CN117700559A

CN117700559A - anti-V5 tag nano antibody, product and application thereof

Info

Publication number: CN117700559A
Application number: CN202311638855.2A
Authority: CN
Inventors: 姬满祎
Original assignee: Shanghai Lingyin Biotechnology Co ltd
Current assignee: Shanghai Lingyin Biotechnology Co ltd
Priority date: 2023-12-01
Filing date: 2023-12-01
Publication date: 2024-03-15

Abstract

The invention discloses a nanometer antibody of an anti-V5 label, a product and application thereof, and relates to the technical field of single-domain heavy chain antibodies. The 3 kinds of anti-V5-labeled nano-antibodies provided by the invention have specific recognition and binding capacities for V5 antigen, so that the nano-antibodies provided by the invention have high specific binding activity. In addition, the nano antibody provided by the invention has excellent performance in the purification of the V5 tag protein, and the characteristic can be used for developing a V5 tag protein affinity purification reagent, so that the nano antibody can be used for separating and purifying a V5 tag protein, a V5 antigen, a fusion protein with a V5 tag, a V5 interaction protein or a specific protein-DNA complex with a V5 tag. The nano antibody provided by the invention can be produced by a large amount of culture, expression and purification through a biological method, can be repeatedly used for a plurality of times, and still has high recovery rate after being repeatedly used for a plurality of times, and has wide application prospect.

Description

anti-V5 tag nano antibody, product and application thereof

Technical Field

The invention relates to the technical field of single-domain heavy chain antibodies, in particular to a nanometer antibody for resisting a V5 label, a product and application thereof.

Background

With the development of proteomics, the separation and purification technology of recombinant proteins plays a very important role, and a suitable protein purification method is selected to specifically and rapidly separate and purify a target protein from a complex sample, wherein the most commonly used protein purification method is an affinity chromatography (Affinity chromatography, AC) method, which is commonly used for fusion expression of a protein and an affinity tag by using a fusion tag technology, and the target protein is further purified by using the affinity tag. Many protein tags with different functions have been developed successively by researchers, including: glutathione transferase (GST), polyhistidine (poly-His), V5 tag, MBP tag, mCherry tag, flag polypeptide, fc segment of immunoglobulin, and the like. The use of the fusion tag facilitates the expression and purification of the target protein, and provides a convenient means for researching the structure and function of the target protein.

The V5 tag is a commonly used protein labeling tool, commonly used for detection, purification and localization of proteins in biological research and production. The V5 tag protein is a peptide segment containing 14 amino acids, namely GKPIPNPLLGLDST, which is derived from amino acids 95-108 of the alpha subunit of the simian virus 5RNA polymerase, and is commonly added at the C-terminus or N-terminus of the target protein for purification, detection and tracing of the target protein due to its low amino acid number, which does not affect the nature of the target protein itself, and which has high affinity for specific antibodies. The V5 tag is mainly used in the following aspects: 1) Protein expression detection: in protein expression systems, the attachment of a V5 tag to the N or C terminus of a protein can make the protein easier to detect during expression. Protein expression can be detected by Western blot, ELISA or other techniques. 2) Protein purification: since the V5 tag has characteristics of easy detection and purification, it can be used for purifying proteins. In summary, the V5 tag is a very practical tool for the purification, detection and tracing of proteins.

Based on the outstanding characteristics of the V5 tag in biological experiments such as protein detection, purification, localization and the like, the development of antibodies aiming at the V5 tag protein is particularly important.

In 1993, hamers Casterman et al found that a class of heavy chain dimer (H2) only antibodies, principally of the IgG2 and IgG3 types, were found in camelids (camels, dromedaries and llamas), and that such antibodies were termed heavy chain only antibodies (Heavy chain only like Antibody, HCAbs) due to the lack of light chains, whereas their antigen binding sites consisted of one domain, termed the VHH region, and were termed single domain antibodies or single domain antibodies (sdabs). Since this type of antibody is a variable region sequence with the constant region removed, the molecular weight is only 15kDa, and the diameter is about 10 nanometers, and thus is also called nanobody (Nbs).

Nanobodies have important competitive advantages over other antibody molecules due to their unique structural, functional, and biophysical properties. First, the specificity is strong. Nanobodies have a molecular weight of 12-15 kD and are currently known antibodies with antigen binding capacity and minimal molecular weight. Due to the small volume, the nano antibody can enter an epitope which cannot be reached by other antibodies or a relatively hidden epitope, so that the specificity of the nano antibody is greatly increased. In addition, due to the specific three-dimensional structure of nanobodies, the longer CDR3 regions of nanobodies can enter into the cavities of molecular targets, such as the active sites of enzymes, which are not available with conventional antibodies. Second, the structure is stable. Because disulfide bonds exist in the nano antibody, the structure of the nano antibody is more stable, and researches prove that 80% of the antibody still remains the binding activity after the nano antibody is placed for one week at 37 ℃, and the nano antibody can still renature even at a high temperature of more than 90 ℃, has excellent thermal stability and greatly increases the times of recycling the nano antibody. Third, it is easy to produce. Once the specific nano antibody gene is obtained, the specific nano antibody gene can be produced in a large quantity through a eukaryotic/prokaryotic expression system, so that the difference of antibodies among different batches is avoided, and meanwhile, the production cost is greatly reduced. Therefore, the preparation of the nano antibody of the V5 tag protein lays a material foundation for further establishing an antigen-antibody specific V5 affinity chromatography system. In addition, as the molecular weight of the nano antibody is small, more antibody fragments can be coupled on the surface of the magnetic beads or the medium to increase the strength of signals, thereby improving the sensitivity of the interaction between proteins; the dissociation constant is low, and the method can be combined with target protein rapidly, thereby greatly saving the experiment time and the like, and is used for immune (co) precipitation (IP/CoIP), chromatin immunoprecipitation (ChIP), pull-down experiments and the like.

The method of combining nanobody technology with tag proteins is currently being gradually applied to the field of biological research. Among them, the Nano-Trap technology has been developed by the company Chromotek in germany, which covalently couples nanobodies of tag proteins to agarose gel or magnetic beads to form a series of products. Due to the high-quality characteristic of the Nano antibody, the Nano-Trap shortens the experiment time and reduces the nonspecific binding, thereby greatly promoting the progress of protein function research. In view of the functional properties of V5 tags and the advantages of nanobodies, it is believed necessary to develop V5 tagged nanobodies to facilitate the study of purification, detection and tracking of fusion proteins containing V5 tags.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a nanometer antibody for resisting a V5 label, a product and application thereof, wherein the nanometer antibody can fully exert the excellent performance of the nanometer antibody, namely, on one hand, the nanometer antibody has excellent specific antigen binding capacity, and on the other hand, the nanometer antibody can overcome the inherent defects of insufficient thermal stability, high production cost and the like of the traditional antibody. The nanobody provided by the invention can be used for purification, detection and tracing through an affinity purification technology.

The invention is realized in the following way:

in a first aspect, the present invention provides a nanobody against a V5 tag, the nanobody comprising:

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.

In a second aspect, the present invention also provides a multivalent nanobody, multispecific nanobody, or fusion nanobody, comprising a nanobody as described above with an anti-V5 tag.

In an alternative embodiment, the multivalent nanobody comprises at least two of the following CDR 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.

In a third aspect, the present invention also provides an antibody comprising the above anti-V5-tagged nanobody or the above multivalent nanobody, multispecific nanobody or fusion nanobody.

In a fourth aspect, the invention also provides a nucleic acid molecule or a recombinant vector comprising a nucleic acid molecule encoding a nanobody against the V5 tag described above; or the nucleic acid molecule encodes the multivalent nanobody, the multispecific nanobody or the fusion nanobody; or a nucleic acid molecule encodes an antibody as described above.

In a fifth aspect, the invention also provides a host cell comprising the recombinant vector described above.

In a sixth aspect, the present invention also provides a nanobody for preparing the anti-V5 tag described above; or the multivalent nanobody, multispecific nanobody, or fusion nanobody described above; or a method of the above antibody, comprising: culturing the host cell described above.

In a seventh aspect, the present invention also provides a nanobody against a V5 tag; or the multivalent nanobody, multispecific nanobody, or fusion nanobody described above; or the use of the above antibody in the preparation of a V5 detection product, a V5 enrichment or purification product, a V5 tagged fusion protein enrichment or purification product, a product for isolation or purification of a V5 tagged protein interacting protein, or a product for isolation or purification of a V5 tagged specific protein-DNA complex.

In an eighth aspect, the present invention also provides a nanobody with an anti-V5 tag as described above; or the multivalent nanobody, multispecific nanobody, or fusion nanobody described above; or the antibody product is nano-particles, magnetic beads, agarose gel microspheres, silica gel microspheres, latex microspheres, test tubes, EP tubes, concave holes of a porous plate micro-reaction plate, NC films, PDMS films or chips.

In a ninth aspect, the invention also provides a method of isolating V5, a V5 tagged fusion protein, a V5 interacting protein or a V5 tagged specific protein-DNA complex, comprising: the above-mentioned anti-V5-tagged nanobody, the above-mentioned multivalent nanobody, multispecific nanobody or fusion nanobody; the antibody or the product is mixed with a sample to be separated and eluted.

The invention has the following beneficial effects:

(1) The 3 kinds of anti-V5-labeled nano-antibodies provided by the invention have specific recognition and binding capacities for V5 antigen, so that the nano-antibodies provided by the invention have high specific binding activity.

(2) The invention also provides an affinity purification material (such as magnetic beads containing anti-V5 nano-antibodies, agarose gel, sepharose beads and the like) containing the anti-V5 tag nano-antibodies, which shows excellent performance in the purification of V5 tag proteins, and the property can be used for developing V5 tag protein affinity purification reagents, thereby being used for the separation and purification of V5 tag proteins, V5 antigens, fusion proteins with V5 tags, V5 interaction proteins or specific protein-DNA complexes with V5 tags.

(3) The nano antibody provided by the invention can be produced by a large amount of culture, expression and purification through a biological method, so that complicated production steps such as purification of ascites of mice are avoided, the production cost is greatly reduced, the nano antibody can be repeatedly used for a plurality of times, and the nano antibody still has high recovery rate after repeated use for a plurality of times and has a wide application prospect.

(4) The nano antibody provided by the invention has strong thermal stability and still has strong biological activity at the temperature of 70 ℃.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an electrophoretogram of 3V 5-tagged nanobodies (V5-06, V5-17, and V5-24);

FIG. 2 is a graph showing the statistical result of the binding condition of 3V 5-labeled nanobodies and antigens;

FIG. 3 is a graph showing the statistical result of the thermal stability of 3V 5-labeled nanobodies;

FIG. 4 is a graph showing the recovery rate statistics of the V5-labeled nanobody agarose gel (V5-06) for repeated use;

FIG. 5 is a graph showing the recovery rate statistics of the V5-labeled nanobody agarose gel (V5-24) for repeated use.

Detailed Description

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

EVQLVESGGGSVQAGGSLRLSCAASGYTFTAYCMGWVRQAPGKEREGVAKLVSVAGRTYYAESVKGRFTVSRDNAKNIL YLQMNNLKPEDTAMYYCAATSDPICWSPTLVYWGHGTQVTVSS。

the amino acid sequence of SEQ ID NO.5 is as follows:

QLQLQASGGGLVQPGGSLRLSCAASGFASFGDTIGWVRQAPGKGREFVSAISNQGGDSHYYAESVKGRFTISRDNSKNTV YLQMNSLRAEDTATYYCAEWGGTRPEFITPYWGQGTQVTVSS。

the amino acid sequence of SEQ ID NO.6 is as follows:

EVKLVESGGGSVQAGGSLRLSCAVSGYSFDNYCMGWVRAGSGKEREGVAKINPSGRYTDYVESVKGRFTISKDNAKNILY LQMNNLKPEDTAMYYCAAVRTSACGSTVITDWGQGTQVTVSS。

in the present invention, the term "complementarity determining regions", "CDRs" or "CDRs" refers to the highly variable regions of the heavy chain of an immunoglobulin, and refers to regions comprising 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 an antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy chain of the antibody. "CDR" in the present invention is synonymous with "complementarity determining region".

In the present invention, the heavy chain complementarity determining region is represented by a CDR, which includes CDR1, CDR2 and CDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, and the 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. 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 Kabat numbering schemes, which are generally considered as widely adopted criteria for numbering antibody residues. The present invention uses Chothia annotation standard to mark CDR regions, but other methods to mark CDR regions are also within the scope of the present invention.

The invention screens and obtains 3 anti-V5 label nano-antibodies, and the 3 nano-antibodies have specific recognition and binding capacity to V5 antigen, so that the nano-antibodies provided by the invention have high specific binding activity. Based on the above, anti-V5 tag nanobody affinity purification materials can be prepared for separation or purification of V5 antigen, separation or purification of V5 tag protein, and separation of protein-DNA complex.

The nano antibody provided by the invention can be produced by a large amount of culture, expression and purification through a biological method, so that complicated production steps such as purification of ascites of mice are avoided, the production cost is greatly reduced, the nano antibody can be repeatedly used for a plurality of times, the recovery rate of target proteins is high, the stability is good, and the application prospect is wide.

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 sequence as SEQ ID No.7 to 9.

The amino acid sequence of SEQ ID NO.7 is as follows: GYTFTAYC.

The amino acid sequence of SEQ ID NO.8 is as follows: LVSVAGRT.

The amino acid sequence of SEQ ID No.9 is as follows: AATSDPICWSPTLVY.

In an alternative 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 sequence as SEQ ID Nos. 10 to 12;

The amino acid sequence of SEQ ID No.10 is as follows: GFASFGDT.

The amino acid sequence of SEQ ID No.11 is as follows: ISNQGGDSH.

The amino acid sequence of SEQ ID No.12 is as follows: AEWGGTRPEFITPY.

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 sequence as SEQ ID Nos. 13 to 15; the amino acid sequence of SEQ ID No.13 is as follows: GYSFDNYC.

The amino acid sequence of SEQ ID No.14 is as follows: INPSGRYT.

The amino acid sequence of SEQ ID No.15 is as follows: AVRTSACGSTVITD.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region, which refers to a region other than CDRs in a heavy chain variable region of a nanobody; the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising FR1, FR2, FR3 and FR4 framework regions.

The heavy chain variable region structure in the invention is as follows:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。

in an alternative embodiment, the framework regions in the heavy chain variable region shown in SEQ ID NO.4 comprise heavy chain framework regions FR1, FR2, FR3 and FR4 shown in sequence as SEQ ID NO. 16-19;

SEQ ID No.16：EVQLVESGGGSVQAGGSLRLSCAAS。

SEQ ID No.17：MGWVRQAPGKEREGVAK。

SEQ ID No.18：

YYAESVKGRFTVSRDNAKNILYLQMNNLKPEDTAMYYC。

SEQ ID No.19：

WGHGTQVTVSS。

in an alternative embodiment, the framework regions in the heavy chain variable region shown in SEQ ID NO.5 include the heavy chain framework regions FR1, FR2, FR3 and FR4 shown in sequence as SEQ ID NO. 20-23;

SEQ ID No.20：QLQLQASGGGLVQPGGSLRLSCAAS。

SEQ ID No.21：IGWVRQAPGKGREFVSA。

SEQ ID No.22：

YYAESVKGRFTISRDNSKNTVYLQMNSLRAEDTATYYC。

SEQ ID No.23：WGQGTQVTVSS。

In an alternative embodiment, the framework regions in the heavy chain variable region shown in SEQ ID NO.6 comprise heavy chain framework regions FR1, FR2, FR3 and FR4 shown in sequence in SEQ ID NO. 24-27.

SEQ ID No.24：EVKLVESGGGSVQAGGSLRLSCAVS。

SEQ ID No.25：MGWVRAGSGKEREGVAK。

SEQ ID No.26：

DYVESVKGRFTISKDNAKNILYLQMNNLKPEDTAMYYC。

SEQ ID No.27：WGQGTQVTVSS。

In an alternative embodiment, the nanobody is a monovalent nanobody.

Noun interpretation:

monovalent nanobody: the antigen-specific nanobody is obtained by screening specific antigen from a nanobody library, can maintain a strict monomer structure due to a large number of hydrophilic residues on the surface of the nanobody, and can be combined with the antigen with high specificity and high affinity only in a monomer form.

In an alternative embodiment, when the nanobody is a monovalent nanobody, the amino acid sequence of the heavy chain variable region of the nanobody is as shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO. 6.

Multivalent nanobody: multivalent antibodies are polymers of monovalent antibodies that recognize the same epitope, with higher antigen affinity than the corresponding monovalent nanobody. Multispecific antibodies are polymers of monovalent antibodies that recognize different epitopes, can bind to different targets or different epitopes of the same target, and have higher antigen recognition capabilities than monovalent antibodies. The nanobody has a simple structure, only has one structural domain, and can be polymerized together through a short connecting sequence, so that the nanobody is converted into a multivalent and multispecific form.

Fusion nanobody: the nano antibody has strict monomer characteristics and small relative molecular mass, and can be easily combined with other structures (such as BSA, igG-Fc and the like) to form new fusion molecules, such as enzymes, antibacterial peptides or developing substances and the like for prolonging half-life of the nano antibody. In the novel fusion molecule, the nanobody is bound with the target antigen thereof in a directional manner, and the part fused with the nanobody can play a corresponding function.

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).

Antibodies include, but are not limited to, any of heavy chain antibodies, full length antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like), murine antibodies, humanized antibodies, or antigen-binding fragments.

The "chimeric antibody" according to the present invention is an antibody in which a variable region of a non-human antibody is fused with a constant region or a framework region of a human antibody, and can reduce an immune response induced by the non-human antibody.

Such antibodies include, but are not limited to, functional fragments of antibodies, generally having 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 the antibody, so long as they exhibit the desired antigen binding activity. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

The above antibodies or nanobodies may also be obtained synthetically by recombinant genetic techniques also known to those skilled in the art or by e.g. automated peptide synthesizers such as those sold by Applied BioSystems.

In an alternative 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.

Considering the degeneracy of codons, the sequence of the genes encoding the above antibodies may be modified in the coding region thereof without changing the amino acid sequence to obtain genes encoding the same antibodies; the modified genes can also be artificially synthesized according to the codon preference of the host for expressing the antibody so as to improve the expression efficiency of the antibody.

The recombinant vector is an expression vector or cloning vector, preferably an expression vector, and may refer to any recombinant polynucleotide construct that can be used to introduce a DNA fragment of interest directly or indirectly (e.g., packaged into a virus) into a host cell by transformation, transfection or transduction for expression of the gene of interest.

One type of vector is a plasmid, i.e., a circular double stranded DNA molecule, into which a DNA fragment of interest can be ligated into a plasmid loop. Another type of vector is a viral vector, which can ligate and package the DNA fragment of interest into the viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter host cells, expression of the gene of interest can be performed.

The host cell is selected from at least one of a prokaryotic host cell, a eukaryotic host cell, and a phage;

in an alternative embodiment, the prokaryotic host cell is E.coli, streptomyces, bacillus subtilis, or Mycobacterium;

in an alternative embodiment, the eukaryotic host cell is an animal cell, a plant cell, or a fungus; fungi such as pichia pastoris, and the like.

In an alternative embodiment, the animal cell is selected from a mammalian cell, an insect cell, or a caenorhabditis elegans;

the mammalian cells are 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 common mammalian cells for producing antibodies or recombinant proteins, and are well known to those of ordinary skill in the art.

On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, etc.), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of a recombinant cell capable of recombinantly expressing the antibody or the functional fragment thereof as described in any one of the above, and on the basis of this, the antibody or the functional fragment thereof of the present invention is prepared by any technique, which falls within the scope of the present invention.

The 3 nano antibodies have higher affinity with V5, so that the nano antibodies can be used for detecting, enriching and purifying V5, enriching and purifying fusion proteins with V5 labels, separating or purifying interaction proteins of proteins with V5 labels and separating or purifying specific protein-DNA complexes with V5 labels.

In an alternative embodiment, the detection product is selected from the group consisting of a reagent, a kit or a chip.

In an alternative embodiment, the kit is a chemiluminescent immunoassay kit, a radioimmunoassay kit, an enzyme linked immunoassay kit, a fluorescent immunoassay kit or a time resolved fluorescent immunoassay kit.

In an alternative embodiment, the kit is a magnetic particle chemiluminescent detection kit.

In an alternative embodiment, the ChIP is a chromatin co-immunoprecipitation ChIP (Chromatin Immunoprecipitation-ChIP, chIP-ChIP), immunoprecipitation ChIP, or V5 fusion protein sedimentation ChIP (e.g., based on a Pull down assay).

In an alternative embodiment, the V5 enriched or purified product, the enriched or purified product of the V5 tagged fusion protein, the product for isolation or purification of the V5 tagged interacting protein, or the product for isolation or purification of the V5 tagged specific protein-DNA complex comprises: a carrier and a nanobody or antibody on the carrier.

In an alternative embodiment, the carrier is selected from the group consisting of nanoparticles, magnetic beads, agarose gel microspheres, silica gel microspheres, latex microspheres, chips, or porous materials.

For example, the activated beads are incubated with the antibody such that the beads are coated with the antibody. For example, coupling nano-antibodies on agarose gel microspheres or silica gel microspheres to prepare corresponding immunoaffinity adsorption materials, and placing the immunoaffinity adsorption materials in a chromatographic column to prepare corresponding affinity columns for V5 enrichment or purification or for enrichment and purification of fusion proteins with V5 tags.

In an alternative embodiment, the nanoparticles are selected from the group consisting of organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

The magnetic beads are also called magnetic microspheres, and the magnetic microspheres are magnetic composite microspheres with a certain structure, wherein organic polymers and inorganic magnetic nanoparticles are combined by a proper method to form the magnetic composite microspheres with a special structure. Magnetic beads include, but are not limited to, nano magnetic beads and micro magnetic microspheres. In an alternative embodiment, the magnetic beads include, but are not limited to: carboxyl magnetic beads, amino magnetic beads, oleylamine modified magnetic beads, silicon hydroxyl magnetic beads, sulfonic acid magnetic microspheres, sulfhydryl magnetic microspheres, PEG modified magnetic beads, non-modified ferroferric oxide magnetic beads, monodisperse silicon-coated magnetic beads, epoxy magnetic beads, monodisperse mesoporous silicon-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 silicon dioxide magnetic microspheres.

Latex microspheres are spherical polymer particles in the colloidal size range formed from an amorphous polymer (typically polystyrene), such as particles less than 100nm in diameter, or particles ranging from 0.3 to 0.5 μm in diameter, or particles exceeding 1 μm in diameter.

The latex microspheres include, but are not limited to, latex microspheres having at least one functional group of hydroxyl, carboxyl or tostal modified on the surface.

Agarose gel microspheres with nanobodies or antibodies are prepared, for example, by the following method:

and (3) reacting the agarose gel with a coupling reagent to form an activated agarose gel microsphere with epoxy groups, and then coupling the activated agarose gel microsphere with the V5 nano antibody or the amino group of the antibody to prepare the agarose gel-V5 adsorption material. Wherein the coupling reagent is a halogenated epoxy compound and/or a diglycidyl ether reagent.

In other embodiments, cyanogen bromide activated sepharose gel can be pumped to dryness in a sand core funnel with corresponding specification, HCl is stirred uniformly and pumped to dryness, then coupling buffer solution is added for treatment, and then V5 nano antibody or antibody is reacted with cyanogen bromide activated sepharose gel to obtain sepharose gel microsphere with nano antibody or antibody.

In an alternative embodiment, the nanoparticles are selected from the group consisting of organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

In an alternative embodiment, the multiwell plate is an elisa plate; in other embodiments, other solid support plates may be used.

In an alternative embodiment, the nanobody or antibody is conjugated and/or physically linked to the product; physical attachment includes, but is not limited to, electrostatic attraction, and the like. Physical connections such as immunonephelometric platforms.

In an alternative embodiment, the nanobody or antibody is further labeled with a detectable label.

In an alternative embodiment, the detectable label is selected from at least one of biotin, a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a colloid.

Fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-photoprotein (FAM), tetrachlorophotoprotein (TET), and the like, or analogs thereof, rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof, for example, including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazoxanthin (chlorophyll), and the like, for example.

In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

In alternative embodiments, the radioisotope includes, but is 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 Ru、 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。

in an alternative embodiment, the chemiluminescent reagent is selected from at least one of acridinium esters, luminol, lucigenin, crustacean fluorescein, ruthenium bipyridine, dioxane, lomustine, isoluminol, and peroxyoxalate.

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

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

In an alternative embodiment, elution with glycine is used.

The separation principle of the specific protein-DNA complex with the V5 tag is as follows: the nanobody of the V5 tag is combined with the same V5 tag carried in the target protein, nuclease (such as micrococcus nuclease) is carried to the binding site of the target protein and the chromatin, and the chromatin is cracked through activation of the nuclease, so that the protein-DNA complex is released.

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The present example performs the construction and screening of nanobody phage display libraries.

1.1 alpaca immunization and lymphocyte separation

Through the induced expression of the escherichia coli, the V5 protein is purified from the escherichia coli, and then buffer is replaced by ultrafiltration, so that excessive harmful substances such as endotoxin, foreign protein, salt ions and the like are removed, and the next step of immunization of the alpaca is facilitated. Selecting one healthy adult alpaca, mixing 1mg of purified V5 protein with Freund's adjuvant according to the ratio of 1:1, immunizing alpaca by adopting a back subcutaneous multipoint injection mode for 7 times, using Freund's complete adjuvant for the first time, using Freund's incomplete adjuvant for the other 6 times, and immunizing for 1 week at intervals. Then, alpaca peripheral blood was collected for constructing phage display library. Separating lymphocytes from collected alpaca peripheral blood by using the operation of a kit instruction of a camel peripheral blood lymphocyte separation solution, wherein each time the lymphocytes are 2.5X10 ⁷ 1mL of RNA isolation reagent was added to each living cell, 1mL was taken for RNA extraction, and the rest was stored at 80 ℃.

1.2 total RNA extraction and reverse transcription to cDNA

RNA extraction was performed by referring to the TAKARA Total RNA extraction kit. The first strand of cDNA was synthesized using RNA as a template and oligo-dT as a primer, and referring to TAKARA company reverse transcriptase instructions.

1.3 antibody variable region Gene amplification

The cDNA obtained by reverse transcription was used as a template for PCR reaction. Amplification was performed in two rounds, and the primer sequences for the first round of PCR were as follows:

CALL001：GTCCTGGCTGCTCTTCTACAAGG

CALL002：GGTACGTGCTGTTGAACTGTTCC

the PCR reaction conditions and procedures were: 95 ℃ for 5 minutes; 95℃for 30 seconds, 57℃for 30 seconds, 72℃for 30 seconds, 30 cycles; 7 minutes at 72 ℃. The band of about 700bp was recovered by using agarose gel recovery kit gel, and finally the nucleic acid concentration was adjusted to 5 ng/. Mu.L with water. The primer sequences for the second round of PCR were as follows:

VHH Back：GATGTGCAGCTGCAGGAGTCTGGRGGAGG

VHH For：CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

the PCR reaction conditions and procedures were: 95 ℃ for 5 minutes; 95℃for 30 seconds, 55℃for 30 seconds, 72℃for 30 seconds, 15 cycles; 7 minutes at 72 ℃. The PCR product was purified using a PCR product recovery kit.

1.4 vector construction

pMES4 was digested simultaneously with PstI and BstEII, and 1.5. Mu.g of the digested vector and 450ng of the digested second PCR were digested, respectively, and 15. Mu. L T4 DNA ligase was added thereto, and the mixture was allowed to mix overnight at 16℃with additional buffer and water to a total volume of 150. Mu.L, and the ligation product was recovered. The PCR product recovery kit was used for product recovery, eluting with 20. Mu.L water. Double digestion results of pMES4 vector were detected by 1% agarose electrophoresis gel.

1.5 electric conversion and storage Capacity determination

10. Mu.L of the purified ligation product was added to a pre-chilled electrocuvette containing 50. Mu.L of E.coli TG1 competent cells, and electrotransformation was performed using an electrotransformation apparatus (ECM 630 electrotransformation apparatus of BTX, USA), the electrocuvette was removed, and the transformant was resuscitated and cultured. Clones were randomly picked and colony PCR identified. The stock capacity was estimated from the PCR positive rate (stock capacity = number of clones x dilution x PCR identification positive rate x 10). The primer sequences were as follows:

MP57：TTATGCTTCCGGCTCGTATG

GIII：CCACAGACAGCCCTCATAG

1.6 phage amplification

Inoculating the resuscitated bacterial solution into YT AG culture medium, and culturing at 37deg.C and 200rpm until the culture OD ₆₀₀ =0.5. 10mL of the bacterial liquid was taken out and added to 4X 10 ¹⁰ VCSM13 (purchased from beijing starter culture) was statically infected at 37 ℃ for 30 minutes. Centrifugation at 4000rpm for 10 min at normal temperature and removal of supernatant. The cells were resuspended in 2 XYTAK (ampicillin and kanamycin) -medium and incubated overnight at 37℃at 200 rpm. The supernatant was centrifuged in a 40mL tube, 10mL of PEG/NaCl (20%/2.5M) solution was added and thoroughly mixed, the supernatant was discarded by centrifugation, the pellet was washed with 1mL of ice PBS and centrifuged, 250. Mu.L of pre-chilled PEG/NaCl was taken, thoroughly mixed and washed for resuspension.

Phage titer was determined: culturing TG1 to OD ₆₀₀ Phage were diluted in gradient with LB medium, mixed with phage TG1 culture diluted in a double ratio, plaque formation in the culture plate was observed the next day, the number of plaques was counted on a dilution gradient plate of 30.300 and phage titer (pfu) was calculated according to the following formula.

Phage titer (pfu/mL)) =dilution x number of plaques x 100

1.7 nanobody screening

Positive clones were screened for antigen by ELISA method. ELISA plates were coated with antigen, blocked with 5% BSA, and washed with PBST. 100. Mu.L of phage supernatant was added to each well and left at 37℃for 1 hour. The supernatant was discarded, and HRP-labeled secondary antibody against M13 was added and left at 37 ℃ for 1 hour. The supernatant was discarded, TMB solution was added, incubated at room temperature for 5 hours, 2M sulfuric acid stop solution was added to each well, and the wells were read with a microplate reader at 450 nm. The clone identified as positive in ELISA was picked up and sent to sequencing company for sequencing.

Expression and purification of 1.8 nanobody in E.coli

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

The anti-V5 nanobody is screened out through alpaca immunization, lymphocyte separation, phage library construction and nanobody screening. Analysis of antibody light and heavy chain genes was performed on the sequencing results using Vector NTI software to determine the framework regions (Framework Regions, FR) and complementarity determining regions (Complementary Determining Regions, CDR) of the variable regions.

As a result, it was found that there were 3 DNA sequences in total, and these 3 antibodies were designated V5-06, V5-17 and V5-24 according to their clone numbers, their nucleotide sequences were shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and their amino acid sequences were shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

Example 2

The present example performs the expression and purification of nanobodies.

2.1 nanobody original Strain TG1 amplification and nanobody recombinant plasmid transformation E.coli BL21 (DE 3)

The original strain TG1 glycerol bacteria containing nano antibody nucleic acid is prepared according to the following steps of 1:1000 ratio was inoculated into 5mL fresh LB A medium and cultured overnight at 37℃and 200 rpm. The following day, plasmids were extracted according to the instructions using a plasmid extraction kit (purchased from OMEGA). After verification, 1. Mu.L of the above plasmid was transformed into 100. Mu.L 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. 600. Mu.L of LB medium was added to the centrifuge tube, and the tube was incubated at 37℃for 60 minutes with shaking. 100. Mu.L of the supernatant was spread on an LB A plate using a triangular spreader, and cultured overnight at 37℃in an inverted state.

2.2 Induction of expression of nanobodies

The above monoclonal colonies were picked up in LB A medium and cultured overnight at 37℃with shaking. The next day, 100mL of fresh LB A culture medium is added into the bacterial liquid according to the proportion of 1:100, and the bacterial liquid is cultured for 3 hours at 37 ℃ in a shaking way until the bacterial liquid OD ₆₀₀ About=0.8, 1mm iptg was added to the final concentration and induced overnight at 30 ℃. On the third day, 8000rpm, the cells were collected by centrifugation for 10 minutes, and 1.5mL of pre-chilled TES buffer was added to resuspend the pellet. After 2 minutes of ice bath, the cycle was repeated 6 times with gentle shaking for 30 seconds. 3.0mL of TES/4 (4-fold dilution of TES with water) was added, and after gentle shaking for 30 seconds, the ice bath was allowed to stand for 2 minutes, and the shaking and standing steps were repeated 6 times as much. Centrifugation was performed at 9000rpm at 4℃for 10 minutes, and about 4.5mL of the supernatant (periplasmic extract) was collected.

2.3 purification and identification of nanobodies

The above samples were purified using His-tag agarose gel (manufactured by Shanghai Reinecke Biotechnology Co., ltd.), washed with 20mM imidazole, eluted and collected using 300mM imidazole, secondarily purified using molecular sieves, and analyzed by SDS-PAGE.

The SDS-PAGE electrophoresis diagram is shown in figure 1, and finally the purified nano antibody is dialyzed into PBS and is preserved at 2-8 ℃ for standby.

Example 3

In this example, the binding capacity of V5 nanobody to V5 protein was verified by ELISA.

The purified V5 nanobody was subjected to antigen binding assay using enzyme-linked immunoassay (ELISA). First according to EZ-Link ^TM Instructions for the sulfonhs LC-biotinylation kit purified V5 nanobodies were conjugated to biotin. Next, V5 protein (1. Mu.g/well) was coated on an ELISA plate, blocked with 3% BSA at 4℃overnight, and incubated at room temperatureIncubation was carried out for 2h, with 5 washes of 1min each with PBST. Biotinylated nanobody (1 μg/well) was added, incubated for 1h at room temperature, washed 5 times with PBST for 1min each, and unbound antibody was washed away. HRP-conjugated streptavidin (1:10000) was added and incubated for 1h at room temperature, washing with PBST 5 times for 1min each. Finally, 100 mu L of TMB color development liquid is added, and color development is carried out for 20min at room temperature and in a dark place. The reaction was terminated by adding 50. Mu.L of 2M concentrated sulfuric acid, and the absorbance at a wavelength of 450nm was measured on an ELISA.

The results are shown in FIG. 2. The 3V 5 nano antibodies have better binding capacity with the V5 protein, and the binding capacities of V5-06 and V5-24 are relatively close.

Example 4

This example conducted a thermal stability experiment of anti-V5 nanobodies.

The thermal stability of the purified V5 nanobody was tested using enzyme-linked immunoassay (ELISA). The V5 protein was coated at a concentration of 5. Mu.g/mL, 100. Mu.L/well, coated at 4℃for 24h, and the plate washed 5 times. Plates were washed 5 times with 1% bsa blocking overnight. The diluted V5 nano antibody is added into the ELISA plate, incubated for 30min at 37 ℃, and the plate is washed for 5 times. Adding 1:2000 ratio of diluted HRP-labeled goat anti-llama IgG and goat anti-mouse IgG (purchased from Abcam), incubated at 37 ℃ for 30min, and plate washed 5 times. TMB color development was added, incubated at 25, 37, 50, 60, 70, 80, 90℃for 30min,2M sulfuric acid was used to terminate the reaction, and absorbance at 450nm was read. The residual antigen binding activity of each antibody was determined by ELISA at different temperatures, 3 parallel replicate wells were set up for each group, the average absorbance value for each replicate was calculated using the binding value after room temperature (25 ℃) treatment as 100% binding control, and the relative activity was calculated as shown in FIG. 3.

Relative activity (%) =od 450 _{Treatment at different temperatures} /OD450 _{Treatment at 25 DEG C} ×100％

The results show that compared with the traditional anti-V5 antibody (purchased from Abcam and product number ab 27671), the 3-strain anti-V5 nano antibody still has stronger biological activity at the temperature of 70 ℃, has better thermal stability and has wide application prospect.

Example 5

This example uses anti-V5 nanobodies for the preparation of affinity purification materials. anti-V5 nanobody magnetic beads were prepared in this example.

Measuring a certain amount of carboxyl magnetic beads (from Shanghai-Fender Biotechnology Co., ltd.), washing the carboxyl magnetic beads with 0.1M MES buffer with pH of 5.0 for 2-3 times, activating with excessive EDC and NHS under acidic condition for 30min, adding magnetic field after activation to separate the magnetic beads from the liquid, discarding supernatant, washing with MES buffer to wash out excessive activator, washing for 3-4 times; adding an anti-V5 nano antibody for coupling, uniformly mixing by using a rotary mixer at room temperature, and reacting for 2h; after the reaction, a magnetic field was applied to separate the beads from the liquid, the supernatant was discarded, and the beads were blocked with a blocking solution containing 3% BSA (0.1M Tris-HCl at pH 8.0) for 3 hours to preserve the anti-V5 nanobody beads, so that the final concentration of the anti-V5 nanobody beads was 10mg/mL, and the beads were preserved at 2 to 8℃for further use.

Example 6

In this example, an anti-V5 nanobody was used to prepare an anti-V5 nanobody agarose gel.

A certain amount of CNBr activated agarose gel (from Shanghai-Reinecke Biotechnology Co.) is measured and pumped to a sand core funnel with corresponding specification, and 0.01M HCl with 2 times of gel volume is added, and the mixture is stirred uniformly and pumped and dried, and the steps are repeated for 2-3 times. And adding coupling buffer solution (PBS, pH 7.2), stirring, drying, repeating for 2-3 times, placing in a reaction container, adding anti-V5 nanometer antibody (5 mg antibody/g agarose gel), and reacting at room temperature for 2h. After the reaction was completed, the reaction mixture was washed twice with PBS having a gel volume of 2 times, and a blocking solution (0.1M Tris-HCl at pH 8.0) was added thereto for reaction at room temperature for 3 hours to block unreacted active groups. After washing 5 times with PBS of 2 times of gel volume, adding 20% ethanol water solution to store the anti-V5 nanometer antibody agarose gel, wherein the final concentration is 50%, and storing at 2-8 ℃ for standby.

Example 7

In this example, anti-V5 nanobody was prepared as anti-V5 nanobody sepharose beads.

A certain amount of NHS-activated agarose beads (from the company of the biotechnology of upper haelsholtzia) was measured into a reaction vessel, a magnetic field was applied to separate the beads from the liquid, the supernatant was discarded, and the supernatant was washed 2 times with MES buffer (0.1 m, ph 6.0) and removed. anti-V5 nanobody (2 mg antibody/mL agarose beads) was added and reacted at room temperature for 2h. After the reaction was completed, the supernatant was removed by magnetic separation, and a blocking solution (0.1M Tris-HCl at pH 8.0) was added to react for 3 hours at room temperature to block unreacted active groups. After washing 5 times with PBS, adding 20% ethanol water solution to store the anti-V5 nanometer antibody agarose magnetic beads, wherein the final concentration is 50%, and storing at 2-8 ℃ for standby.

Example 8

This example uses the anti-V5 nanobody sepharose prepared in example 6 for affinity purification.

Purification of V5 tag protein was performed using the anti-V5 nanobody sepharose prepared in example 6. Adding the anti-V5 nano antibody agarose gel into a chromatographic column, flowing out a preservation solution, washing the chromatographic column for a plurality of times by using PBS (pH 7.2), adding a V5 tag protein sample solution, placing the chromatographic column in a rotary mixer for incubation for 30-60 min, and collecting an effluent liquid, so that the sample can be repeatedly loaded to increase the binding efficiency. Eluting the impurity by using PBS with the volume of 5-10 times of the column volume, eluting the specific V5 tag protein by using glycine (pH2.2) after removing the nonspecifically adsorbed impurity protein, and collecting the eluent, namely the purified protein solution. The recovery rate was calculated by repeating the process 10 times more, as shown in fig. 4 and 5.

Recovery (%) = (actual recovery/initial addition) ×100%.

The result shows that the 2 anti-V5 nano antibody agarose gel (V5-06 and V5-24) can specifically purify the V5 tag protein, and the recovery rate is still more than 80% after 10 times of repeated use, so that the method has good stability and wide application prospect.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanobody against a V5 tag, wherein the nanobody comprises:

2. The anti-V5 tagged nanobody of claim 1, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region shown in SEQ ID No.4 are shown in sequence as SEQ ID nos. 7 to 9;

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 sequence as SEQ ID No. 10-12;

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 No. 13-15 in sequence;

preferably, the framework regions in the heavy chain variable region shown in SEQ ID NO.4 comprise heavy chain framework regions FR1, FR2, FR3 and FR4 shown in sequence as SEQ ID NO. 16-19;

preferably, the framework regions in the heavy chain variable region shown in SEQ ID No.5 comprise heavy chain framework regions FR1, FR2, FR3 and FR4 shown in sequence as SEQ ID No. 20-23;

Preferably, the framework regions in the heavy chain variable region shown in SEQ ID No.6 include heavy chain framework regions FR1, FR2, FR3 and FR4 shown in sequence in SEQ ID No. 24-27.

3. A multivalent nanobody, multispecific nanobody or fusion nanobody, comprising a nanobody according to any one of claims 1-2 against V5 tag;

preferably, the multivalent nanobody comprises at least two of the following CDR regions:

4. An antibody comprising the anti-V5-tagged nanobody of any one of claims 1-2 or the multivalent nanobody, multispecific nanobody, or fusion nanobody of claim 3.

5. A nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the anti-V5 tagged nanobody of any one of claims 1-2; or the nucleic acid molecule encodes the multivalent nanobody, multispecific nanobody, or fusion nanobody of claim 3; or the nucleic acid molecule encodes the antibody of 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 comprising the recombinant vector of claim 5.

7. Preparing a nanobody of the anti-V5 tag of any one of claims 1-2; or the multivalent nanobody, multispecific nanobody, or fusion nanobody of claim 3; or the method of the antibody according to claim 4, comprising: culturing the host cell of claim 6.

8. A nanobody of an anti-V5 tag according to any one of claims 1-2; or the multivalent nanobody, multispecific nanobody, or fusion nanobody of claim 3; or the antibody of claim 4 in the preparation of a V5 detection product, a V5 enrichment or purification product, a V5 tagged fusion protein enrichment or purification product, a product for isolation or purification of a V5 tagged protein-interacting protein, or a product for isolation or purification of a V5 tagged specific protein-DNA complex;

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 immunoassay kit, a fluorescent immunoassay kit or a time-resolved fluoroimmunoassay kit;

preferably, the kit is a magnetic particle chemiluminescent detection kit;

preferably, the chip is a chromatin co-immunoprecipitation chip, an immunoprecipitation chip or an MBP fusion protein sedimentation chip;

preferably, the V5 enriched or purified product, the enriched or purified product of the V5 tagged fusion protein, the product for isolation or purification of the V5 tagged protein-interacting protein, or the product for isolation or purification of the V5 tagged specific protein-DNA complex comprises: a carrier and nanobody or antibody on the carrier;

preferably, the carrier is selected from the group consisting of nanoparticles, magnetic beads, agarose gel microspheres, silica gel microspheres, latex microspheres, chips or porous materials.

9. A nanobody bearing the anti-V5 tag of any one of claims 1-2; or the multivalent nanobody, multispecific nanobody, or fusion nanobody of claim 3; or the antibody product of claim 4, wherein the product is a nanoparticle, a magnetic bead, an agarose gel microsphere, a silica gel microsphere, a latex microsphere, a test tube, an EP tube, a well of a multiwell plate microplate, an NC film, a PDMS film or a chip;

Preferably, the nanoparticles are selected from organic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

preferably, the porous 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 further labeled with a detectable label;

preferably, the detectable label is selected from at least one of biotin, a fluorescent dye, an enzyme that catalyzes the development of a substrate, a radioisotope, a chemiluminescent reagent, and a colloid.

10. A method for isolating V5, a V5-tagged fusion protein, a V5-interacting protein, or a V5-tagged specific protein-DNA complex, comprising: combining the anti-V5-tagged nanobody of any one of claims 1-2, the multivalent nanobody of claim 3, the multispecific nanobody, or the fusion nanobody; the antibody of claim 4 or the product of claim 9, which is mixed with the sample to be separated, and eluted.