Antibody specifically bound with TIF1-gamma protein or antigen binding fragment thereof and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides an antibody specifically bound with TIF1-gamma protein or an antigen binding fragment thereof and application thereof, a 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; a light chain variable region of the antibody or the antigen binding fragment thereof 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 obtained by screening is high in activity, good in stability and relatively high in specificity; the antibody can quantitatively detect the anti-TIF1-gamma autoantibody level in a DM patient, and has important value in developing a kit for detecting idiopathic inflammatory myopathy.

Description

Technical Field

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

[0002] With the research on idiopathic inflammatory myopathy (IIM), MSAs have gradually been detected in the sera of IIM patients. MSAs include anti-aminoacyl-tRNA synthetase (ARS) antibodies, anti-Mi-2 antibodies, anti-signal recognition particles (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, anti-small ubiquitin-like modified activating enzyme (SAE) antibodies, etc. Among them, the anti-TIF1-γ antibody is an autoantibody first discovered in the sera of myositis patients by immunoprecipitation in 2006. Subsequent studies have confirmed that the anti-TIF1-γ antibody can be used as an indicator for predicting DM complicated with malignant tumors.

[0003] TIF1-γ, also known as TRIM33, is one of the members of the tripartite motif (TRIM) protein family. The N-terminus of TRIM contains three domains, including the ring finger, B-box, and coiled-coil domains, also known as the RBCC family, which is a member 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 in cells. TIF1-γ is a novel regulator of the TGF-β signaling pathway and is associated with tumors, inflammation, and autoimmune diseases. TIF1-γ can competitively bind to Smad4 with phosphorylated Smad2 / Smad3, monoubiquitinate the activated Smad4 molecule, and migrate Smad4 from the nucleus to the cytoplasm, preventing it from forming a transcriptional complex with other molecules in the nucleus, thereby inhibiting epithelial-mesenchymal transition and regulating the proliferation, metabolism, and metastasis of cancer cells. Some research reports have shown that p53 is overexpressed in the adenocarcinoma tissues of DM (dermatomyositis) patients with tumors, suggesting that there may be an interaction between the TIF1-γ protein and the tumor suppressor gene p53. Clinical studies have also found that patients positive for anti-TIF1-γ antibodies are prone to heliotrope rash and dysphagia, and less likely to have fever, mechanic's hand, and Raynaud's phenomenon. Therefore, recombinant human TIF1-γ protein can not only provide a reliable means for the diagnosis of DM but also be used to further study its role in the occurrence of cancer.

[0004] The gold standard for detecting anti-TIF1-γ antibodies is immunoprecipitation, which has high specificity and controllability 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, appropriate techniques and methods need to be selected according to the specific experimental purpose, and at the same time, quality control and result verification need to be strengthened to improve the accuracy and credibility of the experimental results, and it is not suitable for clinical detection.

[0005] Currently, the most commonly used method for detecting anti-TIF1-γ antibodies in clinical practice is the anti-myositis antibody spectrum IgG detection kit (Euroimmun blotting method). The advantages of immunoblotting are multiplex detection and high cost performance, which well meet the clinical needs in China. The disadvantage is that the detection time is slightly longer. Most products react at room temperature, and the results are easily affected by changes in room temperature, and the repeatability is average. Some products use visual judgment to read the results, resulting in large human errors. If the incubation temperature is strictly controlled, the repeatability can be effectively improved. However, due to the lack of a unified standard for specificity and sensitivity among different methodologies, there will be certain differences in the detection results. Therefore, providing a new anti-TIF1-γ monoclonal antibody is of great significance for the disease prediction, prognosis evaluation, and treatment of IIM patients. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide an antibody or its antigen-binding fragment that specifically binds to the TIF1-γ protein and its application. The present invention provides an antibody that binds to the TIF1-γ protein with high affinity, and the antibody can quantitatively detect the level of anti-TIF1-γ autoantibody in DM patients, which has important application value in the diagnosis of DM.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

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

[0009] The light-chain variable region of the antibody or its antigen-binding fragment includes CDR1 shown in SEQ ID NO:6, the amino acid sequence of CDR2 is DDS, and CDR3 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 BL21(DE3) competent cells for culture, and the TIF1-γ antigen protein is purified.

[0011] The present invention constructs a phage human antibody library with PBMC from TIF1-γ antibody-positive patients, and screens antibody clones specific to the antigen by specifically binding to the TIF1-γ antigen protein. After three rounds of screening of the antibody phage library, the clones with an antigen group greater than 3 times that of the control group are defined as positive clones, and these monoclonal antibodies are sequenced and analyzed. After excluding incorrect antibody sequences and repeated antibody sequences, and combining the specific binding ability of antigen and antibody reflected by the ELISA experiment, a high-affinity antibody named TIF1-γ-R7P1-G7 is finally obtained. The antibody screened by the present invention has high activity, good stability, and strong specificity, and can quantitatively detect the level of anti-TIF1-γ autoantibody in DM patients.

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

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

[0015] Preferably, the nucleotide sequence of the nucleic acid molecule is as 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, and 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 expressing the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein described in the first aspect, and the engineered cell contains at least one copy of the recombinant plasmid vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into its genome.

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

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

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

[0021] In a sixth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof that specifically binds to the TIF1-γ protein described in the first aspect or the TIF1-γ protease-linked immunosorbent assay kit 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 applied in methodologies such as immunohistochemistry, magnetic particle chemiluminescence, immunoblotting, western-blot, immunofluorescence, etc., and has important application prospects.

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

[0024] The antibody obtained by screening in the present invention has high activity, good stability, and strong specificity; when the antibody is placed at 37°C for 7 days, its activity, i.e., the binding ability with the antigen, has no significant difference. Specificity refers to the specific binding with the antigen, which is judged by ELISA experiment. The higher the OD value, the better the binding ability. And by diluting the concentration of the antibody, the OD value shows a linear relationship with the antibody concentration. High-concentration antibody can reduce the loss of substances caused by adsorption on the container surface. The concentration of this antibody is greater than 0.5 mg / mL, which is higher than the industry level. The said antibody can also quantitatively detect the level of anti-TIF1-γ autoantibody in DM patients and has important value in developing a kit for detecting idiopathic inflammatory myopathy. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 It is the PCR agarose gel electrophoresis map of monoclonal bacteria of the KH phage library.

[0027] Figure 3 It is the PCR agarose gel electrophoresis map of monoclonal bacteria of the λH phage library.

[0028] Figure 4 It is the OD values of the TIF1-γ-R7P1-G7 antibody under different dilution concentrations. DETAILED DESCRIPTION OF THE INVENTION

[0029] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the said embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0030] For those not specifying specific techniques or conditions in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0031] The sources of the reagent materials used in the following specific embodiments 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 Fast Amplification PCR Premix, product number TSE005, manufacturer TSINGKE.

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

[0036] FastPure Plasmid Extraction Mini Kit, product number DC201, manufacturer TSINGKE.

[0037] DL2000 DNA Marker, product number TSJ011 - 500, manufacturer TSINGKE.

[0038] 1kb DNA Marker, product number TSJ102 - 100, manufacturer TSINGKE.

[0039] TG1 Electrocompetent Cells, product number 60502, manufacturer Lucigen.

[0040] pATA - scFv - 2 Vector, manufacturer 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 thirty years of development and improvement, it has been widely used in the establishment of antigen - antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigen epitope research, and cell signal transduction research, etc. Phage antibody library technology is to prepare a human antibody library, express the Fab fragment or single - chain antibody (ScFv) on the surface of phage, and then screen and enrich specific antibodies. The possibility of screening almost all recombinant human monoclonal antibodies that specifically react with antigens from a single - pot antibody library system has been proposed. Therefore, when using phage antibody technology, various antibody fragments (Fab or ScFv) applicable to in - vivo diagnosis or treatment can be obtained. In this example, a phage human antibody library was constructed using PBMC from TIF1 - γ antibody - positive patients, and specific antibody clones against the antigen were screened by specifically 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 positive for TIF1-γ antibody, and RNA was extracted. The RNA of patients positive for TIF1-γ antibody was reverse transcribed into cDNA using the HiScript® III First Strand cDNA Synthesis Kit (+gDNA wiper), and the VH and VL fragments of DNA were amplified.

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

[0048] Table 1

[0049]

[0050] Among them, 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 is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 56°C for 15 s; first extension at 72°C for 20 s; the three steps of denaturation, annealing, and first extension are repeated 30 times; second extension at 72°C for 5 min; incubation at 4°C.

[0073] (2) Vector construction

[0074] Insert the amplified VK and Vλ gene fragments 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 digested with NheI and NotI enzymes to obtain the vector backbone; the amplified VK and Vλ gene fragments were digested with NheI and NotI enzymes respectively to obtain VK or Vλ fragments; the vector backbone was ligated with the VK fragment to obtain the pATA-VK plasmid, and the vector backbone was ligated with the Vλ fragment to obtain the pATA-Vλ plasmid.

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

[0077] Table 2

[0078]

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

[0080] Table 3

[0081]

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

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

[0084] Table 4

[0085]

[0086] The ligation reaction conditions were: incubate overnight at 16 °C and inactivate by heating at 65 °C for 10 min.

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

[0088] Pre-cool the electroporation cuvette (0.2 cm gap) and microcentrifuge tubes on ice. Thaw the TG1 competent cells on ice. Add 6 μL of the DNA mixture to the chilled electroporation cuvette without generating bubbles. Flick the tube quickly downwards with the wrist to deposit the cells at the bottom; perform electroporation at 600 Ω, 10 μF, and 2.5 kV. Immediately add 2 mL of pre-warmed (pre-warmed at 37 °C) SOC medium (Sigma, S1797) to each tube within 10 seconds of the pulse. Shake at 37 °C and 250 rpm for 1 hour; collect all the electro-transformed medium. Serial dilute 10 μL of the culture into 90 μL of SOC medium and spread on LB / Amp / glucose agar plates. Incubate overnight at 37 °C. Calculate the total number of transformants by counting the number of colonies, multiplying by the culture volume, and dividing by the volume plated.

[0089] (4)Colony PCR verification

[0090] Pick single colonies from the LB / Amp / glucose agar plates for colony PCR to verify whether the plasmid has been transformed into the cells.

[0091] The PCR primers include: forward primer (F): agcggataacaatttcacacagga (SEQ ID NO:29). Reverse 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 as follows:

[0096] Pre-denaturation, 98 °C, 3 min; denaturation, 98 °C, 10 s; annealing, 56 °C, 10 s; extension (1), 72 °C, 20 s; repeat the three steps of denaturation, annealing, and extension (1) 30 times; extension (2), 72 °C, 2 min; hold at 4 °C.

[0097] The results of agarose gel electrophoresis after colony PCR are as Figures 1 - 3 shown.

[0098] Figure 1 is the agarose gel electrophoresis map of monoclonal bacteria PCR of the VL phage library; among them, lane M: DL2000, lanes 1-24 pATA-VK, lanes 25-48 pATA-Vλ.

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

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

[0101] 2. First round of panning

[0102] (1) Solid-phase panning in immunotubes

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

[0104] Use 1×10 12 pfu phage library to perform negative selection at room temperature for 30 minutes with 5% skim milk or 1% casein (dissolved in PBST) + 100 μg of control his protein (an irrelevant protein with the same his tag). Add the negatively selected library to the immunotubes and incubate at 30°C for 2 hours. Discard the liquid in the centrifuge tubes and wash 4-6 times with 5 mL of 0.05% PBST. Elute the phages with 1 mL of glycine-hydrochloric acid (pH = 2.2), incubate with shaking at room temperature for about 6-8 minutes, and add 120-130 μL of Tris-HCl (pH = 9.6) neutralization solution to pH = 7.0-8.0. Dilute the eluted phages and infect the logarithmic-phase Escherichia coli TG1, and plate to determine the titer.

[0105] (2) Amplification of the eluted phages

[0106] Absorb the eluted phages and add them to the logarithmic-phase Escherichia coli TG1 bacterial solution. After standing at 37°C for 30 minutes, culture at 220 rpm for 30 minutes to 1 hour. Add the antibiotic Amp to the culture medium and culture at 37°C and 220 rpm until the OD of the bacterial solution is about 0.4 - 0.6. Add helper phages to the bacterial solution, stand at 37°C for 30 minutes, and then culture at 220 rpm for 45 minutes to 1 hour. Centrifuge the bacterial solution at 3000 - 5000 rpm, discard the supernatant, resuspend the bacterial cells with the same volume of 2YT-Amp-Kan culture medium, and culture at 30°C and 220 rpm overnight. The next day, centrifuge the bacterial solution at 4°C and 8000 rpm for 20 minutes, transfer the supernatant to a new centrifuge tube; add 1 / 4 volume of 5×PEG / NaCl solution, mix well, and place on ice or at 4°C for 1 - 2 hours. Centrifuge at 4°C and 8000 rpm for 30 minutes, discard the supernatant; resuspend the precipitate with about 1 mL of PBS; centrifuge at 12000 rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube. Dilute the amplified phages, infect the logarithmic-phase TG1, 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 round of panning

[0109] Use the amplified phages for the second round of panning. The steps are the same as those in the first round of panning, and repeat the panning 7 times.

[0110] 4. Monoclonal phage ELISA screening

[0111] Select an appropriate round, dilute the eluted phages to an appropriate concentration, infect the logarithmic-phase TG1 cells, and plate.

[0112] The next day, pick 96 (or more) monoclonal colonies from the plate, inoculate them into a 96-deep well plate, and culture with shaking at 37°C and 250 rpm until the OD of the bacterial solution is 0.4 - 0.6. Add helper phages to the culture medium in the 96-deep well plate, stand at 37°C for 30 minutes; culture with shaking at 37°C and 250 rpm for 45 minutes to 1 hour. Centrifuge the 96-deep well plate at 4000 rpm for 5 minutes, discard the supernatant; resuspend the bacterial solution in each well with 2YT-Amp-Kan culture medium, and culture with shaking at 30°C and 250 rpm overnight.

[0113] The next day, centrifuge the 96-well deep well plate at 4000 rpm for 10 - 15 minutes, and take the supernatant for ELISA experiment. Coat the ELISA plate and incubate overnight at 4°C; Antigen group: 100 μL / well of TIF1-γ protein (4 μg / mL), Control group 1: 100 μL / well of N-His (4 μg / mL), Control group 2: 100 μL / well of PBS. Discard the liquid in the ELISA 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 2 - 3 times with 300 μL of PBST. Add 100 μL of phage supernatant to each well, incubate at 30°C for 1 hour, and wash 4 - 6 times with 300 μL of PBST. Add 100 μL of anti-M13-HRP antibody (1:6000) diluted with the blocking solution to each well, incubate at 30°C for 1 hour, and 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 an ELISA 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] Perform ELISA verification on the selected positive clones again.

[0117] After sequencing the positive clones obtained by monoclonal screening, remove the double-peak sequences and repetitive sequences to obtain the final positive clones. Perform secondary ELISA detection and verification on the positive clones to ensure the authenticity of the positive results.

[0118] Pick positive monoclonal colonies from the plate and inoculate them into a 96-well deep well plate, and shake culture at 37°C and 250 rpm until the OD of the bacterial solution is 0.4 - 0.6. Add helper phage to the medium in the 96-well deep well plate, and let it stand at 37°C for 30 minutes; then shake culture at 37°C and 250 rpm for 1 hour. Centrifuge the 96-well deep well plate at 4000 rpm for 5 minutes, discard the supernatant; resuspend the bacterial solution in each well with 2YT-Amp-Kan medium, and shake culture overnight at 30°C and 250 rpm.

[0119] The next day, centrifuge the 96-well deep well plate at 4000 rpm for 10 - 15 minutes, and take the supernatant for ELISA experiment. Coat the ELISA plate and incubate overnight at 4°C; Antigen group: 100 μL / well of TIF1-γ protein (4 μg / mL), Control group 1: 100 μL / well of N-His (4 μg / mL), Control group 2: 100 μL / well of PBS. Discard the liquid in the ELISA 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 2 - 3 times with 300 μL PBST. Add 100 μL of phage supernatant to each well, incubate at 30°C for 1 hour, and wash 4 - 6 times with 300 μL PBST. Add 100 μL of anti-M13-HRP antibody (1:6000) diluted with the blocking solution to each well, incubate at 30°C for 1 hour, and wash 4 - 6 times with 300 μL 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] Read the values using an ELISA 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 heavy chain base sequence 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. The stability was tested by a thermal acceleration experiment: the antibody was placed at 37°C for 7 days, and its activity, i.e., the binding ability to the antigen, showed no significant difference.

[0141] Example 2

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

[0143] Example 3

[0144] In this example, the TIF1-γ protease-linked immunosorbent assay kit in Example 2 was used to detect the OD values under different dilution concentrations of the antibody. The specific steps are as follows:

[0145] (1) Coat the enzyme-linked immunosorbent assay (ELISA) plate with the TIF1-γ protein solution. The concentration of the TIF1-γ protein solution is 4 μg / mL, and the coating volume is 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. Incubate at 37°C for 2 hours. The blocking agent is 5% skim milk (dissolved in PBS).

[0147] (3) Gradient dilute TIF1-γ-R7P1-G7 with gradients 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. Add 100 μL of the diluted antibody solution to each well. Incubate at 37°C for 1 hour. After incubation, discard the liquid in the ELISA plate. Wash each well 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 with the blocking solution. Add 100 μL of the diluted secondary antibody to each well. Incubate at 37°C for 30 minutes. After incubation, discard the liquid in the ELISA plate. 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 an ELISA reader to read the values at 450 nm - 620 nm. As Figure 4 shown, Figure 4 are the OD values under different dilution concentrations of the TIF1-γ-R7P1-G7 antibody.

[0151] The higher the OD value, the better the binding ability. And by diluting the concentration of the antibody, the OD value shows a linear relationship with the antibody concentration. Figure 4 The results of

[0152] In summary, the present invention constructs a phage human antibody library using PBMC from patients positive for TIF1-γ antibody, 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 autoantibody in DM patients.

[0153] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope 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 sequence of the nucleic acid molecule is shown in SEQ ID NO: 10 or 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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