An anti-TIF1-γ monoclonal antibody and its application

Abstract

The present invention belongs to the technical field of biomedicine, and provides an anti-TIF1-γ monoclonal antibody and its application. The present invention uses phage antibody library technology to screen out a human-derived anti-TIF1-γ monoclonal antibody with good specificity and strong affinity. By specifically binding to TIF1-γ, the antibody can quantitatively detect the level of anti-TIF1-γ autoantibodies in DM patients, thereby monitoring the disease activity, and can be used for the clinical diagnosis of DM patients. The research of the present invention provides a theoretical and experimental basis for the specific treatment of DM, and also provides a new research direction for the treatment of autoimmune diseases with autoantibodies as the main pathogenic mechanism.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an anti-TIF1-γ monoclonal antibody and an application thereof. Background Art

[0002] Idiopathic inflammatory myopathy (IIM) is a group of systemic autoimmune connective tissue diseases characterized by symmetrical proximal muscle weakness in the limbs. Dermatomyositis (DM) is the most common subtype of inflammatory myopathy. Multiple specific autoantibodies are present in the serum of IIM patients, including myositis-specific autoantibodies (MSAs) and myositis-related antibodies (MAAs). Anti-transcription intermediary factor 1-γ (TIF1-γ) antibodies are novel autoantibodies specific for DM and belong to the group of MSAs. Studies have shown that the sensitivity of anti-TIF1-γ antibodies for diagnosing DM with cancer is 50.0%-71.4% and the specificity is 89.0%-91.3%. TIF1-γ antibodies can serve as a reliable biomarker for monitoring secondary malignancies in adult patients with DM.

[0003] Dermatomyositis is mainly treated with hormones and immunosuppressants. Hormones can improve the patient's inflammatory state and suppress immune disorders. In addition, the use of large doses of hormones in the early stages of the disease can also suppress the patient's overly excited immune state. Some patients are prone to relapse during the process of reducing hormones and need to be treated with immunosuppressants. The choice of immunosuppressant should be determined according to the severity of muscle, skin or lung involvement. If the patient develops gastrointestinal symptoms such as acid reflux, nausea, and vomiting while using hormones and immunosuppressants, some drugs that prevent excessive gastric acid secretion or protect the gastric mucosa can also be used for treatment.

[0004] Anti-TIF1-γ antibodies are an autoantibody first discovered 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 of DM combined with malignancy. However, currently available antibodies are limited in scope and difficult to meet testing needs. Therefore, the development of high-affinity anti-TIF1-γ antibodies is of great value in the diagnosis and testing of idiopathic inflammatory myopathies. Summary of the Invention

[0005] In response to the shortcomings of the prior art, the present invention aims to provide an anti-TIF1-γ monoclonal antibody and its application. The present invention constructs a phage-derived human antibody library using PBMC from patients with TIF1-γ antibodies. By specifically binding to TIF1-γ, humanized anti-TIF1-γ monoclonal antibodies that bind to TIF1-γ are screened. By specifically binding to TIF1-γ, these antibodies can quantitatively detect anti-TIF1-γ autoantibody levels in DM patients, thereby monitoring disease activity and providing clinical diagnosis for DM patients.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-TIF1-γ monoclonal antibody, wherein the heavy chain variable region CDR1 of the monoclonal antibody is shown in SEQ ID NO: 1, CDR2 is shown in SEQ ID NO: 2, and CDR3 is shown in SEQ ID NO: 3;

[0008] The light chain variable region CDR1 of the monoclonal antibody is shown in SEQ ID NO: 4, CDR2 is DDY, and CDR3 is shown in SEQ ID NO: 5;

[0009] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 7.

[0010] Monoclonal antibodies are the largest and fastest-growing group of therapeutic proteins. Currently, over 500 therapeutic antibodies and their derivatives are in clinical trials, with a focus on cancer treatment and autoimmune diseases. This method involves isolating PBMCs from patients with positive TIF1-γ antibodies, extracting RNA, and performing quality control. Qualified RNA is reverse-transcribed into cDNA using RT-PCR, and all antibody VH and VL gene segments are amplified. The in vitro amplified VK and Vλ gene segments are then cloned into the pATA-scFv-2 vector to construct a combinatorial antibody library.

[0011] The antibody gene combinatorial library is inserted immediately downstream of the leader series of gene III (g3) or gene VIII (g8) of the membrane protein encoded by the phage. By superinfection of the helper phage, the polypeptide expressed by the exogenous antibody gene can be displayed as a fusion protein at the N-terminus of the phage coat protein pIII or pVIII. Each phage particle encodes and presents a different antibody, which contains billions of individual clones. In these antibody libraries, the genes encoding those antibodies that can bind to the antigen are enriched by affinity enrichment-gentle elution-phage amplification for the antigen in vitro, and the above enrichment and screening process is repeated until an antibody phage library with good specificity and strong affinity is obtained after several cycles, and positive clones are screened from the antibody phage library. The positive clones are identified by ELISA, and finally fully human antibodies with good specificity and strong affinity are screened from them.

[0012] Preferably, the heavy chain amino acid sequence of the monoclonal antibody comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:8.

[0013] Preferably, the light chain amino acid sequence of the monoclonal antibody comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:9.

[0014] Preferably, the heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO: 8, and the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO: 9.

[0015] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-TIF1-γ monoclonal antibody according to the first aspect.

[0016] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0017] Fourthly, the present invention provides a host cell containing at least one copy of the expression vector described in the third aspect; or the host cell has the nucleic acid molecule described in the second aspect integrated into its chromosome.

[0018] In a fifth aspect, the present invention provides a composition for detecting TIF1-γ in a sample, wherein the composition comprises the anti-TIF1-γ monoclonal antibody described in the first aspect.

[0019] In a sixth aspect, the present invention provides a kit for detecting TIF1-γ in a sample, the kit comprising the anti-TIF1-γ monoclonal antibody described in the first aspect or the composition for detecting TIF1-γ in a sample described in the fifth aspect.

[0020] In the seventh aspect, the present invention provides the use of the anti-TIF1-γ monoclonal antibody described in the first aspect, the composition for detecting TIF1-γ in a sample described in the fifth aspect, or the kit for detecting TIF1-γ in a sample described in the sixth aspect in the preparation of a product for diagnosing and / or detecting idiopathic inflammatory myopathy.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention screened the antibody phage library three times, identifying clones with antigen expression levels greater than three times that of the control group as positive clones. These clones were then sequenced and analyzed. By eliminating erroneous and duplicated antibody sequences and combining the antigen-antibody specific binding capacity demonstrated by ELISA, the TIF1-γ-R7P1-C2 antibody was ultimately obtained. This antibody exhibits high activity, good stability, and strong specificity, enabling quantitative detection of anti-TIF1-γ autoantibody levels in patients with DM, and possesses significant application value in the detection and diagnosis of idiopathic inflammatory myopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the agarose gel electrophoresis diagram of the monoclonal PCR of the VL phage library.

[0024] Figure 2 This is the agarose gel electrophoresis diagram of the monoclonal PCR of the KH phage library.

[0025] Figure 3 This is the agarose gel electrophoresis diagram of monoclonal bacterial PCR of the λH phage library.

[0026] Figure 4 This is the SDS-PAGE electrophoresis diagram of purified TIF1-γ.

[0027] Figure 5 These are the OD values ​​of TIF1-γ-R7P1-C2 antibody at different dilution concentrations. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] Example 1

[0031] Method for constructing a human ScFv phage display library

[0032] The main reagents used in this embodiment are shown in Table 1.

[0033] Table 1

[0034] 1. Library construction

[0035] 1.1 Assembling the heavy chain variable region (VH) and light chain variable region (VL)

[0036] RNA from patients positive for TIF1-γ antibodies was reverse-transcribed into cDNA using the HiScript® III First-Strand cDNA Synthesis Kit (with gDNA Cleaner). VH and VL fragments of the DNA were amplified. PCR reaction conditions and procedures are shown in Table 2.

[0037] Table 2

[0038] Among them, the three steps of denaturation, annealing, and extension (1) were repeated 30 times.

[0039] The primer sequences are as follows:

[0040] Heavy chain variable region upstream primer (F):

[0041] 5'L-VH 1: acaggtgcccactcccaggtgcag. (SEQ ID NO:12)

[0042] 5'L-VH 3:aaggtgtccagtgtgargtgcag. (SEQ ID NO:13)

[0043] 5'L-VH 4 / 6: cccagatgggtcctgtcccaggtgcag. (SEQ ID NO:14)

[0044] 5'L-VH 5 / 7:caaggagtctgttccgaggtgcag. (SEQ ID NO:15)

[0045] Light chain variable region upstream primer (F):

[0046] 5'L VK 1 / 2: atgaggstcccygctcagctgctgg. (SEQ ID NO:16)

[0047] 5'L VK 3:ctcttcctcctgctactctggctcccag. (SEQ ID NO:17)

[0048] 5'L VK 4 / 5:atttctctgttgctctggatctctg. (SEQ ID NO:18)

[0049] 5'L Vλ 1: ggtcctgggcccagtctgtgctg. (SEQ ID NO:19)

[0050] 5' L Vλ 2: ggtcctgggcccagtctgccctg. (SEQ ID NO:20)

[0051] 5'L Vλ 3: gctctgtgacctcctatgagctg. (SEQ ID NO:21)

[0052] 5'L Vλ 4 / 5:ggtctctctcscagcytgtgctg. (SEQ ID NO:22)

[0053] 5'L Vλ 6:gttcttgggccaattttatgctg. (SEQ ID NO:23)

[0054] 5'L Vλ 7:ggtccaattcycaggctgtggtg. (SEQ ID NO:24)

[0055] 5'L Vλ 8 / 9 / 10: gagtggattctcagactgtggtg. (SEQ ID NO:25)

[0056] Light chain variable region downstream primer (R):

[0057] 3'CK: tgctgtccttgctgtcctgct. (SEQ ID NO:26)

[0058] 3'Cλ: caccagtgtggccttgttggcttg. (SEQ ID NO:27)

[0059] Heavy chain variable region downstream primer (R):

[0060] 3'VH:ACTCGAGACGGTGACCAGGGTGCC. (SEQ ID NO:28)

[0061] 1.2 Construction of light chain variable region phage display library

[0062] 1.2.1 Preparation of pATA-scFv-2 vector for library cloning

[0063] The VK and Vλ gene fragments obtained from in vitro amplification were cloned into the pATA-scFv-2 vector using cloning technology to form VK and Vλ libraries.

[0064] 1.2.2 The reaction system for digesting the vector and PCR product is shown in Table 3.

[0065] Table 3

[0066] 1.2.3 The connection system is shown in Table 4.

[0067] Table 4

[0068] The above ligation system was incubated at 16°C overnight and inactivated by heating at 65°C for 10 min.

[0069] 1.2.4 Electroporation

[0070] 1.2.4.1 Preparation of TG1 competent cells.

[0071] 1.2.4.2 Pre-warm 1 mL of SOC medium (Sigma, S1797) at 37°C. Place an electroporation cuvette (0.1 cm gap) and a microcentrifuge tube on ice (one cuvette and one microcentrifuge tube per transformation reaction).

[0072] 1.2.4.3 Remove the electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed (10-15 minutes). After thawing, gently mix the cells. Transfer 50 μL of cells to a frozen microcentrifuge tube on ice.

[0073] 1.2.4.4 Carefully add 3 μL of the DNA mixture to the chilled electroporation cuvette without creating bubbles. Quickly flick the cuvette downward with your wrist to allow the cells to settle to the bottom.

[0074] 1.2.4.5 Electroporate at 600 Ω, 10 μF, and 1.8 kV. Within 10 seconds of the pulse, immediately add 1 mL of pre-warmed SOC medium to each tube. Incubate at 37°C, shaking at 250 rpm for 1 hour.

[0075] 1.2.4.6 Collect all electroporation media. Serially dilute 10 μL of 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 inoculum volume.

[0076] 1.3 Construction of VL-VH phage display library

[0077] 1.3.1 Digestion of vector and PCR product, the digestion reaction system is shown in Table 5.

[0078] Table 5

[0079] 1.3.2 The ligation reaction system is shown in Table 6.

[0080] Table 6

[0081] The above-mentioned ligation reaction system was incubated at 16°C overnight and then inactivated by heating at 65°C for 10 min.

[0082] 1.3.3 Electroporation

[0083] 1.3.3.1 Preparation of TG1 competent cells.

[0084] 1.3.3.2 Preheat 4 mL of SOC medium (Sigma, S1797) to 37℃. Place the electroporation cuvettes (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube per conversion reaction).

[0085] 1.3.3.3 Remove the electrocompetent cells from the -80°C freezer and place them on ice until they are completely thawed (10-15 minutes). After thawing, gently mix the cells.

[0086] 1.3.3.4 Carefully add 6 μL of the DNA mixture to the chilled electroporation cuvette, avoiding the creation of bubbles. Quickly flick the cuvette downward with your wrist to allow the cells to settle to the bottom.

[0087] 1.3.3.5 Electroporation was performed at 600 Ω, 10 μF, and 2.5 kV. Within 10 seconds of the pulse, 2 mL of preheated SOC medium was immediately added to each tube. The tubes were then incubated at 37°C and 250 rpm for 1 hour with shaking.

[0088] 1.3.3.6 Collect all electroporation culture 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 inoculum volume.

[0089] 1.4 Library evaluation

[0090] 1.4.1 Colony PCR: Perform PCR using the constructed library as a template. The PCR reaction conditions are shown in Table 7.

[0091] Table 7

[0092] The three steps of denaturation, annealing, and extension (1) were repeated 30 times. The primer sequences are as follows: upstream primer (F) for identification of pATA-scFv-2 vector: agcggataacaatttcacacagga (SEQ ID NO: 29). downstream primer (R) for identification of pATA-scFv-2 vector: gcccccttattagcgtttgccatc (SEQ ID NO: 30). The results of agarose gel electrophoresis after PCR are shown in Figure 2. Figure 1-Figure 3 shown. Figure 1 It is the monoclonal bacterial PCR agarose gel electrophoresis diagram of the VL phage library; wherein, lane M: DL2000, lanes 1-16 pATA-VK, lanes 17-32 pATA-Vλ. Figure 2 This is a PCR agarose gel electrophoresis image of a single clone of the KH phage library; lane M: DL2000, lanes 1-48 pATA-scFv-KH. Figure 3 It is the agarose gel electrophoresis diagram of the monoclonal bacterial PCR of the λH phage library; wherein, lane M: DL2000, lanes 1-48: pATA-scFv-λH.

[0093] 1.4.2 Sequencing: Positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. Sequencing quality control results are shown in Tables 8 and 9. Table 8 shows the IGLV germline gene analysis, and Table 9 shows the IGHV germline gene analysis. The tables show that the library's heavy and light chain diversity is relatively comprehensive, covering most major families of germline genes.

[0094] Table 8

[0095] Table 9

[0096] 1.5 Expression of TIF1-γ protein

[0097] The TIF1-γ gene sequence was artificially synthesized and recombined into the expression vector plasmid pET28b to obtain the TIF1-γ-pET28b expression vector; the cloning site was Nde1 / Xho1, and the amino acid sequence of TIF1-γ was shown in SEQ ID NO: 31. The gene sequence was shown in SEQ ID NO: 32.

[0098] The TIF1-γ-pET28b expression vector was transfected into BL21 (DE3) competent cells and cultured. The precipitate was collected and subjected to His tag affinity chromatography to obtain TIF1-γ protein. The purified TIF1-γ also needed to be subjected to SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis) to verify its purity. The SDS-PAGE electrophoresis of the purified TIF1-γ is shown in the figure below. Figure 4 As shown, the protein size is 47.18 KDa and the purity is greater than 90%.

[0099] Example 2

[0100] Preparation of monoclonal antibodies specifically binding to TIF1-γ

[0101] The main reagents used in this example are shown in Table 10.

[0102] Table 10

[0103] 1First round of selection

[0104] 1.1 Immunotube solid phase panning

[0105] 1.1.1 Coating: Coat the centrifuge tubes and incubate overnight at 4°C. Antigen group: 1 mL TIF1-γ transfection solution (50 μg / mL); control group: 500 μL transfection solution (0 μg / mL).

[0106] 1.1.2 Washing: Discard the liquid in the centrifuge tube and wash three times with 5 mL 0.05% PBST.

[0107] 1.1.3 Blocking: Add 5 mL of 5% skim milk or 1% casein (dissolved in PBST) to the tube and block at 37°C for 1 hour.

[0108] 1.1.4 Washing: Discard the liquid in the centrifuge tube and wash once with 5 mL PBS.

[0109] 1.1.5 Incubation: 1×10 12 The pfu phage library (or the amplified phage from the previous round) was negatively screened with 5% skim milk or 1% casein (dissolved in PBST) + 100 μg control his protein (an irrelevant protein with the same his tag) at room temperature for 30 minutes. The negatively screened library was added to the immunotube and incubated at 30°C for 2 hours.

[0110] 1.1.6 Washing: Discard the liquid in the centrifuge tube and wash 4-6 times with 5 mL 0.05% PBST.

[0111] 1.1.7 Elution: Elute the phage with 1 mL of glycine-HCl (pH 2.2), incubate at room temperature with shaking for about 6-8 minutes, and add 120-130 μL of Tris-HCl (pH 9.6) to neutralize the solution to pH 7.0-8.0.

[0112] 1.1.8 Dilute the eluted phage and infect E. coli TG1 in the logarithmic phase. Plate and determine the titer.

[0113] 1.2 Amplification of eluted phage

[0114] 1.2.1 Pipette the eluted phage and add it to the logarithmic phase E. coli TG1 culture. After standing at 37°C for 30 minutes, incubate at 220 rpm for 30 minutes to 1 hour.

[0115] 1.2.2 Add antibiotic Amp to the culture medium and culture at 37°C and 220 rpm until the bacterial solution OD is about 0.4-0.6.

[0116] 1.2.3 Add helper phage to the bacterial solution, incubate at 37°C for 30 minutes, and then incubate at 220 rpm for 45 minutes to 1 hour.

[0117] 1.2.4 Centrifuge the bacterial suspension at 3000-5000 rpm, discard the supernatant, resuspend the bacteria in an equal volume of 2YT-Amp-Kan medium, and culture overnight at 30°C and 220 rpm.

[0118] 1.2.5 The next day, centrifuge the bacterial suspension at 8000 rpm at 4°C for 20 minutes. Transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5× PEG / NaCl solution, mix thoroughly, and place on ice or at 4°C for 1-2 hours.

[0119] Centrifuge at 8000 rpm at 64°C for 30 minutes and discard the supernatant. Resuspend the pellet in about 1 mL of PBS. Centrifuge at 12000 rpm for 5 minutes and transfer the supernatant to a new centrifuge tube.

[0120] 1.2.7 Dilute the amplified phage, infect TG1 in the logarithmic phase, and plate to determine the titer.

[0121] 2 Second to eighth rounds of selection

[0122] The amplified phages were used for the second round of panning, following the same steps as 1.1 and 1.2. The panning was repeated for multiple rounds. The panning results are shown in Table 11.

[0123] Table 11

[0124] 3. Polyclonal phage ELISA detection

[0125] 3.1 Coat the ELISA 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.

[0126] 3.2 Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0127] 3.3 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.

[0128] 3.4 Dilute the amplified phage after each round with PBS in 3-fold increments. The initial concentration is 1 × 1012 pfu / mL. Add 100 μL of diluted amplified phage to each well; incubate at 30°C for 1 hour. Wash 4–6 times with 300 μL of PBST.

[0129] 3.5 Add 100 μL of anti-M13-HRP antibody (1:6000) diluted in blocking buffer to each well, incubate at 30°C for 1 hour, and wash 4-6 times with 300 μL of PBST.

[0130] 3.6 Add 100 μL of TMB to each well, incubate at room temperature for 3-8 minutes, and then add 100 μL of 2 M HCl to each well to terminate the reaction.

[0131] 3.7 The values ​​were read using a microplate reader at 450 nm-620 nm. Table 12 shows the results of the polyclonal phage ELISA.

[0132] Table 12

[0133] 4 Monoclonal phage ELISA screening

[0134] 4.1 Select the appropriate round number, dilute the eluted phage to an appropriate concentration, infect TG1 cells in the logarithmic phase, and plate them.

[0135] 4.2 The next day, pick 96 (or more) single colonies from the plate, inoculate into a 96-deep-well plate, and culture at 37°C, 250 rpm, with shaking until the bacterial suspension reaches an OD value of 0.4-0.6.

[0136] 4.3 Add helper phage to the culture medium in a 96-well plate and incubate at 37°C for 30 minutes. Then, shake and culture at 37°C and 250 rpm for 45 minutes to 1 hour.

[0137] 4.4 Centrifuge the 96-well plate at 4000 rpm for 5 minutes and discard the supernatant. Resuspend the bacterial solution in each well with 2YT-Amp-Kan medium and culture with shaking at 30°C and 250 rpm overnight.

[0138] 4.5 The next day, centrifuge the 96-well plate at 4000 rpm for 10-15 minutes and collect the supernatant for ELISA assay.

[0139] 4.6 Coat the ELISA 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.

[0140] 4.7 Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0141] 4.8 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.

[0142] 4.9 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.

[0143] 4.10 Add 100 μL of anti-M13-HRP antibody (1:6000) diluted in blocking buffer to each well, incubate at 30°C for 1 hour, and wash 4-6 times with 300 μL of PBST.

[0144] 4.11 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.

[0145] 4.12 Read the values ​​using a microplate reader at 450 nm-620 nm and sequence the highly specific clones.

[0146] 5 positive clones secondary validation ELISA

[0147] 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 secondary ELISA testing according to steps 4.2-4.12 of step 4 to ensure the authenticity of the positive results. Table 13 shows the results of the positive monoclonal phage ELISA.

[0148] Table 13

[0149] The full sequence sequencing results of the TIF1-γ-R7P1-C2 antibody are as follows:

[0150] TIF1-γ-R7P1-C2 antibody:

[0151] The heavy chain variable region CDR1 is shown in SEQ ID NO: 1: GYTFTGYY.

[0152] The heavy chain variable region CDR2 is shown in SEQ ID NO: 2: MNPNSGNT.

[0153] The heavy chain variable region CDR3 is shown in SEQ ID NO: 3: AAEGAYGDSGDV.

[0154] The light chain variable region CDR1 is shown in SEQ ID NO: 4: NIGNKR.

[0155] The light chain variable region CDR2 is DDY.

[0156] The light chain variable region CDR3 is shown in SEQ ID NO: 5: QLWDDDSDHVV.

[0157] The amino acid sequence of the heavy chain variable region of the TIF1-γ-R7P1-C2 antibody is shown in SEQ ID NO: 6:

[0158] EVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVAMTRDTSISTAYMELSGLTSDDTAVYYCAAEGAYGDSGDVWGQGTMVTVSS.

[0159] The amino acid sequence of the light chain variable region of the TIF1-γ-R7P1-C2 antibody is shown in SEQ ID NO: 7:

[0160] SYELTQPLSVSAAPGTTATFTCGGDNIGNKRVHWYQQRPGQAPVLVVSDDYDRPSGIPERFSGSHSGEAAILTISGVEAGDEADYYCQLWDDDSDHVVFGGGTQLTVL.

[0161] The base sequence of the heavy chain of the TIF1-γ-R7P1-C2 antibody is SEQ ID NO: 10.

[0162] The amino acid sequence of the heavy chain of the TIF1-γ-R7P1-C2 antibody is SEQ ID NO:8.

[0163] The base sequence of the light chain of the TIF1-γ-R7P1-C2 antibody is SEQ ID NO:11.

[0164] The light chain amino acid sequence of the TIF1-γ-R7P1-C2 antibody is SEQ ID NO:9.

[0165] Example 3

[0166] OD values ​​of antibodies at different dilution concentrations detected by ELISA

[0167] The experimental steps of enzyme-linked immunosorbent assay (ELISA) are as follows:

[0168] 1. Coating: Coat the microplate with 100 μL of TIF1-γ protein (4 μg / mL) per well and incubate overnight at 4°C.

[0169] 2. Washing: Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0170] 3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0171] 4. Positive antibody incubation: dilute the TIF1-γ-R7P1-C2 antibody in a serial dilution series, add 100 μL of the diluted antibody solution to each well, and incubate at 37°C for 1 hour.

[0172] 5. Washing: Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0173] 6. Secondary antibody incubation: Dilute Goat Anti-Human IgG (H+L) antibody (Jackson, code: 109-035-088) 10000 times with blocking buffer, add 100 μL of diluted secondary antibody to each well, and incubate at 37°C for 30 minutes.

[0174] 7. Washing: Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0175] 8. Color development: Add 100 μL TMB to each well, incubate at 37°C for 10 minutes, and then add 50 μL 2 M HCl to each well to terminate the reaction.

[0176] 9. Read the plate: Use a microplate reader to read the values ​​at 450 nm-620 nm, such as Figure 5 shown.

[0177] Figure 5 The results showed that the screened human anti-TIF1-γ monoclonal antibody had a strong ability to specifically bind to TIF1-γ.

[0178] In summary, this invention has screened and obtained a high-affinity antibody, named TIF1-γ-R7P1-C2. This antibody exhibits high activity, good stability, and strong specificity, enabling quantitative detection of anti-TIF1-γ autoantibody levels in DM patients, and has significant application value in the diagnosis and detection of dermatomyositis.

[0179] The applicant declares that the above description is only a specific embodiment 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 those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-TIF1-γ monoclonal antibody, characterized in that: The heavy chain variable region CDR1 of the monoclonal antibody is shown in SEQ ID NO: 1, CDR2 is shown in SEQ ID NO: 2, and CDR3 is shown in SEQ ID NO: 3; The light chain variable region CDR1 of the monoclonal antibody is as shown in SEQ ID NO:4, CDR2 is DDY, and CDR3 is as shown in SEQ ID NO:5; The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

7.

2. The anti-TIF1-γ monoclonal antibody according to claim 1, characterized in that The heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO:8, and the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO:

9.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-TIF1-γ monoclonal antibody according to claim 1 or 2.

4. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that The host cell contains at least one copy of the expression vector of claim 4; or the nucleic acid molecule of claim 3 is integrated into the chromosome of the host cell.

6. A composition for detecting TIF1-γ in a sample, characterized in that: The composition comprises the anti-TIF1-γ monoclonal antibody according to claim 1 or 2.

7. A kit for detecting TIF1-γ in a sample, characterized in that: The kit comprises the anti-TIF1-γ monoclonal antibody according to claim 1 or 2 or the composition for detecting TIF1-γ in a sample according to claim 6.

8. Use of the anti-TIF1-γ monoclonal antibody according to claim 1 or 2, the composition for detecting TIF1-γ in a sample according to claim 6, or the kit for detecting TIF1-γ in a sample according to claim 7 in the preparation of a product for diagnosing and / or detecting idiopathic inflammatory myopathy.

Images