Antibody for identifying TIF1-gamma and application thereof

Abstract

The invention belongs to the field of biological medicine, and provides an antibody for identifying TIF1-gamma and application thereof, and the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID NO: 1; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 5; the heavy chain variable region comprises a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3 and a CDR3 as shown in SEQ ID NO: 4; the light chain variable region comprises CDR1 as shown in SEQ ID NO: 6, CDR2 is DVN, and CDR3 as shown in SEQ ID NO: 7. The antibody disclosed by the invention has the characteristics of good specificity and strong affinity, can be used for quantitatively detecting the level of the anti-TIF1-gamma autoantibody in a DM patient so as to monitor the illness state activity, and can be used for clinical diagnosis of the DM patient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to an antibody for identifying TIF1-γ and its application. Background Art

[0002] TIF1-γ (Transcriptional Intermediary Factor 1 Gamma), also known as TRIM33 (Tripartite Motif Containing 33), is a transcriptional regulatory protein belonging to the TIF1 family. It plays an important role in cell differentiation, development and tumorigenesis by participating in chromatin remodeling and transcriptional regulation. Early studies found that TIF1-γ plays a key role in embryonic development and cell differentiation. Further research revealed that TIF1-γ regulates gene expression by binding to chromatin and transcription factors, and it participates in the TGF-β signaling pathway, affecting cell proliferation and differentiation.

[0003] TIF1-γ antibodies have shown potential in cancer diagnosis and prognosis assessment, especially in the immunotherapy of autoimmune diseases and cancers. Anti-TIF1-γ antibodies are novel autoantibodies specific for DM, also known as anti-p155 or anti-155 / 140 antibodies. TIF1-γ has received extensive attention because it can act as a transcriptional cofactor to block the TGF-β / Smad signaling pathway. Clinically, anti-TIF1-γ antibodies in adult DM patients are associated with the occurrence of malignant tumors. The typical TGF-β signaling pathway is that TGF-β first binds to the type II TGF-β receptor (TGFβRII), and then forms a complex with the type I TGF-β receptor (TGFβRI) to activate it. TGFβRI phosphorylates and activates the members of R-Smad (Smad1, 2, 3, 5, 8), and R-Smad then binds to Co-Smad (Smad4) to form a complex that translocates into the nucleus to regulate the transcription of target genes.

[0004] The TIF1-γ protein belongs to the TRIMs (Tripartite motif-containing) family of proteins and has a RING domain, which can act as a ubiquitin ligase E3 and plays an important role in the process of tumorigenesis, such as participating in the regulation of the apoptosis-related protein P53 and inhibiting the activity of the TGF-β signaling pathway. It is speculated that autoantibodies against these proteins may be produced during tumorigenesis, thus promoting the occurrence of cancer. Therefore, measuring the level of anti-TIF1-γ autoantibodies in DM patients lays an experimental foundation for the study of the pathogenesis of DM complicated with malignant tumors, and provides important clues for clinical disease prediction, prognosis assessment and treatment plan selection for IIM patients.

[0005] Both the detection methods and treatment regimens for patients with TIF1-γ antibody-positive DM lack specificity, with poor efficacy and a long treatment course. In China, the detection methods for anti-TIF1-γ antibodies are all qualitative immunoblotting and immunoblot strip methods, and the consistency of the detection results is poor, and the level of anti-TIF1-γ antibodies in the patient's serum cannot be quantitatively detected. At present, there is a lack of effective detection means for quickly and accurately diagnosing such diseases and quantitatively detecting the level of anti-TIF1-γ antibodies in the patient's serum. 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 that recognizes TIF1-γ and its application. The present invention constructs a phage human antibody library with PBMC from patients positive for TIF1-γ antibody, and through specific binding to TIF1-γ, a human-derived anti-TIF1-γ antibody that specifically binds to TIF1-γ is screened. The antibody has high activity, good stability, and strong specificity, and can quantitatively detect the level of anti-TIF1-γ autoantibodies in DM patients.

[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 that recognizes TIF1-γ. The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:5;

[0009] The heavy chain variable region includes CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4;

[0010] The light chain variable region includes CDR1 as shown in SEQ ID NO:6, CDR2 is DVN, and CDR3 as shown in SEQ ID NO:7.

[0011] The present invention isolated PBMC from patients positive for TIF1-γ antibody and extracted RNA, reverse transcribed the RNA with qualified quality inspection into cDNA by RT-PCR technology, amplified all antibody VH and VL gene fragments therein, and constructed an antibody library; high-affinity antibodies were expressed and screened by phage display technology.

[0012] Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO:9.

[0013] In a second aspect, the present invention provides a method for preparing the antibody that recognizes TIF1-γ described in the first aspect, the method comprising: synthesizing a nucleic acid molecule encoding the antibody that recognizes TIF1-γ described in the first aspect, constructing a recombinant expression vector, and introducing the recombinant expression vector into a host cell, screening positive host cells for culture to obtain the antibody that recognizes TIF1-γ.

[0014] In a third aspect, the present invention provides a nucleic acid molecule that encodes the antibody that recognizes TIF1-γ described in the first aspect.

[0015] In a fourth aspect, the present invention provides an expression cassette that contains the nucleic acid molecule encoding the antibody that recognizes TIF1-γ described in the first aspect.

[0016] In a fifth aspect, the present invention provides a recombinant expression vector that contains the nucleic acid molecule described in the third aspect or the expression cassette described in the fourth aspect.

[0017] In a sixth aspect, the present invention provides a recombinant cell that expresses the antibody that recognizes TIF1-γ described in the first aspect.

[0018] In a seventh aspect, the present invention provides an immunohistochemistry kit for detecting TIF1-γ, the kit containing the antibody that recognizes TIF1-γ described in the first aspect, and any one or at least two combinations of antigen retrieval buffer, blocking solution, enzyme-labeled secondary antibody, chromogenic agent or hematoxylin counterstain solution.

[0019] In an eighth aspect, the present invention provides an enzyme-linked immunosorbent assay kit for detecting TIF1-γ, the kit containing the antibody that recognizes TIF1-γ described in the first aspect, and any one or at least two combinations of blocking solution, enzyme-labeled secondary antibody, chromogenic solution or termination solution.

[0020] In a ninth aspect, the present invention provides the use of any one or at least two combinations of the antibody that recognizes TIF1-γ described in the first aspect, the immunohistochemistry kit for detecting TIF1-γ described in the seventh aspect or the enzyme-linked immunosorbent assay kit for detecting TIF1-γ described in the eighth aspect in the preparation of products for diagnosing and / or detecting idiopathic inflammatory myopathy.

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

[0022] Through multiple rounds of screening of antibody phage libraries and combined with ELISA identification, the present invention screened a high-affinity antibody named TIF1-γ-R8P1-B2. The antibody has high activity, good stability, and strong specificity, and can quantitatively detect the level of anti-TIF1-γ autoantibodies in DM patients, and has important application value in the detection and diagnosis of idiopathic inflammatory myopathy. Brief Description of the Drawings

[0023] Figure 1 It is the OD value result under different dilution concentrations of the antibody detected by ELISA. Detailed Embodiments

[0024] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0025] For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be purchased through regular channels.

[0026] Example 1

[0027] 1. Construction of a human ScFv phage display library

[0028] (1) Isolation of PBMC from TIF1-γ antibody-positive patients

[0029] Take a sterile centrifuge tube, add 5 mL of lymphocyte separation medium, and then slowly add the blood sample. Centrifuge at 500 g for 25 minutes at 20 °C. After centrifugation, carefully aspirate the circular milky white (human) lymphocyte layer in the centrifuge tube and transfer it to a new centrifuge tube. Add 10 mL of PBS to the centrifuge tube containing (human) lymphocytes, mix the cells evenly. Centrifuge at 250 g for 10 minutes, and discard the supernatant. Resuspend the obtained cells with 5 mL of PBS, centrifuge at 250 g for 10 minutes, and discard the supernatant. Resuspend the obtained cells with 5 mL of PBS, centrifuge at 250 g for 10 minutes, and discard the supernatant. Resuspend the obtained cells with 0.5 mL of PBS for standby.

[0030] (2) RNA extraction and RNA quality control

[0031] Thaw the PBMC (Trizol) stored at -80°C at room temperature. Add 1 / 5 volume of chloroform to each tube. Vortex for 15 s to fully emulsify and make the solution milky white. Let it stand for 5 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes. Take the colorless supernatant and add an equal volume of isopropanol. Invert the tube up and down to mix well and let it stand at -20°C for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 10 minutes and discard the supernatant. Slowly add 75% ethanol along the tube wall. Gently invert the centrifuge tube up and down. Centrifuge at 12,000 rpm at 4°C for 5 minutes and discard the supernatant. Dry at room temperature in a laminar flow hood for 2 - 5 minutes and add an appropriate amount of RNase-free water to dissolve.

[0032] (3)RNA Reverse Transcription

[0033] Prepare the reverse transcription amplification PCR reaction system. The enzyme used for amplification is Phanta®Max Ultra-Fidelity DNA Polymerase (Phanta®Max Ultra-Fidelity DNA Polymerase, product number P505-d3, manufacturer Vazyme). The amplification reaction system is shown in Table 1.

[0034] Table 1

[0035]

[0036] Among them, the heavy chain variable region upstream primer F includes:

[0037] 5’L-VH 1: acaggtgcccactcccaggtgcag. (SEQ ID NO:12)

[0038] 5’L-VH 3: aaggtgtccagtgtgargtgcag. (SEQ ID NO:13)

[0039] 5’L-VH 4 / 6: cccagatgggtcctgtcccaggtgcag. (SEQ ID NO:14)

[0040] 5’L-VH 5 / 7: caaggagtctgttccgaggtgcag. (SEQ ID NO:15)

[0041] The light chain variable region upstream primer F includes:

[0042] 5’L VK 1 / 2: atgaggstcccygctcagctgctgg. (SEQ ID NO:16)

[0043] 5’L VK 3: ctcttcctcctgctactctggctcccag. (SEQ ID NO:17)

[0044] 5’L VK 4 / 5: atttctctgttgctctggatctctg. (SEQ ID NO:18)

[0045] 5’L Vλ 1: ggtcctgggcccagtctgtgctg. (SEQ ID NO:19)

[0046] 5’ L Vλ 2: ggtcctgggcccagtctgccctg. (SEQ ID NO:20)

[0047] 5’L Vλ 3: gctctgtgacctcctatgagctg. (SEQ ID NO:21)

[0048] 5’L Vλ 4 / 5: ggtctctctcscagcytgtgctg. (SEQ ID NO:22)

[0049] 5’L Vλ 6: gttcttgggccaattttatgctg. (SEQ ID NO:23)

[0050] 5’L Vλ 7: ggtccaattcycaggctgtggtg. (SEQ ID NO:24)

[0051] 5’L Vλ 8 / 9 / 10: gagtggattctcagactgtggtg. (SEQ ID NO:25)

[0052] The downstream primer R of the heavy chain variable region includes:

[0053] 3’VH: ACTCGAGACGGTGACCAGGGTGCC. (SEQ ID NO:26)

[0054] The downstream primer R of the light chain variable region includes:

[0055] 3’CK: tgctgtccttgctgtcctgct. (SEQ ID NO:27)

[0056] 3’Cλ: caccagtgtggccttgttggcttg. (SEQ ID NO:28)

[0057] The procedure of reverse transcription amplification PCR 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; extension (1) at 72°C for 20 s; extension (2) at 72°C for 5 min; incubation at 4°C. Among them, the three steps of denaturation, annealing, and extension (1) are repeated 30 times.

[0058] (4) Construction of VK library and Vλ library

[0059] Vector fragment:

[0060] Digest the pATA-scFv-2 plasmid (manufacturer ProteoGenix): Digest the pATA-scFv plasmid with NheI + NotI at 37°C for 5 h, cut the gel and recover the digested vector.

[0061] PCR product fragment:

[0062] Digest the PCR products of VK and Vλ fragments: Digest the Vκ and Vλ fragments obtained by PCR with NheI + NotI at 37°C for 5 h, cut the gel and recover the digested fragments.

[0063] Ligation of vector and PCR product fragments: Ligate the digested pATA-scFv-2 plasmid with the VK fragment to obtain the pATA-VK plasmid. Ligate the digested pATA-scFv-2 vector with the Vλ fragment to obtain the pATA-Vλ plasmid.

[0064] Transform TG1 competent cells (TG1 electrocompetent cells, product number 60502, manufacturer Lucigen): Electroporate the obtained pATA-VK plasmid and pATA-Vλ plasmid into TG1 competent cells respectively, and collect all the electroporation media. Continuously dilute 10 μL of the culture into 90 μL of SOC medium, and spread it 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 inoculated on the plate.

[0065] (5) Construction of VL-VH library

[0066] Vector fragment:

[0067] Digest the pATA-scFv-VK plasmid and pATA-scFv-Vλ plasmid: Digest the pATA-scFv-VK plasmid and pATA-scFv-Vλ with sfiI and XhoI respectively, cut the gel and recover the digested vector.

[0068] PCR product fragment:

[0069] Digest the PCR product of the VH fragment: Digest the VH fragment obtained by PCR with sfiI and XhoI, cut the gel and recover the digested fragment.

[0070] Ligate the vector and the PCR product fragment: Ligate the digested pATA-scFv-VK plasmid with the VH fragment to obtain the pATA-scFv-KH plasmid; Ligate the digested pATA-scFv-Vλ plasmid with the VH fragment to obtain the pATA-scFv-λH plasmid.

[0071] Transform TG1 competent cells: Electroporate the obtained pATA-scFv-KH plasmid and pATA-scFv-λH plasmid into TG1 competent cells, and collect all the electroporation media. Continuously dilute 10 μL of the culture into 90 μL of SOC medium, and spread it on an LB / Amp / glucose agar plate. 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 inoculated on the plate.

[0072] (6)Library evaluation

[0073] Colony PCR: Use the constructed library as a template for PCR. The PCR system is shown in Table 2, and the PCR program is shown in Table 3.

[0074] Table 2

[0075]

[0076] Table 3

[0077]

[0078] Forward primer (F) for pATA-scFv-2 vector identification: agcggataacaatttcacacagga (SEQ ID NO:29).

[0079] Reverse primer (R) for pATA-scFv-2 vector identification: gcccccttattagcgtttgccatc (SEQ ID NO:30).

[0080] After colony PCR, the results showed that the sizes of the PCR fragments were all around 1.1 - 1.3 kb as expected, indicating that the insertion rate of the library reached 100%.

[0081] (7)Prepare phage particles displaying antibodies.

[0082] Inoculate the original bacterial solution into 2YT-Amp-Glucose medium to make the OD of the bacterial solution = 0.1, and culture at 37°C and 220 rpm until the OD 600 reaches 0.4 - 0.6.

[0083] Add helper phage (M13KO7 helper phage, catalog number N0315S, manufacturer NEB) to the culture medium. After standing at 37 °C for 30 minutes, culture at 220 rpm for 45 - 60 minutes.

[0084] Centrifuge the bacterial liquid and discard the supernatant. Resuspend the cells with an equal volume of 2YT - Amp - Kan medium. Culture overnight at 30 °C and 220 rpm.

[0085] Centrifuge the bacterial liquid, transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5×PEG / NaCl solution. Mix well and let stand on ice for 1 - 2 h. Centrifuge at 10000 g for 30 minutes, collect the precipitate and resuspend it with PBS buffer. Store it short - term at 4 °C or add glycerol and store it long - term at - 80 °C.

[0086] 2. Antibody screening

[0087] (1) Immunotube solid - phase panning

[0088] Antigen coating: Coat centrifuge tubes and incubate overnight at 4 °C. Antigen group: 1 mL of TIF1 - γ transfection solution (50 μg / mL), control group: 500 μL of transfection solution (0 μg / mL). The next day, discard the liquid in the centrifuge tubes and wash three times with 5 mL of 0.05% PBST.

[0089] Blocking: 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.

[0090] Binding of phage library to antigen: 1×10 12 pfu phage library (or the amplified phage from the previous round) is pre - incubated with 5% skim milk or 1% casein (dissolved in PBST) + 100 μg of control his protein (an irrelevant protein with the same his - tag) at room temperature for 30 minutes for negative selection. Then 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.

[0091] Elution: Elute the phage 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 adjust the pH to 7.0 - 8.0. Dilute the eluted phage and infect exponentially - growing Escherichia coli TG1, and plate to determine the titer.

[0092] (2) Amplification of eluted phage

[0093] Aspirate the eluted phages and add them to the exponentially growing 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 medium and culture at 37 °C and 220 rpm until the OD of the bacterial solution reaches 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 pellet in an equal volume of 2YT - Amp - Kan 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 and let stand for 1 - 2 hours. Centrifuge at 4 °C and 8000 rpm for 30 minutes, 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. Dilute the amplified phages, infect exponentially growing TG1, and plate to determine the titer.

[0094] The amino acid sequence of the TIF1 - γ protein in the TIF1 - γ transfection solution is shown in SEQ ID NO:31.

[0095] Repeat the above steps of immunotube solid - phase panning and amplification of eluted phages.

[0096] 3. Monoclonal isolation and identification

[0097] (1) Pick monoclonal colonies from the last round of screening for amplification and verify the positive monoclonal phages by ELISA.

[0098] Dilute the eluted phage from the last round to an appropriate concentration, infect exponentially growing TG1, and plate.

[0099] The next day, pick monoclonal colonies from the plate and inoculate them into 600 μL of 2YT - Amp - Glucose medium, culture at 37 °C and 250 rpm with shaking for 2 h. After the OD of the bacterial solution reaches 0.4 - 0.6, add helper phages. Stand at 37 °C for 30 minutes, then culture at 250 rpm with shaking for 45 minutes. Centrifuge for 5 minutes and discard the supernatant. Resuspend the bacterial pellet in 2YT - Amp - Kan medium in each well, and culture at 30 °C and 250 rpm with shaking overnight.

[0100] The next day, centrifuge for 10 - 15 minutes and take the supernatant for ELISA experiment. Coat the immunoplates: antigen protein (4 μg / mL in PBS buffer), 100 μL, overnight at 4°C. Coat the control wells with 100 μL PBS. Wash 3 times with 300 μL PBST. Block with 300 μL of 5% skim milk / PBST at 30°C for 1 hour. Wash 3 times with 300 μL PBST. Add 100 μL of phage supernatant to each well and incubate at 30°C for 1 hour. Wash 3 times with 300 μL PBST. Add 100 μL of secondary antibody dilution (anti - M13 - HRP, 1:5000) and incubate at 30°C for 1 hour. Wash 3 times with 300 μL PBST. Add 100 μL of chromogenic solution TMB and develop color in the dark for 5 minutes, then add 100 μL of 2M HCl to terminate the reaction. Read the absorbance with an ELISA reader (450 - 620 nm).

[0101] The results are shown in Table 4.

[0102] Table 4

[0103]

[0104] In the table, 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.

[0105] From the above results, it can be seen that TIF1 - γ - R8P1 - B2 has obvious specific binding to the antigen.

[0106] The selected phage positive clones were subjected to whole - sequence sequencing to obtain the corresponding antibody heavy and light chains, and the whole sequence is as follows:

[0107] The heavy - chain variable region of the TIF1 - γ - R8P1 - B2 antibody is shown in SEQ ID NO:1:

[0108] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGSGSPRGGLDIWGQGTMVTVSS.

[0109] The CDR1 - 3 of the heavy - chain variable region of the TIF1 - γ - R8P1 - B2 antibody are shown as follows:

[0110] CDR1, SEQ ID NO:2: GYTFTSYY.

[0111] CDR2, SEQ ID NO:3: INPSGGST.

[0112] CDR3, SEQ ID NO:4: ARGSGSPRGGLDI.

[0113] The light chain variable region of the TIF1-γ-R8P1-B2 antibody is as shown in SEQ ID NO:5:

[0114] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVNNRPSGVAYRFSGSKSGNTASLSISGLQAEDEADYYCSSFTSSRTYVFGTGTKVTVL.

[0115] The CDR1-3 of the light chain variable region of the TIF1-γ-R8P1-B2 antibody are as follows:

[0116] CDR1, SEQ ID NO:6: SSDVGGYNY.

[0117] CDR2, DVN.

[0118] CDR3, SEQ ID NO:7: SSFTSSRTYV.

[0119] The heavy chain amino acid sequence of the TIF1-γ-R8P1-B2 antibody is as shown in SEQ ID NO:8.

[0120] The heavy chain base sequence of the TIF1-γ-R8P1-B2 antibody is as shown in SEQ ID NO:10.

[0121] The light chain amino acid sequence of the TIF1-γ-R8P1-B2 antibody is as shown in (SEQ ID NO:9).

[0122] The light chain base sequence of the TIF1-γ-R8P1-B2 antibody is as shown in SEQ ID NO:11.

[0123] Example 2

[0124] ELISA was used to detect the OD values under different dilution concentrations of the antibody. The experimental steps of the enzyme-linked immunosorbent assay (ELISA) are as follows:

[0125] 1. Coating: Coat the enzyme-linked immunosorbent assay (ELISA) plate with 100 μL / well of TIF1-γ protein (4 μg / mL) and incubate overnight at 4°C.

[0126] 2. Washing: Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0127] 3. Blocking: Add 300 μL of 5% skim milk (dissolved in PBS) to each well and block at 37°C for 2 hours.

[0128] 4. Incubation with primary antibody: Gradient dilute the TIF1-γ-R8P1-B2 antibody and add 100 μL of the diluted antibody solution to each well. Incubate at 37°C for 1 hour.

[0129] 5. Washing: Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0130] 6. Incubation with secondary antibody: Dilute the 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 and incubate at 37°C for 30 minutes.

[0131] 7. Washing: Discard the liquid in the ELISA plate and wash each well three times with 300 μL of 0.05% PBST.

[0132] 8. Color development: Add 100 μL of TMB to each well and incubate at 37°C for 10 minutes. Then add 50 μL of 2 M HCl to each well to terminate the reaction.

[0133] 9. Reading the plate: Read the values using an ELISA reader at 450 nm - 620 nm, as Figure 1 shown.

[0134] Figure 1 The results of... indicate that the screened human-derived anti-TIF1-γ monoclonal antibody has a strong ability to specifically bind to TIF1-γ.

[0135] Example 3

[0136] The experimental steps for detecting the immunohistochemistry of TIF1-γ are as follows:

[0137] 1. Sample preparation: Prepare cell samples, perform FFPE embedding and sectioning. Perform antigen retrieval using a sodium citrate antigen retrieval solution in a microwave oven.

[0138] 2. Block with 3% BSA solution.

[0139] 3. Discard the blocking solution, add the TIF1-γ antibody dropwise, and place the sections flat in a wet box and incubate overnight at 4°C.

[0140] 4. Wash the sections with PBS, add the secondary antibody dropwise to cover the tissue, and incubate at room temperature for 30 minutes.

[0141] 5. After washing the sections with PBS, add freshly prepared DAB chromogenic solution dropwise and control the chromogenic time under the microscope. When it turns yellowish brown, immediately rinse the sections with tap water to terminate the chromogenesis.

[0142] 6. Counterstain with hematoxylin for about 3 minutes and rinse with tap water. Then differentiate with hematoxylin differentiating solution for several seconds and rinse again. Finally, blue with hematoxylin bluing solution and rinse with running water.

[0143] 7. Dehydrate and clear the sections in sequence, and then mount the sections with neutral balsam.

[0144] 8. Judge the staining intensity under the microscope.

[0145] In the experimental results, the control cells did not express, while the positive cells expressed, indicating that the TIF1-γ antibody provided by the present invention can be used for immunohistochemical detection.

[0146] In summary, the present invention provides an antibody that recognizes TIF1-γ and its application. The antibody has high activity, good stability, and strong specificity, and has important application value in the detection of TIF1-γ.

[0147] The applicant declares that the above 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 within the technical scope disclosed by the present invention by those skilled in the art all fall within the protection scope and the disclosure scope of the present invention.

Claims

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

7.

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

9.

3. A method for preparing the antibody recognizing TIF1-γ according to claim 1 or 2, characterized in that: The method comprises: synthesizing a nucleic acid molecule encoding the antibody recognizing TIF1-γ according to claim 1 or 2, constructing a recombinant expression vector, introducing the recombinant expression vector into a host cell, screening positive host cells for culturing, and obtaining the antibody recognizing TIF1-γ.

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

5. An expression cassette, characterized in that The expression cassette contains a nucleic acid molecule encoding the antibody recognizing TIF1-γ according to claim 1 or 2.

6. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 4 or the expression cassette according to claim 5.

7. A recombinant cell, characterized in that The recombinant cell expresses the antibody recognizing TIF1-γ according to claim 1 or 2.

8. An immunohistochemistry kit for detecting TIF1-γ, characterized in that: The kit contains the antibody for recognizing TIF1-γ according to claim 1 or 2; and any one or a combination of at least two of an antigen repair buffer, a blocking solution, an enzyme-labeled secondary antibody, a color developer or a hematoxylin counterstaining solution.

9. An enzyme-linked immunosorbent assay kit for detecting TIF1-γ, characterized in that: The kit contains the antibody for recognizing TIF1-γ according to claim 1 or 2; and any one or a combination of at least two of a blocking solution, an enzyme-labeled secondary antibody, a color developing solution or a stop solution.

10. Use of any one of the antibodies recognizing TIF1-γ according to claim 1 or 2, the immunohistochemistry kit for detecting TIF1-γ according to claim 8, or the enzyme-linked immunosorbent assay kit for detecting TIF1-γ according to claim 9, or a combination of at least two thereof, in the preparation of a product for diagnosing and / or detecting idiopathic inflammatory myopathy.

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