An antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein and its applications

Abstract

The present invention belongs to the field of biomedical technology, and provides an antibody or its antigen-binding fragment that specifically binds to the TIF1-γ protein and its application. The heavy-chain variable region of the antibody or its antigen-binding fragment comprises CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:2, and CDR3 as shown in SEQ ID NO:3; the light-chain variable region of the antibody or its antigen-binding fragment comprises CDR1 as shown in SEQ ID NO:6, the amino acid sequence of CDR2 is DDS, and CDR3 as shown in SEQ ID NO:7. The antibody screened in the present invention has high activity, good stability, and strong specificity; the antibody can quantitatively detect the level of anti-TIF1-γ autoantibodies in DM patients, and has important value in the development of a kit for detecting idiopathic inflammatory myopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an antibody or antigen-binding fragment thereof that specifically binds to TIF1-γ protein and applications thereof. Background Art

[0002] With the development of research into idiopathic inflammatory myopathy (IIM), MSAs have been increasingly detected in the sera of IIM patients. These include anti-aminoacyl-tRNA synthetase (ARS) antibodies, anti-Mi-2 antibodies, anti-signal recognition particle (SRP) antibodies, anti-nuclear matrix protein 2 (NXP2) antibodies, anti-melanoma differentiation-associated gene 5 (MDA5) antibodies, anti-transcription intermediate factor (TIF) 1-γ antibodies, and anti-small ubiquitin-like modified activating enzyme (SAE) antibodies. Anti-TIF1-γ was first identified in the serum of myositis patients in 2006 through immunoprecipitation. Subsequent studies have confirmed that anti-TIF1-γ antibodies can be used as a predictor for the development of malignant tumors in DM.

[0003] TIF1-γ, also known as TRIM33, is a member of the tripartite motif (TRIM) protein family. The N-terminus of TRIM contains three domains: a ring finger, a B-box, and a coiled-coil domain. TRIMs, also known as the RBCC family, are members of the RING finger family of E3 ubiquitin ligases. The transforming growth factor-β (TGF-β) signaling pathway has been shown to induce epithelial-mesenchymal transition (EMT). TIF1-γ is a novel regulator of the TGF-β signaling pathway and is implicated in tumors, inflammation, and autoimmune diseases. TIF1-γ competitively binds to phosphorylated Smad2 / Smad3 for Smad4, resulting in monoubiquitination of activated Smad4 and translocation of Smad4 from the nucleus to the cytoplasm, preventing it from forming transcriptional complexes with other molecules in the nucleus. This inhibits EMT and regulates cancer cell proliferation, metabolism, and metastasis. Some studies have reported overexpression of p53 in adenocarcinoma tissues of patients with DM (dermatomyositis, DM) and tumors, suggesting that TIF1-γ may interact with the tumor suppressor gene p53. Clinical studies have also found that individuals with anti-TIF1-γ antibodies are more likely to experience heliotropism and dysphagia, and less likely to experience fever, mechanic's hands, or Raynaud's phenomenon. Therefore, recombinant human TIF1-γ protein not only provides a reliable tool for diagnosing DM but also allows for further research into its role in cancer development.

[0004] The gold standard for detecting anti-TIF1-γ antibodies is the immunoprecipitation method, which is highly specific and controllable and can be used to study protein interactions and structural characteristics. However, it also has disadvantages such as obtaining low-abundance target proteins and poor specificity. Therefore, when conducting this experiment, it is necessary to select appropriate techniques and methods according to the specific experimental purpose, and at the same time strengthen quality control and result verification to improve the accuracy and credibility of the experimental results. It is not suitable for clinical testing.

[0005] Currently, the most commonly used immunoblotting method for detecting anti-TIF1-γ antibodies in clinical practice is the Anti-Myositis Antibody Spectrum IgG Detection Kit (Immuno Western Blot). The advantages of the immunoblotting method are its ability to perform multiple tests simultaneously and its high cost-effectiveness, which is highly consistent with clinical needs in China. However, its disadvantage is its slightly longer detection time. Most products operate at room temperature, making the results susceptible to room temperature fluctuations and resulting in average reproducibility. Some products rely on visual interpretation, which can lead to significant human error. Strict control of the incubation temperature can effectively improve reproducibility. However, due to the varying specificity and sensitivity of different methodologies, there is a lack of standardized test results, leading to some variability. Therefore, the development of a new anti-TIF1-γ monoclonal antibody is of great significance for the prognosis, assessment, and treatment of IIM patients. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide an antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein and its use. The present invention provides an antibody that binds to the TIF1-γ protein with high affinity. The antibody is capable of quantitatively detecting anti-TIF1-γ autoantibody levels in patients with diabetes, and has important application value in the diagnosis of diabetes.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TIF1-γ protein, wherein the heavy chain variable region of the antibody or the antigen-binding fragment thereof comprises CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3;

[0009] The light chain variable region of the antibody or antigen-binding fragment thereof includes CDR1 as shown in SEQ ID NO: 6, the amino acid sequence of CDR2 is DDS, and CDR3 as shown in SEQ ID NO: 7.

[0010] The present invention recombines the gene of the artificially synthesized TIF1-γ domain into the expression vector plasmid pET28b to obtain the TIF1-γ-pET28b expression vector. The TIF1-γ-pET28b expression vector is transformed into a BL21 (DE3) competent cell culture medium and purified to obtain the TIF1-γ antigen protein.

[0011] The present invention constructs a phage human antibody library using PBMC from patients with positive TIF1-γ antibodies. Antibody clones targeting the specific antigen are screened by specifically binding to the TIF1-γ antigen protein. After three rounds of screening of the antibody phage library, clones whose antigen group expression was greater than three times that of the control group were identified as positive clones, and these monoclonal clones were sequenced and analyzed. By eliminating erroneous and duplicated antibody sequences and combining the antigen-antibody specific binding ability as determined by ELISA, a high-affinity antibody, named TIF1-γ-R7P1-G7, was ultimately obtained. The antibodies screened by the present invention have high activity, good stability, and strong specificity, and are capable of quantitatively detecting anti-TIF1-γ autoantibody levels in DM patients.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 8.

[0013] Preferably, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO: 9.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein according to the first aspect.

[0015] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 10 or SEQ ID NO: 11.

[0016] In a third aspect, the present invention provides a recombinant plasmid vector, comprising a vector backbone and an exogenous DNA sequence, wherein the exogenous DNA sequence comprises the nucleic acid molecule described in the second aspect.

[0017] In a fourth aspect, the present invention provides an engineered cell that expresses the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein as described in the first aspect, wherein the engineered cell contains at least one copy of the recombinant plasmid vector as described in the third aspect, or the nucleic acid molecule as described in the second aspect is integrated into its genome.

[0018] In a fifth aspect, the present invention provides a TIF1-γ enzyme-linked immunosorbent assay kit, which comprises the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein as described in the first aspect.

[0019] Preferably, the kit further comprises any one or a combination of at least two of an enzyme-labeled plate, an enzyme-labeled antibody, a buffer, a blocking solution, a standard, a developer or a terminator.

[0020] In the present invention, the kit is the first kit in China for detecting anti-TIF1γ antibodies, providing a new method for TIF1γ detection and filling a gap in the market; because the antibody has strong specific binding ability with the antigen, the time required for enzyme-linked immunosorbent assay is shorter, saving time.

[0021] In a sixth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein as described in the first aspect or the TIF1-γ enzyme-linked immunosorbent assay kit as described in the fifth aspect in the preparation of a product for diagnosing and / or detecting idiopathic inflammatory myopathy.

[0022] In the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein can be used in immunohistochemistry, magnetic particle chemiluminescence, immunoblotting, western-blot, immunofluorescence and other methodologies, and has important application prospects.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The antibodies obtained by screening in the present invention have high activity, good stability, and strong specificity; when the antibodies are placed at 37°C for 7 days, there is no significant difference in their activity, that is, their ability to bind to the antigen. Specificity refers to the specific binding to the antigen, which is judged by ELISA experiments. The higher the OD value, the better the binding ability. And by diluting the concentration of the antibody, the OD value is linearly related to the antibody concentration. High concentrations of antibodies can reduce material losses caused by adsorption on the container surface. The antibody concentration is greater than 0.5 mg / mL, which is higher than the industry level. The antibody can also quantitatively detect the level of anti-TIF1-γ autoantibodies in DM patients, and is of great value in the development of a kit for detecting idiopathic inflammatory myopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the agarose gel electrophoresis diagram of the monoclonal PCR of the VL phage library.

[0026] Figure 2 This is the agarose gel electrophoresis diagram of the monoclonal PCR of the KH phage library.

[0027] Figure 3 This is the agarose gel electrophoresis diagram of monoclonal bacterial PCR of the λH phage library.

[0028] Figure 4 These are the OD values ​​of TIF1-γ-R7P1-G7 antibody at different dilution concentrations. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] The sources of the reagents and materials used in the following specific examples are as follows:

[0032] HiScript® III First-Strand cDNA Synthesis Kit, product number R312-02, manufacturer Vazyme.

[0033] Phanta®Max ultra-fidelity DNA polymerase, product number P505-d3, manufacturer Vazyme.

[0034] 2×T5 rapid amplification PCR master mix, product number TSE005, manufacturer TSINGKE (Qingke Biotechnology).

[0035] FastPure Gel DNA Extraction Mini Kit, product number DC301, manufacturer Vazyme.

[0036] FastPure Plasmid Extraction Mini Kit, catalog number DC201, manufacturer TSINGKE (Qingke Biotechnology).

[0037] DL2000 DNA marker, product number TSJ011-500, manufacturer TSINGKE (Qingke Biotechnology).

[0038] 1kb DNA marker, product number TSJ102-100, manufacturer TSINGKE (Qingke Biotechnology).

[0039] TG1 electroporated competent cells, product number 60502, manufacturer Lucigen.

[0040] pATA-scFv-2 vector, manufactured by ProteoGenix.

[0041] M13KO7 helper phage, product number N0315S, manufacturer NEB.

[0042] Example 1

[0043] Phage display technology was first established by Smith in 1985. After more than 30 years of development and refinement, it has been widely used in the construction of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, epitope research, and cell signaling research. Phage display antibody library technology involves preparing a human antibody library and expressing Fab fragments or single-chain Fv (ScFv) on the surface of phage, thereby screening and enriching specific antibodies. It has been proposed that single-pot antibody library systems can screen for nearly any recombinant human monoclonal antibody that specifically reacts with an antigen. Therefore, using phage display antibody technology, a variety of antibody fragments (Fab or ScFv) can be obtained for in vivo diagnostic or therapeutic applications. In this example, a phage display human antibody library was constructed using PBMC from patients positive for TIF1-γ antibodies. Antibody clones specific to the antigen were screened for specific binding to TIF1-γ.

[0044] 1. Library construction

[0045] (1) Amplification of VH and VL fragments

[0046] Lymphocytes were isolated from the blood of patients with TIF1-γ antibody positive results, and RNA was extracted. RNA from TIF1-γ antibody positive patients was reverse transcribed into cDNA using the HiScript® III First Strand cDNA Synthesis Kit (with gDNA Cleanup Reagent), and the VH and VL fragments of the DNA were amplified.

[0047] The amplification reaction system is shown in Table 1.

[0048] Table 1

[0049]

[0050] The primers are as follows:

[0051] Heavy chain variable region upstream primer (F):

[0052] 5'L-VH 1: acaggtgcccactcccaggtgcag. (SEQ ID NO:12)

[0053] 5'L-VH 3:aaggtgtccagtgtgargtgcag. (SEQ ID NO:13)

[0054] 5'L-VH 4 / 6: cccagatgggtcctgtcccaggtgcag. (SEQ ID NO:14)

[0055] 5'L-VH 5 / 7:caaggagtctgttccgaggtgcag. (SEQ ID NO:15)

[0056] Light chain variable region upstream primer (F):

[0057] 5'L VK 1 / 2: atgaggstcccygctcagctgctgg. (SEQ ID NO:16)

[0058] 5'L VK 3:ctcttcctcctgctactctggctcccag. (SEQ ID NO:17)

[0059] 5'L VK 4 / 5:atttctctgttgctctggatctctg. (SEQ ID NO:18)

[0060] 5'L Vλ 1: ggtcctgggcccagtctgtgctg. (SEQ ID NO:19)

[0061] 5' L Vλ 2: ggtcctgggcccagtctgccctg. (SEQ ID NO:20)

[0062] 5'L Vλ 3: gctctgtgacctcctatgagctg. (SEQ ID NO:21)

[0063] 5'L Vλ 4 / 5:ggtctctctcscagcytgtgctg. (SEQ ID NO:22)

[0064] 5'L Vλ 6:gttcttgggccaattttatgctg. (SEQ ID NO:23)

[0065] 5'L Vλ 7:ggtccaattcycaggctgtggtg. (SEQ ID NO:24)

[0066] 5'L Vλ 8 / 9 / 10: gagtggattctcagactgtggtg. (SEQ ID NO:25)

[0067] Light chain variable region downstream primer (R):

[0068] 3'CK: tgctgtccttgctgtcctgct. (SEQ ID NO:26)

[0069] 3'Cλ: caccagtgtggccttgttggcttg. (SEQ ID NO:27)

[0070] Heavy chain variable region downstream primer (R):

[0071] 3'VH:ACTCGAGACGGTGACCAGGGTGCC. (SEQ ID NO:28)

[0072] The amplification program was as follows: pre-denaturation, 95°C, 3 min; denaturation, 95°C, 15 s; annealing, 56°C, 15 s; first extension, 72°C, 20 s; denaturation, annealing, first extension, these three steps were repeated 30 times; second extension, 72°C, 5 min; and insulation at 4°C.

[0073] (2) Vector construction

[0074] The amplified VK and Vλ gene fragments were inserted into the pATA-scFv-2 vector to obtain the VK library and the Vλ library. The specific steps include:

[0075] The pATA-scFv-2 vector was enzymatically digested with NheI and NotI enzymes to obtain a vector backbone; the amplified VK and Vλ gene fragments were enzymatically digested with NheI and NotI enzymes, respectively, to obtain VK or Vλ fragments; the vector backbone was ligated with the VK fragment to obtain a pATA-VK plasmid, and the vector backbone was ligated with the Vλ fragment to obtain a pATA-Vλ plasmid.

[0076] The reaction system for enzyme digestion of pATA-scFv-2 vector is shown in Table 2.

[0077] Table 2

[0078]

[0079] The reaction system for enzyme digestion of VK and Vλ gene fragments is shown in Table 3.

[0080] Table 3

[0081]

[0082] The reaction system in Table 3 was reacted under the following conditions: 37°C for 5 h, 65°C for 10 min, and 4°C for incubation. KpnI enzyme was then added and the reaction was continued under the following conditions: 37°C for 1 h, 65°C for 10 min, and 4°C for incubation.

[0083] The connection system is shown in Table 4.

[0084] Table 4

[0085]

[0086] The ligation reaction conditions were as follows: incubation at 16°C overnight and heat inactivation at 65°C for 10 min.

[0087] (3) Transform the pATA-VK plasmid and pATA-Vλ plasmid into TG1 competent cells

[0088] Pre-chill electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice. Thaw TG1 competent cells on ice. Add 6 μL of the DNA mixture to the chilled electroporation cuvette, avoiding bubbles. Quickly flick the tube downward with your wrist to sediment the cells at the bottom; electroporate at 600 Ω, 10 μF, and 2.5 kV. Within 10 seconds of the pulse, immediately add 2 mL of pre-warmed (pre-warmed at 37°C) SOC medium (Sigma, S1797) to each tube. Shake at 37°C at 250 rpm for 1 hour; collect all electroporation medium. Serially dilute 10 μL of culture into 90 μL of SOC medium and plate onto LB / Amp / glucose agar plates. Incubate at 37°C overnight. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the plate inoculation volume.

[0089] (4) Colony PCR verification

[0090] Single colonies were picked from LB / Amp / glucose agar plates for colony PCR to verify whether the plasmid was transformed into cells.

[0091] PCR primers included: upstream primer (F): agcggataacaatttcacacagga (SEQ ID NO: 29) and downstream primer (R): gcccccttattagcgtttgccatc (SEQ ID NO: 30).

[0092] The PCR reaction system is shown in Table 5.

[0093] Table 5

[0094]

[0095] The PCR reaction program is:

[0096] Pre-denaturation, 98℃, 3 min; denaturation, 98℃, 10 s; annealing, 56℃, 10 s; extension (1), 72℃, 20 s; denaturation, annealing, extension (1) these three steps, repeated 30 times; extension (2), 72℃, 2 min; keep warm at 4℃.

[0097] The results of agarose gel electrophoresis after colony PCR were as follows Figure 1-Figure 3 shown.

[0098] Figure 1 It is the monoclonal bacterial PCR agarose gel electrophoresis diagram of the VL phage library; wherein, lane M: DL2000, lanes 1-24 pATA-VK, lanes 25-48 pATA-Vλ.

[0099] Figure 2It is the agarose gel electrophoresis diagram of the monoclonal bacteria PCR of the KH phage library; wherein, lane M: DL2000, lanes 1-24: pATA-scFv-KH.

[0100] Figure 3 It is the agarose gel electrophoresis diagram of the monoclonal bacterial PCR of the λH phage library; wherein, lane M: DL2000, lanes 25-48: pATA-scFv-λH.

[0101] 2. First round of selection

[0102] (1) Immunotube solid phase panning

[0103] Coat a centrifuge tube with 1 mL of TIF1-γ transfection solution (50 μg / mL) and incubate overnight at 4°C to serve as the antigen group. Coat a centrifuge tube with 500 μL of transfection solution (0 μg / mL) and incubate overnight at 4°C to serve as the control group. Discard the contents of the tube and wash three times with 5 mL of 0.05% PBST. Add 5 mL of 5% skim milk or 1% casein (in PBST) to the tube and block at 37°C for 1 hour. Discard the contents of the tube and wash once with 5 mL of PBS.

[0104] Using 1×10 12 Negatively screen the pfu phage library with 5% skim milk or 1% casein (in PBST) plus 100 μg of a control His protein (an unrelated protein with the same His tag) at room temperature for 30 minutes. Add the negatively screened library to an immunoblot tube and incubate at 30°C for 2 hours. Discard the liquid from the centrifuge tube and wash 4-6 times with 5 mL of 0.05% PBST. Elute the phage with 1 mL of glycine-HCl (pH 2.2) and incubate at room temperature with shaking for approximately 6-8 minutes. Add 120-130 μL of Tris-HCl (pH 9.6) to neutralize the solution to a pH of 7.0-8.0. Dilute the eluted phage and infect Escherichia coli TG1 cells in logarithmic phase. Plate the solution and determine the titer.

[0105] (2) Amplification of eluted phage

[0106] Aspirate the eluted phage and add it to a logarithmic-phase E. coli TG1 culture. Incubate at 37°C for 30 minutes, then incubate at 220 rpm for 30 minutes to 1 hour. Add the antibiotic Amp to the culture medium and incubate at 37°C and 220 rpm until the culture reaches an OD of approximately 0.4-0.6. Add the helper phage to the culture medium, incubate at 37°C for 30 minutes, then incubate at 220 rpm for 45 minutes to 1 hour. Centrifuge the culture at 3000-5000 rpm, discard the supernatant, and resuspend the cells in an equal volume of 2YT-Amp-Kan medium. Incubate at 30°C and 220 rpm overnight. The next day, centrifuge the culture at 4°C and 8000 rpm for 20 minutes. Transfer the supernatant to a fresh centrifuge tube and add 1 / 4 volume of 5× PEG / NaCl solution. Mix thoroughly and incubate on ice or at 4°C for 1-2 hours. Centrifuge at 8000 rpm at 4°C for 30 minutes, discard the supernatant, resuspend the pellet in approximately 1 mL of PBS, centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. Dilute the amplified phage, infect logarithmic-phase TG1 cells, and plate to determine the titer.

[0107] The amino acid sequence of the TIF1-γ protein in the TIF1-γ transfection solution is shown in SEQ ID NO: 31.

[0108] 3. Second to eighth rounds of selection

[0109] The amplified phages were used for the second round of panning, following the same steps as the first round, and the panning was repeated for 7 rounds.

[0110] 4. Monoclonal phage ELISA screening

[0111] Select the appropriate round number, dilute the eluted phage to a suitable concentration, infect TG1 cells in the logarithmic phase, and plate them.

[0112] The next day, pick 96 (or more) single colonies from the plate and seed them into a 96-deep-well plate. Culture at 37°C, 250 rpm, and shake until the bacterial suspension reaches an OD value of 0.4-0.6. Add helper phage to the 96-deep-well plate medium and incubate at 37°C for 30 minutes. Incubate at 37°C, 250 rpm, and shake for 45 minutes to 1 hour. Centrifuge the 96-deep-well plate at 4000 rpm for 5 minutes, discard the supernatant, and resuspend the bacterial suspension in each well in 2YT-Amp-Kan medium. Culture at 30°C, 250 rpm, and shake overnight.

[0113] The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 minutes and remove the supernatant for ELISA. Coat the plate and incubate overnight at 4°C. Antigen group: 100 μL / well TIF1-γ protein (4 μg / mL); control group 1: 100 μL / well N-His (4 μg / mL); control group 2: 100 μL / well PBS. Discard the plate and wash each well three times with 300 μL of 0.05% PBST. Add 300 μL of 5% skim milk (dissolved in PBST) to each well, block at 30°C for 1 hour, and wash two to three times with 300 μL of PBST. Add 100 μL of phage supernatant to each well, incubate at 30°C for 1 hour, and wash four to six times with 300 μL of PBST. Add 100 μL of anti-M13-HRP antibody (1:6000) diluted in blocking buffer to each well, incubate at 30°C for 1 hour, wash 4-6 times with 300 μL of PBST, add 100 μL of TMB to each well, incubate at room temperature for 3-8 minutes, and then add 100 μL of 2M HCl to each well to terminate the reaction.

[0114] Use a microplate reader to read the values ​​at 450 nm-620 nm, and collect highly specific positive clones for the next verification experiment.

[0115] 5. ELISA verification

[0116] The positive clones screened were verified again by ELISA.

[0117] After sequencing the positive clones obtained from the monoclonal screening, doublets and repetitive sequences were removed to obtain the final positive clones. The positive clones were then verified by a secondary ELISA test to ensure the authenticity of the positive results.

[0118] Positive single colonies were picked from the plate and plated into a 96-well plate. Culture was shaken at 37°C, 250 rpm, until the OD value of the bacterial suspension reached 0.4-0.6. Helper phage was added to the 96-well plate culture medium and the culture was allowed to stand at 37°C for 30 minutes. Then, the culture was shaken at 37°C, 250 rpm, for 1 hour. The 96-well plate was centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded. The bacterial suspension was resuspended in 2YT-Amp-Kan medium in each well and cultured at 30°C, 250 rpm, with shaking overnight.

[0119] The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 minutes and remove the supernatant for ELISA. Coat the plate and incubate overnight at 4°C. Antigen group: 100 μL / well TIF1-γ protein (4 μg / mL); control group 1: 100 μL / well N-His (4 μg / mL); control group 2: 100 μL / well PBS. Discard the plate and wash each well three times with 300 μL of 0.05% PBST. Add 300 μL of 5% skim milk (dissolved in PBST) to each well, block at 30°C for 1 hour, and wash two to three times with 300 μL of PBST. Add 100 μL of phage supernatant to each well, incubate at 30°C for 1 hour, and wash four to six times with 300 μL of PBST. Add 100 μL of anti-M13-HRP antibody (1:6000) diluted in blocking buffer to each well, incubate at 30°C for 1 hour, wash 4-6 times with 300 μL of PBST, add 100 μL of TMB to each well, incubate at room temperature for 3-8 minutes, and then add 100 μL of 2M HCl to each well to terminate the reaction.

[0120] The values ​​were read using a microplate reader at 450 nm-620 nm. Table 6 shows the results of positive monoclonal phage ELISA.

[0121] Table 6

[0122]

[0123] The full sequence sequencing results of the screened phage positive clone TIF1-γ-R7P1-G7 are as follows:

[0124] The amino acid sequence of the heavy chain variable region of the positive clone TIF1-γ-R7P1-G7 is shown in SEQ ID NO: 4:

[0125] QVQLVQSGSELKKPGASVRVSCEALGYTFTDFYIHWVRQAPGQSLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAREGDGDYPDYWGQGTLVTVSS.

[0126] The heavy chain variable region of the positive clone TIF1-γ-R7P1-G7 includes:

[0127] CDR1: SEQ ID NO:1: GYTFTDFY;

[0128] CDR2: SEQ ID NO:2: MNPNSGNT;

[0129] CDR3: SEQ ID NO:3: AREGDGDYPDY.

[0130] The amino acid sequence of the light chain variable region of the positive clone TIF1-γ-R7P1-G7 is shown in SEQ ID NO: 8:

[0131] QPVLTQPPSVSVAPGKTATITCGGNNMAIKSVHWYQQKPGQAPILVVYDDSDRPPEIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDRYVFGTGTKLTVL.

[0132] The light chain variable region of the positive clone TIF1-γ-R7P1-G7 includes:

[0133] CDR1:SEQ ID NO:6:NMAIKS;

[0134] CDR2: DDS;

[0135] CDR3: SEQ ID NO:7: QVWDSSSDRYV.

[0136] The base sequence of the heavy chain of the positive clone TIF1-γ-R7P1-G7 is shown in SEQ ID NO:10.

[0137] The heavy chain amino acid sequence of the positive clone TIF1-γ-R7P1-G7 is shown in SEQ ID NO:5.

[0138] The light chain base sequence of the positive clone TIF1-γ-R7P1-G7 is shown in SEQ ID NO:11.

[0139] The light chain amino acid sequence of the positive clone TIF1-γ-R7P1-G7 is shown in SEQ ID NO:9.

[0140] The antibody has good stability, which can be verified by a heat acceleration test: when the antibody is placed at 37°C for 7 days, there is no significant difference in its activity, i.e., its ability to bind to the antigen.

[0141] Example 2

[0142] This example provides a TIF1-γ enzyme-linked immunosorbent assay kit, which includes the positive clone TIF1-γ-R7P1-G7 screened in Example 1, an ELISA plate, an enzyme-labeled antibody, a buffer, a blocking solution, a standard, a color developer, and a terminator. The buffer is 0.05% PBST, the blocking solution is 5% skim milk (dissolved in PBS), the color developer is TMB, and the terminator is HCl.

[0143] Example 3

[0144] This example uses the TIF1-γ enzyme-linked immunosorbent assay kit in Example 2 to detect the OD values ​​of the antibody at different dilution concentrations. The specific steps include:

[0145] (1) Coat the ELISA plate with TIF1-γ protein solution at a concentration of 4 μg / mL and a coating volume of 100 μL per well. Incubate overnight at 4°C. Wash three times with 300 μL of 0.05% PBST.

[0146] (2) Block the washed ELISA plate with 300 μL of blocking agent per well at 37°C for 2 hours. The blocking agent is 5% skim milk (dissolved in PBS).

[0147] (3) TIF1-γ-R7P1-G7 was diluted in a gradient of 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, and 0 μg / mL. 100 μL of the diluted antibody solution was added to each well and incubated at 37°C for 1 hour. After incubation, the liquid in the ELISA plate was discarded and each well was washed three times with 300 μL of 0.05% PBST.

[0148] (4) Dilute the secondary antibody Goat Anti-Human IgG (H+L) antibody (Jackson, code: 109-035-088) 10,000-fold in blocking solution, add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 30 minutes. After incubation, discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0149] (5) Add 100 μL of TMB to each well, incubate at 37°C for 10 minutes, and then add 50 μL of 2 M HCl to each well to terminate the reaction.

[0150] (6) Use a microplate reader to read the values ​​at 450 nm-620 nm, such as Figure 4 As shown, Figure 4 OD values ​​of TIF1-γ-R7P1-G7 antibody at different dilution concentrations.

[0151] The higher the OD value, the better the binding ability. And by diluting the concentration of the antibody, the OD value is linearly related to the antibody concentration. Figure 4 The results showed that the TIF1-γ-R7P1-G7 antibody had a strong specific binding ability to TIF1-γ.

[0152] In summary, the present invention constructs a phage human antibody library using PBMC of patients positive for TIF1-γ antibodies, and screens the antibody TIF1-γ-R7P1-G7 that specifically binds to the antigen by specifically binding to TIF1-γ; the antibody has strong specificity and can quantitatively detect the level of anti-TIF1-γ autoantibodies in DM patients.

[0153] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TIF1-γ protein, characterized in that: The heavy chain variable region of the antibody or antigen-binding fragment thereof comprises CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; The light chain variable region of the antibody or antigen-binding fragment thereof includes CDR1 as shown in SEQ ID NO:6, the amino acid sequence of CDR2 is DDS, and CDR3 as shown in SEQ ID NO:

7.

2. The antibody or antigen-binding fragment thereof that specifically binds to TIF1-γ protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:4; the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof that specifically binds to TIF1-γ protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO:5; the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO:

9.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleotide sequences of the nucleic acid molecules are shown in SEQ ID NO:10 and SEQ ID NO:

11.

6. A recombinant plasmid vector, characterized in that: The recombinant plasmid vector comprises a vector backbone and an exogenous DNA sequence, and the exogenous DNA sequence comprises the nucleic acid molecule according to claim 4 or 5.

7. An engineered cell expressing the antibody or antigen-binding fragment thereof that specifically binds to TIF1-γ protein according to any one of claims 1 to 3, characterized in that: The engineered cell contains at least one copy of the recombinant plasmid vector of claim 6, or the nucleic acid molecule of claim 4 or 5 is integrated into its genome.

8. A TIF1-γ ELISA kit, characterized in that: The kit comprises the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein according to any one of claims 1 to 3.

9. The TIF1-γ ELISA kit according to claim 8, characterized in that: The kit also includes any one or a combination of at least two of an ELISA plate, an enzyme-labeled antibody, a buffer, a blocking solution, a standard substance, a color developer or a terminator.

10. Use of the antibody or antigen-binding fragment thereof that specifically binds to TIF1-γ protein according to any one of claims 1 to 3 or the TIF1-γ ELISA kit according to claim 8 or 9 in the preparation of a product for diagnosing and / or detecting idiopathic inflammatory myopathy.

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