Specific antibody of sumo tagged protein and its preparation method and application

Abstract

The application relates to an antigen binding protein, an antibody or an antibody active fragment obtained by immunizing a camelid with a Sumo tag protein. The application relies on the immune system of the camelid to screen, identify and prepare an antibody specifically recognizing and binding the Sumo tag protein. The obtained antibody has high specificity and can be used for target antigen detection, and has potential clinical diagnosis and treatment values. The antibody provided by the application has simple structure, is easy to be genetically engineered, is easy to be humanized, has high stability, has low mass production cost, and is beneficial to realize large-scale production.

Description

TECHNICAL FIELD

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

[0002] Sumo (small ubiquitin-like modifier) tag protein is a small ubiquitin-like modifier protein. Studies have found that Sumo can be used as a fusion tag and a molecular chaperone for recombinant protein expression, which can further improve the expression of fusion proteins, and has the functions of resisting protease hydrolysis, promoting the correct folding of target proteins, and improving the solubility of recombinant proteins.

[0003] Sumo fusion tag is widely used in prokaryotic expression system, and is used not only for the expression of conventional proteins, but also for the expression of toxic proteins, antibacterial peptides, and protein dimers.

[0004] Sumo protein was first discovered in yeast in 1996, and later found that ubiquitination-modified proteins exist from yeast to eukaryotic cells. Ubiquitination modification is a very common post-translational modification of proteins, and is also a current research hotspot. Current studies have found that there are various Sumo proteins in eukaryotic cells, Sumo modification mainly modifies lysine residues of proteins, and Sumo modification is a dynamic and reversible process, which dynamically adjusts protein structure through modification dissociation to maintain different physiological functions.

[0005] Targeting Sumo tag protein to develop specific nanobodies plays an important role in the application of purification, expression detection, etc. of fusion Sumo tag protein.

[0006] Therefore, it is necessary to develop an antibody that can recognize the complex spatial structure on the surface of an antigen and produce high specificity and high affinity. SUMMARY

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

[0008] In a first aspect, the present application 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, vicuna, alpaca and paca, 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 with a Kd value below 500 nM, preferably below 400 nM, more preferably below 300 nM. D In some embodiments, the antigen binding protein, antibody or antibody active fragment binds to the Sumo tag protein with a Kd value below 500 nM, preferably below 400 nM, more preferably below 300 nM.

[0013] In some embodiments, the present application provides a nanobody or an active fragment thereof that specifically recognizes the Sumo tag protein.

[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 steps of: 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 tag protein is available through commercial channels.

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

[0017] (1) immunizing a camelid with the Sumo tag protein as an antigen, collecting the peripheral venous blood of the immunized animal, and isolating lymphocytes;

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

[0019] (3) inserting the DNA obtained by amplification 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, vicuna, alpaca and guanaco, preferably alpaca.

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

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

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

[0024] In a third aspect, the present application provides an antibody library obtained by the method for constructing an antibody library described above, or polyclonal antibodies produced by expression of the antibody library.

[0025] In a fourth aspect, the present application provides a method for constructing an antigen-specific antibody library, comprising the following steps: screening the antibody library of 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 viruses with antigens, removing viruses that do not specifically bind to the antigens, and retaining viruses that specifically bind to the antigens;

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

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

[0031] In a fifth aspect, the present application provides an antigen-specific antibody library obtained by the method for constructing an antigen-specific antibody library described above, or polyclonal antibodies that specifically bind to antigens produced by expression of the antigen-specific antibody library.

[0032] In a sixth aspect, the present application provides a method for preparing an antigen-binding protein, an antibody, or an active fragment of an antibody, comprising the following steps: screening the antibody library of 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 active fragment of an antibody comprises the following steps:

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

[0035] (b) incubating the viruses with antigens, removing viruses that do not specifically bind to the antigens, and retaining viruses that specifically bind to the antigens;

[0036] (c) infecting bacteria with the viruses that specifically bind to the antigens, spreading the infected bacteria on plate culture medium, and picking single colonies.

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

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

[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 a monoclonal antibody.

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

[0041] In an eighth aspect, the present application provides an antigen binding protein, an antibody or an antibody active fragment specifically recognizing a Sumo tagged 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] a CDR1 as set forth in SEQ ID NO: 3;

[0043] a CDR2 as set forth in SEQ ID NO: 4; and

[0044] a CDR3 as set forth in SEQ ID NO: 5.

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

[0046] In some embodiments, the heavy chain variable region has: an amino acid sequence as set forth 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, the antibody or the 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 application provides a nucleic acid molecule encoding an amino acid sequence as set forth in any one of SEQ ID NO: 3 to SEQ ID NO: 6 or the antigen binding protein, the antibody or the antibody active fragment as 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 set forth in SEQ ID NO: 7.

[0051] In a tenth aspect, the present application provides an expression vector comprising the nucleic acid molecule as 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 comprises a nucleotide sequence encoding a phage coat protein pill.

[0054] In an eleventh aspect, the present application provides a virus into which the expression vector as described above is introduced.

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

[0056] In a twelfth aspect, the present application provides a host cell comprising the nucleic acid molecule as described above, or the host cell is infected with the virus as described above.

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

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

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

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

[0061] In a sixteenth aspect, the present application provides a protein conjugate comprising the antigen binding protein, antibody or antibody active fragment as described above or the humanized antigen binding protein, antibody or antibody active fragment as 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 application provides a kit for detecting a Sumo-tagged protein in a sample, comprising the antigen binding protein, antibody or antibody active fragment as described above or the humanized antigen binding protein, antibody or antibody active fragment as described above.

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

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

[0066] In some embodiments, the sample is animal serum, preferably human serum.

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

[0068] In some embodiments, the sample is animal serum, preferably human serum.

[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 application has the following advantages: the antibody provided by the present application can be used for detecting target antigens in serum, and further for detecting proto-oncogenes, judging lesions, and has potential clinical diagnostic and therapeutic value. The antibody provided by the present application has a simple structure, is easy to genetically engineer, and has a mature optimization strategy for enhancing the affinity of nanobodies, prolonging the half-life in vivo, and coupling with other molecules for drug development, such as connecting radioisotopes, coupling drug delivery, CART and fluorescent labeling high-resolution imaging, etc. The antibody sequence provided by the present application has high homology with the sequence of the VH region of human IgG, and a small number of amino acid mutations can realize humanization of single-domain antibodies. The antibody provided by the present application has high stability, can avoid the requirement of low-temperature storage and transportation of conventional antibodies, is conducive to large-scale popularization and application, has low production cost, and is easy to mass-produce. The monoclonal nanobody designed by the present application can be well recombinantly expressed in the cost-effective E. coli expression system, has low production cost, and the yield can be as high as several milligrams per liter of E. coli. The E. coli recombinant expression system is mature in technology and simple in quality control, which is conducive to reducing production cost and realizing large-scale production.

[0071] The development of Sumo targeting monoclonal antibodies can provide a powerful tool for researchers, help to further study the function and regulation mechanism of Sumo in immune regulation, inflammation, infection and other biological processes, help to better understand the mechanism of these biological processes, provide new direction and target for the treatment strategy of related diseases, and also provide rich materials for basic biological research.

[0072] Compared with the traditional method of separating antibodies from animal serum or lymphocytes such as mice and rabbits, the technical scheme of the present application can long-term store all nanobody fragments (i.e. library) of the alpaca, and can continuously support the subsequent screening and development of nanobodies. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 Figure 2 is a schematic diagram of the antigen affinity detection results of the monoclonal Sumo_nbsu. DETAILED DESCRIPTION

[0074] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the following will further describe the present application with examples. The specific examples described herein are only used to explain the present application, and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0075] Definitions

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein and, unless otherwise specified, terms used in the singular will include the plural and vice versa, as appropriate for the purposes of explaining the present application.

[0077] Unless the context clearly indicates otherwise, as used herein, the construction "a" or "an" is taken to include plural referents unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells as well as equivalents thereof known to those skilled in the art, and so forth.

[0078] As used herein, the term "about" means ±20% of the numerical value of the number that follows it. In some embodiments, the term "about" means ±10% of the numerical value of the number that follows it. In some embodiments, the term "about" means ±5% of the numerical value of the number that follows it.

[0079] Sumo (small ubiquitin-like modifier)Sumo tag protein is a small ubiquitin-like modifier protein, research finds that Sumo can be used as a fusion tag and molecular chaperone of recombinant protein expression, which can not only further improve the expression of fusion protein, but also has the functions of resisting protease hydrolysis, promoting correct folding of target protein, and improving solubility of recombinant protein.

[0080] K D values Dissociation constant (K D ) is a specific type of equilibrium constant, which is used to measure the tendency of a larger object to separate (dissociate) from another smaller component, is the reciprocal of association constant, and the unit is mol / L (M) or nmol / L (nM). D The smaller the K D value, the stronger the binding ability of the two substances.

[0081] Nanobody The natural missing light chain antibody existing in the peripheral blood of camelidae animals only contains one heavy chain variable region (VHH) and two conventional CH2 regions and CH3 regions, but is not easy to stick together or even aggregate into a block like artificially modified single-chain antibody fragments; the VHH structure cloned and expressed alone has structural stability and binding activity with antigens comparable to original heavy chain antibodies, and is the smallest unit known to bind target antigens; the VHH crystal is 2.5 nm long and 4 nm long, and the molecular weight is only about 15 kD, so it is also called nanobody (Nb). Compared with traditional mice, rabbits and other animals that can only recognize the polypeptide on the surface of the antigen, the immune system in camelidae animals can recognize the complex spatial structure on the surface of the antigen, and can produce nanobodies with high specificity and high affinity.

[0082] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys and sheep, the technical scheme of the present application is to rely on the antibodies produced by the immune system of the llama, which is called "nanobody". Nanobody is a small antibody fragment isolated from immunoglobulin in animals such as camels, which has the same antigen binding capacity and structural stability as complete antibodies, and is the smallest unit known to bind target antigens, with a relative molecular mass of only about 15 kD. Compared with traditional mice, rabbits and other animals that can only recognize the polypeptide on the surface of the antigen, the immune system in camelidae animals can recognize the complex spatial structure on the surface of the antigen, and can produce nanobodies with high specificity and high affinity.

[0083] According to the technical scheme of the present application, some amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, see Table 1 below:

[0084] Table 1

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

[0086] In addition, because of the degeneracy of bases, substitutions can be made to the bases of the polynucleotide sequence without changing the activity or function of the polynucleotide sequence, see Table 2 below:

[0087] Table 2

[0088]

[0089]

[0090] The following examples and drawings are provided to assist in understanding the present application. It should be understood, however, that these examples and drawings are provided only for the purpose of illustration and are not to be construed as limiting the present application in any way. The scope of the present application is set forth in the claims. It should be understood that any modifications and variations of the examples and drawings can be made without departing from the spirit of the present application.

[0091] Example 1. Preparation of antigen

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

[0093] (2) Transfect the Sumo tag protein recombinant expression plasmid into BL21(DE3) competent cells to culture a monoclonal strain expressing the Sumo tag protein;

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

[0095] (4) Collect all bacteria, and after processes such as lysis, centrifugation, affinity chromatography, and gel filtration chromatography, obtain the recombinantly expressed Sumo tag protein.

[0096] The amino acid sequence of the fully expressed recombinant Sumo tag 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. Immunization of alpaca

[0106] This example immunizes the alpaca with the antigen recombinant Sumo-tag protein of Example 1. The specific steps are as follows:

[0107] (1) Divide the antigen in Example 1 into 4 parts, each about 0.5 mg; immunize the alpaca 4 times, and inject the antigen subcutaneously into the animal, with the first immunization being the first day, and the subsequent immunizations being the 11th day, the 21st day, and the 31st day;

[0108] (2) On the 30th day, collect about 200 mL of alpaca peripheral venous blood before the fourth immunization;

[0109] (3) On the 45th day, i.e. 14 days after the fourth immunization, collect about 200 mL of alpaca peripheral venous blood.

[0110] Compared with the traditional immunization technical scheme of animal antibodies such as mice and rabbits, the technical advantage of the present application lies in collecting a large amount of alpaca peripheral venous blood, which is conducive to subsequent screening of highly diverse nanobodies.

[0111] Example 3. Construction of alpaca nanobody library

[0112] Two batches of alpaca peripheral venous blood collected in Example 2 are used as raw materials to construct a high-diversity nanobody library. The processing methods of the two batches of alpaca peripheral venous blood are the same, and the specific steps are as follows:

[0113] (1) Using methods such as density gradient centrifugation, lymphocytes are isolated from the peripheral blood of alpaca veins;

[0114] (2) Total mRNA of the lymphocytes is extracted and reverse transcribed into cDNA;

[0115] (3) Using appropriate DNA primers (see Table 3 below), the above-mentioned cDNA is used as a template to amplify the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e. the DNA fragments of nanobodies, by polymerase chain reaction (PCR);

[0116] Table 3. Primers used for constructing the nanobody library

[0117]

[0118]

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

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

[0121] Example 4. Phage surface display screening of specific nanobodies

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

[0123] (1) Take an appropriate amount of frozen nanobody library and inoculate into bacterial culture medium, and after appropriate culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S), and continue to culture under appropriate conditions;

[0124] (2) Extract the amplified phage in the bacterial culture supernatant by PEG-NaC method;

[0125] (3) Incubate the phage with the antigen, and the Sumo antigen is pre-fixed on the immunotube (Maxisorp immunotube, ThermoFisher Scientific);

[0126] (4) Washing: discard the phage, and then rinse the antigen with PBS buffer for an appropriate number of times, wash and remove the phage that does not specifically bind to the antigen, and retain the phage that specifically binds to the antigen;

[0127] (5) Elution: Elute the phage with acidic glycine solution to dissociate the phage from the antigen and retain.

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

[0129] (6) Conversion into a specific nanobody library: The phage are again infected into E. coli cultured to an appropriate state, but the helper phage is not added again. After the phage infection is complete, the specific nanobodies exist in the form of DNA plasmids in the E. coli. The total E. coli are collected, which become an antigen-specific nanobody library. The library can be used as raw material to return to step (1) for the next round of phage surface display screening;

[0130] (7) Conversion into a monoclonal nanobody colony: About 0.5% of the total volume of the phage obtained in step (5) is diluted and then again infected into E. coli cultured to an appropriate state, but the helper phage is not added again. After the phage infection is complete, the E. coli are evenly smeared on a bacterial culture dish and incubated at 37°C overnight to obtain a monoclonal colony containing a nanobody DNA plasmid. The monoclonal colony is used as raw material to identify a positive monoclonal nanobody.

[0131] Example 5. Identification of a positive monoclonal nanobody

[0132] This example uses the bacterial culture dish with the monoclonal colony obtained in step (7) of Example 4 to identify a positive monoclonal nanobody. The specific steps are as follows:

[0133] (1) Pick the monoclonal colony to a microplate for culture;

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

[0135] (3) Collect the bacterial culture supernatant containing the nanobody, incubate with the antigen Sumo-tag protein, and the antigen is previously immobilized on a 96-microplate (Maxisorp transparent microplate, ThermoFisher Scientific);

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

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

[0138] b. Blocking: The next day, discard the antigen, add 100 μL / well of PBS-2% BSA, incubate at room temperature for 1 hour with shaking;

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

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

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

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

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

[0144] h. Add ELISA color developing substrate, incubate at room temperature for 30 min in the dark;

[0145] i. Read OD450nM.

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

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

[0148]

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

[0150] (1) The monoclonal nanobodies capable of specifically recognizing and binding to the antigen obtained in Example 5 are transformed into BL21(DE3) competent cells, and the small batch expression and purification of the monoclonal nanobodies are performed by means of the E. coli expression system, and the batch production capacity is about several milligrams.

[0151] (2) The ELISA method is used to incubate nanobodies of different concentrations, and the affinity of the nanobodies to the antigen is measured according to the binding ability of the nanobodies to the Sumo tag protein.

[0152] A group of monoclonal colonies corresponding to antibodies are obtained, and the detection results of the affinity of the antibodies to the Sumo tag protein antigen are as shown in Figure 1 The affinity values K D The results are shown in Table 5.

[0153] Table 5: Affinity test results

[0154] K D (nM) Sumo_nbsu 253.3

[0155] From the above results, it can be seen that the antibody obtained by selecting the monoclonal colony binds to the Sumo tag protein with a K D value below 500 nM, preferably below 400 nM, more preferably below 300 nM.

[0156] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the scope of protection of the present application.

Claims

1. A nanobody or its active fragment that specifically recognizes the Sumo-tagged protein, characterized in that, The nanobody or its active fragment contains a heavy chain variable region having: 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.

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

6.

3. A nucleic acid molecule encoding the nanobody or its active fragment as described in claim 1 or 2.

4. An expression vector containing the nucleic acid molecule as described in claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A method for expressing nanobodies or their active fragments using the host cells described in claim 5.

7. The humanized nanobody or its active fragment obtained by humanizing the nanobody or its active fragment as described in claim 1 or 2.

8. A protein conjugate that specifically binds to a Sumo-tagged protein, characterized in that, The conjugate comprises the nanobody or its active fragment as described in claim 1 or 2, or the humanized nanobody or its active fragment as described in claim 7; the protein conjugate further comprises a ligand, wherein the ligand is selected from radioisotopes, fluorescent groups, and delivery carriers.

9. A kit for detecting Sumo-tagged proteins in samples, characterized in that, It comprises the nanobody or its active fragment as described in claim 1 or 2, or the humanized nanobody or its active fragment as described in claim 7.

10. The use of the nanobody or its active fragment as described in claim 1 or 2, or the humanized nanobody or its active fragment as described in claim 7, in a kit for preparing a Sumo-tagged protein for detecting in a sample.

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