Targeting light-activated fluorescent protein Dronpa antibody and related application thereof

Abstract

The invention discloses an antibody targeting light activated fluorescent protein Dronpa and related application thereof.The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in a heavy chain variable region of the antibody are shown as SEQ ID NO.4-6 respectively, and the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 in a light chain variable region of the antibody are shown as SEQ ID NO.7-9 respectively. According to the antibody and the application thereof, accurate tool support is provided for detection and analysis of Dronpa protein, and the antibody can be applied to detection of Dronpa protein. Meanwhile, a key technical guarantee is provided for life science experiments (such as living cell imaging and molecular mechanism research) and potential clinical application based on the Dronpa protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody that targets the photoactivated fluorescent protein Dronpa and its related applications. Background Technology

[0002] Photoactivatable Fluorescent Proteins (PA-FP) technology induces conformational or chemical changes in fluorescent proteins using light of a specific wavelength, transforming them from a non-fluorescent or low-fluorescence state to a high-fluorescence state. This unique light-controlled switching characteristic gives PA-FP numerous advantages over traditional techniques in protein labeling. In intracellular dynamic tracking scenarios, PA-FP enables precise tracking of protein dynamics, avoiding interference from whole-cell fluorescence signals. In developmental biology research, it can track the spatiotemporal trajectory of cell differentiation and tissue formation in real time. Moreover, PA-FP is non-fluorescent when unactivated, emitting light only after activation in the target region, significantly reducing background noise. In live-cell super-resolution imaging, combined with single-molecule localization technology, it can achieve ultra-high resolution at the 20 nm level with near-zero background signal. PA-FP technology plays an irreplaceable and crucial role in cutting-edge fields such as super-resolution microscopy, in vivo dynamic research, and precision medicine.

[0003] Dronpa, a novel photoresponsive fluorescent protein derived from corals (Pectiniidae family), is an important member of the PA-FP family. Its core characteristic is its unique and stable reversible photo-switching property. The photophysical mechanism of this protein is based on a photoinduced change in chromophore conformation: in its inactive state, Dronpa exists primarily in a non-fluorescent form. Upon excitation with 405 nm violet light, its internal chromophore structure changes, allowing it to efficiently switch to a fluorescently activated state and stably emit green fluorescence with a peak at 518 nm under 488 nm blue light excitation. When exposed to high-intensity 488 nm blue light again, the fluorescent state can rapidly reverse back to the initial non-fluorescent state, and this activation-inactivation cycle can be repeated multiple times without significant loss of fluorescent activity. Dronpa is suitable for spatiotemporal dynamic tracking of proteins in living cells, super-resolution microscopy imaging of subcellular structures (such as STED and PALM techniques), real-time monitoring of protein-protein interactions, cell lineage tracing, and multicolor fluorescence imaging experiments. It provides a powerful tool to deeply understand the dynamic behavior of biomolecules in living organisms, signaling pathway regulation mechanisms, and the molecular mechanisms of disease development. It is one of the indispensable core reagents in modern cell biology and molecular biology research.

[0004] Currently, conventional methods for detecting the photoactivated fluorescent protein Dronpa mainly include direct detection based on fluorescence characteristics and traditional antibody detection. Direct detection based on fluorescence characteristics utilizes the characteristic that Dronpa emits fluorescence when excited by light of a specific wavelength. This allows for direct observation and measurement of Dronpa in the sample using equipment such as fluorescence microscopes and fluorescence spectrophotometers. This method is relatively simple to operate and provides a direct view of Dronpa's fluorescence signal, and was widely used in early related studies. Traditional antibody detection utilizes the binding of specific antibodies to the Dronpa protein, and then uses techniques such as immunofluorescence and immunoblotting to detect the complex, thereby indirectly determining the presence and content of Dronpa. In immunofluorescence experiments, a secondary antibody labeled with fluorescein interacts with a primary antibody bound to Dronpa, exhibiting a fluorescence signal under a fluorescence microscope, thus enabling the localization and semi-quantitative analysis of Dronpa.

[0005] However, these conventional detection methods have many limitations. Direct detection based on fluorescence characteristics often lacks sensitivity; when the Dronpa content in a sample is low, the weak fluorescence signal is easily masked by background noise, making accurate detection difficult. Furthermore, the autofluorescence characteristics of different biological samples vary, interfering with the specificity of the fluorescence signal and making it difficult to accurately distinguish Dronpa fluorescence from background fluorescence, significantly impacting the accuracy of the detection results. While traditional antibody detection has a certain degree of specificity, in practical applications, due to the inconsistent quality of antibodies, non-specific binding often occurs, leading to false positive results and reducing the reliability of the detection. Moreover, most of these traditional detection methods struggle to accurately quantify Dronpa, failing to provide sufficient data support for studies requiring precise understanding of Dronpa content changes. Some detection methods, such as prolonged high-intensity light excitation during fluorescence microscopy, can damage Dronpa and cells in the sample, affecting the sample's biological activity and limiting its application in fields such as live cell dynamics studies. Therefore, developing a novel detection technology that is highly sensitive, specific, accurate in quantification, and causes minimal damage to samples has become one of the key technical problems that urgently need to be solved in the current Dronpa research field. Summary of the Invention

[0006] In order to overcome the above-mentioned technical problems existing in the field, the purpose of this invention is to provide an antibody that targets the photoactivated fluorescent protein Dronpa and its related applications.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] In a first aspect, the present invention provides an antibody against the photoactivated fluorescent protein Dronpa or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;

[0009] The heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3, whose corresponding amino acid sequences are shown in SEQ ID NO:4-6, respectively.

[0010] The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, whose corresponding amino acid sequences are shown in SEQ ID NO:7-9, respectively.

[0011] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2.

[0012] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3.

[0013] In this invention, the complementarity-determining region (CDR) is a key segment in the antibody variable region that directly binds to the antigen, determining the antibody's specificity and affinity. Specifically, a complete antibody molecule contains six CDR segments: CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region (VH) and CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region (VL). These six CDR segments synergistically form an antigen-binding pocket through spatial conformation, jointly recognizing antigen epitopes. Besides the complementarity-determining region (CDR), the relatively conserved portion of the variable region is called the backbone region (FR). The backbone region is designed to provide structural support for the CDR and maintain the spatial conformation stability of the antibody variable region. The variable regions of the natural heavy chain and light chain each contain four backbone region segments (FR1, FR2, FR3, FR4). The structure of the heavy chain variable region is usually HFR1―CDR-H1―HFR2―CDR-H2―HFR3―CDR-H3―HFR4; the structure of the light chain variable region is usually LFR1―CDR-L1―LFR2―CDR-L2―LFR3―CDR-L3―LFR4.

[0014] In some embodiments, the antibody CDR can be determined using various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, etc. These coding systems are known in the art. For example, the amino acid sequence numbering of the antigen-binding protein involved herein can be used to determine the CDR sequence of the antibody according to any of the IMGT, CCG, Kabat, AbM, or Chothia numbering schemes. It should be noted that the CDR sequence determined by encoding the heavy chain variable region and light chain variable region of the antibody provided by this invention using any coding system is within the scope of protection of this invention.

[0015] In some embodiments, amino acid sequences that have more than 75% homology with the amino acid sequences corresponding to the heavy chain variable region and light chain variable region of the antibody described in this invention are all within the protection scope of this invention.

[0016] In some embodiments, the antibodies described in this invention can be prepared using methods conventional in the art, such as hybridoma technology, phage display technology, and cell line expression well-known in the art. Furthermore, this invention also provides antibody fusion proteins comprising the antibodies described in this invention or their antigen-binding fragments as expression targets, active molecules, or targeting molecules. Fusion proteins can be formed by adding tags (e.g., His6 tags) to both ends of the antibodies described in this invention to facilitate protein expression or purification. The tags do not affect the function of the target protein and can be easily cleaved.

[0017] In a second aspect, the present invention provides a nucleic acid molecule that encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0018] In some embodiments, the nucleic acid molecule may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The invention also includes degenerate variants of the nucleotide sequence encoding the antibody or its antigen-binding fragment, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0019] In some embodiments, the coding sequence of the antibody or fragment thereof described in this invention can be readily obtained by those skilled in the art based on the amino acid sequence and codons. Those skilled in the art can also modify the expression of the peptide in different species through codon optimization; codon preferences for different species are a conventional technique in the art.

[0020] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention;

[0021] Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector;

[0022] Optionally, the expression vector is a plasmid vector, a bacteriophage vector, a viral vector, or a yeast expression vector;

[0023] Optionally, the viral vector is a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, or herpesvirus vector.

[0024] In this invention, the expression vector refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the above-described functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0025] The present invention does not impose any particular limitation on the specific type of expression vector. Those skilled in the art can make conventional selections according to actual needs. Any type of expression vector containing the nucleic acid molecule described in the second aspect of the present invention is within the protection scope of the present invention.

[0026] In some implementations, the plasmid vector may specifically include conventional vectors in the art such as the pcDNA series (pcDNA3.1 vector, pcDNA3.4 vector), pUC series (pUC18, pUC19), pBluescript series (pBluescript II KS (+), pBluescript II SK (-)), pCMV series (pCMV-HA, pCMV-Myc), pET series (pET-28a, pET-32a), and pGEX series (pGEX-4T-1, pGEX-6P-1). Among them, the pcDNA series vectors are suitable for eukaryotic expression in mammalian cells and can efficiently drive the expression of antibody genes in cells such as HEK293; the pET and pGEX series vectors are often used in prokaryotic expression systems, which facilitates the efficient preparation of antibody fragments or full-length antibodies in Escherichia coli; and the pUC and pBluescript series vectors are often used as tool vectors for gene cloning and subcloning due to their simple construction and high copy number.

[0027] In some implementations, the phage vectors can be specifically divided into two main categories: λ phage vectors (such as λgt10, λgt11, EMBL3, EMBL4) and M13 phage vectors (such as M13mp18, M13mp19). λ phage vectors can accommodate larger fragments of exogenous nucleic acid molecules, making them suitable for the construction and screening of antibody gene libraries. Some vectors also carry the lacZ gene, allowing for rapid identification of positive clones through blue-white screening. M13 phage vectors are single-stranded circular DNA vectors that generate double-stranded replicating DNA during replication, facilitating gene manipulation. Furthermore, the single-stranded phage particles they secrete can be used for DNA sequencing, site-directed mutagenesis, and other experiments, making them widely used in the cloning and sequence analysis of antibody variable region genes.

[0028] In some implementations, the viral vector may specifically include lentiviral vectors (such as pLVX-Puro, pLVX-IRES-ZsGreen1), adenoviral vectors (such as pAdEasy-1, pAdTrack-CMV), adeno-associated virus vectors (such as pAAV-MCS, pAAV-IRES-EGFP), retroviral vectors (such as pLXSN, pBabe-Puro), and herpesvirus vectors (such as pHSV-1, pHSV-TK). These vectors achieve efficient delivery of exogenous genes by utilizing the virus's own infection mechanism. Among them, lentiviral vectors can integrate the target gene into the host cell genome to achieve long-term stable expression; adenoviral vectors and adeno-associated virus vectors have a wide host range and low immunogenicity, making them suitable for gene delivery in various cell types in vivo and in vitro; retroviral vectors are mainly targeted at dividing cells and are often used for antibody gene expression in actively dividing cells such as tumor cells; herpesvirus vectors can accommodate large nucleic acid fragments and are suitable for the delivery of full-length antibody genes or multi-gene tandem expression cassettes.

[0029] In some implementation schemes, the yeast expression vectors can be specifically divided into Pichia pastoris expression vectors (such as pPIC3.5K, pPIC9K, pPICZα-A), Saccharomyces cerevisiae expression vectors (such as pYES2, pYES3, pESC series), and Hansenula polymorpha expression vectors (such as pHWO10, pPink-HC). Among them, Pichia pastoris expression vectors often carry the strong AOX1 promoter, which can achieve efficient induction of exogenous gene expression, and Pichia pastoris can perform post-translational modifications (such as glycosylation) on the expressed antibody to ensure antibody activity; Saccharomyces cerevisiae expression vectors are often regulated by the GAL1 promoter, which is suitable for rapid expression and functional verification of small molecule antibody fragments; Hansenula polymorpha expression vectors have nutritional auxotroph selection markers and high protein secretion efficiency, which has a cost advantage in large-scale antibody preparation.

[0030] Fourthly, the present invention provides a recombinant host cell, wherein the recombinant host cell comprises the expression vector described in the third aspect of the present invention;

[0031] Optionally, the host cell is a eukaryotic cell;

[0032] Optionally, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell;

[0033] Optionally, the host cell is an NS / 0 myeloma cell, a 293 cell, a CHO cell, a HeLa cell, a Cap cell, or a COS cell.

[0034] In some implementations, the recombinant host cell is a cell line obtained by introducing an expression vector carrying the target gene into the original host cell using genetic engineering techniques. Key construction steps include: host cell preparation (e.g., competent cell preparation, adherent cell digestion), expression vector introduction (e.g., liposome transfection, electroporation, virus-mediated transfection), positive cell selection (using resistance genes, fluorescent markers, etc. on the vector), and stable cell line acclimatization (long-term passage culture to verify stability).

[0035] In some implementations, the host cell is preferably a eukaryotic cell, which has a complete post-translational modification system (such as glycosylation, proper folding of disulfide bonds, etc.) to ensure that the antibody targeting Dronpa maintains its native conformation and specific binding activity, avoiding protein folding abnormalities or loss of activity that may occur due to prokaryotic cell expression.

[0036] Among these, eukaryotic cells can specifically be mammalian cells, plant cells, or yeast cells. Mammalian cells are the preferred host cell type, especially suitable for the large-scale preparation and functional verification of recombinant antibodies. Specific types of mammalian cells include NS / 0 myeloma cells, 293 cells, CHO cells, HeLa cells, Cap cells, and COS cells. 293 cells have high transfection efficiency and fast growth rate, making them suitable for transient and efficient antibody expression. CHO cells have stable genetic characteristics, high expression levels, and good adaptability to large-scale culture, making them a classic cell line for recombinant antibody production in the biopharmaceutical field. NS / 0 myeloma cells do not secrete endogenous immunoglobulins, avoiding interference with the purification of the target antibody, making them suitable for the preparation of high-purity antibodies. HeLa cells, Cap cells, and COS cells are often used for preliminary verification of antibody expression, cell localization experiments, and protein-protein interaction studies due to their ease of culture and strong proliferative capacity.

[0037] In some implementations, the host cell may also be a plant cell or a yeast cell: plant cells can achieve low-cost, large-scale expression of antibodies through genetic engineering and have natural biosafety advantages, making them suitable for preparing antibody reagents for scientific research; yeast cells (such as Pichia pastoris and Saccharomyces cerevisiae) combine the characteristics of easy culture and rapid growth of prokaryotic cells with the post-translational modification capabilities of eukaryotic cells, enabling them to efficiently secrete and express antibodies, and their culture cost is lower than that of mammalian cells, making them a suitable alternative for small-scale laboratory preparation or industrial-scale production of antibodies.

[0038] Fifthly, the present invention provides an antibody conjugate comprising the antibody or its antigen-binding fragment as described in the first aspect of the present invention and a conjugate thereto;

[0039] Optionally, the conjugate is a purification tag, a detection tag conjugate, a nanomaterial, or a signal amplifying molecule;

[0040] Optionally, the purification tag is a peptide tag or a protein tag;

[0041] Optionally, the detection marker conjugate is a fluorescent dye, an enzyme label, or a radionuclide;

[0042] Optionally, the nanomaterial is a quantum dot, gold nanoparticle, magnetic nanoparticle, carbon nanotube, or graphene;

[0043] Optionally, the signal amplifying molecule is a streptavidin-enzyme complex, luciferase, or quantum dot-antibody complex.

[0044] In some implementations, the peptide tags include His tags (such as 6×His, 8×His), Flag tags, Myc tags, HA tags, Strep tags (such as Strep-tag II), etc.; protein tags include glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (Trx), ubiquitin, SUMO tags, etc.

[0045] In some embodiments, the fluorescent dyes include fluorescein-type dyes (such as FITC, FAM), rhodamine-type dyes (such as TRITC, Rhodamine B), cyanine dyes (such as Cy3, Cy5, Cy7), Alexa Fluor series dyes (such as Alexa Fluor 488, 594, 647), luciferase substrates (such as fluorescein), etc.; enzyme labeling includes horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (β-Gal), glucose oxidase (GOD), etc.; radionuclides include... 125 I, 32 P, 35 S,99m Tc, etc. Antibodies labeled with fluorescent dyes can be directly detected by instruments such as fluorescence microscopy and flow cytometry, and are suitable for experiments such as immunofluorescence and cell localization; HRP and AP labeled antibodies are often used in conjunction with chromogenic substrates (such as TMB, BCIP / NBT) for qualitative and quantitative detection such as ELISA and Western blotting; radionuclide labeled antibodies have extremely high sensitivity and are suitable for the detection of trace amounts of low-abundance Dronpa protein.

[0046] In some implementations, the quantum dots include core-shell quantum dots such as CdSe / ZnS, CdTe, and InP / ZnS, which have advantages such as high fluorescence quantum yield, strong photostability, and tunable emission spectra, making them suitable for multicolor fluorescence imaging and super-resolution microscopy detection. Gold nanoparticles (10-100 nm in diameter) have a unique surface plasmon resonance effect, enabling visual detection through color changes. They can also be combined with immunochromatography for rapid detection and can be used as an enhancer to increase the intensity of the detection signal. Magnetic nanoparticles (such as Fe3O4 nanoparticles) can be modified with carboxyl, amino, and other groups on their surface, facilitating covalent binding with antibodies. A magnetic field can be used to achieve rapid separation and enrichment of target proteins, making them suitable for the purification and detection of Dronpa protein in complex samples. Carbon nanotubes (single-walled / multi-walled) and graphene have large specific surface areas, high conductivity, and biocompatibility, making them suitable as sensor substrate materials to construct highly sensitive Dronpa protein detection platforms, suitable for rapid quantitative analysis of trace proteins.

[0047] In some embodiments, the streptavidin-enzyme complex (such as streptavidin-HRP, streptavidin-AP) utilizes the high affinity of streptavidin for biotin (dissociation constant up to 10). -15 M) enables cascaded signal amplification, and when used with biotin-labeled secondary antibodies, it can significantly improve the detection sensitivity of experiments such as ELISA and Western blotting; luciferases (such as firefly luciferase and kidney luciferase) generate fluorescent signals by catalyzing specific substrates, featuring no background interference and high signal intensity, and can be used for real-time dynamic monitoring of Dronpa protein in live cells; quantum dot-antibody complexes refer to the direct coupling of quantum dots with the antibodies of this invention, combining the strong fluorescence characteristics of quantum dots with the specificity of antibodies to achieve integrated signal amplification and targeted detection, suitable for super-resolution imaging and single-molecule detection; TSA complexes (horseradish peroxidase-tyramine signal amplification complexes) catalyze the deposition of tyramine molecules around the target protein through HRP catalysis, and combined with luciferin-labeled tyramine, can achieve efficient signal amplification, especially suitable for the localization detection of low-abundance Dronpa proteins.

[0048] Sixthly, the present invention provides any of the following products:

[0049] (1) A photoactivated fluorescent protein Dronpa detection reagent, wherein the detection reagent comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention;

[0050] (2) A photoactivated fluorescent protein Dronpa detection product, wherein the detection product comprises the antibody or its antigen-binding fragment as described in the first aspect of the present invention or the detection reagent;

[0051] Optionally, the detection reagent is a flow cytometry reagent, an immunoblotting reagent, or a live imaging probe;

[0052] Optionally, the flow cytometry-specific reagent is the antibody labeled with fluorescein or its antigen-binding fragment;

[0053] Optionally, the immunoblotting reagent is an enzyme-labeled or fluorescently labeled antibody or its antigen-binding fragment;

[0054] Optionally, the in vivo imaging probe is an antibody probe modified with a penetrating peptide, and the antibody is the antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0055] Optionally, the detection product is a detection kit, a protein detection chip, an immunosensor chip, or a test strip.

[0056] In some implementations, the detection kit also includes an integrated experimental tool kit containing the aforementioned detection reagents. In addition to the core antibody / reagent, it also includes auxiliary reagents required for the experiment (such as blocking buffer, washing buffer, chromogenic solution / substrate solution, secondary antibody, standards, etc.), and some kits also include experimental consumables (such as 96-well plates, reaction membranes, etc.). Depending on the application scenario, it can be categorized into ELISA kits, immunofluorescence kits, and immunoblotting kits, providing standardized experimental procedures, enabling rapid detection of Dronpa protein, suitable for routine testing and batch sample analysis in research laboratories, ensuring the stability and reproducibility of experimental results.

[0057] In some embodiments, the protein detection chip is a miniature detection tool formed by immobilizing the anti-Dronpa antibody or its antigen-binding fragment of the present invention on a chip substrate (such as a glass slide or nitrocellulose membrane). It can simultaneously immobilize multiple antibody sites or multiple target antibodies. After incubation with the chip, the Dronpa protein can specifically bind to the antibody on the chip. The signal is detected by fluorescently labeled secondary antibody, chemiluminescence, or other methods, achieving high-throughput and rapid detection of Dronpa protein in the sample. It can also simultaneously analyze the expression of multiple samples or multiple proteins, making it suitable for large-scale sample screening and multi-protein joint analysis scenarios.

[0058] In some embodiments, the immunosensor chip is a novel detection chip combining biosensing technology and the principle of immune recognition. The anti-Dronpa antibody or its antigen-binding fragment of this invention is immobilized on the sensor surface (such as a gold electrode or optical sensing interface). When the Dronpa protein in the sample specifically binds to the antibody on the chip surface, it causes a change in the physical signals (such as resistance, refractive index, and mass) on the sensor surface. This change is converted into a quantifiable electrical or optical signal by a signal conversion system, achieving highly sensitive and rapid quantitative detection of the Dronpa protein. This eliminates the need for complex signal labeling and is suitable for rapid quantitative analysis of Dronpa protein in trace samples, such as clinical samples or precious biological samples.

[0059] In some implementations, the test strip is a rapid detection tool based on immunochromatography, using a nitrocellulose membrane as a carrier. A test line (T line, coated with the anti-Dronpa antibody described in this invention) and a control line (C line, coated with anti-antibody or antigen) are pre-set on the membrane. Samples (such as cell lysates or body fluids) migrate onto the test strip via capillary action. If the sample contains Dronpa protein, it forms a complex with the labeled antibody (such as a secondary antibody labeled with gold nanoparticles) on the test strip, resulting in enrichment and color development at the test line. Color development at the control line indicates a valid test. This test strip is easy to operate, requires no specialized equipment, and provides rapid detection (results within minutes), making it suitable for rapid on-site qualitative screening of Dronpa protein, such as preliminary verification of experimental samples or emergency testing scenarios.

[0060] In a seventh aspect, the present invention provides any of the following methods:

[0061] (1) A method for detecting photoactivated fluorescent protein Dronpa in a non-diagnostic and non-therapeutic purpose, the method comprising: contacting a sample to be tested with an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, and detecting the formation of an antigen-antibody immune complex;

[0062] (2) A method for preparing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the method comprising: culturing the recombinant host cell according to the fourth aspect of the present invention, inducing antibody expression, separating and purifying the culture product, and obtaining the antibody or antigen-binding fragment thereof.

[0063] Eighthly, the present invention provides any of the following applications:

[0064] (1) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention in the preparation of a detection reagent for detecting the photoactivated fluorescent protein Dronpa;

[0065] (2) The use of the antibody or its antigen-binding fragment described in the first aspect of the present invention or the detection reagent described in the sixth aspect of the present invention in the preparation of a detection product for detecting the photoactivated fluorescent protein Dronpa;

[0066] (3) The application of the antibody or its antigen-binding fragment described in the first aspect of the present invention in live cell imaging, subcellular structure super-resolution microscopy imaging, protein-protein interaction monitoring, cell lineage tracing or multicolor fluorescence imaging experiments;

[0067] (4) The application of the antibody or its antigen-binding fragment described in the first aspect of the present invention in the analysis of dynamic behavior of biomolecules, the study of signal pathway regulation mechanisms, or the study of molecular mechanisms of disease occurrence and development;

[0068] Optionally, the detection reagent is a flow cytometry reagent, an immunoblotting reagent, or a live imaging probe;

[0069] Optionally, the flow cytometry-specific reagent is the antibody labeled with fluorescein or its antigen-binding fragment;

[0070] Optionally, the immunoblotting reagent is an enzyme-labeled or fluorescently labeled antibody or its antigen-binding fragment;

[0071] Optionally, the in vivo imaging probe is an antibody probe modified with a penetrating peptide, and the antibody is the antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0072] Optionally, the detection product is a detection kit, a protein detection chip, an immunosensor chip, or a test strip.

[0073] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0074] This invention discloses a novel antibody 6D7 targeting the photoactivated fluorescent protein Dronpa. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region of antibody 6D7 are shown in SEQ ID NO:4-6, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region of antibody 6D7 are shown in SEQ ID NO:7-9. It can specifically recognize and bind to Dronpa protein, providing a precise tool for its detection and analysis. It solves the problems of large background interference and insufficient specificity of traditional fluorescence detection. At the same time, it can be adapted to various experimental scenarios such as immunoblotting, immunofluorescence, and immunoprecipitation, accurately locating the distribution of Dronpa in cells or tissues. It provides key technical support for life science experiments (such as live cell imaging and molecular mechanism research) and potential clinical applications based on Dronpa protein. Attached Figure Description

[0075] Figure 1 Electrophoresis results of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa;

[0076] Figure 2 HPLC results of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa;

[0077] Figure 3 Figure: ELISA results of antibody 6D7 binding activity against the photoactivated fluorescent protein Dronpa. Detailed Implementation

[0078] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0079] Example 1: Screening for antibodies targeting the photoactivated fluorescent protein Dronpa

[0080] 1. Immunogen Recombinant Expression

[0081] (1) Synthesize the photoactivated fluorescent protein Dronpa expression protein and sequence, construct it into the pCDNA3.1 vector; extract the plasmid for transfection; transfect it into HEK293 cells and culture the cells for 7 days; harvest the supernatant, purify it with Ni column, and obtain the photoactivated fluorescent protein Dronpa expression protein after concentration and replacement buffer.

[0082] The sequence information of the recombinant photoactivated fluorescent protein Dronpa is as follows:

[0083] MHHHHHHGSMSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVKEGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNATNDITL DGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDVNMALSLEGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQAK (SEQ ID NO:1).

[0084] 2. Immunization of mice, SP2 / 0 fusion, selection, and subcloning.

[0085] (1) Immunization of mice: The first immunization was performed with Freund's complete adjuvant, 100 μg per mouse, by intraperitoneal injection, with a total dose of 0.5 mL / mouse. The second immunization was performed 3 weeks later. From the second immunization onwards, Freund's incomplete adjuvant was used, with a dose of 50 μg / 0.5 mL / mouse. The third immunization was performed 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0086] (2) SP2 / 0 fusion: Take feeder cells, which can be 10 5 One hole per use, laid up 10 plates the day before fusion. 5 100 μL / well; mouse immune spleen cells and prepared myeloma cells were fused with PEG fusion agent and seeded into 96 cell culture plates containing feeder cells, 100 μL / well.

[0087] (3) Positive screening: Positive wells were screened by ELISA. Recombinant photoactivated fluorescent protein Dronpa was incubated overnight. The plates were washed, blocked with skim milk powder, and incubated at 37°C for 1 h. The plates were washed again, and 100 μL of 96-well culture supernatant was added. The plates were incubated at 37°C for 1 h. The plates were washed again, and HRP-labeled goat anti-mouse secondary antibody was added. The plates were incubated at 37°C for 30 min. The plates were washed again, and chromogenic solution was added. The plates were developed for 10 min. The stop solution was added, and the OD450 value was read. Cell lines with high expression levels were screened for subcloning culture.

[0088] 3. Sequence Fishing

[0089] Cells were collected, and total RNA was extracted using Trizol. cDNA was generated by reverse transcription using oligo(dT)20 primers. Then, the heavy and light chain variable regions were amplified by PCR using specific primers. After electrophoresis purification, the PCR products were transformed into a TA clone insertion vector, and positive clones were selected for sequencing.

[0090] 4. Experimental Results

[0091] Antibody 6D7 targeting the photoactivated fluorescent protein Dronpa was obtained through screening, and its corresponding sequence information is shown in Tables 1 and 2.

[0092] Table 1. Sequences of the heavy chain and light chain variable regions of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa.

[0093]

[0094] Table 2. CDR sequence of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa.

[0095]

[0096] Example 2: Detection of the affinity and specificity of antibodies targeting the photoactivated fluorescent protein Dronpa.

[0097] 1. Antibody expression

[0098] The amino acid sequence of 6D7 as described in Example 1 was optimized according to the human codon, sent to GenScript for synthesis, and constructed into the pcDNA3.4 vector. The plasmid was then prepared for use.

[0099] One day before transfection, healthy 293 cells (70-80% adherent cell confluence, 3×10⁶ suspension cell density) were transfected. 5 DNA (cells / mL) was seeded into culture dishes. On the day of transfection, endotoxin-free plasmids were prepared at an HC:LC ratio of 1:1-1.2. The amount of PEI was calculated with N / P = 3:1. DNA and PEI were diluted separately with serum-free medium and mixed. The mixture was incubated at room temperature for 15-20 min to form a complex. The complex was added to the cells and cultured at 37°C and 5% CO2. The medium was changed after 6-12 h. The supernatant was collected after 48-96 h of culture. The antibody concentration and activity were detected by UV 280 nm, ELISA, and SDS-PAGE.

[0100] The results showed that the concentration of the 6D7 clone was 318 mg / L.

[0101] 2. Physicochemical properties

[0102] (1) Antibody purity was determined by SDS-PAGE.

[0103] Main instruments: Chemiluminescence imaging system, Tanon-5200, purchased from Tanon; electrophoresis apparatus, Powererpac Basic, purchased from BIO-RAD; electrophoresis tank, DYC-Mini4, purchased from BIO-RAD.

[0104] Main reagents: 1 M Tris-HCl buffer, purchased from Beijing Solarbio Science & Technology Co., Ltd.; 1.5 M Tris-HCl buffer, purchased from Beijing Solarbio Science & Technology Co., Ltd.; 10% SDS, purchased from Beijing Solarbio Science & Technology Co., Ltd.; FastStain, purchased from Gene Universal; 30% gel mixing solution (29:1), purchased from Beijing Solarbio Science & Technology Co., Ltd.; Rainbow 180 broad-spectrum protein marker, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0105] Sample preparation: Take 20 μL of sample and mix it evenly with 5 μL of 5× reducing buffer, heat it at 95℃ for 5 min, and then cool it; take 20 μL of sample and mix it evenly with 5 μL of 5× non-reducing buffer.

[0106] Electrophoresis: Prepare the gel, add an appropriate amount of electrophoresis buffer, load the sample, and perform electrophoresis.

[0107] Staining and destaining: After electrophoresis, place the gel in an appropriate amount of Coomassie Brilliant Blue staining solution and stain at room temperature for 1 hour or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue destaining solution, and destain at room temperature for 4-24 hours. After destaining, soak in ddH2O, compare with the unstained gel using the marker protein as a reference, cut off the gel containing the desired protein component, collect it, and separate the protein to be purified from the gel.

[0108] (2) The purity of the antibody was determined by HPLC.

[0109] Main instruments: Liquid chromatograph, purchased from Agilent Technologies, model 1100 / 1200 / 1260; Liquid chromatography column, purchased from Tosoh Corporation, model WieTSKgeL G3000SWxl, 7.8 mm I.D. × 30 cm; pH meter, purchased from Sartorius Scientific Instruments Co., Ltd.; Electronic balance, purchased from Sartorius Scientific Instruments Co., Ltd.

[0110] Main reagents: dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0111] Mobile phase preparation: Add potassium dihydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride to approximately 900 mL of purified water, stir to dissolve, and bring the volume to 1 L. Measure the pH using a pH meter to ensure it is between 6.2 and 0.1. Filter through a 0.22 μm filter membrane and store at room temperature.

[0112] Sample preparation: System suitability sample: Standard diluted to 2 mg / mL with mobile phase; Test sample: Sample to be tested diluted to 2 mg / mL with mobile phase.

[0113] Chromatographic conditions: injection volume 25 µL, injector temperature 6℃, run time 30 min, flow rate 0.5 mL / min, column temperature 30℃, detection wavelength 280 nm (VWD) or detection wavelength 280 nm with bandwidth 4 nm, reference wavelength 360 nm with bandwidth 80 nm (DAD), peak width 0.025 min (0.5 s).

[0114] The electrophoresis and liquid chromatography results of the antibody 6D7 targeting the photoactivated fluorescent protein Dronpa are shown below. Figure 1 (From left to right, the bands are marker and restoration band, respectively.) Figure 2 As shown in the figure, the detection purity of the 6D7 is greater than 95%.

[0115] 3. ELISA detection of antibody binding activity

[0116] Coating: Dilute the antigen (Dronpa, a photoactivated fluorescent protein) to 2 μg / mL with coating buffer, mix well, add 100 μL / well to a 96-well coated plate, seal with a membrane, and incubate overnight at 4°C. Wash the plate three times with a plate washer, ensuring no liquid remains on the plate after the last wash, and pat dry the surface of the plate with absorbent paper.

[0117] Blocking: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37℃ for 1 h, and wash the plate 3 times as described above. Serially dilute the antibody to 100 μL / well, react at 37℃ for 1 h, and wash the plate 3 times as described above.

[0118] Secondary antibody: Dilute with DPBS at a ratio of 1:2000, add 100 μL / well to a 96-well plate, react at 37°C for 1 h, and wash the plate 3 times according to the above steps.

[0119] Color development: Add TMB, 100 μL / well, and develop color at room temperature in the dark for 10 min.

[0120] Termination: Add 100 μL of 2N H2SO4 per well. Measure OD using a microplate reader. 450 Detection within 10 minutes.

[0121] The ELISA results for detecting the binding activity of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa are shown in the figure below. Figure 3 As shown, the results indicate that the antibody 6D7 can specifically bind to the photoactivated fluorescent protein Dronpa in a concentration-dependent manner, with corresponding EC50 values. 50 The concentrations were 0.1206 μg / mL, indicating high affinity.

[0122] 4. Specific detection of antibodies

[0123] Coated with consistent concentrations of Dronpa, GFP (green fluorescent protein), EGFP (enhanced green fluorescent protein), dEGFP (unstable enhanced green fluorescent protein), and GCaMP (green fluorescent Ca). 2+ Several common green fluorescent proteins (indicators) are used, along with antibodies and secondary antibodies, for color development, reading, and data analysis.

[0124] The specificity detection results of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa are shown in Table 3 below. The results show that only the photoactivated fluorescent protein Dronpa showed normal color development, while the other antigens did not show positive reactions. That is, antibody 6D7 targeting the photoactivated fluorescent protein Dronpa can specifically bind to the photoactivated fluorescent protein Dronpa and has high specificity.

[0125] Table 3. Specificity detection results of antibody 6D7 targeting the photoactivated fluorescent protein Dronpa.

[0126]

Claims

1. An antibody against the photoactivated fluorescent protein Dronpa or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3, whose corresponding amino acid sequences are shown in SEQ ID NO:4-6, respectively. The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3, whose corresponding amino acid sequences are shown in SEQ ID NO:7-9, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4; Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; Optionally, the expression vector is a plasmid vector, a bacteriophage vector, a viral vector, or a yeast expression vector; Optionally, the viral vector is a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, or herpesvirus vector.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the expression vector according to claim 5; Optionally, the host cell is a eukaryotic cell; Optionally, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell; Optionally, the host cell is an NS / 0 myeloma cell, a 293 cell, a CHO cell, a HeLa cell, a Cap cell, or a COS cell.

7. An antibody conjugate, characterized in that, The antibody conjugate comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 and the conjugate thereof; Optionally, the conjugate is a purification tag, a detection tag conjugate, a nanomaterial, or a signal amplifying molecule; Optionally, the purification tag is a peptide tag or a protein tag; Optionally, the detection marker conjugate is a fluorescent dye, an enzyme label, or a radionuclide; Optionally, the nanomaterial is a quantum dot, gold nanoparticle, magnetic nanoparticle, carbon nanotube, or graphene; Optionally, the signal amplifying molecule is a streptavidin-enzyme complex, luciferase, or quantum dot-antibody complex.

8. Any of the following products, characterized in that, The products include: (1) A photoactivated fluorescent protein Dronpa detection reagent, wherein the detection reagent comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-3; (2) A photoactivated fluorescent protein Dronpa detection product, wherein the detection product comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 or the detection reagent; Optionally, the detection reagent is a flow cytometry reagent, an immunoblotting reagent, or a live imaging probe; Optionally, the flow cytometry-specific reagent is the antibody labeled with fluorescein or its antigen-binding fragment; Optionally, the immunoblotting reagent is an enzyme-labeled or fluorescently labeled antibody or its antigen-binding fragment; Optionally, the in vivo imaging probe is an antibody probe modified with a penetrating peptide, and the antibody is any one of claims 1-3 or its antigen-binding fragment; Optionally, the detection product is a detection kit, a protein detection chip, an immunosensor chip, or a test strip.

9. The following method, characterized in that, The method includes: (1) A method for detecting photoactivated fluorescent protein Dronpa for non-diagnostic and non-therapeutic purposes, the method comprising: contacting a sample to be tested with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3, and detecting the formation of an antigen-antibody immune complex; (2) A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-3, the method comprising: culturing the recombinant host cell according to claim 6, inducing antibody expression, separating and purifying the culture product, and obtaining the antibody or antigen-binding fragment thereof.

10. The following application, characterized in that, The applications include: (1) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 in the preparation of a reagent for detecting the photoactivated fluorescent protein Dronpa; (2) The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-3 or the detection reagent as described in claim 8 in the preparation of a detection product for detecting the photoactivated fluorescent protein Dronpa; (3) The application of the antibody or antigen-binding fragment of any one of claims 1-3 in live cell imaging, subcellular structure super-resolution microscopy imaging, protein-protein interaction monitoring, cell lineage tracing or multicolor fluorescence imaging experiments; (4) The application of the antibody or antigen-binding fragment of any one of claims 1-3 in the analysis of dynamic behavior of biomolecules, the study of signaling pathway regulation mechanisms, or the study of molecular mechanisms of disease occurrence and development; Optionally, the detection reagent is a flow cytometry reagent, an immunoblotting reagent, or a live imaging probe; Optionally, the flow cytometry-specific reagent is the antibody labeled with fluorescein or its antigen-binding fragment; Optionally, the immunoblotting reagent is an enzyme-labeled or fluorescently labeled antibody or its antigen-binding fragment; Optionally, the in vivo imaging probe is an antibody probe modified with a penetrating peptide, and the antibody is any one of claims 1-3 or its antigen-binding fragment; Optionally, the detection product is a detection kit, a protein detection chip, an immunosensor chip, or a test strip.

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