Nano antibody for resisting mCherry tag, antibody and application of nano antibody

Abstract

The invention discloses an anti-mCherry tag nano antibody, an antibody and application thereof, and relates to the technical field of nano antibodies. According to the invention, three anti-mCherry tag nano antibodies are obtained through screening, and the three nano antibodies have specific recognition and binding capacities for mCherry antigens, so that the nano antibodies provided by the invention have highly specific binding activity. On the basis, an anti-mCherry tag nano antibody affinity purification material can be prepared and used for separation or purification of mCherry antigens, separation or purification of mCherry tag proteins and separation of protein-DNA compounds. The nano antibody provided by the invention can be massively cultured, expressed and purified by a biological method, and has the advantages of low production cost, repeated use for multiple times, high recovery rate of target protein after repeated use, good stability and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of nano antibodies, and in particular to an anti-mCherry label nano antibody, an antibody and applications thereof. Background Art

[0002] With the progress of proteomics, the separation and purification technology of genetically engineered recombinant proteins plays a very important role. If you want to specifically and quickly separate and purify the target protein from a complex sample, you must choose a suitable protein purification method. Among them, the most commonly used protein purification method is affinity chromatography (AC). This method often uses fusion tag technology to fuse the protein with the affinity tag and further purify the target protein using the affinity tag. At present, researchers have successively developed many protein tags with different functions, including: glutathione transferase (GST), polyhistidine (poly-His), V5 tag, MBP tag, mCherry tag, Flag peptide, and Fc segment of immunoglobulin. The use of fusion tags facilitates the expression and purification of target proteins and provides a convenient means for studying the structure and function of target proteins.

[0003] mCherry is a red fluorescent tag derived from a variant of red fluorescent protein (RFP) in coral (Discosoma). It can be used as a cell marker and visualization tool in a variety of biological systems, including yeast, mammalian cells, and small vertebrates such as zebrafish. mCherry has high photostability and can withstand multiple excitations, making it suitable for long-term observation and imaging. It has high fluorescence intensity, allowing it to be clearly and accurately detected under various observation conditions. In addition, mCherry has higher absorption and emission wavelengths than the common green fluorescent protein (GFP), making it more resistant to photobleaching and background interference in some cases. mCherry can form a good spectral match with blue, green, and yellow fluorescent proteins to achieve multi-color co-localization and time-lapse imaging. It can also be combined with antibodies or other tags for applications such as immunofluorescence, protein interaction studies, and cell tracking. In plant biology research, mCherry is used as a reporter gene to monitor gene expression and protein localization. In summary, mCherry is a very useful red fluorescent tag with high stability and sensitivity, suitable for a variety of biological systems and fluorescence imaging applications.

[0004] Based on the outstanding characteristics of the mCherry tag in biological experiments such as protein expression and purification, immunofluorescence, protein interaction research, and cell tracking, it is particularly important to develop antibodies against mCherry-tagged proteins.

[0005] 1993, Hamers Casterman et al. found that a class of antibodies with only heavy chain dimers (H2) were found in camelids (camels, dromedaries and llamas), which were mainly IgG2 and IgG3 types. Since these antibodies lack light chains, they are called heavy chain only antibodies (HCAbs), and their antigen binding sites are composed of a domain, called the VHH region, so these antibodies are also called single domain antibodies or single domain antibodies (sdAb). Since these antibodies are variable region sequences after the constant region is removed, the molecular weight is only 15kDa, and the diameter is about 10 nanometers, so they are also called nano antibodies (Nbs).

[0006] At present, the method of combining nano-antibody technology with tagged proteins is gradually being applied to the field of biological research. Among them, Chromotek in Germany has taken the lead in launching Nano-Trap technology, which covalently couples nano-antibodies of tagged proteins with agarose gel or magnetic beads to form a series of products. Due to the excellent characteristics of nano-antibodies, Nano-Trap shortens the experimental time and reduces non-specific binding, which greatly promotes the progress of protein function research. In view of the functional characteristics of the mCherry tag and the advantages of nano-antibodies, we believe that it is necessary to develop nano-antibodies with mCherry tags to facilitate the study of fusion proteins containing mCherry tags or proteins that interact with mCherry.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a nanobody against mCherry tags to give full play to the superior performance of nanobodies. The nanobody has excellent specific antigen binding ability and can overcome the inherent defects of traditional antibodies such as insufficient thermal stability and high production cost. And further provide its application in protein interaction through affinity purification technology.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a nanobody against mCherry tag, the nanobody comprising:

[0011] CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.4;

[0012] CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.5;

[0013] Or CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.6.

[0014] In a second aspect, the present invention further provides a multivalent nanobody, a multispecific nanobody or a fusion nanobody, which comprises the above-mentioned anti-mCherry tag nanobody.

[0015] In a third aspect, the present invention further provides an antibody comprising the above-mentioned anti-mCherry tag nanobody or the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody.

[0016] In a fourth aspect, the present invention also provides a nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned anti-mCherry tag nanobody; or the nucleic acid molecule encodes the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; or the nucleic acid molecule encodes the above-mentioned antibody.

[0017] In a fifth aspect, the present invention also provides a host cell containing the above-mentioned recombinant vector.

[0018] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned anti-mCherry tag Nanobody; or the above-mentioned multivalent Nanobody, multispecific Nanobody or fusion Nanobody; or the above-mentioned antibody, which comprises: culturing the above-mentioned host cell.

[0019] In a seventh aspect, the present invention further provides an anti-mCherry tag nanobody; or the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; or the use of the above-mentioned antibody in any of the following items,

[0020] (1) Preparation of mCherry detection products;

[0021] (2) preparing mCherry enriched or purified products;

[0022] (3) preparing an enriched product or purified product of a fusion protein with an mCherry tag;

[0023] (4) preparing products for separation or purification of interacting proteins with mCherry-tagged proteins;

[0024] (5) preparing an isolated or purified product of a specific protein-DNA complex with an mCherry tag;

[0025] (6) Application in the preparation of tracing detection products for fusion proteins or cells with mCherry tags.

[0026] In an eighth aspect, the present invention also provides a nanobody with the above-mentioned anti-mCherry tag; or the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; or the product of the above-mentioned antibody, the product is nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, EP tubes, multi-well plate micro reaction plate recesses, membranes, PDMS membranes or chips.

[0027] In the ninth aspect, the present invention also provides a method for separating mCherry, a fusion protein with an mCherry tag, an mCherry interacting protein or a specific protein-DNA complex with an mCherry tag, which comprises: mixing the above-mentioned anti-mCherry-tagged nanoantibody, the above-mentioned multivalent nanoantibody, multispecific nanoantibody or fusion nanoantibody; the above-mentioned antibody or product with the sample to be separated and eluting.

[0028] The present invention has the following beneficial effects:

[0029] (1) The three anti-mCherry tag nanobodies provided by the present invention have specific recognition and binding capabilities for mCherry antigens, thus showing that the nanobodies provided by the present invention have highly specific binding activity.

[0030] (2) The present invention also provides an affinity purification material containing an anti-mCherry-tagged nanobody (such as anti-mCherry nanobody magnetic beads, agarose gel, agarose magnetic beads, etc.), which shows excellent performance in the purification of mCherry-tagged proteins. This property can be used to develop mCherry-tagged protein affinity purification reagents, thereby being used for the separation and purification of mCherry-tagged proteins, mCherry antigens, fusion proteins with mCherry tags, mCherry interacting proteins or specific protein-DNA complexes with mCherry tags.

[0031] (3) The nanobodies provided by the present invention can be mass-produced by biological methods for expression and purification, thus avoiding cumbersome production steps such as purification using mouse ascites, greatly reducing production costs, and can be reused multiple times with a high recovery rate, thus having broad application prospects.

[0032] (4) The nanoantibodies provided by the present invention have strong thermal stability and still have strong biological activity at a temperature of 70°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 The electrophoresis diagrams of three mCherry-tagged nanobodies (mCherry12, mCherry19, and mCherry28);

[0035] Figure 2 It is a statistical diagram of the binding of 3 mCherry-tagged nanobodies to antigens;

[0036] Figure 3 This is a statistical diagram of the thermal stability of three mCherry-tagged nanobodies;

[0037] Figure 4 This is a statistical result diagram of the recovery rate of repeated use of mCherry-tagged nanoantibody agarose gel (mCherry19);

[0038] Figure 5 This is a statistical graph of the recovery rate of repeated use of mCherry-tagged nanoantibody agarose gel (mCherry28). DETAILED DESCRIPTION

[0039] In a first aspect, the present invention provides a nanobody against mCherry tag, the nanobody comprising:

[0040] CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.4;

[0041] CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.5;

[0042] Or CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.6.

[0043] The amino acid sequence of SEQ ID NO.4 is as follows:

[0044] EVKLVESGGGLVQAGGSLRLSCAASGITFSKYAMGWVRQAPGKEREGVATITSRGSDTYYAESVEGRFTVSRDNAKKTLYLQMDSLKSEDTAQ YYCAAPPYFRPWHPVWVRTQSLCARWGHGTQVTVSS.

[0045] The amino acid sequence of SEQ ID NO.5 is as follows:

[0046] EVQLVESGGDLVKPGGSLKLSCAASGFTFSHYAIGWLRQAPGKGLEFVSCIGSRGTYTYYADSVKGDFTISRDNAKNTVYLQMDSLKPEDTAVY ACVADPYGRSVHPLWARTLYVYWGQGTQVTVSS.

[0047] The amino acid sequence of SEQ ID NO.6 is as follows:

[0048] EVKLVESGGGLVQPGESLRLSCAASGFRFSYYAMSWVRQAPGKGREGVAAISMNGTYTYYPDSVKGRFTISKDNTKNTVYLQMNSPKPEDTAV YYCARKRGPICTFVESAYDSRHWGQGTQVTVSS.

[0049] "CDR" in the present invention is synonymous with "complementarity determining region". In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the hypervariable region of the heavy chain of an immunoglobulin, and refers to a region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the hypervariable region of the heavy chain of a nanobody.

[0050] In the present invention, the heavy chain complementary determining region is represented by CDR, which includes CDR1, CDR2 and CDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Chothia annotation standard to label the CDR region, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0051] The present invention screened and obtained three anti-mCherry tag nano antibodies, and these three nano antibodies all have specific recognition and binding capabilities to mCherry antigens, thus showing that the nano antibodies provided by the present invention have highly specific binding activity. Based on this, anti-mCherry tag nano antibody affinity purification materials can be prepared for the separation or purification of mCherry antigens, the separation or purification of mCherry tag proteins, and the separation of protein-DNA complexes.

[0052] The nano-antibodies provided by the present invention can be mass-cultured, expressed, purified and produced by biological methods, avoiding cumbersome production steps such as purification using mouse ascites, greatly reducing production costs, and can be reused multiple times. The recovery rate of the target protein is high after repeated use, the stability is good, and the application prospects are broad.

[0053] In a preferred embodiment of the present invention, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO.4 are shown in SEQ ID Nos.7 to 9, respectively.

[0054] The amino acid sequence of SEQ ID NO.7 is as follows: GITFSKYA.

[0055] The amino acid sequence of SEQ ID NO.8 is as follows: ITSRGSDT.

[0056] The amino acid sequence of SEQ ID No.9 is as follows:

[0057] AAPPYFRPWHPVWVRTQSLCAR.

[0058] In an optional embodiment, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO.5 are shown in SEQ ID Nos.10 to 12, respectively;

[0059] The amino acid sequence of SEQ ID No. 10 is as follows: GFTFSHYA.

[0060] The amino acid sequence of SEQ ID No. 11 is as follows: IGSRGTYT.

[0061] The amino acid sequence of SEQ ID No.12 is as follows:

[0062] VADPYGRSVHPLWARTLYVY.

[0063] In an alternative embodiment, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO.6 are shown in SEQ ID Nos.13 to 15, respectively;

[0064] The amino acid sequence of SEQ ID No. 13 is as follows: GFRFSYYA.

[0065] The amino acid sequence of SEQ ID No. 14 is as follows: ISMNGTYT.

[0066] The amino acid sequence of SEQ ID No. 15 is as follows: ARKRGPICTFVESAYDSRH.

[0067] In an optional embodiment, the framework region in the heavy chain variable region shown in SEQ ID NO.4 includes the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID Nos.16 to 19, respectively.

[0068] SEQ ID No. 16: EVKLVESGGGLVQAGGSLLRLSCAAS.

[0069] SEQ ID No. 17: MGWVRQAPGKEREGVAT.

[0070] SEQ ID No.18:

[0071] YYAESVEGRFTVSRDNAKKTLYLQMDSLKSEDTAQYYC.

[0072] SEQ ID No. 19: WGHGTQVTVSS.

[0073] In an optional embodiment, the framework region in the heavy chain variable region shown in SEQ ID NO.5 includes the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID Nos.20 to 23, respectively.

[0074] SEQ ID No. 20: EVQLVESGGDLVKPGGSLKLSCAAS.

[0075] SEQ ID No. 21: IGWLRQAPGKGLEFVSC.

[0076] SEQ ID No.22:

[0077] YYADSVKGDFTISRDNAKNTVYLQMDSLKPEDTAVYAC.

[0078] SEQ ID No. 23: WGQGTQVTVSS.

[0079] In an optional embodiment, the framework region in the heavy chain variable region shown in SEQ ID NO.6 includes the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID Nos.24 to 27, respectively.

[0080] SEQ ID No. 24: EVKLVESGGGLVQPGESLRLSCAAS.

[0081] SEQ ID No. 25: MSWVRQAPGKGREGVAA.

[0082] SEQ ID No.26:

[0083] YYPDSVKGRFTISKDNTKNTVYLQMNSPKPEDTAVYYC.

[0084] SEQ ID No. 27: WGQGTQVTVSS.

[0085] The heavy chain variable regions shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 correspond to the following nanoantibody numbers: mCherry12, mCherry19, and mCherry28, respectively.

[0086] In an optional embodiment, the above-mentioned Nanobody is a monovalent Nanobody.

[0087] Glossary:

[0088] Monovalent nanoantibodies: Antigen-specific nanoantibodies obtained by screening from a nanoantibody library using specific antigens. Because of the large number of hydrophilic residues on their surface, they can maintain a strict monomer structure and can bind to their antigens with high specificity and high affinity only in this monomer form.

[0089] In a second aspect, the present invention further provides a multivalent nanobody, a multispecific nanobody or a fusion nanobody, which comprises the above-mentioned anti-mCherry tag nanobody.

[0090] Multivalent nanobodies: Multivalent antibodies are polymers of monovalent antibodies that recognize the same epitope and have higher antigen affinity than the corresponding monovalent nanobodies. Multispecific antibodies are polymers of monovalent antibodies that recognize different epitopes and can bind to different targets or different epitopes of the same target, and have higher antigen recognition ability than monovalent antibodies. Nanobodies have a simple structure and only one domain, which can be aggregated together through short connecting sequences to convert into multivalent and multispecific forms.

[0091] Fusion nanobodies: Nanobodies have strict monomer characteristics and their relative molecular mass is very small. They can easily be combined with other structures (such as BSA, IgG-Fc, etc.) through genetic engineering technology to form new fusion molecules, such as enzymes that can extend their half-life, antimicrobial peptides or imaging substances. In the new fusion molecule, the nanobody binds to its target antigen in a directional manner, and the part fused with the nanobody can play the corresponding function.

[0092] In an alternative embodiment, the multivalent Nanobody comprises at least two of the following CDRs regions:

[0093] (1) CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.4;

[0094] (2) CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.5;

[0095] (3) CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.6.

[0096] For example, the multivalent Nanobody is a polymer of (1) and (2), the multivalent Nanobody is a polymer of (1) and (3), the multivalent Nanobody is a polymer of (2) and (3), or the multivalent Nanobody is a polymer of (1), (2) and (3).

[0097] In a third aspect, the present invention further provides an antibody comprising the above-mentioned anti-mCherry tag nanobody or the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody.

[0098] Antibodies include, but are not limited to, any of heavy chain antibodies, full-length antibodies, chimeric antibodies, multispecific antibodies (eg, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), murine antibodies, humanized antibodies, or antigen-binding fragments.

[0099] The "chimeric antibody" described in the present invention is an antibody formed by fusing the variable region of a non-human antibody with the constant region or framework region of a human antibody, which can reduce the immune response induced by the non-human antibody.

[0100] The above antibodies include but are not limited to functional fragments of antibodies, which usually have the same binding specificity as the antibody from which they are derived, and are selected from any one of F(ab')2, Fab', Fab, Fv and scFv of antibodies, as long as they exhibit the desired antigen binding activity. It is easy for those skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above functional fragments.

[0101] The above antibodies or Nanobodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0102] In a fourth aspect, the present invention also provides a nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned anti-mCherry tag nanobody; or the nucleic acid molecule encodes the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; or the nucleic acid molecule encodes the above-mentioned antibody.

[0103] In an optional embodiment, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0104] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0105] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can directly or indirectly (such as packaging into a virus) introduce the target DNA fragment into the host cell through transformation, transfection or transduction to express the target gene.

[0106] One type of vector is a plasmid, which is a circular double-stranded DNA molecule that can connect the target DNA fragment to the plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0107] In a fifth aspect, the present invention also provides a host cell containing the above-mentioned recombinant vector.

[0108] The host cell is selected from at least one of a prokaryotic host cell, a eukaryotic host cell and a bacteriophage;

[0109] In an alternative embodiment, the prokaryotic host cell is Escherichia coli, Streptomyces, Bacillus subtilis or Mycobacterium;

[0110] In an alternative embodiment, the eukaryotic host cell is an animal cell, a plant cell or a fungus; the fungus is, for example, Pichia pastoris.

[0111] In an alternative embodiment, the animal cell is selected from a mammalian cell, an insect cell or a Caenorhabditis elegans cell;

[0112] The mammalian cell is selected from any one of 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, mouse L cells, LNCaP cells, 633 cells, Vero, BHK cells, CV1 cells, Hela cells, MDCK cells, Hep-2 cells and Per6 cells. Among them, 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

[0113] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned anti-mCherry tag Nanobody; or the above-mentioned multivalent Nanobody, multispecific Nanobody or fusion Nanobody; or the above-mentioned antibody, which comprises: culturing the above-mentioned host cell.

[0114] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, etc.) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0115] In a seventh aspect, the present invention further provides an anti-mCherry tag nanobody; or the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; or the use of the above-mentioned antibody in any of the following items,

[0116] (1) Preparation of mCherry detection products;

[0117] (2) preparing mCherry enriched or purified products;

[0118] (3) preparing an enriched product or purified product of a fusion protein with an mCherry tag;

[0119] (4) preparing products for separation or purification of interacting proteins with mCherry-tagged proteins;

[0120] (5) preparing an isolated or purified product of a specific protein-DNA complex with an mCherry tag;

[0121] (6) Application in the preparation of tracing detection products for fusion proteins or cells with mCherry tags.

[0122] mCherry detection products include but are not limited to mCherry fluorescent immunoassay products, chemiluminescent immunoassay products, enzyme-linked immunosorbent assay products or time-resolved fluorescent immunoassay products.

[0123] Since the aforementioned three nanoantibodies all have a high affinity for mCherry, they can be used for the detection, enrichment and purification of mCherry, as well as for the enrichment and purification of fusion proteins with mCherry tags, for the separation or purification of interacting proteins of proteins with mCherry tags, and for the separation or purification of specific protein-DNA complexes with mCherry tags.

[0124] In an optional embodiment, the detection product is selected from a reagent, a kit or a chip.

[0125] In an optional embodiment, the kit is a chemiluminescent immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a time-resolved fluorescent immunoassay kit.

[0126] In an optional embodiment, the kit is a magnetic particle chemiluminescence detection kit;

[0127] In an optional embodiment, the chip is a chromatin immunoprecipitation chip (Chromatin Immunoprecipitation-chip, ChIP-chip), an immunoprecipitation chip, an immunoprecipitation chip or an mCherry fusion protein sedimentation chip (such as based on Pull down analysis).

[0128] In an optional embodiment, the mCherry enrichment or purification product, the enrichment or purification product of the fusion protein with an mCherry tag, the product for separating or purifying the interacting protein with the mCherry-tagged protein, or the separation or purification product of the specific protein-DNA complex with the mCherry tag comprises: a vector and a nanobody or antibody on the vector.

[0129] In an alternative embodiment, the Nanobody or antibody on the carrier is coupled to the carrier as a ligand and / or physically linked to the carrier.

[0130] In an optional embodiment, in a certain mCherry enrichment or purification product, an enrichment product or purification product of a fusion protein with an mCherry tag, a product for separation or purification of an interacting protein with an mCherry-tagged protein, or a product for separation or purification of a specific protein-DNA complex with an mCherry tag, the type of ligand includes but is not limited to the above-mentioned nano antibodies or antibodies. Those skilled in the art can make adjustments as needed. For example, in order to improve the efficiency of separation or purification, those skilled in the art can easily think of adding other ligands for coordination, including but not limited to sugar-binding ligands, protein-binding ligands, dye ligands, amino acid affinity ligands, nucleotides and nucleotide analog ligands, and biomimetic ligands.

[0131] Those skilled in the art can also adjust the density and quantity of the ligands as needed to meet the efficiency and effect of the affinity chromatography.

[0132] In an optional embodiment, the carrier is selected from nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, chips or porous materials.

[0133] For example, the activated magnetic beads are incubated with antibodies to coat the magnetic beads with antibodies. For example, nano antibodies are coupled to agarose gel microspheres or silica microspheres to prepare corresponding immunoaffinity adsorption materials, which are placed in a chromatography column to prepare corresponding affinity columns for mCherry enrichment or purification or for enrichment and purification of fusion proteins with mCherry tags.

[0134] In an alternative embodiment, the nanoparticles are selected from organic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0135] In an eighth aspect, the present invention also provides a nanobody with the above-mentioned anti-mCherry tag; or the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; or the product of the above-mentioned antibody, the product is nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, test tubes, EP tubes, multi-well plate micro reaction plate recesses, membranes, PDMS membranes or chips.

[0136] Magnetic beads are also called magnetic microspheres. Magnetic microspheres refer to composite microspheres with certain magnetism that are formed by combining organic polymers and inorganic magnetic nanoparticles with a special structure through appropriate methods. Magnetic beads include but are not limited to nanomagnetic beads and micron magnetic microspheres. In an optional embodiment, magnetic beads include but are not limited to: carboxyl magnetic beads, amino magnetic beads, oleylamine-modified magnetic beads, silicon hydroxyl magnetic beads, sulfonic acid-based magnetic microspheres, thiol-based magnetic microspheres, PEG-modified magnetic beads, unmodified ferroferric oxide magnetic beads, monodisperse silica-coated magnetic beads, epoxy-based magnetic beads, monodisperse mesoporous silica-coated magnetic beads, gold-coated magnetic nanoparticles, streptavidin-modified magnetic beads, polylysine-modified magnetic beads, nickel magnetic beads, magnetic polystyrene microspheres, magnetic polyacrylamide microspheres, and silica magnetic microspheres.

[0137] Latex microspheres are spherical polymer particles formed from an amorphous polymer (usually polystyrene) in the colloidal size range, for example particles with a diameter less than 100 nm, or particles with a size of 0.3-0.5 μm, or particles with a size greater than 1 μm.

[0138] The latex microspheres include but are not limited to latex microspheres whose surfaces are modified with at least one functional group of hydroxyl, carboxyl or Toysal.

[0139] Sepharose microspheres with nanobodies or antibodies are prepared, for example, by the following method:

[0140] The agarose gel is reacted with a coupling reagent to form activated agarose gel microspheres with epoxy groups, which are then coupled with the amino groups of mCherry nanobodies or antibodies to prepare agarose gel-mCherry adsorption materials, wherein the coupling reagent is a halogenated epoxy compound and / or a diglycidyl ether reagent.

[0141] In other embodiments, the cyanogen bromide-activated agarose gel can also be drained into a sand core funnel of corresponding specifications, stirred evenly with HCl and then drained, and then treated with coupling buffer, and the mCherry nanobody or antibody is reacted with the cyanogen bromide-activated agarose gel to obtain agarose gel microspheres with nanobody or antibody.

[0142] In an alternative embodiment, the nanoparticles are selected from organic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0143] In an optional embodiment, the multi-well plate is an ELISA plate; in other embodiments, it may be other solid support plates.

[0144] In an optional embodiment, the nanobody or antibody is coupled and / or physically connected to the product; the physical connection includes but is not limited to electrostatic adsorption, etc. The physical connection is such as an immunoturbidimetric platform.

[0145] In an optional embodiment, the nanobody or antibody is also labeled with a detectable marker; in other embodiments, the nanobody or antibody may not be labeled with a detectable marker. It is easy for technicians in this field to think that the anti-mCherry nanobody can qualitatively, quantitatively, trace, separate or purify target proteins, DNA or cells and other substances according to the fluorescence signal of mCherry after binding to mCherry.

[0146] In an optional embodiment, the detectable label is selected from at least one of biotin, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents and colloids.

[0147] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0148] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0149] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb,67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0150] In an optional embodiment, the chemiluminescent agent is selected from at least one of acridinium ester, luminol, lucigenin, crustacean fluorescein, bipyridine ruthenium, dioxetane, lophanine, isoluminol and peroxyoxalate.

[0151] Colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0152] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0153] In the ninth aspect, the present invention also provides a method for separating mCherry, a fusion protein with an mCherry tag, an mCherry interacting protein or a specific protein-DNA complex with an mCherry tag, which comprises: mixing the above-mentioned anti-mCherry-tagged nanoantibody, the above-mentioned multivalent nanoantibody, multispecific nanoantibody or fusion nanoantibody; the above-mentioned antibody or product with the sample to be separated and eluting.

[0154] In an alternative embodiment, glycine is used for elution.

[0155] The separation principle of specific protein-DNA complexes with mCherry tags is as follows: mCherry-tagged nanoantibodies are used to bind to the same mCherry tag in the target protein, bringing nucleases (such as micrococcal nuclease) to the binding site between the target protein and chromatin, cleaving the chromatin through the activation of the nuclease, and releasing the protein-DNA complex.

[0156] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0157] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0158] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0159] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0160] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0161] Example 1

[0162] This example constructs and screens a nanobody phage display library.

[0163] 1.1 Alpaca immunization and lymphocyte isolation

[0164] Through the induced expression of Escherichia coli, the mCherry protein was purified from it, and then its buffer was replaced by ultrafiltration to remove endotoxins, impurities, salt ions and other unnecessary harmful substances, so as to facilitate the next step of immunization of alpacas. A healthy adult alpaca was selected, 1 mg of purified mCherry protein was mixed with Freund's adjuvant in a ratio of 1:1, and the alpaca was immunized by multiple subcutaneous injections on the back. A total of 7 immunizations were performed, with Freund's complete adjuvant used for the first time and Freund's incomplete adjuvant used for the remaining 6 times. The immunization interval was 1 week. After that, peripheral blood of the alpaca was collected for the construction of a phage display library. The collected alpaca peripheral blood was used to separate lymphocytes using the instructions of the camel peripheral blood lymphocyte separation kit, with 2.5×10 7 Add 1 mL of RNA isolation reagent to each living cell, take 1 mL for RNA extraction, and Store at 80℃.

[0165] 1.2 Total RNA extraction and reverse transcription to synthesize cDNA

[0166] RNA was extracted according to the instructions of TAKARA Total RNA Extraction Kit. Using RNA as template and oligo-dT as primer, the first strand of cDNA was synthesized according to the instructions of TAKARA reverse transcriptase.

[0167] 1.3 Antibody variable region gene amplification

[0168] The cDNA obtained by reverse transcription was used as a template for PCR reaction. Two rounds of amplification were performed, and the primer sequences for the first round of PCR were as follows:

[0169] CALL001:GTCCTGGCTGCTCTTCTACAAGG

[0170] CALL002:GGTACGTGCTGTTGAACTGTTCC

[0171] The PCR reaction conditions and procedures are: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 7 minutes. Use an agarose gel recovery kit to recover a band of about 700 bp, and finally adjust the nucleic acid concentration to 5 ng / μL with water. The primer sequences for the second round of PCR are as follows:

[0172] VHH Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

[0173] VHHFor:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

[0174] The PCR reaction conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 15 cycles; 72°C for 7 minutes. The PCR product was purified using a PCR product recovery kit.

[0175] 1.4 Vector construction

[0176] pMES4 and the second PCR product were double-digested with PstI and BstEII, respectively. 1.5 μg of the digested vector and 450 ng of the second PCR product were taken, 15 μL of T4 DNA ligase was added, and the buffer and water were supplemented to a total volume of 150 μL. The ligation was carried out at 16°C overnight and the ligation product was recovered. The product was recovered using a PCR product recovery kit and eluted with 20 μL of water. The double digestion results of the pMES4 vector were detected on a 1% agarose electrophoresis gel.

[0177] 1.5 Electroporation and storage capacity determination

[0178] Take 10 μL of the purified ligation product, add it to a pre-cooled electroporation cup containing 50 μL of E. coli TG1 competent cells, place it in an electroporator (ECM630 electroporator from BTX, USA) for electroporation, take out the electroporation cup, revive and culture the transformants. Randomly select clones and perform colony PCR identification. Calculate the library capacity based on the PCR positive rate (liquidity capacity = number of clones × dilution factor × PCR identification positive rate × 10). The primer sequences are as follows:

[0179] MP57:TTATGCTTCCGGCTCGTATG;

[0180] GIII:CCACAGACAGCCCTCATAG.

[0181] 1.6 Phage amplification

[0182] Take the revived bacterial solution and inoculate it into YT AG medium, and culture it at 37℃200rpm until the culture OD reaches 600 =0.5. Take out 10mL of bacterial solution and add 4×10 10VCSM13 (purchased from Beijing Qiyan Biotechnology) was statically infected at 37°C for 30 minutes. Centrifuged at 4000rpm for 10 minutes at room temperature and the supernatant was removed. The bacteria were resuspended in 2×YTAK medium (containing ampicillin and kanamycin) and cultured overnight at 37°C 200rpm. The supernatant was taken from the centrifugation tube and placed in a 40mL tube. 10mL of PEG / NaCl (20% / 2.5M) solution was added and mixed thoroughly. The supernatant was discarded after centrifugation. The precipitate was washed and centrifuged with 1mL of ice PBS. 250μL of pre-cooled PEG / NaCl was taken from the supernatant and mixed thoroughly and washed and resuspended.

[0183] Determine the phage titer: culture TG1 to OD 600 =0.4, gradient dilute the phage with LB medium, take the diluted phage TG1 culture and mix them, observe the plaque formation in the culture plate the next day, and 300 dilution gradient plates were counted and the phage titer (pfu) was calculated according to the following formula.

[0184] Phage titer (pfu / mL) = dilution factor × number of plaques × 100

[0185] 1.7 Nanoantibody Screening

[0186] Screen positive clones with antigen by ELISA method. Coat ELISA plate with antigen, block with 5% BSA, and wash with PBST. Add 100 μL phage supernatant to each well and place at 37°C for 1 hour. Discard the supernatant, add HRP-labeled mouse anti-M13 secondary antibody, and place at 37°C for 1 hour. Discard the supernatant, add TMB solution, incubate at room temperature for 5 hours, add 2M sulfuric acid stop solution to each well, and read with an enzyme reader at 450nm. Pick the clones identified as positive in ELISA, shake the plasmid and send it to the sequencing company for sequencing.

[0187] 1.8 Expression and purification of nanobodies in Escherichia coli

[0188] The clones with positive phage ELISA results were selected, the plasmids were extracted and transformed into the BL21 competent cells, the nanobody protein expression was induced with IPTG, the supernatant (periplasmic extract) was collected, and the periplasmic extract was dialyzed into PBS, purified using His-tag agarose gel (produced by Shanghai Lingyin Biotechnology Co., Ltd.), eluted and collected using different concentrations of imidazole, the collected samples were subjected to reduced protein electrophoresis analysis, and finally the nanobody was dialyzed into PBS.

[0189] Through alpaca immunization, lymphocyte isolation, phage library construction, and nanoantibody screening, anti-mCherry nanoantibodies were screened. The sequencing results were analyzed using Vector NTI software to analyze the antibody light chain and heavy chain genes to determine the framework regions (FR) and complementary determining regions (CDR) of the variable region.

[0190] The results showed that there were three DNA sequences in total. The three antibodies were named mCherry12, mCherry19 and mCherry28 according to their clone numbers. Their nucleotide sequences were shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and their amino acid sequences were shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0191] Example 2

[0192] This example provides a detailed process for the expression and purification of nanobodies.

[0193] 2.1 Amplification of the original strain TG1 of nanoantibody and transformation of the recombinant plasmid of nanoantibody into Escherichia coli BL21 (DE3)

[0194] The original strain TG1 glycerol bacteria containing nanobody nucleic acid was inoculated into 5mL of fresh LB medium at a ratio of 1:1000 and cultured overnight at 37℃200rpm. The next day, the plasmid was extracted according to the instructions using a plasmid extraction kit (purchased from OMEGA). After verification, 1μL of the above plasmid was transformed into 100μL of competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a 42℃ water bath for 90 seconds, and cooled in an ice bath for 3 minutes. Add 600μL of LB medium to the centrifuge tube and culture at 37℃ with shaking for 60 minutes. Take 100μL of the supernatant, spread it on the LB plate with a triangular spreader, and culture it upside down at 37℃ overnight.

[0195] 2.2 Inducible expression of nanobodies

[0196] Pick the above monoclonal colony in LB medium and culture at 37℃ overnight with shaking. The next day, take the bacterial solution and add 100mL fresh LB medium at a ratio of 1:100, culture at 37℃ with shaking for 3 hours until the bacterial solution OD600=0.8, add IPTG with a final concentration of 1mM, and induce at 30℃ overnight. On the third day, centrifuge at 8000rpm for 10 minutes to collect the bacteria, and add 1.5mL of pre-cooled TES buffer to resuspend the precipitate. After ice bathing for 2 minutes, gently shake for 30 seconds, and repeat this cycle 6 times. Add 3.0mL TES / 4 (dilute TES 4 times with water), shake gently for 30 seconds, let stand on ice for 2 minutes, and repeat the shaking and standing steps for a total of 6 times. Centrifuge at 9000rpm and 4℃ for 10 minutes, and collect about 4.5mL of supernatant (periplasmic extract).

[0197] 2.3 Purification and identification of nanobodies

[0198] The above samples were purified using His-tag agarose gel (produced by Shanghai Lingyin Biotechnology Co., Ltd.), washed with 20mM imidazole, eluted and collected with 300mM imidazole, and then purified again using molecular sieves. The purified samples were analyzed by SDS-PAGE electrophoresis.

[0199] The results of SDS-PAGE electrophoresis are shown in the figure Figure 1 As shown, the purified nanobody was finally dialyzed into PBS and stored at 2-8°C for later use.

[0200] Example 3

[0201] In this example, the ELISA method was used to verify the binding ability of the mCherry nanobody to the mCherry protein.

[0202] The purified mCherry nanobody was tested for antigen binding using enzyme-linked immunosorbent assay (ELISA). TM According to the instructions of the Sulfo-NHS-LC-biotinylation kit, the purified mCherry nanobody was coupled to biotin. Secondly, the mCherry protein (1μg / well) was coated on the ELISA plate, incubated at 4℃ overnight, blocked with 3% BSA, incubated at room temperature for 2h, and washed with PBST 5 times, 1min each time. Add biotinylated nanoantibody (1μg / well), incubate at room temperature for 1h, wash with PBST 5 times, 1min each time, and wash away the unbound antibody. Then add HRP-coupled streptavidin (1:10000), incubate at room temperature for 1h, wash with PBST 5 times, 1min each time. Finally, add 100μL TMB colorimetric solution and color at room temperature in the dark for 20min. Add 50μL 2M concentrated sulfuric acid to terminate the reaction, and measure the absorbance value at a wavelength of 450nm on the ELISA reader.

[0203] The results are as follows Figure 2 As shown in the figure, all three mCherry nanobodies have good binding ability with mCherry protein, especially mCherry19 has the strongest binding ability.

[0204] Example 4

[0205] In this example, the thermal stability test of anti-mCherry nanobody was performed.

[0206] The thermal stability of the purified mCherry nanobody was tested by enzyme-linked immunosorbent assay (ELISA). The mCherry protein was coated at a concentration of 5 μg / mL, 100 μL / well, coated at 4°C for 24 hours, and the plate was washed 5 times. Blocked overnight with 1% BSA as a blocking agent, and the plate was washed 5 times. The diluted mCherry nanobody was added to the ELISA plate, incubated at 37°C for 30 minutes, and the plate was washed 5 times. HRP-labeled goat anti-llama IgG and goat anti-mouse IgG (purchased from Abcam) diluted 1:2000 were added, incubated at 37°C for 30 minutes, and the plate was washed 5 times. TMB colorimetric solution was added, incubated at 25, 37, 50, 60, 70, 80, and 90°C for 30 minutes, 2M sulfuric acid was used to terminate the reaction, and the absorbance value was read at 450nm. ELISA was used to determine the residual antigen binding activity of each antibody after treatment at different temperatures. Three parallel replicate wells were set up in each group. The binding value after treatment at room temperature (25°C) was used as the 100% binding control. The average absorbance value of each group of repeated samples was calculated, and the relative activity was calculated. Figure 3 Relative activity (%) = OD450 不同温度处理 / OD450 25℃处理 ×100%.

[0207] The results showed that compared with traditional anti-mCherry antibodies (purchased from Abcam, catalog number ab125096), the three anti-mCherry nanoantibodies still had strong biological activity at a temperature of 70°C. Among them, the mCherry28 nanoantibody showed higher binding activity when treated at 80 and 90°C than at room temperature, with relative binding activities of 102% and 104% respectively. It has good thermal stability and broad application prospects.

[0208] Example 5

[0209] In this embodiment, anti-mCherry nanoantibody was used to prepare anti-mCherry nanoantibody magnetic beads.

[0210] A certain amount of carboxyl magnetic beads (from Shanghai Lingyin Biotechnology Co., Ltd.) were measured, and the carboxyl magnetic beads were washed with 0.1 M MES buffer at pH 5.0 for 2 to 3 times, and activated with excess EDC and NHS under acidic conditions for 30 minutes. After the activation was completed, a magnetic field was applied to separate the magnetic beads from the liquid, the supernatant was discarded, and the beads were washed with MES buffer to wash away excess activator for 3 to 4 times; anti-mCherry nanoantibodies were added for coupling, and the beads were mixed using a rotary mixer at room temperature for 2 hours; after the reaction was completed, a magnetic field was applied to separate the magnetic beads from the liquid, the supernatant was discarded, and the beads were blocked with a blocking solution containing 3% BSA (0.1 M Tris-HCl at pH 8.0) for 3 hours to preserve the anti-mCherry nanoantibody magnetic beads, so that the final concentration of the anti-mCherry nanoantibody magnetic beads was 10 mg / mL, and they were stored at 2 to 8°C for later use.

[0211] Example 6

[0212] In this example, anti-mCherry nanobody was used to prepare anti-mCherry nanobody agarose gel.

[0213] Measure a certain amount of CNBr activated agarose gel (from Shanghai Lingyin Biotechnology Co., Ltd.) into a sand core funnel of corresponding specifications and drain it, add 2 times the volume of gel 0.01M HCl, stir evenly and drain it, repeat 2-3 times. Then add coupling buffer (PBS, pH7.2) and stir and mix well and drain it, repeat 2-3 times, place it in a reaction vessel, add anti-mCherry nanobody (5mg antibody / g agarose gel), and react at room temperature for 2h. After the reaction is completed, wash twice with 2 times the volume of gel PBS, add blocking solution (0.1M Tris-HCl with pH 8.0) and react at room temperature for 3h to block the unreacted active groups. After washing 5 times with 2 times the volume of gel PBS, add 20% ethanol aqueous solution to preserve the anti-mCherry nanobody agarose gel, the final concentration is 50%, and store at 2-8°C for use.

[0214] Example 7

[0215] In this example, anti-mCherry nanobody was used to prepare anti-mCherry nanobody agarose magnetic beads.

[0216] Measure a certain amount of NHS-activated agarose beads (from Shanghai Lingyin Biotechnology Co., Ltd.) into the reaction container, add a magnetic field to separate the beads from the liquid, discard the supernatant, wash twice with MES buffer (0.1M, pH6.0), and remove the supernatant. Add anti-mCherry nanoantibodies (2mg antibody / mL agarose beads) and react at room temperature for 2h. After the reaction is completed, magnetic separation is performed, the supernatant is removed, and blocking solution (0.1M Tris-HCl with a pH of 8.0) is added to react at room temperature for 3h to block the unreacted active groups. After washing with PBS 5 times, add 20% ethanol aqueous solution to preserve the anti-mCherry nanoantibody agarose beads, the final concentration is 50%, and store at 2-8°C for use.

[0217] Example 8

[0218] In this example, the anti-mCherry nanobody agarose gel prepared in the above example 6 was subjected to affinity purification experiment.

[0219] Use the anti-mCherry nano antibody agarose gel prepared in Example 6 to purify the mCherry tagged protein. Add the anti-mCherry nano antibody agarose gel to the chromatography column, let the preservation solution flow out, wash the chromatography column several times with PBS (pH7.2), add the mCherry tagged protein sample solution, place it in a rotating mixer and incubate for 30 to 60 minutes, then collect the effluent. Repeated loading can increase the binding efficiency. Wash with 5 to 10 times the column volume of PBS to remove non-specifically adsorbed impurities, then use glycine (pH2.2) to elute the specific mCherry tagged protein, and collect the eluate, which is the purified protein solution. Repeat the use for 10 times and calculate the recovery rate, as shown in the following figure. Figure 4 , 5 shown.

[0220] Recovery rate (%) = (actual recovery amount / initial addition amount) × 100%.

[0221] The results showed that the use of two anti-mCherry nanoantibody agarose gels (mCherry19 and mCherry28) can specifically purify mCherry-tagged proteins, and after 10 repeated uses, the recovery rate is still greater than 85%, which has good stability and broad application prospects.

[0222] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanobody against mCherry tag, characterized in that: The nanobody comprises: CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.4; CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.5; Or CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.

6.

2. The anti-mCherry tag nanobody according to claim 1, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO.4 are shown in SEQ ID Nos.7 to 9 respectively; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO.5 are shown in SEQ ID Nos.10 to 12, respectively; Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID NO.6 are shown in SEQ ID Nos.13 to 15, respectively; Preferably, the framework region in the heavy chain variable region shown in SEQ ID NO.4 includes the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID Nos.16 to 19 in sequence; Preferably, the framework region in the heavy chain variable region shown in SEQ ID NO.5 includes the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID Nos.20 to 23 in sequence; Preferably, the framework region in the heavy chain variable region shown in SEQ ID NO.6 includes the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in SEQ ID Nos.24 to 27 in sequence.

3. A multivalent nanobody, a multispecific nanobody or a fusion nanobody, characterized in that: It comprises the anti-mCherry tag nanobody according to any one of claims 1-2; Preferably, the multivalent Nanobody comprises at least two of the following CDRs regions: (1) CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.4; (2) CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.5; (3) CDR1, CDR2 and CDR3 in the heavy chain variable region as shown in SEQ ID NO.

6.

4. An antibody, characterized in that It contains the anti-mCherry tag nanobody according to any one of claims 1-2 or the multivalent nanobody, multispecific nanobody or fusion nanobody according to claim 3.

5. A nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the anti-mCherry tag Nanobody according to any one of claims 1-2; or the nucleic acid molecule encodes the multivalent Nanobody, multispecific Nanobody or fusion Nanobody according to claim 3; or the nucleic acid molecule encodes the antibody according to claim 4; Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

6. A host cell, characterized in that It contains the recombinant vector according to claim 5.

7. A method for preparing an anti-mCherry tag nanobody as described in any one of claims 1-2; or a multivalent nanobody, a multispecific nanobody or a fusion nanobody as described in claim 3; or an antibody as described in claim 4, characterized in that: It includes: Cultivate the host cell according to claim 6.

8. A nanobody against mCherry tags as claimed in any one of claims 1 to 2; or a multivalent nanobody, a multispecific nanobody or a fusion nanobody as claimed in claim 3; or the use of the antibody as claimed in claim 4 in any of the following, characterized in that: (1) Preparation of mCherry detection products; (2) preparing mCherry enriched or purified products; (3) preparing an enriched product or purified product of a fusion protein with an mCherry tag; (4) preparing products for separation or purification of interacting proteins with mCherry-tagged proteins; (5) preparing an isolated or purified product of a specific protein-DNA complex with an mCherry tag; (6) Application in the preparation of tracing detection products for fusion proteins or cells with mCherry tags; Preferably, the detection product is selected from a reagent, a kit or a chip; Preferably, the kit is a chemiluminescent immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a time-resolved fluorescent immunoassay kit; Preferably, the kit is a magnetic microparticle chemiluminescence detection kit or a fluorescent immunoassay kit; Preferably, the chip is a chromatin immunoprecipitation chip, an immunoprecipitation chip, an immunoprecipitation chip or an mCherry fusion protein precipitation chip; Preferably, the mCherry enriched or purified product, the enriched or purified product of the fusion protein with the mCherry tag, the product for separating or purifying the interacting protein with the mCherry-tagged protein, or the product for separating or purifying the specific protein-DNA complex with the mCherry tag comprises: a vector and a nanobody or antibody on the vector; Preferably, the Nanobody or antibody on the carrier is coupled and / or physically linked to the carrier as a ligand; Preferably, the carrier is selected from nanoparticles, magnetic beads, agarose gel microspheres, silica microspheres, latex microspheres, chips or porous materials; Preferably, the tracer detection product is a reagent, a kit or a chip.

9. A nanobody with an anti-mCherry tag as claimed in any one of claims 1 to 2; or a multivalent nanobody, a multispecific nanobody or a fusion nanobody as claimed in claim 3; or an antibody product as claimed in claim 4, characterized in that: The product is a nanoparticle, magnetic bead, agarose gel microsphere, silica microsphere, latex microsphere, test tube, EP tube, multi-well plate microplate concave hole, membrane, PDMS membrane or chip; Preferably, the nanoparticles are selected from organic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles; Preferably, the multi-well plate is an ELISA plate; Preferably, the Nanobody or antibody is coupled and / or physically linked to the product; Preferably, the Nanobody or antibody is also labeled with a detectable marker; Preferably, the detectable marker is selected from at least one of biotin, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents and colloids.

10. A method for separating mCherry, a fusion protein with an mCherry tag, an mCherry interacting protein, or a specific protein-DNA complex with an mCherry tag, characterized in that: The method comprises: mixing the anti-mCherry tag nanobody according to any one of claims 1 to 2, the multivalent nanobody, multispecific nanobody or fusion nanobody according to claim 3; the antibody according to claim 4 or the product according to claim 9 with a sample to be separated, and eluting.

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