Leu Gln Asp

340 345 350

Met Ile Asn Glu Val Asp Ala Asp Gly Asp Gly Thr Ile Asp Phe Pro

355 360 365

Glu Phe Leu Thr Met Met Ala Arg Lys Gly Ser Tyr Arg Asp Thr Glu

370 375 380

Glu Glu Ile Arg Glu Ala Phe Gly Val Phe Asp Lys Asp Gly Asn Gly

385 390 395 400

Tyr Ile Ser Ala Ala Glu Leu Arg His Val Met Thr Asn Leu Gly Glu

405 410 415

Lys Leu Thr Asp Glu Glu Val Asp Glu Met Ile Arg Glu Ala Asp Ile

420 425 430

Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val Gln Met Met Thr

435 440 445

Ala Lys

450

<210> 2

<211> 417

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Val Ser Lys Gly Glu Glu Glu Asn Met Ala Ser Leu Pro Ala Thr

1 5 10 15

His Glu Leu His Ile Phe Gly Ser Ile Asn Gly Ile Asp Phe Asp Met

20 25 30

Val Gly Gln Gly Thr Gly Asn Pro Asn Asp Gly Tyr Glu Glu Leu Asn

35 40 45

Leu Lys Ser Thr Met Gly Asp Leu Gln Phe Ser Pro Trp Ile Leu Val

50 55 60

Pro His Ile Gly Tyr Gly Phe His Gln Tyr Leu Pro Tyr Pro Asp Gly

65 70 75 80

Met Ser Pro Phe Gln Ala Ala Met Val Asp Gly Ser Gly Tyr Gln Val

85 90 95

His Arg Thr Met Gln Phe Glu Asp Gly Ala Ser Leu Thr Val Asn Tyr

100 105 110

Arg Tyr Thr Tyr Glu Gly Ser His Ile Lys Gly Glu Ala Gln Val Glu

115 120 125

Gly Thr Gly Phe Pro Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr

130 135 140

Ala Glu Ala His Asp Gln Leu Thr Glu Glu Gln Ile Ala Glu Phe Lys

145 150 155 160

Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Thr Ile Thr Thr

165 170 175

Lys Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu

180 185 190

Ala Glu Leu Arg Val Met Ile Ile Glu Val Asp Ala Asp Gly Asp Gly

195 200 205

Thr Leu Asp Phe Pro Glu Phe Leu Ala Met Met Ala Arg Lys Met Lys

210 215 220

Tyr Arg Asp Thr Glu Glu Glu Ile Arg Glu Ala Phe Gly Val Phe Asp

225 230 235 240

Lys Asp Gly Asn Gly Tyr Ile Gly Ala Ala Glu Leu Arg His Val Met

245 250 255

Thr Asn Leu Gly Glu Lys Leu Thr Asp Glu Glu Val Gly Glu Leu Ile

260 265 270

Arg Glu Ala Asp Ile Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe

275 280 285

Val Gln Met Met Thr Ala Lys Gly Gly Ser Gly Gly Gly Ser Ser Ser

290 295 300

Arg Arg Lys Trp Asn Lys Ala Gly His Ala Val Arg Ala Ile Gly Arg

305 310 315 320

Leu Ser Ser Met Tyr Phe Ala Asp Trp Cys Val Ser Lys Lys Thr Cys

325 330 335

Pro Asn Asp Lys Thr Ile Val Ser Thr Phe Lys Trp Ala Phe Ile Thr

340 345 350

Asp Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr Thr Phe

355 360 365

Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met Tyr Val

370 375 380

Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn Phe Lys

385 390 395 400

Glu Trp Gln Lys Ala Phe Thr Asp Val Met Gly Met Asp Glu Leu Tyr

405 410 415

Lys

<210> 3

<211> 441

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Leu Gln Asn Glu Leu Ala Leu Lys Leu Ala Gly Leu Asp Ile Asn

1 5 10 15

Lys Thr Gly Gly Gly Ser His His His His His His Val Ser Lys Gly

20 25 30

Glu Glu Glu Asn Met Ala Ser Leu Pro Ala Thr His Glu Leu His Ile

35 40 45

Phe Gly Ser Ile Asn Gly Ile Asp Phe Asp Met Val Gly Gln Gly Thr

50 55 60

Gly Asn Pro Asn Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Met

65 70 75 80

Gly Asp Leu Gln Phe Ser Pro Trp Ile Leu Val Pro His Ile Gly Tyr

85 90 95

Gly Phe His Gln Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gln

100 105 110

Ala Ala Met Val Asp Gly Ser Gly Tyr Gln Val His Arg Thr Met Gln

115 120 125

Phe Glu Asp Gly Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu

130 135 140

Gly Ser His Ile Lys Gly Glu Ala Gln Val Glu Gly Thr Gly Phe Pro

145 150 155 160

Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr Ala Glu Ala His Asp

165 170 175

Gln Leu Thr Glu Glu Gln Ile Ala Glu Phe Lys Glu Ala Phe Ser Leu

180 185 190

Phe Asp Lys Asp Gly Asp Gly Thr Ile Thr Thr Lys Glu Leu Gly Thr

195 200 205

Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu Leu Arg Val

210 215 220

Met Ile Ile Glu Val Asp Ala Asp Gly Asp Gly Thr Leu Asp Phe Pro

225 230 235 240

Glu Phe Leu Ala Met Met Ala Arg Lys Met Lys Tyr Arg Asp Thr Glu

245 250 255

Glu Glu Ile Arg Glu Ala Phe Gly Val Phe Asp Lys Asp Gly Asn Gly

260 265 270

Tyr Ile Gly Ala Ala Glu Leu Arg His Val Met Thr Asn Leu Gly Glu

275 280 285

Lys Leu Thr Asp Glu Glu Val Gly Glu Leu Ile Arg Glu Ala Asp Ile

290 295 300

Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val Gln Met Met Thr

305 310 315 320

Ala Lys Gly Gly Ser Gly Gly Ser Arg Arg Lys Trp Asn Lys Ala Gly

325 330 335

His Ala Val Arg Ala Ile Gly Arg Leu Ser Ser Ile Tyr Phe Ala Asp

340 345 350

Trp Cys Val Ser Lys Lys Thr Cys Pro Asn Asp Lys Thr Ile Val Ser

355 360 365

Thr Phe Lys Trp Ala Phe Ile Thr Asp Asn Gly Lys Arg Tyr Arg Ser

370 375 380

Thr Ala Arg Thr Thr Tyr Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr

385 390 395 400

Leu Lys Asn Gln Pro Met Tyr Val Phe Arg Lys Thr Glu Leu Lys His

405 410 415

Ser Lys Thr Glu Leu Asn Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp

420 425 430

Val Met Gly Met Asp Glu Leu Tyr Lys

435 440

<210> 4

<211> 419

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met His His His His His His Val Ser Lys Gly Glu Glu Glu Asn Met

1 5 10 15

Ala Ser Leu Pro Ala Thr His Glu Leu His Ile Phe Gly Ser Ile Asn

20 25 30

Gly Ile Asp Phe Asp Met Val Gly Gln Gly Thr Gly Asn Pro Asn Asp

35 40 45

Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Met Gly Asp Leu Gln Phe

50 55 60

Ser Pro Trp Ile Leu Val Pro His Ile Gly Tyr Gly Phe His Gln Tyr

65 70 75 80

Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gln Ala Ala Met Val Asp

85 90 95

Gly Ser Gly Tyr Gln Val His Arg Thr Met Gln Phe Glu Asp Gly Ala

100 105 110

Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His Ile Lys

115 120 125

Gly Glu Ala Gln Val Glu Gly Thr Gly Phe Pro Ala Asp Gly Pro Val

130 135 140

Met Thr Asn Ser Leu Thr Ala Glu Ala His Asp Gln Leu Thr Glu Glu

145 150 155 160

Gln Ile Ala Glu Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly

165 170 175

Asp Gly Thr Ile Thr Thr Lys Glu Leu Gly Thr Val Met Arg Ser Leu

180 185 190

Gly Gln Asn Pro Thr Glu Ala Glu Leu Arg Val Met Ile Ile Glu Val

195 200 205

Asp Ala Asp Gly Asp Gly Thr Leu Asp Phe Pro Glu Phe Leu Ala Met

210 215 220

Met Ala Arg Lys Met Lys Tyr Arg Asp Thr Glu Glu Glu Ile Arg Glu

225 230 235 240

Ala Phe Gly Val Phe Asp Lys Asp Gly Asn Gly Tyr Ile Gly Ala Ala

245 250 255

Glu Leu Arg His Val Met Thr Asn Leu Gly Glu Lys Leu Thr Asp Glu

260 265 270

Glu Val Gly Glu Leu Ile Arg Glu Ala Asp Ile Asp Gly Asp Gly Gln

275 280 285

Val Asn Tyr Glu Glu Phe Val Gln Met Met Thr Ala Lys Gly Gly Gly

290 295 300

Gly Ser Arg Arg Lys Trp Asn Lys Ala Gly His Ala Val Arg Ala Ile

305 310 315 320

Gly Arg Leu Ser Ser Ile Tyr Phe Ala Asp Trp Cys Val Ser Lys Lys

325 330 335

Thr Cys Pro Asn Asp Lys Thr Ile Val Ser Thr Phe Lys Trp Ala Phe

340 345 350

Ile Thr Asp Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr

355 360 365

Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met

370 375 380

Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn

385 390 395 400

Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp Val Met Gly Met Asp Glu

405 410 415

Leu Tyr Lys

<210> 5

<211> 440

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Leu Gln Asn Glu Leu Ala Leu Lys Leu Ala Gly Leu Asp Ile Asn

1 5 10 15

Lys Thr Gly Gly Gly Ser His His His His His His Val Ser Lys Gly

20 25 30

Glu Glu Glu Asn Met Ala Ser Leu Pro Ala Thr His Glu Leu His Ile

35 40 45

Phe Gly Ser Ile Asn Gly Ile Asp Phe Asp Met Val Gly Gln Gly Thr

50 55 60

Gly Asn Pro Asn Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Met

65 70 75 80

Gly Asp Leu Gln Phe Ser Pro Trp Ile Leu Val Pro His Ile Gly Tyr

85 90 95

Gly Phe His Gln Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gln

100 105 110

Ala Ala Met Val Asp Gly Ser Gly Tyr Gln Val His Arg Thr Met Gln

115 120 125

Phe Glu Asp Gly Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu

130 135 140

Gly Ser His Ile Lys Gly Glu Ala Gln Val Glu Gly Thr Gly Phe Pro

145 150 155 160

Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr Ala Glu Ala His Asp

165 170 175

Asp Leu Thr Glu Glu Gln Ile Ala Glu Phe Lys Glu Glu Phe Ser Leu

180 185 190

Phe Asp Lys Asp Gly Asp Gly Thr Ile Thr Thr Lys Glu Leu Gly Thr

195 200 205

Val Phe Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu Leu Arg Val

210 215 220

Met Ile Ile Glu Val Asp Ala Asp Gly Asp Gly Thr Leu Asp Phe Pro

225 230 235 240

Glu Phe Leu Ala Met Met Ala Arg Lys Met Lys Tyr Arg Asp Thr Glu

245 250 255

Glu Glu Ile Arg Glu Ala Phe Gly Val Phe Asp Lys Asp Gly Asn Gly

260 265 270

Tyr Ile Gly Ala Ala Glu Leu Arg His Val Met Thr Asn Leu Gly Glu

275 280 285

Lys Leu Thr Asp Glu Glu Val Gly Glu Leu Ile Arg Glu Ala Asp Ile

290 295 300

Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val Gln Met Met Thr

305 310 315 320

Ala Lys Gly Gly Gly Gly Ser Arg Arg Lys Trp Asn Lys Ala Gly His

325 330 335

Ala Val Arg Ala Ile Gly Arg Leu Ser Ser Ile Tyr Phe Ala Asp Trp

340 345 350

Cys Val Ser Lys Lys Thr Cys Pro Asn Asp Lys Thr Ile Val Ser Thr

355 360 365

Phe Lys Trp Ala Phe Ile Thr Asp Asn Gly Lys Arg Tyr Arg Ser Thr

370 375 380

Ala Arg Thr Thr Tyr Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu

385 390 395 400

Lys Asn Gln Pro Met Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser

405 410 415

Lys Thr Glu Leu Asn Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp Val

420 425 430

Met Gly Met Asp Glu Leu Tyr Lys

435 440

<210> 6

<211> 453

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Leu Gln Asn Glu Leu Ala Leu Lys Leu Ala Gly Leu Asp Ile Asn

1 5 10 15

Lys Thr Gly Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Met His

20 25 30

His His His His His Val Ser Lys Gly Glu Glu Glu Asn Met Ala Ser

35 40 45

Leu Pro Ala Thr His Glu Leu His Ile Phe Gly Ser Ile Asn Gly Ile

50 55 60

Asp Phe Asp Met Val Gly Gln Gly Thr Gly Asn Pro Asn Asp Gly Tyr

65 70 75 80

Glu Glu Leu Asn Leu Lys Ser Thr Met Gly Asp Leu Gln Phe Ser Pro

85 90 95

Trp Ile Leu Val Pro His Ile Gly Tyr Gly Phe His Gln Tyr Leu Pro

100 105 110

Tyr Pro Asp Gly Met Ser Pro Phe Gln Ala Ala Met Val Asp Gly Ser

115 120 125

Gly Tyr Gln Val His Arg Thr Met Gln Phe Glu Asp Gly Ala Ser Leu

130 135 140

Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His Ile Lys Gly Glu

145 150 155 160

Ala Gln Val Glu Gly Thr Gly Phe Pro Ala Asp Gly Pro Val Met Thr

165 170 175

Asn Ser Leu Thr Ala Glu Ala His Asp Gln Leu Thr Glu Glu Gln Ile

180 185 190

Ala Glu Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly

195 200 205

Thr Ile Thr Thr Lys Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln

210 215 220

Asn Pro Thr Glu Ala Glu Leu Arg Val Met Ile Ile Glu Val Asp Ala

225 230 235 240

Asp Gly Asp Gly Thr Leu Asp Phe Pro Glu Phe Leu Ala Met Met Ala

245 250 255

Arg Lys Met Lys Tyr Arg Asp Thr Glu Glu Glu Ile Arg Glu Ala Phe

260 265 270

Gly Val Phe Asp Lys Asp Gly Asn Gly Tyr Ile Gly Ala Ala Glu Leu

275 280 285

Arg His Val Met Thr Asn Leu Gly Glu Lys Leu Thr Asp Glu Glu Val

290 295 300

Gly Glu Leu Ile Arg Glu Ala Asp Ile Asp Gly Asp Gly Gln Val Asn

305 310 315 320

Tyr Glu Glu Phe Val Gln Met Met Thr Ala Lys Gly Gly Ser Gly Gly

325 330 335

Gly Ser Ser Ser Arg Arg Lys Trp Asn Lys Ala Gly His Ala Val Arg

340 345 350

Ala Ile Gly Arg Leu Ser Ser Ile Tyr Phe Ala Asp Trp Cys Val Ser

355 360 365

Lys Lys Thr Cys Pro Asn Asp Lys Thr Ile Val Ser Thr Phe Lys Trp

370 375 380

Ala Phe Ile Thr Asp Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr

385 390 395 400

Thr Tyr Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln

405 410 415

Pro Met Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu

420 425 430

Leu Asn Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp Val Met Gly Met

435 440 445

Asp Glu Leu Tyr Lys

450

<210> 7

<211> 1350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg 60

ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt 120

cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag 180

aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc 240

cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc 300

ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg 360

aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 420

atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag 480

ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 540

ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc 600

ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 660

ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 720

ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac 780

ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 840

gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 900

aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta 960

tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct 1020

ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac 1080

ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa agggagctac 1140

agggacacgg aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc 1200

tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat 1260

gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac 1320

gaagagtttg tacaaatgat gacagcgaag 1350

<210> 8

<211> 1251

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggtgagca agggcgagga ggagaacatg gccagcctgc ctgctacaca tgaactgcac 60

atttttggaa gcatcaacgg catcgacttc gacatggtgg gccagggcac tggaaatcct 120

aatgacggat acgaggagct gaatctgaag agcaccatgg gcgacctgca gttcagcccc 180

tggatactgg tgcctcatat tggctatggc ttccaccaat acctgcccta ccccgacgga 240

atgagcccct ttcaagctgc catggtggat ggcagcggat accaagtgca tagaaccatg 300

cagttcgagg acggcgccag ccttacagtg aattatagat atacctacga gggcagccac 360

atcaagggcg aggcacaagt ggaaggcaca ggatttcccg ctgatggccc cgtgatgacc 420

aatagcctga ccgctgaagc ccacgaccag ctgacagaag aacagatcgc cgaattcaag 480

gaggccttta gcctgttcga caaggacggc gacggcacca ttaccacaaa agagctgggc 540

acagtgatga gaagcctggg ccaaaatccc acagaagctg agctgagagt gatgatcatc 600

gaggtggacg ccgacggcga tggaacactg gattttcctg aatttctggc catgatggcc 660

agaaagatga agtacagaga caccgaggag gagatcagag aggccttcgg cgtgtttgac 720

aaggacggca atggatacat tggcgccgct gaactgagac acgtgatgac caatctgggc 780

gagaagctga ccgacgagga ggtgggagaa ctgatcagag aggctgacat tgacggcgac 840

ggccaggtga attatgagga gtttgtgcag atgatgaccg ccaagggcgg cagcggagga 900

ggaagcagca gcagaagaaa atggaataag gccggccacg ccgtgagagc cattggcaga 960

ctttctagca tgtacttcgc cgactggtgc gtgagcaaga agacctgccc taacgacaag 1020

accatcgtga gcaccttcaa gtgggccttc atcaccgaca acggcaagag atacagaagc 1080

accgccagaa ccacctacac cttcgccaaa cccatggccg ctaactacct gaagaaccag 1140

cccatgtacg tgttcagaaa gaccgagctg aagcacagca agaccgagct gaacttcaag 1200

gagtggcaga aggccttcac cgacgtgatg ggcatggacg agctgtacaa g 1251

<210> 9

<211> 1323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgctgcaga acgagcttgc tcttaagttg gctggacttg atattaacaa gactggagga 60

ggttctcatc atcatcatca tcatgtgagc aagggcgagg aggagaacat ggccagcctg 120

cctgctacac atgaactgca catttttgga agcatcaacg gcatcgactt cgacatggtg 180

ggccagggca ctggaaatcc taatgacgga tacgaggagc tgaatctgaa gagcaccatg 240

ggcgacctgc agttcagccc ctggatactg gtgcctcata ttggctatgg cttccaccaa 300

tacctgccct accccgacgg aatgagcccc tttcaagctg ccatggtgga tggcagcgga 360

taccaagtgc atagaaccat gcagttcgag gacggcgcca gccttacagt gaattataga 420

tatacctacg agggcagcca catcaagggc gaggcacaag tggaaggcac aggatttccc 480

gctgatggcc ccgtgatgac caatagcctg accgctgaag cccacgacca gctgacagaa 540

gaacagatcg ccgaattcaa ggaggccttt agcctgttcg acaaggacgg cgacggcacc 600

attaccacaa aagagctggg cacagtgatg agaagcctgg gccaaaatcc cacagaagct 660

gagctgagag tgatgatcat cgaggtggac gccgacggcg atggaacact ggattttcct 720

gaatttctgg ccatgatggc cagaaagatg aagtacagag acaccgagga ggagatcaga 780

gaggccttcg gcgtgtttga caaggacggc aatggataca ttggcgccgc tgaactgaga 840

cacgtgatga ccaatctggg cgagaagctg accgacgagg aggtgggaga actgatcaga 900

gaggctgaca ttgacggcga cggccaggtg aattatgagg agtttgtgca gatgatgacc 960

gccaagggcg gcagcggagg aagcagaaga aaatggaata aggccggcca cgccgtgaga 1020

gccattggca gactttctag catctacttc gccgactggt gcgtgagcaa gaagacctgc 1080

cctaacgaca agaccatcgt gagcaccttc aagtgggcct tcatcaccga caacggcaag 1140

agatacagaa gcaccgccag aaccacctac accttcgcca aacccatggc cgctaactac 1200

ctgaagaacc agcccatgta cgtgttcaga aagaccgagc tgaagcacag caagaccgag 1260

ctgaacttca aggagtggca gaaggccttc accgacgtga tgggcatgga cgagctgtac 1320

aag 1323

<210> 10

<211> 1257

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgcatcatc atcatcatca tgtgagcaag ggcgaggagg agaacatggc cagcctgcct 60

gctacacatg aactgcacat ttttggaagc atcaacggca tcgacttcga catggtgggc 120

cagggcactg gaaatcctaa tgacggatac gaggagctga atctgaagag caccatgggc 180

gacctgcagt tcagcccctg gatactggtg cctcatattg gctatggctt ccaccaatac 240

ctgccctacc ccgacggaat gagccccttt caagctgcca tggtggatgg cagcggatac 300

caagtgcata gaaccatgca gttcgaggac ggcgccagcc ttacagtgaa ttatagatat 360

acctacgagg gcagccacat caagggcgag gcacaagtgg aaggcacagg atttcccgct 420

gatggccccg tgatgaccaa tagcctgacc gctgaagccc acgaccagct gacagaagaa 480

cagatcgccg aattcaagga ggcctttagc ctgttcgaca aggacggcga cggcaccatt 540

accacaaaag agctgggcac agtgatgaga agcctgggcc aaaatcccac agaagctgag 600

ctgagagtga tgatcatcga ggtggacgcc gacggcgatg gaacactgga ttttcctgaa 660

tttctggcca tgatggccag aaagatgaag tacagagaca ccgaggagga gatcagagag 720

gccttcggcg tgtttgacaa ggacggcaat ggatacattg gcgccgctga actgagacac 780

gtgatgacca atctgggcga gaagctgacc gacgaggagg tgggagaact gatcagagag 840

gctgacattg acggcgacgg ccaggtgaat tatgaggagt ttgtgcagat gatgaccgcc 900

aagggcggcg gaggcagcag aagaaaatgg aataaggccg gccacgccgt gagagccatt 960

ggcagacttt ctagcatcta cttcgccgac tggtgcgtga gcaagaagac ctgccctaac 1020

gacaagacca tcgtgagcac cttcaagtgg gccttcatca ccgacaacgg caagagatac 1080

agaagcaccg ccagaaccac ctacaccttc gccaaaccca tggccgctaa ctacctgaag 1140

aaccagccca tgtacgtgtt cagaaagacc gagctgaagc acagcaagac cgagctgaac 1200

ttcaaggagt ggcagaaggc cttcaccgac gtgatgggca tggacgagct gtacaag 1257

<210> 11

<211> 1320

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgctgcaga acgagcttgc tcttaagttg gctggacttg atattaacaa gactggagga 60

ggttctcatc atcatcatca tcatgtgagc aagggcgagg aggagaacat ggccagcctg 120

cctgctacac atgaactgca catttttgga agcatcaacg gcatcgactt cgacatggtg 180

ggccagggca ctggaaatcc taatgacgga tacgaggagc tgaatctgaa gagcaccatg 240

ggcgacctgc agttcagccc ctggatactg gtgcctcata ttggctatgg cttccaccaa 300

tacctgccct accccgacgg aatgagcccc tttcaagctg ccatggtgga tggcagcgga 360

taccaagtgc atagaaccat gcagttcgag gacggcgcca gccttacagt gaattataga 420

tatacctacg agggcagcca catcaagggc gaggcacaag tggaaggcac aggatttccc 480

gctgatggcc ccgtgatgac caatagcctg accgctgaag cccacgacga tctgacagaa 540

gaacagatcg ccgaattcaa ggaggagttt agcctgttcg acaaggacgg cgacggcacc 600

attaccacaa aagagctggg cacagtgttc agaagcctgg gccaaaatcc cacagaagct 660

gagctgagag tgatgatcat cgaggtggac gccgacggcg atggaacact ggattttcct 720

gaatttctgg ccatgatggc cagaaagatg aagtacagag acaccgagga ggagatcaga 780

gaggccttcg gcgtgtttga caaggacggc aatggataca ttggcgccgc tgaactgaga 840

cacgtgatga ccaatctggg cgagaagctg accgacgagg aggtgggaga actgatcaga 900

gaggctgaca ttgacggcga cggccaggtg aattatgagg agtttgtgca gatgatgacc 960

gccaagggcg gcggaggcag cagaagaaaa tggaataagg ccggccacgc cgtgagagcc 1020

attggcagac tttctagcat ctacttcgcc gactggtgcg tgagcaagaa gacctgccct 1080

aacgacaaga ccatcgtgag caccttcaag tgggccttca tcaccgacaa cggcaagaga 1140

tacagaagca ccgccagaac cacctacacc ttcgccaaac ccatggccgc taactacctg 1200

aagaaccagc ccatgtacgt gttcagaaag accgagctga agcacagcaa gaccgagctg 1260

aacttcaagg agtggcagaa ggccttcacc gacgtgatgg gcatggacga gctgtacaag 1320

<210> 12

<211> 1359

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgctgcaga acgagcttgc tcttaagttg gctggacttg atattaacaa gactggaggg 60

atcactctcg gcatggacga gctttacaag atgcatcatc atcatcatca tgtgagcaag 120

ggcgaggagg agaacatggc cagcctgcct gctacacatg aactgcacat ttttggaagc 180

atcaacggca tcgacttcga catggtgggc cagggcactg gaaatcctaa tgacggatac 240

gaggagctga atctgaagag caccatgggc gacctgcagt tcagcccctg gatactggtg 300

cctcatattg gctatggctt ccaccaatac ctgccctacc ccgacggaat gagccccttt 360

caagctgcca tggtggatgg cagcggatac caagtgcata gaaccatgca gttcgaggac 420

ggcgccagcc ttacagtgaa ttatagatat acctacgagg gcagccacat caagggcgag 480

gcacaagtgg aaggcacagg atttcccgct gatggccccg tgatgaccaa tagcctgacc 540

gctgaagccc acgaccagct gacagaagaa cagatcgccg aattcaagga ggcctttagc 600

ctgttcgaca aggacggcga cggcaccatt accacaaaag agctgggcac agtgatgaga 660

agcctgggcc aaaatcccac agaagctgag ctgagagtga tgatcatcga ggtggacgcc 720

gacggcgatg gaacactgga ttttcctgaa tttctggcca tgatggccag aaagatgaag 780

tacagagaca ccgaggagga gatcagagag gccttcggcg tgtttgacaa ggacggcaat 840

ggatacattg gcgccgctga actgagacac gtgatgacca atctgggcga gaagctgacc 900

gacgaggagg tgggagaact gatcagagag gctgacattg acggcgacgg ccaggtgaat 960

tatgaggagt ttgtgcagat gatgaccgcc aagggcggca gcggaggagg aagcagcagc 1020

agaagaaaat ggaataaggc cggccacgcc gtgagagcca ttggcagact ttctagcatc 1080

tacttcgccg actggtgcgt gagcaagaag acctgcccta acgacaagac catcgtgagc 1140

accttcaagt gggccttcat caccgacaac ggcaagagat acagaagcac cgccagaacc 1200

acctacacct tcgccaaacc catggccgct aactacctga agaaccagcc catgtacgtg 1260

ttcagaaaga ccgagctgaa gcacagcaag accgagctga acttcaagga gtggcagaag 1320

gccttcaccg acgtgatggg catggacgag ctgtacaag 1359

Claims

1. A calcium indicator protein which is one or more of:

(1) NEMOm: the amino acid sequence is shown as SEQ ID NO. 3;

(2) NEMOc: the amino acid sequence is shown as SEQ ID NO. 4;

(3) NEMOf: the amino acid sequence is shown as SEQ ID NO. 5;

(4) NEMOs: the amino acid sequence is shown in SEQ ID NO. 6.

2. A product for detecting calcium signaling or screening for drugs targeting calcium channels, comprising the calcium indicator protein of claim 1.

3. The product of claim 2, wherein the product is a kit.

4. Use of the calcium indicator protein of claim 1 or the product of claim 2 or 3 for detecting calcium signals or screening for drugs targeting calcium channels.

5. A method of detecting a calcium signal, wherein the method comprises using the calcium indicator protein of claim 1 or the product of claim 2 or 3.

6. The method of claim 5, wherein the method is applied to detect calcium signals in living cells.

7. A method of screening for a drug targeting a calcium channel, wherein the method comprises using the calcium indicator protein of claim 1 or the product of claim 2 or 3.

8. A biomaterial, being:

(1) A nucleic acid molecule encoding the calcemic indicator protein of claim 1;

(2) A plasmid or cell comprising the nucleic acid molecule of (1).