WO2024077672A1 - 一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒 - Google Patents

一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒 Download PDF

Info

Publication number
WO2024077672A1
WO2024077672A1 PCT/CN2022/128591 CN2022128591W WO2024077672A1 WO 2024077672 A1 WO2024077672 A1 WO 2024077672A1 CN 2022128591 W CN2022128591 W CN 2022128591W WO 2024077672 A1 WO2024077672 A1 WO 2024077672A1
Authority
WO
WIPO (PCT)
Prior art keywords
flamp2b
flamp2
camp
probe
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/128591
Other languages
English (en)
French (fr)
Inventor
储军
王亮
刘文峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Publication of WO2024077672A1 publication Critical patent/WO2024077672A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the invention relates to the technical field of biomedicine, and in particular to cAMP fluorescent probes G-Flamp2 and G-Flamp2b and applications and kits thereof.
  • Cyclic adenosine monophosphate is the downstream messenger molecule of the G protein-coupled receptor (GPCR) family, which is currently the largest drug target. Fluorescence imaging of cAMP at the cellular and in vivo levels is an important direction for basic research and drug development of GPCR signaling pathways.
  • cAMP fluorescent probes are mainly divided into fluorescence resonance energy transfer probes based on fluorescent proteins and probes based on single fluorescent proteins. The latter have a larger dynamic range and are easier to use than the former.
  • cAMP probes based on single fluorescent proteins are divided into two subcategories: green and red.
  • the former mainly include Flamindo2, cADDis, cAMPr and G-Flamp1, and the latter mainly include Pink Flamindo, Red cADDis, R-FlincA, R-Flamp1m and R-Flamp2m.
  • cAMP fluorescence imaging in living cells refers to expressing cAMP fluorescent probes in cells and then using a fluorescence microscope to detect changes in the intensity of probe fluorescence. Fluorescent probes are the key to cAMP fluorescence imaging analysis. In recent years, internationally renowned laboratories have developed cAMP single fluorescent protein probes with different performance and spectra using different fluorescent proteins, different cAMP sensing modules and different fusion sites, including Flamindo/Flamindo2/Pink Flamindo probes developed by Dr. Tetsuya Kitaguchi, cADDis/Red cADDis probes developed by Dr. Anne Marie Quinn, R-FlincA probes developed by Dr. Kazuki Horikawa, and cAMPr probes developed by Dr. Justin Blau. For the green cAMP probe, the applicant has developed a higher performance probe G-Flamp1 (CN201911251920.X and CN202010354936.X).
  • the object of the present invention is to overcome the above-mentioned technical problems and provide a cAMP fluorescent probe G-Flamp2 and G-Flamp2b with a large dynamic range, high fluorescence brightness, and high detection sensitivity, which can detect changes in cAMP concentration after cell stimulation and can be used in combination with existing probes of other fluorescence bands (such as red) or red light-sensitive tool proteins to perform dual-color imaging or optical control/fluorescence imaging at the same time, as well as their applications and kits.
  • fluorescence bands such as red
  • red light-sensitive tool proteins to perform dual-color imaging or optical control/fluorescence imaging at the same time, as well as their applications and kits.
  • the purpose of the present invention is to provide a cAMP fluorescent probe G-Flamp2b with a large dynamic range and high fluorescence brightness, which can detect changes in cAMP concentration after cells are stimulated and can be used in combination with existing probes of other fluorescence bands (such as red) or red light-sensitive tool proteins to perform dual-color imaging or optical control/fluorescence imaging at the same time, as well as its application and kit.
  • a cAMP fluorescent probe G-Flamp2b with a large dynamic range and high fluorescence brightness, which can detect changes in cAMP concentration after cells are stimulated and can be used in combination with existing probes of other fluorescence bands (such as red) or red light-sensitive tool proteins to perform dual-color imaging or optical control/fluorescence imaging at the same time, as well as its application and kit.
  • the present invention provides a cAMP fluorescent probe G-Flamp2 and G-Flamp2b, wherein the amino acid sequence of G-Flamp2 is shown in SEQ ID NO: 1;
  • the amino acid sequence of G-Flamp2b is shown in SEQ ID NO:2.
  • the optimal single-photon excitation wavelength of the fluorescent probes G-Flamp2 and G-Flamp2b is 440 ⁇ 10 nm.
  • the optimal two-photon excitation wavelength of the fluorescent probes G-Flamp2 and G-Flamp2b is 900-940 nm.
  • the present invention provides a method for preparing cAMP fluorescent probes R-Flamp2 and G-Flamp2b.
  • a method for preparing a cAMP fluorescent probe G-Flamp2 comprises: performing the following mutations on the amino acids of G-Flamp1: K118N, K126E, S207R, T248S, E316V, I333T, to obtain a G-Flamp2 probe;
  • the amino acid sequence of G-Flamp2 is shown in SEQ ID NO: 1.
  • a preparation method of a cAMP fluorescent probe G-Flamp2b comprises the following steps: subjecting the amino acids of G-Flamp1 to the following mutations: G42E, G61D, V66D, K118N, K126E, F183Y, S207R, T248S, E316V, I333T, R350N, V351S, E355K, S378I, to obtain a G-Flamp2b probe.
  • the amino acid sequence of G-Flamp2b is shown in SEQ ID NO: 2.
  • the present invention provides a use of a cAMP fluorescent probe G-Flamp2 or G-Flamp2b in detecting cAMP.
  • the present invention provides a use of a cAMP fluorescent probe G-Flamp2 or G-Flamp2b in detecting cAMP in living cells and/or living animals.
  • the present invention provides a kit for the fluorescent probes G-Flamp2 and G-Flamp2b.
  • the present invention provides a method for detecting cAMP by fluorescence imaging in living cells.
  • a method for detecting cAMP by fluorescence imaging in living cells comprises the following steps:
  • the transfected cells were cultured overnight. After starving the cells for 2 to 4 hours, the culture medium was replaced with a colorless and transparent live cell fluorescence imaging buffer.
  • the present invention provides a method for detecting cAMP by fluorescence imaging in mammalian cells.
  • a method for detecting cAMP by fluorescence imaging in mammalian cells comprises the following steps:
  • the culture medium is DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature is 37°C, and the CO2 content is 5%;
  • G-Flamp2 or G-Flamp2b plasmid was transfected
  • the fluorescent probe G-Flamp2 provided by the present invention is obtained by performing the following mutations on the amino acids of G-Flamp1: K118N, K126E, S207R, T248S, E316V, I333T.
  • the brightness is 60% higher than that of G-Flamp1
  • the dynamic range ( ⁇ F/F 0 ) is 12.
  • the ⁇ F/F 0 of G-Flamp2 is higher than the ⁇ F/F0 ( ⁇ 10) of G-Flamp1, and it is a cAMP green fluorescent probe with the largest dynamic range. Therefore, under the same expression amount, the probe has a higher fluorescence brightness and a larger response amplitude, that is, a higher sensitivity.
  • the fluorescent probe G-Flamp2 provided by the present invention is a green cAMP probe, and the optimal single-photon excitation wavelength is 440 ⁇ 10nm, and the optimal two-photon excitation wavelength is 900-940nm.
  • the fluorescent probe G-Flamp2 is a green probe that can be used in combination with a large number of existing red probes or other tool proteins to simultaneously perform dual-color imaging or optical control/fluorescence imaging.
  • the fluorescent probe G-Flamp2 provided by the present invention is expressed in mammalian cells, and the change in cAMP concentration after the cells are stimulated by a common fluorescence microscope can be detected.
  • the fluorescent probe G-Flamp2b provided by the present invention is obtained by mutating several amino acids of G-Flamp1 as follows: G42E, G61D, V66D, K118N, K126E, F183Y, S207R, T248S, E316V, I333T, R350N, V351S, E355K, S378I, and its fluorescence brightness in HEK293T cells is 210% higher than that of G-Flamp1. Therefore, the fluorescence brightness of the probe is higher at the same expression level, that is, it can be used at a lower expression level.
  • the fluorescent probe G-Flamp2b provided by the present invention is a green cAMP probe, and the optimal single-photon excitation wavelength is 440 ⁇ 10nm, and the optimal two-photon excitation wavelength is 900-940nm.
  • the fluorescent probe G-Flamp2b provided by the present invention is a green probe that can be used in combination with a large number of existing red probes or other tool proteins to simultaneously perform dual-color imaging or optical control/fluorescence imaging.
  • the fluorescent probe G-Flamp2b provided by the present invention is expressed in mammalian cells, and a common fluorescence microscope can be used to detect changes in cAMP concentration after cells are stimulated by specific stimuli.
  • the cAMP fluorescent probes G-Flamp2 and G-Flamp2b provided by the present invention and their applications and kits have the following advantages:
  • FIG1 is a schematic diagram of the design of two fluorescent probes provided by the present invention.
  • FIG2 is an affinity curve of two fluorescent probes provided by the present invention.
  • Figure A is the affinity curve of G-Flamp2 probe
  • Figure B is the affinity curve of G-Flamp2b probe
  • HEPES buffer at pH 7.2 the normalized fluorescence response of G-Flamp2 or G-Flamp2b probe at different cAMP concentrations
  • FIG3 shows the fluorescence excitation and emission spectra of G-Flamp2 (A) and G-Flamp2b (B) probes;
  • FIG4 is a comparison of the brightness of G-Flamp1 and G-Flamp2 and G-Flamp1 and G-Flamp2b in HEK293T cells; wherein, statistics were performed using a t-test; *** means P ⁇ 0.001, ** means P ⁇ 0.05;
  • Figure 5 shows the response of G-Flamp1, G-Flamp2 and G-Flamp2b probes in HEK293T cells under single-photon excitation; wherein, the excitation wavelength is 440 ⁇ 10nm, and the fluorescence receiving wavelength is 530 ⁇ 15nm; the curve data represents: mean ⁇ standard error of mean (SEM).
  • the G-Flamp2 probe is obtained, whose amino acid sequence is shown in SEQ ID NO:1.
  • the G-Flamp2b probe was obtained, whose amino acid sequence is shown in SEQ ID NO:2.
  • the G-Flamp2 probe was expressed in bacteria, cultured at room temperature for 3 days, and then the bacteria were collected and disrupted by ultrasonication in HEPES buffer (containing 150 mM KCl and 50 mM HEPES) at pH 7.2, and then centrifuged to obtain the supernatant (containing the probe).
  • HEPES buffer containing 150 mM KCl and 50 mM HEPES
  • the excitation wavelength of fluorescence is 450 nm and the receiving wavelength is 515 nm.
  • the G-Flamp2b probe was expressed in bacteria, cultured at room temperature for 3 days, and then the bacteria were collected and ultrasonically disrupted in HEPES buffer (containing 150 mM KCl and 50 mM HEPES) at pH 7.2, and then centrifuged to obtain the supernatant (containing the probe).
  • HEPES buffer containing 150 mM KCl and 50 mM HEPES
  • the excitation wavelength of fluorescence is 450 nm and the receiving wavelength is 515 nm.
  • HEK293T cells were cultured in a 12-well plate in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, at 37°C and 5% CO2 .
  • the same mass of plasmids expressing G-Flamp1 and G-Flamp2 were transfected into cells in different wells using the Lipofectamine 2000 kit.
  • the cells were washed once with a colorless and transparent live cell fluorescence imaging buffer, and then the cells were resuspended in 200 ⁇ l of live cell fluorescence imaging buffer, transferred to a 96-well ELISA plate, and after standing at 37°C for 10 minutes, the fluorescence intensity of the cells was detected using an ELISA reader, and FIG4A was obtained.
  • HEK293T cells were cultured in a 12-well plate in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, at 37°C and 5% CO2 .
  • the same mass of plasmids expressing G-Flamp1 and G-Flamp2b were transfected into cells in different wells using the Lipofectamine 2000 kit.
  • the cells were washed once with a colorless and transparent live cell fluorescence imaging buffer, and then the cells were resuspended in 200 ⁇ l of live cell fluorescence imaging buffer, transferred to a 96-well ELISA plate, and after standing at 37°C for 10 minutes, the fluorescence intensity of the cells was detected using an ELISA instrument, and Figure 4B was obtained.
  • HEK293T cells were cultured in glass-bottomed culture dishes in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 .
  • G-Flamp1 or G-Flamp2 plasmid was transfected using Lipofectamine 2000 kit. After overnight culture, cells were starved for 2 to 4 hours with serum-free and phenol red-free culture medium (purchased from GIBCO), and then the culture medium was replaced with colorless and transparent live cell fluorescence imaging buffer, and imaging analysis was performed using the IX83 single-photon fluorescence microscope (excitation wavelength of about 450nm, maximum emission wavelength of about 530nm) built by our laboratory.
  • the imaging frequency was 15s/frame (15 seconds per frame), and Forskolin (purchased from Beyotime Biotechnology Co., Ltd.) was added in the tenth frame.
  • the final concentration of Forskolin was 60 ⁇ M (for example: there were 500 ⁇ L of live cell fluorescence imaging buffer in the culture dish, 4 ⁇ L of 12mM stock solution was diluted in 300 ⁇ L of live cell fluorescence imaging buffer, and at the tenth frame, the diluted 300 ⁇ L Forskolin solution was dropped into the cell dish being imaged).
  • HEK293T cells were cultured in glass-bottomed culture dishes in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2 .
  • G-Flamp1 or G-Flamp2b plasmid was transfected using Lipofectamine 2000 kit. After overnight culture, cells were starved for 2 to 4 hours with serum-free and phenol red-free medium (purchased from GIBCO), and then the medium was replaced with colorless and transparent live cell fluorescence imaging buffer, and imaging analysis was performed using the IX83 single-photon fluorescence microscope (excitation wavelength of about 450nm, maximum emission wavelength of about 530nm) built by our laboratory.
  • the imaging frequency was 15s/frame (15 seconds per frame), and Forskolin (purchased from Bio-Technology Co., Ltd.) was added in the tenth frame, and the final concentration of Forskolin was 60 ⁇ M (for example: there were 500 ⁇ l of live cell fluorescence imaging buffer in the culture dish, 4 ⁇ l of 12mM stock solution was diluted in 300 ⁇ l of live cell fluorescence imaging buffer, and the diluted Forskolin solution was dropped into the cell dish being imaged in the tenth frame).
  • Forskolin purchased from Bio-Technology Co., Ltd.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

提供cAMP绿色荧光探针G-Flamp2和 G-Flamp2b及其应用和试剂盒。一种cAMP荧光探针G-Flamp2，所述的G-Flamp2的氨基酸序列如SEQ ID NO:1所示。另一种cAMP荧光探针G-Flamp2b，所述的G-Flamp2的氨基酸序列如SEQ ID NO:2所示。优点:(1) 检测范围广。(2)可以用在活细胞和活动物体的cAMP信号检测中。(3)可用于药物筛选。(4) 可与其他颜色的荧光探针或者光敏蛋白工具联合使用。

Description

一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒 技术领域
本发明涉及生物医学技术领域，具体涉及一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒。
背景技术
环磷酸腺苷(cAMP)是目前最大药物靶标G蛋白偶联受体(GPCR)家族的下游信使分子，细胞及活体水平的cAMP荧光成像是GPCR信号通路的基础研究和药物开发的重要方向。cAMP荧光探针主要分为基于荧光蛋白的荧光共振能量转移探针及基于单个荧光蛋白的探针，后者动态范围较前者大且使用简单。目前基于单个荧光蛋白的cAMP探针分为绿色和红色2小类，前者主要有Flamindo2、cADDis、cAMPr和G-Flamp1，后者主要有Pink Flamindo、Red cADDis、R-FlincA、R-Flamp1m和R-Flamp2m。
活细胞中cAMP荧光成像是指将cAMP荧光探针表达在细胞中，然后利用荧光显微镜检测探针荧光的强度变化。荧光探针是cAMP荧光成像分析的关键。近几年，国际上知名实验室利用不同荧光蛋白、不同cAMP感应模块及不同的融合位点，开发了不同性能和光谱的cAMP单荧光蛋白探针，包括Tetsuya Kitaguchi博士开发的Flamindo/Flamindo2/Pink Flamindo探针、Anne Marie Quinn博士开发的cADDis/Red cADDis探针、Kazuki Horikawa博士开发的R-FlincA探针及Justin Blau博士开发的cAMPr探针。对于绿色cAMP探针，申请人已开发更高性能的探针G-Flamp1(CN201911251920.X和CN202010354936.X)。
综上，开发高性能的绿色cAMP荧光探针，提高其在实际应用中的亮度和动态范围，对于提高探测灵敏度和多色成像具有重要意义。
发明内容
本发明的目的在于克服上述技术问题，提供一种动态范围大、荧光亮度高、 检测灵敏度高，可检测细胞受刺激后cAMP浓度改变，可与已有的其他荧光波段(如红色)探针或者红光敏感的工具蛋白联合使用，同时进行双色成像或者进行光学控制/荧光成像联用的cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒。
本发明的目的在于提供一种动态范围较大、荧光亮度高，可检测细胞受刺激后cAMP浓度改变，可与已有的其他荧光波段(如红色)探针或者红光敏感的工具蛋白联合使用，同时进行双色成像或者进行光学控制/荧光成像联用的cAMP荧光探针G-Flamp2b及其应用和试剂盒。
为实现上述目的，本发明采用的技术方案如下。
第一方面，本发明提供一种cAMP荧光探针G-Flamp2和G-Flamp2b，所述的G-Flamp2的氨基酸序列如SEQ ID NO：1所示；
所述的G-Flamp2b的氨基酸序列如SEQ ID NO:2所示。
进一步的，所述的荧光探针G-Flamp2和G-Flamp2b最佳的单光子的激发波长为440±10nm。
进一步的，所述的荧光探针G-Flamp2和G-Flamp2b最佳的双光子的激发波长为900～940nm。
第二方面，本发明提供一种cAMP荧光探针R-Flamp2和G-Flamp2b的制备方法。
一种cAMP荧光探针G-Flamp2的制备方法为：对G-Flamp1的氨基酸进行如下突变：K118N，K126E，S207R，T248S，E316V，I333T，得到G-Flamp2探针；
G-Flamp2的氨基酸序列如SEQ ID NO：1所示。
一种cAMP荧光探针G-Flamp2b的制备方法为：对G-Flamp1的氨基酸进行如下突变：G42E，G61D，V66D，K118N，K126E，F183Y，S207R，T248S，E316V，I333T，R350N，V351S，E355K，S378I，得到G-Flamp2b探针。
G-Flamp2b的氨基酸序列如SEQ ID NO：2所示。
第三方面，本发明提供一种cAMP荧光探针G-Flamp2或G-Flamp2b在检测cAMP中的应用。
第四方面，本发明提供一种cAMP荧光探针G-Flamp2或G-Flamp2b在活细胞和/或动物活体中检测cAMP中的应用。
第五方面，本发明提供一种荧光探针G-Flamp2和G-Flamp2b的试剂盒。
第六方面，本发明提供一种活细胞中cAMP荧光成像检测方法。
一种活细胞中cAMP荧光成像检测方法包括以下步骤：
(1)活细胞培养，细胞密度约50％-80％时，转染G-Flamp2或G-Flamp2b质粒；
(2)转染后的细胞培养过夜，饥饿细胞2～4小时后，将培养基换为无色透明的活细胞荧光成像缓冲液；
(3)单光子的激发波长440±10nm下，荧光显微镜成像分析。
第七方面，本发明提供一种哺乳动物细胞中cAMP荧光成像检测方法。
一种哺乳动物细胞中cAMP荧光成像检测方法包括以下步骤：
(1)哺乳动物细胞培养，培养基为含10％胎牛血清和1％penicillin-streptomycin的DMEM培养液，培养温度为37℃，CO 2含量为5％；
细胞密度约50％-80％时，转染G-Flamp2或G-Flamp2b质粒；
(2)转染后的哺乳动物细胞培养过夜，用不含血清和酚红的培养基饥饿细胞2～4小时后，将培养基换为无色透明的活细胞荧光成像缓冲液；
(3)单光子的激发波长440±10nm，荧光显微镜成像分析，成像频率为15s/frame(15秒每帧)，在第十帧加入Forskolin，至Forskolin终浓度60μM，检测G-Flamp2或G-Flamp2b探针荧光的强度变化。
本发明提供的荧光探针G-Flamp2，是G-Flamp1的氨基酸进行如下突变获得的：K118N，K126E，S207R，T248S，E316V，I333T，在37℃生理温度培养哺乳动物细胞中，亮度较G-Flamp1提高60％，动态范围(ΔF/F 0)为12。G-Flamp2的ΔF/F 0要高于G-Flamp1的ΔF/F0(～10)，为动态范围最大的cAMP绿色荧光探针。因此，该探针在同等表达量下，荧光亮度更高，响应幅度更大，即灵敏度更高。本发明提供的荧光探针G-Flamp2为绿色cAMP探针，最适单光子的激发波长为440±10nm，最适双光子的激发波长为900～940nm。荧光探针G-Flamp2是绿色探针，能够与已有的大量红色探针或者其他工具蛋白联合使用，可同时进行双色成像或者进行光学控制/荧光成像联用。将本发明提供的荧光探针G-Flamp2表达在哺乳动物细胞中，利用普通的荧光显微镜，即可检测细胞受特定刺激后cAMP浓度改变。
本发明提供的荧光探针G-Flamp2b，是G-Flamp1的若干氨基酸进行如下突变获得的：G42E，G61D，V66D，K118N，K126E，F183Y，S207R，T248S，E316V，I333T，R350N，V351S，E355K，S378I，其在HEK293T细胞中荧光亮度较G-Flamp1提高210％，因此，该探针在同等表达量下，荧光亮度更高，即可以在更低的表达量下使用。本发明提供的荧光探针G-Flamp2b为绿色cAMP探针，最适单光子的激发波长为440±10nm，最适双光子的激发波长为900～940nm。本发明提供的荧光探针G-Flamp2b是绿色探针，能够与已有的大量红色探针或者其他工具蛋白联合使用，可同时进行双色成像或者进行光学控制/荧光成像联用。将本发明提供的荧光探针G-Flamp2b表达在哺乳动物细胞中，利用普通的荧光显微镜，即可检测细胞受特定刺激后cAMP浓度改变。
与现有技术相比，本发明提供的一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒的优点：
(1)荧光亮度高、灵敏度高。
(2)可以用于溶液中cAMP浓度检测。
(3)可以用于活细胞和活动物体的cAMP信号检测。
(4)应用范围广，可与红色探针或红色光敏蛋白工具联合使用。
附图说明
图1为本发明提供的2种荧光探针的设计示意图；
图2为本发明提供的2种荧光探针探针的亲和力曲线；
图中A为G-Flamp2探针的亲和力曲线；B为G-Flamp2b探针的亲和力曲线；在pH 7.2的HEPES缓冲液中，G-Flamp2或G-Flamp2b探针在不同cAMP浓度下的归一化的荧光响应；
图3为G-Flamp2(A图)和G-Flamp2b(B图)探针的荧光激发和发射光谱；
图4为G-Flamp1和G-Flamp2以及G-Flamp1和G-Flamp2b在HEK293T细胞中的亮度比较；其中，统计用t检验；***为P<0.001,**为P<0.05；
图5为单光子激发下，G-Flamp1、G-Flamp2和G-Flamp2b探针在HEK293T细胞中的响应；其中，激发波长为440±10nm，荧光接收波长为530±15nm；曲线数据表示：平均值±均值标准误差(Standard error of mean，SEM)。
具体实施方式
为使本领域的技术人员更好地理解本发明的技术方案，以下实施例对本发明的作进一步详细描述，以下实施例仅用于说明发明，但不用来限制本发明的范围。
实施例1
对G-Flamp1的若干氨基酸进行突变，即得到G-Flamp2探针，其氨基酸序列如SEQ ID NO:1所示。
对G-Flamp1的若干氨基酸进行突变，即得到G-Flamp2b探针，其氨基酸序列如SEQ ID NO:2所示。
实施例2
将G-Flamp2探针表达在细菌中，室温培养3天收集菌体，在pH＝7.2的HEPES缓冲液(含150mM KCl及50mM HEPES)中超声破碎，然后离心获取上 清液(含探针)。
取120μL探针溶液，利用多功能酶标仪Infinite M1000 PRO检测探针对不同cAMP浓度的的响应。荧光的激发波长为450nm，接收波长为515nm。
获取ΔF/F 0与cAMP浓度的曲线，即可得G-Flamp2在不同cAMP浓度下荧光变化，及解离常数K d(图2A)。同时可以获取荧光激发和发射光谱(图3A)。
将G-Flamp2b探针表达在细菌中，室温培养3天收集菌体，在pH＝7.2的HEPES缓冲液(含150mM KCl及50mM HEPES)中超声破碎，然后离心获取上清液(含探针)。
取120μL探针溶液，利用多功能酶标仪Infinite M1000 PRO检测探针对不同cAMP浓度的的响应。荧光的激发波长为450nm，接收波长为515nm。
获取ΔF/F0与cAMP浓度的曲线，即可得G-Flamp2b不同cAMP浓度下荧光变化，及解离常数Kd(图2B)。同时可以获取荧光激发和发射光谱(图3B)。
实施例3
HEK293T细胞培养在12孔板中，培养基为含10％胎牛血清和1％penicillin-streptomycin的DMEM培养液，培养温度为37℃，CO 2含量为5％。用Lipofectamine 2000试剂盒转染相同质量的可表达G-Flamp1和G-Flamp2的质粒至不同孔的细胞中。约40小时后，用无色透明的活细胞荧光成像缓冲液清洗细胞一遍，然后将细胞重悬在200μl的活细胞荧光成像缓冲液中，转移至96孔酶标板，37℃静置10分钟后，用酶标仪检测细胞的荧光强度，即得到附图4A。
HEK293T细胞培养在12孔板中，培养基为含10％胎牛血清和1％penicillin-streptomycin的DMEM培养液，培养温度为37℃，CO 2含量为5％。用Lipofectamine 2000试剂盒转染相同质量的可表达G-Flamp1和G-Flamp2b的质粒至不同孔的细胞中。约40小时后，用无色透明的活细胞荧光成像缓冲液清洗细胞一遍，然后将细胞重悬在200μl的活细胞荧光成像缓冲液中，转移 至96孔酶标板，37℃静置10分钟后，用酶标仪检测细胞的荧光强度，即得到附图4B。
实施例4
将HEK293T细胞培养在玻璃底的培养皿中，培养基为含10％胎牛血清和1％penicillin-streptomycin的DMEM，培养温度为37℃，CO 2含量为5％。
细胞密度为50％-80％左右时，用Lipofectamine 2000试剂盒转染G-Flamp1或G-Flamp2质粒。过夜培养后，用不含血清和酚红的培养基(购自GIBCO公司)饥饿细胞2到4小时，然后将培养基换为无色透明的活细胞荧光成像缓冲液，采用本实验室自行搭建的IX83单光子荧光显微镜(激发波长450nm左右，最大发射波长530nm左右)进行成像分析。
成像频率为15s/frame(15秒每帧)，在第十帧加入Forskolin(购自碧云天生物技术公司)，Forskolin终浓度为60μM(例：培养皿中有500μL活细胞荧光成像缓冲液，将4μL 12mM的母液稀释于300μL活细胞荧光成像缓冲液中，第十帧时将此稀释好的300μL Forskolin溶液滴入正在成像的细胞皿中)。
Forskolin刺激后，细胞中G-Flamp1和G-Flamp2荧光强度的变化，见附图5A。至此完成了哺乳动物细胞内cAMP浓度变化的荧光成像步骤。
将HEK293T细胞培养在玻璃底的培养皿中，培养基为含10％胎牛血清和1％penicillin-streptomycin的DMEM，培养温度为37℃，CO 2含量为5％。
细胞密度为50％-80％左右时，用Lipofectamine 2000试剂盒转染G-Flamp1或G-Flamp2b质粒。过夜培养后，用不含血清和酚红的培养基(购自GIBCO公司)饥饿细胞2到4小时，然后将培养基换为无色透明的活细胞荧光成像缓冲液，采用本实验室自行搭建的IX83单光子荧光显微镜(激发波长450nm左右，最大发射波长530nm左右)进行成像分析。
成像频率为15s/frame(15秒每帧)，在第十帧加入Forskolin(购自碧云天生物技术公司)，Forskolin终浓度为60μM(例：培养皿中有500微升活细胞荧光成像缓冲液，将4微升12mM的母液稀释于300微升活细胞荧光成像缓冲液中，第十帧时将此稀释好的Forskolin溶液滴入正在成像的细胞皿中)。
Forskolin刺激后，细胞中G-Flamp1和G-Flamp2b荧光强度的变化，见附图5B。至此完成了哺乳动物细胞内cAMP浓度变化的荧光成像步骤。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种变换，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征和步骤，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (9)

  1. 一种cAMP荧光探针G-Flamp2和G-Flamp2b，其特征在于：所述的G-Flamp2的氨基酸序列如SEQ ID NO:1所示；
    所述的G-Flamp2b的氨基酸序列如SEQ ID NO:2所示。
  2. 根据权利要求1所述的一种cAMP荧光探针G-Flamp2和G-Flamp2b，其特征在于：所述的荧光探针G-Flamp2及G-Flamp2b最佳的单光子的激发波长为440±10nm。
  3. 根据权利要求1或2所述的一种cAMP荧光探针G-Flamp2和G-Flamp2b，其特征在于：所述的荧光探针G-Flamp2及G-Flamp2b最佳的双光子的激发波长为900～940nm。
  4. 一种cAMP荧光探针G-Flamp2和G-Flamp2b的制备方法，其特征在于，所述的方法为：对G-Flamp1的氨基酸进行如下突变：K118N,K126E,S207R,T248S,E316V,I333T，得到G-Flamp2探针；
    对G-Flamp1的氨基酸进行如下突变：G42E，G61D，V66D，K118N，K126E，F183Y，S207R，T248S，E316V，I333T，R350N，V351S，E355K，S378I，得到G-Flamp2b探针。
  5. 一种cAMP荧光探针G-Flamp2和G-Flamp2b在检测cAMP中的应用。
  6. 一种cAMP荧光探针G-Flamp2和G-Flamp2b在活细胞和/或动物活体中检测cAMP中的应用。
  7. 一种包含权利要求1～4任意一项所述的荧光探针G-Flamp2和G-Flamp2b的试剂盒。
  8. 一种活细胞中cAMP荧光成像检测方法，其特征在于，所述的方法包括以下步骤：
    (1)活细胞培养，细胞密度在约50％-80％时，转染G-Flamp2或G-Flamp2b 质粒；
    (2)转染后的细胞培养过夜，饥饿细胞2～4小时后，将培养基换为无色透明的缓冲液；
    (3)单光子的激发波长440±10nm下，荧光显微镜成像分析。
  9. 一种哺乳动物细胞中cAMP荧光成像检测方法，其特征在于，所述的方法包括以下步骤：
    (1)哺乳动物细胞培养，培养基为含10％胎牛血清和1％penicillin-streptomycin的DMEM培养液，培养温度为37℃，CO 2含量为5％；
    细胞密度约50％-80％时，转染G-Flamp2或G-Flamp2b质粒；
    (2)转染后的哺乳动物细胞培养过夜，用不含血清和酚红的培养基饥饿细胞2～4小时后，将培养基换为无色透明的活细胞荧光成像缓冲液；
    (3)单光子的激发波长440±10nm，荧光显微镜成像分析，成像频率为15秒每帧，在第十帧加入Forskolin，至Forskolin终浓度60μM，检测G-Flamp2或G-Flamp2b探针荧光的强度变化。
PCT/CN2022/128591 2022-10-12 2022-10-31 一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒 Ceased WO2024077672A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211247977.4A CN117866067A (zh) 2022-10-12 2022-10-12 一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒
CN202211247977.4 2022-10-12

Publications (1)

Publication Number Publication Date
WO2024077672A1 true WO2024077672A1 (zh) 2024-04-18

Family

ID=90583457

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/128591 Ceased WO2024077672A1 (zh) 2022-10-12 2022-10-31 一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒

Country Status (2)

Country Link
CN (1) CN117866067A (zh)
WO (1) WO2024077672A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070111270A1 (en) * 2004-08-23 2007-05-17 The Johns Hopkins University cAMP reporters and high throughput assays
CN109627344A (zh) * 2018-12-28 2019-04-16 深圳先进技术研究院 cAMP荧光探针及其应用
CN113024674A (zh) * 2019-12-09 2021-06-25 中国科学院深圳先进技术研究院 荧光亮度宽幅变化的环磷酸腺苷荧光探针
CN113567402A (zh) * 2020-04-29 2021-10-29 中国科学院深圳先进技术研究院 一种cAMP荧光探针G-Flamp1的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070111270A1 (en) * 2004-08-23 2007-05-17 The Johns Hopkins University cAMP reporters and high throughput assays
CN109627344A (zh) * 2018-12-28 2019-04-16 深圳先进技术研究院 cAMP荧光探针及其应用
CN113024674A (zh) * 2019-12-09 2021-06-25 中国科学院深圳先进技术研究院 荧光亮度宽幅变化的环磷酸腺苷荧光探针
CN113567402A (zh) * 2020-04-29 2021-10-29 中国科学院深圳先进技术研究院 一种cAMP荧光探针G-Flamp1的应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIAYUAN ZHANG; YUXIN SONG; CHUCHEN WANG; JING WANG: "A Novel Fluorescent Sensor for cAMP Based on E. Coli CAP Protein", JOURNAL OF CHINESE PHARMACEUTICAL SCIENCES, SCHOOL OF PHARMACEUTICAL SCIENCES, BEIJING MEDICAL UNIVERSITY, BEIJING , CN, vol. 28, no. 8, 31 December 2019 (2019-12-31), Beijing , CN , pages 556 - 560, XP009553838, ISSN: 1003-1057 *
SEIKO KAWATAA: "Green Fluorescent cAMP Indicator of High Speed and Specificity Suitable for Neuronal Live-cell Imaging", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 119, no. 28, 6 July 2022 (2022-07-06), pages e2122618119, XP093157583, ISSN: 0027-8424 *

Also Published As

Publication number Publication date
CN117866067A (zh) 2024-04-12

Similar Documents

Publication Publication Date Title
Selenko et al. Looking into live cells with in-cell NMR spectroscopy
CN113501881A (zh) 融合蛋白
CN110702589A (zh) 一种流式细胞仪检测外泌体的方法
Mortati et al. CARS and SHG microscopy to follow collagen production in living human corneal fibroblasts and mesenchymal stem cells in fibrin hydrogel 3D cultures
Saha et al. Dynamics of the HIV gag lattice detected by localization correlation analysis and time-lapse IPALM
Kuhlemann et al. Genetic code expansion and click-chemistry labeling to visualize GABA-A receptors by super-resolution microscopy
Albrecht et al. Rapid 3D-STORM imaging of diverse molecular targets in tissue
CN112480271B (zh) 高性能红色cAMP荧光探针及其应用
Han et al. Multifunctional peptide-oligonucleotide conjugate promoted sensitive electrochemical biosensing of cardiac troponin I
Mao et al. A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein–Protein Interactions
CN117866067A (zh) 一种cAMP荧光探针G-Flamp2和G-Flamp2b及其应用和试剂盒
Stockhammer et al. When less is more: Endogenous tagging with TurboID increases the sensitivity of proximity labelling-based experiments
CN116693689A (zh) cAMP荧光探针R-Flamp2m及其应用和试剂盒
CN116577501B (zh) 一种基于纳米抗体-单抗夹心检测新型冠状病毒核衣壳蛋白的均相方法
WO2021217479A1 (zh) 一种camp荧光探针g-flamp1的应用
CN113567402B (zh) 一种cAMP荧光探针G-Flamp1的应用
CN120118170A (zh) 一种cAMP荧光探针G-Flamp3及其应用
Banerjee et al. Visualizing the conformational dynamics of membrane receptors using single-molecule FRET
CN113024674B (zh) 荧光亮度宽幅变化的环磷酸腺苷荧光探针
CN118150530A (zh) mTORC1荧光探针SEaTOR的设计构建及其应用
US20240069027A1 (en) Use of nitrogen-doped carbon fluorescent quantum dot in preparation of product for detecting aerobic glycolysis
CN116693690A (zh) 一种cGMP G-Flig系列探针及其应用和试剂盒
CN120483938B (zh) 一种监测线粒体-溶酶体互作介导溶酶体酸化过程的方法
Kawamura et al. Electroporation-mediated delivery of protein biosensors for metabolic imaging in differentiated myotubes
CN114058623B (zh) 一种核酸适体识别并结合整合素α3亚基及其相关功能

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22961855

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22961855

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 22961855

Country of ref document: EP

Kind code of ref document: A1