Nano antibody for resisting galectin 3 binding protein as well as preparation method and application of nano antibody

Abstract

The invention belongs to the technical field of nano-antibodies, and particularly relates to a nano-antibody for resisting galectin 3 binding protein as well as a preparation method and application of the nano-antibody. The galectin 3 binding protein is used as a research object, after being expressed and purified, the galectin 3 binding protein is used for immunizing alpaca, a phage display technology is used for screening to obtain the anti-galectin 3 binding protein nano antibody, and the antibody has high sensitivity and capacity of specifically binding with the galectin 3 binding protein. Through the preparation of the anti-galectin 3 binding protein nano antibody, a material is provided for establishing a diagnosis method for related diseases such as systemic lupus erythematosus, Behcet's disease, biliary tract cancer and hepatitis C.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobodies, and in particular relates to a nanobody against a galectin-3 binding protein, and a preparation method and application thereof. BACKGROUND

[0002] Galectin-3 binding protein (Gal-3BP) is a secreted host protein that is expressed in various cell types in the human body. Gal-3BP can interact with collagen, fibronectin, gal-1, and gal-3, and is closely related to the galectin family.

[0003] In the study of various diseases (such as systemic lupus erythematosus, Behcet's disease, biliary tract cancer, hepatitis C, HIV-1), Gal-3BP can be used as a diagnostic marker or predictor. Studies have shown that Gal-3BP can be expressed in various cancer cells (such as breast cancer cells, pancreatic cancer cells, and colon cancer cells), and can mediate cell adhesion, metastasis, and signal transduction, and is considered to be a glycoprotein that plays a role in innate immunity after bacterial and viral infection. It has also been identified as a variety of disease-related antigens, such as metabolic syndrome, systemic lupus erythematosus venous thrombosis. The level of Gal-3BP in the serum of cancer patients is elevated, and it has been confirmed that it is associated with poor prognosis of the disease. In addition, Gal-3BP in metastatic breast cancer cell lines may inhibit the differentiation of fibroblasts derived from monocytes by binding to the CD209 receptor, thereby promoting the metastasis of breast cancer and melanoma.

[0004] There is a naturally light chain-deleted antibody in the peripheral blood of a llama, which only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, and has a molecular weight of only 15 KDa, so it is also called nanobody (Nb). Nanobody has a simple structure, good antigen specificity, and strong tissue penetration, and has broad application prospects in the diagnosis and treatment of diseases. Based on the advantages of nanobody, such as small molecular weight and easy genetic engineering modification, the anti-Gal-3BP nanobody can be fused with an enzyme reporter gene for expression, thereby directly obtaining a fusion protein of nanobody and enzyme, avoiding labeling of the antibody and using a secondary antibody, thereby simplifying the production process and reducing production costs, and having a very broad market prospect in the study of cancer. SUMMARY

[0005] The purpose of the present application is to provide a nanobody against a galectin-3 binding protein, and a preparation method and application thereof, so as to realize high sensitivity and high specificity detection of the galectin-3 binding protein.

[0006] To this end, the present application provides the following technical solutions.

[0007] A first aspect of the present application provides a VHH chain of a Nanobody against a galectin-3 binding protein, the VHH chain being selected from: (1) a VHH chain having an amino acid sequence as set forth in SEQ ID NO: 1; (2) a VHH chain that is sequence homologous to, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to, the amino acid sequence as set forth in SEQ ID NO: 1; (3) a VHH chain that differs from the amino acid sequence as set forth in SEQ ID NO: 1 by no more than 5, 4, 3, 2, or no more than 1 amino acid; and / or (4) a VHH chain that is identical to the amino acid sequence as set forth in SEQ ID NO: 1, including substitution, deletion, and / or insertion of 1 to 5 amino acid residues.

[0008] A second aspect of the present application provides a Nanobody against a galectin-3 binding protein, which is a Nanobody against an epitope of a galectin-3 binding protein, the Nanobody having a VHH chain as previously described, and the VHH chain being selected from: (1) a VHH chain having an amino acid sequence as set forth in SEQ ID NO: 1; (2) a VHH chain that is sequence homologous to, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to, the amino acid sequence as set forth in SEQ ID NO: 1; (3) a VHH chain that differs from the amino acid sequence as set forth in SEQ ID NO: 1 by no more than 5, 4, 3, 2, or no more than 1 amino acid; and / or (4) a VHH chain that is identical to the amino acid sequence as set forth in SEQ ID NO: 1, including substitution, deletion, and / or insertion of 1 to 5 amino acid residues.

[0009] A third aspect of the present application provides a biological material related to a VHH chain of a Nanobody against a galectin-3 binding protein, or a Nanobody against a galectin-3 binding protein, as previously described, the biological material being selected from any one of (1) to (12): (1) a nucleic acid molecule encoding a VHH chain of a Nanobody against a galectin-3 binding protein, or a Nanobody against a galectin-3 binding protein, as previously described; (2) an expression cassette containing the nucleic acid molecule of (1); (3) a recombinant vector containing the nucleic acid molecule of (1); (4) a recombinant vector comprising the expression cassette of (2); (5) a recombinant microorganism comprising the nucleic acid molecule of (1); (6) a recombinant microorganism comprising the expression cassette of (2); (7) a recombinant microorganism comprising the recombinant vector of (3); (8) a recombinant microorganism comprising the recombinant vector of (4); (9) a transgenic animal cell line comprising the nucleic acid molecule of (1); (10) a transgenic animal cell line comprising the expression cassette of (2); (11) a transgenic animal cell line comprising the recombinant vector of (3); (12) a transgenic animal cell line comprising the recombinant vector of (4).

[0010] In a preferred embodiment of the application, the nucleotide sequence of the nucleic acid molecule is selected from: (a) the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 2; (b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide set forth in SEQ ID NO: 2; (c) a nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the nucleotide set forth in SEQ ID NO: 2; (d) a nucleotide sequence encoding an amino acid having at least about 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid set forth in SEQ ID NO: 1; (e) a nucleotide sequence encoding an amino acid having no more than 5, 4, 3, 2, or 1 amino acid residues difference in sequence from SEQ ID NO: 1; and / or (f) a nucleotide sequence encoding an amino acid having the amino acid sequence set forth in SEQ ID NO: 1 including 1 to 5 amino acid residues substitutions, deletions, and / or insertions.

[0011] A fourth aspect of the application provides a method of producing a VHH chain of a Nanobody against a galectin-3 binding protein or a Nanobody against a galectin-3 binding protein, the method comprising the steps of: (1) introducing a VHH chain of a nanobody of an anti-galectin 3 binding protein or a nucleic acid molecule of a nanobody of an anti-galectin 3 binding protein as described above into a recipient cell to obtain a transgenic cell expressing the VHH chain of the nanobody of the anti-galectin 3 binding protein or the nanobody of the anti-galectin 3 binding protein; and (2) culturing the transgenic cell to obtain the VHH chain of the nanobody of the anti-galectin 3 binding protein or the nanobody of the anti-galectin 3 binding protein.

[0012] A fifth aspect of the present application provides a pharmaceutical composition comprising: (a) a VHH chain of a nanobody of an anti-galectin 3 binding protein or a nanobody of an anti-galectin 3 binding protein as described above; and (b) a pharmaceutically acceptable carrier.

[0013] A sixth aspect of the present application provides a recombinant protein comprising: (a) a sequence of a VHH chain of a nanobody of an anti-galectin 3 binding protein or a sequence of a nanobody of an anti-galectin 3 binding protein as described above; and (b) a tag sequence for assisting expression and / or purification.

[0014] In a preferred embodiment of the present application, the tag sequence comprises a 6His tag and / or an HA tag.

[0015] A seventh aspect of the present application provides use of a VHH chain of a nanobody of an anti-galectin 3 binding protein or a nanobody of an anti-galectin 3 binding protein as described above in the preparation of a medicament, reagent, detection plate or kit for detecting an anti-galectin 3 binding protein molecule.

[0016] By means of the above technical solution, the present application has at least the following advantages: The present application takes galectin 3 binding protein as the research object, immunizes a llama after expression and purification of the galectin 3 binding protein, and uses phage display technology to screen a strain of anti-galectin 3 binding protein nanobody. The antibody has the ability of high sensitivity and specificity binding to the galectin 3 binding protein. The present application provides materials for establishing a diagnostic method of related diseases such as systemic lupus erythematosus, Behcet's disease, biliary tract cancer and hepatitis C by preparation of the anti-galectin 3 binding protein nanobody.

[0017] The above description is only a summary of the technical solution of the present application. In order to more clearly understand the technical means of the present application and can be implemented according to the content of the description, the following describes the preferred embodiments of the present application in detail. Attached Figure Description

[0018] Figure 1 The image shows a gel electrophoresis diagram of the purified Gal-3BP protein; Figure 2 The image shows a gel electrophoresis diagram of the PCR amplification product of the nanobody of the present invention; Figure 3 The image shows a gel electrophoresis diagram of the second-round PCR amplification products of the nanobody of the present invention; Figure 4 This diagram illustrates the results of gene insertion efficiency detection in a phage display library. Figure 5 This is a schematic diagram showing the purification results of the nanobody of the present invention; Figure 6 This is a schematic diagram of the affinity detection results of the nanobody of the present invention; Figure 7 This is a schematic diagram of the specific detection results of the nanobody of the present invention; Figure 8 The specificity of the nanobody of the present invention was verified in cell experiments. Detailed Implementation

[0019] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0021] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0022] It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents and instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0023] Unless otherwise specified, the practice of this invention will take place using conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. 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 invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] To a certain extent, the terms “comprising,” “including,” and “having, has, with,” or variations thereof, are used in the specific implementation and / or claims, and these terms are intended to include a manner similar to the term “comprising.”

[0025] To enable effective detection of the disease biomarker galactolectin 3 binding protein, this invention provides a VHH chain of an anti-galactolectin 3 binding protein nanobody, wherein the VHH chain is selected from: (1) Having a VHH chain with an amino acid sequence as shown in SEQ ID NO: 1; (2) A VHH chain that is sequence homologous to the amino acid shown in SEQ ID NO: 1 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; (3) A VHH chain whose sequence difference from the amino acid shown in SEQ ID NO: 1 is no more than 5, 4, 3, 2 or no more than 1 amino acid; and / or (4) The VHH chain as shown in SEQ ID NO: 1, comprising an amino acid sequence of 1 to 5 amino acid residues that have been substituted, deleted and / or inserted.

[0026] The sequence of SEQ ID NO: 1 is shown below: QVQLVESGGGLVQPGGSLRLSCITSGLIFRTATVGWYRQAPGKGREFVSRISSGSTADYVESVKGRFTISRAKNTLYLQMNNLKTEDTAVYLCAAGGSFGQGTQVTVSS According to another aspect of the present invention, a nanobody against galactoglobulin 3 binding protein is provided, which is a nanobody targeting the antigenic epitope of galactoglobulin 3 binding protein, said nanobody having a VHH chain as described above, and said VHH chain is selected from: (1) Having a VHH chain with an amino acid sequence as shown in SEQ ID NO: 1; (2) A VHH chain that is sequence homologous to the amino acid shown in SEQ ID NO: 1 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; (3) A VHH chain whose sequence difference from the amino acid shown in SEQ ID NO: 1 is no more than 5, 4, 3, 2 or no more than 1 amino acid; and / or (4) The VHH chain as shown in SEQ ID NO: 1, comprising an amino acid sequence of 1 to 5 amino acid residues that have been substituted, deleted and / or inserted.

[0027] In this invention, the term "nanobody" refers to the variable region of a cloned antibody heavy chain. A single-domain antibody (VHH) consisting of only one variable region of a heavy chain is constructed, which is the smallest antigen-binding fragment with full function.

[0028] According to another aspect of the invention, a biomaterial is provided relating to a VHH chain of a nanobody against galactoglobulin 3 binding protein as described above, or a nanobody against galactoglobulin 3 binding protein, said biomaterial being selected from any of the following (1) to (12): (1) VHH chain of nanobody encoding anti-galactoglobulin 3 binding protein as described above, or nucleic acid molecule of nanobody encoding anti-galactoglobulin 3 binding protein; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1); (4) A recombinant vector containing the expression cassette described in (2); (5) Recombinant microorganisms containing the nucleic acid molecules described in (1); (6) Recombinant microorganisms containing the expression cassette described in (2); (7) Recombinant microorganisms containing the recombinant vector described in (3); (8) Recombinant microorganisms containing the recombinant vector described in (4); (9) A transgenic animal cell line containing the nucleic acid molecule described in (1); (10) A transgenic animal cell line containing the expression cassette described in (2); (11) A transgenic animal cell line containing the recombinant vector described in (3); (12) A transgenic animal cell line containing the recombinant vector described in (4).

[0029] Furthermore, the nucleotide sequence of this nucleic acid molecule is selected from: (a) The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2; (b) The nucleotide sequence that hybridizes with the nucleotide shown in SEQ ID NO: 2 under stringent conditions; (c) A nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the nucleotide shown in SEQ ID NO: 2; (d) A nucleotide sequence encoding an amino acid, wherein the amino acid is at least about 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid shown in SEQ ID NO: 1; (e) A nucleotide sequence encoding an amino acid, wherein the sequence of said amino acid differs from SEQ ID NO: 1 by no more than 5, 4, 3, 2, or 1 amino acid residue; and / or (f) A nucleotide sequence encoding an amino acid having the amino acid sequence shown in SEQ ID NO: 1, including substitutions, deletions and / or insertions of 1 to 5 amino acid residues.

[0030] The sequence of SEQ ID NO:2 is shown below: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGATCTCTGAGACTCTCCTGTATAACCTCCGGATTGATCTTCAGAACTGCTACCGTGGGTTGGTACCGCCAGGCTccAGGAAAGGGGCGCGAGTTCGTCtCGCGTATTAGTAGTGGAA GTACCGCAGACTATGTAGAGTCTGTGAAGGGCCGATTCACCATTTCCAGAGCCAAGAAtACGCTGTATCTGCAAATGAAtAATCTGAAAACcGAGGACACGGCCGTCTATTTGTGTGCTGCAGGCGGTTCCTTCGGCCAGGGGACCCAGGTCACCGTCTCCTCA In this invention, the terms "having," "comprising," or "including" are open-ended descriptions, containing the specified components or steps described, as well as other specified components or steps that do not substantially affect them. However, when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the said sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activities described in this invention.

[0031] According to another aspect of the present invention, a method for generating a VHH chain of a nanobody against galactoglobulin 3 binding protein or a nanobody against galactoglobulin 3 binding protein is provided, the method comprising the following steps: (1) Introducing the VHH chain of the nanobody encoding the anti-galactoglobulin 3 binding protein as described above, or the nucleic acid molecule of the anti-galactoglobulin 3 binding protein nanobody, into recipient cells to obtain transgenic cells expressing the VHH chain of the anti-galactoglobulin 3 binding protein nanobody or the anti-galactoglobulin 3 binding protein nanobody; and (2) Culture the transgenic cells to obtain the VHH chain of the anti-galactoglobulin 3 binding protein nanobody or the anti-galactoglobulin 3 binding protein nanobody.

[0032] According to another aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (a) The VHH chain of the anti-galactoglobulin 3 binding protein nanobody as described above, or the anti-galactoglobulin 3 binding protein nanobody; and (b) Pharmaceutically acceptable carriers.

[0033] According to another aspect of the present invention, a recombinant protein is provided, the recombinant protein comprising: (a) The sequence of the VHH chain of the anti-galactoglobulin 3 binding protein nanobody as described above, or the sequence of the anti-galactoglobulin 3 binding protein nanobody; and (b) Tag sequences that assist in expression and / or purification.

[0034] In a preferred embodiment, the tag sequence includes a 6His tag and / or an HA tag.

[0035] According to another aspect of the invention, a VHH chain of an anti-galactoglobulin 3 binding protein nanobody as described above, or an anti-galactoglobulin 3 binding protein nanobody, is provided for use in the preparation of a pharmaceutical agent, reagent, detection plate, or kit for detecting anti-galactoglobulin 3 binding protein molecules.

[0036] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0038] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0039] Example 1: Construction of a phage display library for anti-Gal-3BP nanobody 1 Experimental Methods 1.1 Immunizing alpacas 50 μg of purified Gal-3BP protein (its gel electrophoresis image is shown below) Figure 1 (As shown) After mixing and emulsifying with an equal volume of Freund's adjuvant, the mixture was administered to alpacas via subcutaneous injection at multiple sites in the neck. For the initial immunization, Freund's complete adjuvant was used; thereafter, Freund's incomplete adjuvant was used every two weeks for a total of six immunizations. Seven days after the final immunization, blood samples were collected, and the antibody titer in alpaca serum after the sixth immunization was detected using an indirect ELISA (using purified Gal-3BP protein as the coating antigen, 100 ng / well). When the serum antibody titer against Gal-3BP protein was not less than 1:64000, a phage display antibody library was constructed.

[0040] 1.2 Construction and panning of VHH phage antibody library 1.2.1 Isolation of peripheral blood lymphocytes Alpaca peripheral blood lymphocytes (PBMCs) were isolated using human peripheral blood lymphocyte separation medium. The specific operation was performed according to the instructions of the kit (Solepro, catalog number P8610). The isolated PBMCs were used for the extraction of total RNA from the cells.

[0041] 1.2.2 Amplification of the VHH gene fragment Total RNA was extracted from PBMCs using an RNA extraction kit (OMEGA, catalog number R6834-01), and cDNA was obtained by reverse transcription using a reverse transcription kit (TaKaRa, catalog number R026A) with the extracted total RNA as a template. The VHH gene was then amplified by nested PCR. After the first-step PCR amplification (reaction system and program are shown in Tables 1 and 2, respectively), the fragments were identified and separated by 1.5% agarose gel electrophoresis, and the PCR product with a molecular weight of ~700 bp was recovered. The recovered product was then used as a template for a second round of PCR amplification (reaction system and program are shown in Tables 3 and 4, respectively), to obtain the VHH fragment with a molecular weight of ~400 bp, which was then excised and recovered from the gel.

[0042] Table 1 PCR reaction system Upstream primer: 5'-TCCTGGCTGCTCTTCTA-3' SEQ ID NO: 3 Downstream primer: 5'-GGTACGTGCTGTTGAAC-3' SEQ ID NO: 4 Table 2 PCR reaction procedure Table 3 PCR Reaction System Upstream primer: 5'-CTACAAATGCCTATGCATCCCAGGTGCAGCTCGTGGAGTC-3' SEQ ID NO: 5 Downstream primer: 5'-AAACAACTTTCAACAGTGGAGGGGTCTTCGCTGTGGTGCG-3' SEQ ID NO: 6 Table 4 PCR reaction procedure 1.2.3 Construction of VHH phage display vector The VHH product and the linearized phage vector were ligated by homologous recombination, and the specific operation was performed according to the instructions of the homologous recombination kit (Novazan, catalog number C113-01).

[0043] 1.2.4 Preparation of SS320 electrocompetent cells Escherichia coli SS320 (pre-infected with M13KO7 Helper Phage) was cultured to OD. 600nmAfter reaching 0.4, rapidly cool the cells and centrifuge at 4℃ and 6000 g for 5-10 min. Wash the cells repeatedly 3-5 times with pre-cooled ddH2O (sterile). After the last centrifugation, resuspend the SS320 competent cells in 10% glycerol water (sterile), 200 μL / tube, and store at -80℃ for later use.

[0044] 1.2.5 Transformation of ligation products and harvesting of phage antibody libraries Take one tube of SS320 E. coli competent cells from the -80℃ cryopreservation chamber, place it on ice for 5 min to thaw, then gently add the ligation product into the SS320 competent cells, gently mix and add it into an electroporation cuvette, and perform electroporation transformation using an Eppendorf electroporator with the parameters set to 2.5 kV, 25 μF, 200 Ω, and 2 mm. The transformed competent cells were immediately resuspended and transferred to 20 mL of preheated SOC medium at 37°C. After incubation at 37°C and 120 r / min for 1 h with shaking, 100 μL of the stock solution was taken (for later use), and the remaining culture was transferred to 500 mL of 2×YT / Amp-Kan medium. The culture was incubated at 37°C for 12-14 h. After the culture was completed, the culture was centrifuged at 4°C and 10000 g / min for 10 min. The supernatant was collected and 1 / 4 volume of pre-cooled PEG / NaCl solution was added. The mixture was thoroughly mixed and placed in a 4°C refrigerator for 30-60 min. Finally, the mixture was centrifuged, the precipitate was resuspended with 1 mL of PBT, the concentration was measured and diluted to a specific level, and aliquoted and stored at -80°C for later use.

[0045] 1.2.6 Determination of phage display library capacity and diversity The recombinant bacteria activated by electroporation (the above 100 μL stock solution) were serially diluted 10-fold. -3 -10 -5 10 μL of the dilution solution was dropped onto LB / Amp-Kan plates and incubated at 37℃ for 12 h. The transformation efficiency was calculated, and the final library volume was 2 × 10⁻⁶. 9 A phage library of CFU (colony-forming units). Fifteen single clones were randomly selected and identified by PCR using upstream primer VHH-F (5'-CAGGTGCAGCTCGTGGAGTC-3' SEQ ID NO: 7) and downstream primer VHH-R (5'-TGAGGAGACGGTGACCTG-3' SEQ ID NO: 8). The specific reaction system and procedure are shown in Tables 5 and 6, respectively. Positive clones were identified by agarose gel electrophoresis. The target fragment size was approximately 400 bp. The positive clones were then sequenced to determine the diversity of nanobodies.

[0046] Table 5 PCR Reaction System Table 6 PCR Reaction Procedure 1.3 Screening of specific nanobodies against Gal-3BP protein 1.3.1 Panning of recombinant phages specific to anti-Gal-3BP protein The purified Gal-3BP protein was coated onto an ELISA plate, with 5% BSA as an antigen-free control. The prepared phage solution was added to the ELISA plate and incubated at room temperature for 2 h. The phage sample was then discarded. Freshly prepared 0.1M HCl was added to each well, and the plate was gently shaken at room temperature for 10 min. The eluent was then quickly neutralized with an equal volume of 1M Tris-HCl (pH 8.0). The eluent was added to 1 mL of logarithmic-phase TG1 bacteria and incubated at 37°C and 200 rpm for 1 h. Then, M13 helper phage was added, and the plate was incubated at 37°C and 200 rpm for another 1 h. The plate was then transferred to 40 mL of 2×YT / Amp-Kan medium. The above steps were repeated to collect the phage library. The first-round phage library was then screened using the steps described in 1.3.1 for the second and third rounds of screening. The enrichment of the phage was determined by titration in each round.

[0047] 1.3.2 Screening of Nanobodies Ninety single colonies were randomly selected from the enriched plates and inoculated into 96-well deep-well plates, one colony per well. The plates were then incubated overnight in 2×YT / Amp-Kan medium. The bacterial culture was centrifuged to collect the phages from the supernatant. Nanobodies corresponding to the Gal-3BP protein were screened using ELISA. Positive bacterial cultures were identified by Phage ELISA and sequenced to obtain the nanobodies sequences. The specific procedures are as follows: (1) Coat ELISA plates with Gal-3BP protein, 100 ng / well, overnight at 4℃; (2) Invert the ELISA plate to remove the supernatant, add 200 μL of 5% BSA to each well, and add 200 μL of 5% BSA to the negative control group that is not coated with Gal-3BP protein. Block at 37°C for 1 hour. (3) Centrifuge the 96 deep well plate at 4000 rpm and 4℃ for 10 min; (3) After 1.5 h, discard the supernatant in the ELISA plate, wash the plate 3 times with PBST and shake it dry, add 50 μL of phage (i.e. the supernatant of the 96-well plate), and gently shake at room temperature for 2 h. (4) Clean the ELISA plate, wash it 4 times with PBST, add 100 μL of HRP-M13 antibody, and incubate at 37 °C for 1 h; (5) Color development: Discard the supernatant in the ELISA plate, wash the plate 3 times with PBST and shake it dry, add 100 μL of TMB color development solution, and place it at room temperature for 10~15 min to observe the color development. (6) Termination of reaction: Add 100 μL of sulfuric acid to each well to terminate the reaction; and (7) Measure the OD450 value of each well using an ELISA reader at a wavelength of 450 nm. Select 10-15 positive clones with high absorbance values ​​for preservation and sequence verification.

[0048] 1.3.3 Cloning and Expression of Nanobodies The antibody sequence was cloned into the pcDNA 3.1-Fc eukaryotic expression vector (containing the CH2 and CH3 gene fragments of human Fc), the plasmid was extracted and transfected into 293F suspension cells, the supernatant was collected after 5-7 days, dialyzed overnight with PBS buffer, the target antibody was purified using Protein A purification medium, and SDS-PAGE was performed to analyze the results.

[0049] 2. Experimental Results 2.1 First round of PCR amplification Agarose gel electrophoresis results showed that after transcription using oligo dT+ random primers and reverse primers, the first round of amplification using 2 μL as template resulted in a strong, clear band without any impurities. The gel was then excised and the target band was recovered (results are shown below). Figure 2 (As shown).

[0050] 2.2 Second round of PCR amplification After the first round of PCR amplification, approximately 500 bp of the target band was recovered. This band was then used as a template for the second round of PCR amplification, resulting in a final target fragment of approximately 450 bp (see results). Figure 3 (As shown).

[0051] 2.3 Results of phage library construction A phage display library was successfully established with a capacity of 1×10⁻⁶. 9 / mL.

[0052] 2.4 PCR of positive clone colonies Colony PCR agarose gel electrophoresis results showed that all 15 selected single clones were positive, with a positive rate of 100% (structure as shown). Figure 4 As shown in the figure, the positive clone rate of the antibody library meets the requirements.

[0053] 2.5 Sequence Diversity Thirty monoclonal antibodies were randomly selected and sequenced. The sequences were then transcribed and translated into protein sequences using GENtle software. Sequence diversity comparison showed that 27 sequences were independent sequences, with a diversity of 90%, which met the requirements for antibody library diversity.

[0054] 2.6 Nanobody Screening Results The nucleotide sequence of a nanobody (SEQ ID NO: 2) was successfully obtained using the Phage ELISA method.

[0055] 2.7 Results of Cloning and Expression Experiments for Nanobodies Sequencing revealed that the nanobody gene was successfully cloned into the pCDNA 3.1-Fc eukaryotic expression vector. The constructed nanobody plasmid was transfected into 293F suspension cells. After 6 days of culture, the cell supernatant was collected, and the antibody was purified. The results showed that the anti-Gal-3BP protein nanobody (hereinafter referred to as anti-Gal-3BP protein nanobody Nb1 or Nb1) was successfully purified (results are shown below). Figure 5 (As shown).

[0056] Example 2: Characterization of anti-Gal-3BP protein nanobody 1 Experimental Methods 1.1 Affinity Detection of Nanobodies The affinity of the prepared nanobodies was detected using the SPR assay. The specific steps are as follows: Place the CM5 chip into the Biacore 3000 chip well, run the program to activate the chip, determine the optimal pH of the sodium acetate solution for protein binding to the chip, and then conjugate the Gal-3BP protein to the CM5 chip before blocking. Dilute the obtained anti-Gal-3BP protein nanobody Nb1 to 100 nM according to the pre-binding ratio, perform 2-fold serial dilutions, for a total of 9 dilutions. Remove the microplate and place the antibody, PBS, and 10 mM glycine (pH 2.5) into the microplate according to the set positions. Follow the Biacore 3000 user manual to set and run the program to determine the affinity between the anti-Gal-3BP nanobody Nb1 and the Gal-3BP protein.

[0057] 1.2 Nanobody Specific Detection The specificity of the obtained anti-Gal-3BP nanobody Nb1 was analyzed by Western blotting. The specific steps were as follows: (1) Add an appropriate amount of loading buffer to the His-tagged Gal-3BP protein, H7 subtype influenza virus HA protein, H9 subtype influenza virus HA1 protein, and CHI3L1 protein (CH3 protein for short). After boiling and denaturation, add the protein samples to the gel wells, along with pre-stained protein molecular weight standards to determine the molecular weight of the target proteins later. After protein electrophoresis, attach the gel to the nitrocellulose membrane and place it in the transfer tank, ensuring there are no air bubbles between the gel and the membrane to ensure uniform transfer of proteins from the gel to the membrane. After transfer, remove the membrane, block it with 5% skim milk powder solution, and incubate it on a shaker at room temperature for 1-2 hours.

[0058] (2) Antibody incubation: HRP-labeled anti-His and anti-Gal-3BP nanobody Nb1 were used for incubation at room temperature for 1-2 hours.

[0059] (3) Washing: Wash the membrane multiple times with TBST solution to remove unbound antibodies. Each wash lasts 5 minutes and is repeated 5 times.

[0060] (4) Secondary antibody incubation: Add HRP-labeled anti-Fc secondary antibody and incubate at room temperature on a shaker for 30 minutes (Note: HRP-labeled anti-His membrane does not need to be incubated with secondary antibody. After incubation, wash and expose directly).

[0061] (5) Wash again: Wash the membrane several times with TBST solution to remove unbound secondary antibody. Each wash lasts 5 minutes and is repeated 5 times in total.

[0062] (6) Chemiluminescence color development: Add a chemiluminescent substrate, the enzyme catalyzes the substrate to emit light, and the chemiluminescence imaging system is used for detection.

[0063] 1.3 Nanobody Specific Detection The specificity of the obtained anti-Gal-3BP nanobody Nb1 was analyzed by indirect immunofluorescence assay. The specific steps were as follows: The plasmid Gal-3BP-pCDNA3.1, which overexpresses Gal-3BP, was transfected into A549 cells with the Gal-3BP gene knocked out (Gal-3BP overexpression group). The blank plasmid pCDNA3.1 was transfected into A549 cells with the Gal-3BP gene knocked out as the control group (Gal-3BP knockdown group). 24 h after transfection, cells were washed twice with PBS; then fixed with 4% paraformaldehyde for 15 min. After fixation, cells were washed three times with PBS, then blocked at 37°C for 30 min with 2% BSA solution prepared in PBS. After blocking, cells were thoroughly washed with PBS buffer, then permeabilized with 0.1% Triton X-100 for 15 min, followed by thorough washing with PBS buffer. Then, anti-Gal-3BP nanobody Nb1 diluted with 1% BSA solution was added and incubated for 1 h. After incubation, unbound antibodies were washed away. Then, anti-Fc-FITC secondary antibody was added and incubated for 30 min. After incubation, unbound antibodies were washed away. Finally, DAPI was added and detected and photographed using a laser confocal microscope.

[0064] 2. Experimental Results 2.1 Affinity Detection Results of Nanobodies Affinity assay results showed that the anti-Gal-3BP nanobody Nb1 exhibited high affinity activity (1.39 nM) as determined by SPR (see results below). Figure 6 (As shown).

[0065] 2.2 Results of Western Blotting of Nanobodies Western blotting was performed after transfer to a membrane, with negative controls for different proteins included. The results demonstrated that the screened anti-Gal-3BP nanobody Nb1 bound to the Gal-3BP protein, while showing no reaction compared to the control group (results are shown in Figure 1). Figure 7 (As shown).

[0066] 2.3 Results of indirect immunofluorescence assay for nanobodies Indirect immunofluorescence (IFA) assay was performed 24 h post-transfection, with a blank cell control included. The results demonstrated that the anti-Gal-3BP nanobody Nb1 screened in this invention can bind to the Gal-3BP protein (e.g., ...). Figure 8 (As shown).

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A VHH chain of a nanobody against galactoglobulin 3 binding protein, characterized in that, The VHH chain is selected from: (1) Having a VHH chain with an amino acid sequence as shown in SEQ ID NO: 1; (2) A VHH chain that is sequence homologous to the amino acid shown in SEQ ID NO: 1 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; (3) A VHH chain whose sequence difference from the amino acid shown in SEQ ID NO: 1 is no more than 5, 4, 3, 2 or no more than 1 amino acid; and / or (4) The VHH chain as shown in SEQ ID NO: 1, comprising an amino acid sequence of 1 to 5 amino acid residues that have been substituted, deleted and / or inserted.

2. A nanobody against galactoglobulin 3 binding protein, characterized in that, A nanobody targeting the galactoglobulin 3 binding protein antigenic epitope, the nanobody having the VHH chain as described in claim 1, wherein the VHH chain is selected from: (1) Having a VHH chain with an amino acid sequence as shown in SEQ ID NO: 1; (2) A VHH chain that is sequence homologous to the amino acid shown in SEQ ID NO: 1 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; (3) A VHH chain whose sequence difference from the amino acid shown in SEQ ID NO: 1 is no more than 5, 4, 3, 2 or no more than 1 amino acid; and / or (4) The VHH chain as shown in SEQ ID NO: 1, comprising an amino acid sequence of 1 to 5 amino acid residues that have been substituted, deleted and / or inserted.

3. A biomaterial relating to the VHH chain of the anti-galactoglobulin 3-binding protein nanobody of claim 1, or the anti-galactoglobulin 3-binding protein nanobody of claim 2, characterized in that, The biomaterial is selected from any one of the following (1) to (12): (1) The VHH chain of the nanobody encoding the anti-galactoglobulin 3 binding protein of claim 1, or the nucleic acid molecule of the nanobody encoding the anti-galactoglobulin 3 binding protein of claim 2; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1); (4) A recombinant vector containing the expression cassette described in (2); (5) Recombinant microorganisms containing the nucleic acid molecules described in (1); (6) Recombinant microorganisms containing the expression cassette described in (2); (7) Recombinant microorganisms containing the recombinant vector described in (3); (8) Recombinant microorganisms containing the recombinant vector described in (4); (9) A transgenic animal cell line containing the nucleic acid molecule described in (1); (10) A transgenic animal cell line containing the expression cassette described in (2); (11) A transgenic animal cell line containing the recombinant vector described in (3); (12) A transgenic animal cell line containing the recombinant vector described in (4).

4. The biomaterial according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is selected from: (a) The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2; (b) The nucleotide sequence that hybridizes with the nucleotide shown in SEQ ID NO: 2 under stringent conditions; (c) A nucleotide sequence having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the nucleotide shown in SEQ ID NO: 2; (d) A nucleotide sequence encoding an amino acid, wherein the amino acid is at least about 95%, 96%, 97%, 98%, or at least 99% identical to the amino acid shown in SEQ ID NO: 1; (e) A nucleotide sequence encoding an amino acid, wherein the sequence of said amino acid differs from SEQ ID NO: 1 by no more than 5, 4, 3, 2, or 1 amino acid residue; and / or (f) A nucleotide sequence encoding an amino acid having the amino acid sequence shown in SEQ ID NO: 1, including substitutions, deletions and / or insertions of 1 to 5 amino acid residues.

5. A method for generating a VHH chain of a nanobody against galactoglobulin 3 binding protein or a nanobody against galactoglobulin 3 binding protein, characterized in that, The method includes the following steps: (1) Introducing a VHH chain of the nanobody encoding the anti-galactoglobulin 3 binding protein of claim 1, or a nucleic acid molecule of the anti-galactoglobulin 3 binding protein nanobody of claim 2, into recipient cells to obtain transgenic cells expressing the VHH chain of the anti-galactoglobulin 3 binding protein nanobody or the anti-galactoglobulin 3 binding protein nanobody; and (2) Culture the transgenic cells to obtain the VHH chain of the anti-galactoglobulin 3 binding protein nanobody or the anti-galactoglobulin 3 binding protein nanobody.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) The VHH chain of the anti-galactoglobulin 3 binding protein nanobody of claim 1, or the anti-galactoglobulin 3 binding protein nanobody of claim 2; and (b) Pharmaceutically acceptable carriers.

7. A recombinant protein, characterized in that, The recombinant protein comprises: (a) The sequence of the VHH chain of the anti-galactoglobulin 3 binding protein nanobody of claim 1, or the sequence of the anti-galactoglobulin 3 binding protein nanobody of claim 2; and (b) Tag sequences that assist in expression and / or purification.

8. The recombinant protein according to claim 7, characterized in that, The tag sequence includes a 6His tag and / or a HA tag.

9. The use of the VHH chain of the anti-galactoglobulin 3 binding protein nanobody of claim 1, or the anti-galactoglobulin 3 binding protein nanobody of claim 2, in the preparation of pharmaceuticals, reagents, detection plates, or kits for detecting anti-galactoglobulin 3 binding protein molecules.

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