Specific antibody of Sumo tag protein as well as preparation method and application of specific antibody

Abstract

The invention relates to an antigen binding protein, an antibody or an antibody active fragment obtained by immunizing a camelidae animal with a Sumo tag protein. The antibody capable of realizing specific recognition and combining with the Sumo tag protein is screened, identified and prepared by virtue of an immune system of a camelidae animal, and the obtained antibody is high in specificity, can be used for target antigen detection and has potential clinical diagnosis and treatment values; the antibody provided by the invention is simple in structure, easy to carry out genetic engineering modification, easy to realize humanization, high in stability, low in mass production cost and beneficial to realizing large-scale production.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a specific antibody of a Sumo tag protein and a preparation method and application thereof. Background Art

[0002] Sumo (small ubiquitin-like modifier) ​​tag protein is a small molecule ubiquitin-like modifier protein. Studies have found that Sumo can be used as a fusion tag and molecular chaperone for recombinant protein expression. It can not only further increase the expression level of the fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of the target protein, and improving the solubility of the recombinant protein.

[0003] Sumo fusion tags are widely used in prokaryotic expression systems. In addition to being used for the expression of conventional proteins, they are also used for the expression of toxic proteins, antimicrobial peptides, protein dimers, etc.

[0004] Sumo proteins were first discovered in yeast in 1996. Later, it was found that ubiquitinated proteins were found in yeast and eukaryotic cells. Ubiquitination modification is a very common post-translational modification of proteins and is also a hot topic of research. Current studies have found that there are many types of Sumo proteins in eukaryotic cells. Sumoylation mainly modifies the lysine residues of proteins. Sumoylation is a dynamic and reversible process that dynamically regulates protein structure through modification and dissociation to maintain different physiological functions.

[0005] The development of specific nanoantibodies targeting Sumo-tagged proteins plays an important role in the purification, expression detection, and other applications of fused Sumo-tagged proteins.

[0006] Therefore, it is necessary to develop an antibody that can recognize the complex spatial structure of the antigen surface and produce highly specific and high-affinity antibodies. Summary of the invention

[0007] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention provides a specific antibody for Sumo tag protein and a preparation method and application thereof.

[0008] In a first aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by immunizing a camelid with a Sumo tag protein.

[0009] In some embodiments, the camelid is selected from the group consisting of dromedary, Bactrian camel, llama, llama, alpaca and vicuña, preferably alpaca.

[0010] In some embodiments, the antibody is a nanobody and the antibody active fragment is a nanobody active fragment.

[0011] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

[0012] In some embodiments, the antigen binding protein, antibody or antibody active fragment binds to the Sumo tag protein. D The value is below 500 nM, preferably below 400 nM, and more preferably below 300 nM.

[0013] In some embodiments, the present invention provides Nanobodies or active fragments thereof that specifically recognize Sumo tag proteins.

[0014] In some embodiments, the Sumo tag protein has an amino acid sequence as shown in SEQ ID NO: 1. Further preferably, the Sumo tag protein is prepared by a method comprising the following steps: constructing a nucleotide sequence encoding the Sumo tag protein into a vector plasmid; transfecting the vector plasmid into a eukaryotic cell line for expression, and purifying.

[0015] In some embodiments, the Sumo-tagged protein is commercially available.

[0016] In a second aspect, the present invention provides a method for constructing an antibody library, the method comprising the following steps:

[0017] (1) Using Sumo tag protein as an antigen to immunize camelid animals, collecting venous peripheral blood from the immunized animals, and isolating lymphocytes;

[0018] (2) extracting total mRNA from the lymphocytes, reverse transcribing it into cDNA and amplifying it;

[0019] (3) Inserting the amplified DNA into a viral expression vector, transforming into bacteria, collecting colonies, and obtaining an antibody library.

[0020] In some embodiments, the camelid is selected from the group consisting of dromedary, Bactrian camel, llama, llama, alpaca and vicuña, preferably alpaca.

[0021] In some embodiments, the immunization in step (1) is performed by subcutaneous injection. The number of immunizations is preferably 3 to 5 times. The venous peripheral blood is preferably collected before and after the last immunization.

[0022] In some embodiments, the viral expression vector in step (3) is a phage expression vector.

[0023] In some embodiments, the bacteria in step (3) are TG1 competent bacteria.

[0024] In a third aspect, the present invention provides an antibody library obtained by the above method for constructing an antibody library, or a polyclonal antibody expressed by the antibody library.

[0025] In a fourth aspect, the present invention provides a method for constructing an antigen-specific antibody library, the method comprising the following steps: screening the antibody library described in the third aspect to obtain an antigen-specific antibody library.

[0026] In some embodiments, the method for constructing an antigen-specific antibody library comprises the following steps:

[0027] (i) culturing the antibody library to release viruses;

[0028] (ii) incubating the virus with the antigen to remove the virus that non-specifically binds to the antigen and retain the virus that specifically binds to the antigen;

[0029] (iii) infecting bacteria with the virus that specifically binds to the antigen, collecting colonies, and obtaining an antigen-specific antibody library.

[0030] In some embodiments, the bacteria in step (iii) is Escherichia coli.

[0031] In a fifth aspect, the present invention provides an antigen-specific antibody library obtained by the above method for constructing an antigen-specific antibody library, or a polyclonal antibody that specifically binds to an antigen and is expressed and produced by the antigen-specific antibody library.

[0032] In a sixth aspect, the present invention provides a method for preparing an antigen-binding protein, an antibody or an active fragment of an antibody, the method comprising the following steps: screening the antibody library described in the third aspect to obtain an antigen-binding protein, an antibody or an active fragment of an antibody that specifically binds to an antigen.

[0033] In some embodiments, the method for preparing an antigen binding protein, an antibody or an antibody active fragment comprises the following steps:

[0034] (a) culturing the antibody library to release viruses;

[0035] (b) incubating the virus with the antigen to remove the virus that non-specifically binds to the antigen and retain the virus that specifically binds to the antigen;

[0036] (c) Infecting bacteria with the virus that specifically binds to the antigen, smearing the infected bacteria on a plate culture medium for culture, and selecting a single colony.

[0037] In some embodiments, the bacteria in step (c) is Escherichia coli.

[0038] In some embodiments, the single colony can be expanded and then identified for antigen-specific binding.

[0039] In some embodiments, the single colony can be expanded and then subjected to step (d): DNA extraction, transformation into host cells and expression to obtain monoclonal antibodies.

[0040] In a seventh aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by the above-mentioned method for preparing an antigen-binding protein, an antibody or an antibody active fragment.

[0041] In an eighth aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment that specifically recognizes a Sumo tag protein; the antigen-binding protein, the antibody or the antibody active fragment comprises at least one heavy chain variable region; the heavy chain variable region has:

[0042] CDR1 as shown in SEQ ID NO:3;

[0043] CDR2 as shown in SEQ ID NO:4; and

[0044] CDR3 as shown in SEQ ID NO:5.

[0045] In some embodiments, the heavy chain variable region has: a CDR1 as shown in SEQ ID NO:3, a CDR2 as shown in SEQ ID NO:4, and a CDR3 as shown in SEQ ID NO:5.

[0046] In some embodiments, the heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:6.

[0047] In some embodiments, the antigen binding protein, antibody or antibody active fragment comprises one of the heavy chain variable regions and lacks a light chain.

[0048] In some embodiments, the antibody is a nanobody and the antibody active fragment is a nanobody active fragment.

[0049] In a ninth aspect, the present invention provides a nucleic acid molecule encoding an amino acid sequence shown in any one of SEQ ID NO:3 to SEQ ID NO:6 or the antigen-binding protein, antibody or antibody active fragment described above.

[0050] In some embodiments, the nucleic acid molecule encoding the antigen binding protein, antibody or antibody active fragment has a nucleotide sequence as shown in SEQ ID NO:7.

[0051] In a tenth aspect, the present invention provides an expression vector containing the nucleic acid molecule described above.

[0052] In some embodiments, the expression vector is a phage expression vector, preferably a phage surface display screening vector.

[0053] In some embodiments, the expression vector further contains a nucleotide sequence encoding the bacteriophage envelope protein pill.

[0054] In an eleventh aspect, the present invention provides a virus into which the above-mentioned expression vector is exogenously introduced.

[0055] In some embodiments, the virus is a bacteriophage.

[0056] In a twelfth aspect, the present invention provides a host cell, which comprises the nucleic acid molecule described above, or the host cell is infected by the virus described above.

[0057] In some embodiments, the host cell is Escherichia coli.

[0058] In a thirteenth aspect, the present invention provides a method for expressing an antigen-binding protein, an antibody or an active fragment of an antibody using the host cell described above.

[0059] In a fourteenth aspect, the present invention provides an antigen-binding protein, an antibody or an antibody active fragment obtained by expressing the host cell described above.

[0060] In a fifteenth aspect, the present invention provides a humanized antigen-binding protein, antibody or antibody active fragment obtained by humanizing the antigen-binding protein, antibody or antibody active fragment described above.

[0061] In a sixteenth aspect, the present invention provides a protein conjugate comprising the antigen-binding protein, antibody or antibody active fragment described above or the humanized antigen-binding protein, antibody or antibody active fragment described above and a ligand.

[0062] In some embodiments, the ligand is selected from the group consisting of a radioisotope, a fluorescent group, and a delivery vehicle.

[0063] In a seventeenth aspect, the present invention provides a kit for detecting Sumo-tagged proteins in samples, which comprises the antigen-binding protein, antibody or antibody active fragment described above, or the humanized antigen-binding protein, antibody or antibody active fragment described above.

[0064] In some embodiments, the antigen binding protein, antibody or antibody active fragment is labeled with a marker. Preferably, the marker is selected from an enzyme, a chemiluminescent group and an isotope group.

[0065] In some embodiments, the sample is animal serum, preferably human serum.

[0066] In an eighteenth aspect, the present invention provides use of the antigen-binding protein, antibody or antibody active fragment described above, the humanized antigen-binding protein, antibody or antibody active fragment described above, the protein conjugate described above or the kit described above in detecting Sumo-tagged proteins in samples.

[0067] In some embodiments, the sample is animal serum, preferably human serum.

[0068] In a nineteenth aspect, the present invention provides the use of the antigen-binding protein, antibody or antibody active fragment described above, the humanized antigen-binding protein, antibody or antibody active fragment described above in the preparation of a kit for detecting Sumo-tagged proteins in samples.

[0069] In some embodiments, the sample is animal serum, preferably human serum.

[0070] Compared with the prior art, the technical solution provided by the present invention has the following significant advantages: the antibodies provided by the present invention can be used to detect target antigens in serum, and further used to detect proto-oncogenes, judge lesions, and have potential clinical diagnosis and treatment value. The antibody provided by the present invention has a simple structure, is easy to be genetically engineered, and has a mature optimization strategy for enhancing the affinity of nano antibodies, prolonging the half-life in vivo, and coupling with other molecules for drug development, such as connecting radioisotopes, coupling delivery drugs, CART and fluorescent labeling high-resolution imaging, etc. The antibody sequence provided by the present invention has high homology with the VH region sequence of human IgG, and a few amino acid mutations can achieve the humanization of single-domain antibodies. The antibody provided by the present invention has high stability, can avoid the requirements of conventional antibodies for low-temperature storage and transportation, is conducive to large-scale popularization and application, has low mass production costs, and is easy to prepare by large-scale recombinant preparation. The monoclonal nano antibodies designed by the present invention can be well recombinantly expressed in a low-cost Escherichia coli expression system, with low mass production costs and yields of up to several milligrams per liter of Escherichia coli. The Escherichia coli recombinant expression system technology is mature and the quality control is simple, which is conducive to reducing production costs and realizing large-scale production.

[0071] The development of Sumo-targeted monoclonal antibodies can provide researchers with powerful tools to help conduct in-depth studies on the functions and regulatory mechanisms of Sumo in multiple biological processes such as immune regulation, inflammation, and infection. This will help to better understand the mechanisms of these biological processes and provide new directions and targets for the treatment of related diseases. It will also provide rich materials for basic biological research.

[0072] Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, the technical solution of the present invention can preserve all the nano-antibody fragments (i.e., the library) of alpacas for a long time, and can continuously support the subsequent screening and development of nano-antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a schematic diagram of the affinity test results between monoclonal antibody Sumo_nbsu and antigen. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0075] definition

[0076] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0077] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0078] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0079] Sumo (small ubiquitin-like modifier): Sumo tag protein is a small molecule ubiquitin-like modifier protein. Studies have found that Sumo can be used as a fusion tag and molecular chaperone for recombinant protein expression. It can not only further increase the expression level of the fusion protein, but also has the functions of resisting protease hydrolysis, promoting the correct folding of the target protein, and improving the solubility of the recombinant protein.

[0080] K D value :Dissociation constant (dissociation constant, K D ) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) from another smaller component. It is the reciprocal of the association constant and has units of mol / L (M) or nmol / L (nM). K D The smaller the value, the stronger the binding ability of the two substances.

[0081] Nanobody: An antibody that is naturally missing light chains and exists in the peripheral blood of camelids. This antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as artificially modified single-chain antibody fragments. The VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen. The VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only about 15kD, so it is also called a nanobody (Nanobody, Nb). Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune system in camelids can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanobodies.

[0082] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on antibodies produced by the immune system of alpacas, which are called "nanoantibodies". Nanoantibodies are tiny antibody fragments separated from immunoglobulins in animals such as camels. They have the same antigen binding ability and structural stability as complete antibodies. They are the smallest units that can bind to target antigens, with a relative molecular mass of only about 15kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune systems in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanoantibodies.

[0083] According to the technical solution of the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below:

[0084] Table 1

[0085] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu; Val

[0086] In addition, due to the degeneracy of the bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, as shown in Table 2 below:

[0087] Table 2

[0088]

[0089]

[0090] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0091] Example 1. Preparation of antigens

[0092] (1) Constructing a DNA sequence encoding a Sumo protein with a His tag at the N-terminus into a pet-28a Escherichia coli expression vector to form a Sumo-tagged protein recombinant expression plasmid;

[0093] (2) Transfecting the Sumo-tagged protein recombinant expression plasmid into BL21 (DE3) competent cells and culturing to obtain a monoclonal strain expressing the Sumo-tagged protein;

[0094] (3) culturing the strain at 37°C, and then adding a suitable inducer (IPTG, isopropyl-β-D-thiogalactoside) at 27°C to induce the expression of Sumo-tagged protein;

[0095] (4) All bacteria are collected, and after lysis, centrifugation, affinity chromatography, gel filtration chromatography and other processes, the recombinantly expressed Sumo-tagged protein is obtained.

[0096] The amino acid sequence of the fully expressed recombinant Sumo-tagged protein is as follows:

[0097] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGK EMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO: 1).

[0098] The nucleotide sequence corresponding to the recombinant Sumo tag protein is as follows:

[0099] ATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCCAGAGGTCAAGCCAGAAGTC

[0100] AAGCCTGAGACTCACATCAATTTAAAGGTGTCCGATGGATCTTCAGAGATCTTCTTCAA

[0101] GATCAAAAAGACCACTCCTTTAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGT

[0102] AAGGAAATGGACTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGAC

[0103] CCCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAGAACAGATTG

[0104] GTGGT (SEQ ID NO: 2).

[0105] Example 2. Alpaca Immunization

[0106] In this example, the antigen recombinant Sumo tag protein of Example 1 was used to immunize alpacas. The specific steps are as follows:

[0107] (1) The antigen in Example 1 was evenly divided into 4 portions, each portion was about 0.5 mg; the alpacas were immunized 4 times in total, and the antigen was injected subcutaneously into the animal body, with the first immunization being recorded as day 1, and the subsequent immunizations were recorded on days 11, 21, and 31 respectively;

[0108] (2) On day 30, before the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca;

[0109] (3) On day 45, i.e. 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca.

[0110] Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the technical advantage of the present invention lies in the collection of a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nano-antibodies.

[0111] Example 3. Construction of Alpaca Nanobody Library

[0112] The two batches of alpaca venous peripheral blood collected in Example 2 were used as raw materials to construct a highly diverse nanoantibody library. The two batches of alpaca venous peripheral blood were processed in the same way, and the specific steps were as follows:

[0113] (1) Lymphocytes were isolated from alpaca venous peripheral blood using density gradient centrifugation and other methods;

[0114] (2) extracting total mRNA from lymphocytes and reverse transcribed into cDNA;

[0115] (3) using appropriate DNA primers (see Table 3 below) and the above cDNA as a template, amplifying the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e., the DNA fragments of the nanobody, by polymerase chain reaction (PCR);

[0116] Table 3. Primers used to construct the nanobody library

[0117]

[0118]

[0119] (4) connecting the VHH DNA to the phage surface display screening vector phen1 to form a VHH-pIII fusion protein expression vector plasmid library; wherein pIII is a protein present on the flagella on the surface of the phage;

[0120] (5) The DNA ligation product is transformed into TG1 competent bacteria by electroporation, and all colonies are collected after appropriate cultivation, which is the alpaca nanoantibody library.

[0121] Example 4. Screening of specific nanobodies by phage surface display

[0122] This example uses the nanobody library obtained in Example 3 as a source, and obtains antigen-specific nanobodies through phage surface display screening. The specific steps are as follows:

[0123] (1) Taking an appropriate amount of frozen nanobody library, inoculating it into bacterial culture medium, adding an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) after appropriate cultivation, and continuing to culture under appropriate conditions;

[0124] (2) extracting the amplified phages from the bacterial culture supernatant using the PEG-NaC method;

[0125] (3) phages were appropriately incubated with antigens, and Sumo antigens were pre-immobilized in immunotubes (Maxisorp immunotubes, ThermoFisher Scientific);

[0126] (4) Panning: discard the phages, rinse the antigen with PBS buffer for an appropriate number of times, remove the phages that non-specifically bind to the antigen, and retain the phages that specifically bind to the antigen;

[0127] (5) Elution: Use acidic glycine solution to elute the phage, so that the phage and antigen are dissociated and retained.

[0128] At this point, phage expressing specific nanobodies are obtained, and the obtained phages are subjected to the following operations:

[0129] (6) Transformation into a specific nanoantibody library: The phage is again infected with the E. coli cultured to a suitable state, but no helper phage is added. After the phage infection is complete, the specific nanoantibody exists in the E. coli in the form of a DNA plasmid. Collect all these E. coli to form an antigen-specific nanoantibody library. This library can be used as a raw material and returned to step (1) for the next round of phage surface display screening;

[0130] (7) Transformation into monoclonal nanoantibody colonies: Take about 0.5% of the total volume of the phage obtained in step (5), dilute it, and infect the E. coli cultured to a suitable state again, but no helper phage is added. After the phage infection is complete, the E. coli are evenly spread on the bacterial culture dish and cultured at 37°C overnight to obtain monoclonal colonies containing nanoantibody DNA plasmids. These monoclonal colonies are used as raw materials to identify positive monoclonal nanoantibodies.

[0131] Example 5. Identification of positive monoclonal nanobodies

[0132] This example uses step (7) of Example 4 to obtain a bacterial culture dish with monoclonal colonies, and conducts identification of positive monoclonal nanoantibodies. The specific steps are as follows:

[0133] (1) Pick a single clone and culture it in a microplate;

[0134] (2) adding IPTG to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody);

[0135] (3) The bacterial culture supernatant containing nanobodies was collected and incubated with the antigen Sumo-tagged protein, which was pre-immobilized in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific);

[0136] (4) Using enzyme-linked immunosorbent assay (ELISA), detect whether the monoclonal nanobody binds to the Sumo antigen. The main experimental steps are as follows:

[0137] a. Coating: dilute the antigen to 5 μg / mL with PBS, 50 μL / well, and incubate overnight at 4°C with shaking;

[0138] b. Blocking: On the second day, discard the antigen, add 100 μL / well PBS-2% BSA and incubate at room temperature for 1 hour;

[0139] c. Washing: 3 times PBST, 3 times PBS, 150 μL / well;

[0140] d. Add culture supernatant, 50 μL / well, and incubate at room temperature with shaking for 1-2 hours;

[0141] e. Washing: 3 times PBST, 3 times PBS, 150 μL / well;

[0142] f. Add diluted anti-myc HRP and incubate at room temperature for 1 hour;

[0143] g. Washing: 3 times PBST, 3 times PBS, 150 μL / well;

[0144] h. Add ELISA color substrate and incubate at room temperature in the dark for 30 minutes;

[0145] i. Read OD450nM.

[0146] (5) For the monoclonal nanoantibody microbial colonies that can bind to the antigen, after appropriate culturing again, the DNA plasmid is extracted and the nucleotide sequence and amino acid sequence of the nanoantibody are obtained by sequencing, as shown in Table 4.

[0147] Table 4: Amino acid sequence and nucleotide sequence of monoclonal antibody Sumo_nbsu

[0148]

[0149] Example 6. Small batch monoclonal nanobody recombinant expression and purification

[0150] (1) In Example 5, a monoclonal nanobody capable of specifically recognizing and binding to an antigen was obtained. The DNA plasmid encoding the nanobody was transformed into BL21 (DE3) competent cells, and the monoclonal nanobody was expressed and purified in small batches using an E. coli expression system. The batch production was about several milligrams.

[0151] (2) Using the ELISA method, different concentrations of nanoantibodies were incubated, and the affinity of the nanoantibody to the antigen was measured based on the binding ability of the nanoantibody to the Sumo tag protein.

[0152] A group of monoclonal colony corresponding antibodies were obtained, and the affinity test results for the Sumo tag protein antigen were as follows: Figure 1 As shown, the affinity value K D The results are shown in Table 5 below.

[0153] Table 5: Affinity test results

[0154] <![CDATA[K D (nM)]]> Sumo_nbsu 253.3

[0155] From the above results, it can be seen that the Kp1 binding affinity of the antibody obtained by selecting the monoclonal colony in this example and binding to the Sumo tag protein D The value is below 500 nM, preferably below 400 nM, and more preferably below 300 nM.

[0156] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Nanobodies or their active fragments that specifically recognize Sumo-tagged proteins.

2. The nanobody or active fragment thereof according to claim 1, characterized in that The nanobody or its active fragment comprises a heavy chain variable region, and the heavy chain variable region has: CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4 and CDR3 as shown in SEQ ID NO:

5.

3. The Nanobody or active fragment thereof according to claim 1, characterized in that: The heavy chain variable region has: an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

6.

4. A nucleic acid molecule encoding the Nanobody or active fragment thereof according to any one of claims 1 to 3. An expression vector comprising the nucleic acid molecule according to claim 4. A host cell comprising the nucleic acid molecule of claim 4.

7. A method for expressing a nanobody or an active fragment thereof using the host cell according to claim 6.

8. A humanized Nanobody or an active fragment thereof obtained by humanizing the Nanobody or an active fragment thereof according to any one of claims 1 to 3.

9. A protein conjugate, characterized in that Comprising the Nanobody or its active fragment according to any one of claims 1 to 3 or the humanized Nanobody or its active fragment according to claim 8.

10. The protein conjugate according to claim 9, characterized in that The protein conjugate further comprises a ligand, wherein the ligand is selected from the group consisting of a radioisotope, a fluorescent group and a delivery vehicle.

11. A kit for detecting Sumo-tagged proteins in a sample, characterized in that: Comprising the Nanobody or its active fragment according to any one of claims 1 to 3 or the humanized Nanobody or its active fragment according to claim 8.

12. Use of the Nanobody or its active fragment according to any one of claims 1 to 3, or the humanized Nanobody or its active fragment according to claim 8 in the preparation of a kit for detecting Sumo-tagged proteins in a sample.

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