Anti-TIF1-gamma monoclonal antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides an anti-TIF1-gamma monoclonal antibody and application thereof.The human-derived anti-TIF1-gamma monoclonal antibody good in specificity and high in affinity is screened out through a phage antibody library technology, the antibody is specifically combined with TIF1-gamma, and the TIF1-gamma monoclonal antibody with high affinity is obtained. The anti-TIF1-gamma autoantibody level in a DM patient can be quantitatively detected, so that the illness state activity degree is monitored, and the kit can be used for clinical diagnosis of the DM patient. The research of the invention provides theoretical and experimental basis for specific treatment of DM, and also provides a new research direction for treatment of autoimmune diseases with autoantibodies as main pathogenic mechanisms.

Description

Technical Field

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

[0002] Idiopathic inflammatory myopathy (IIM) is a group of systemic autoimmune connective tissue diseases characterized by symmetric proximal limb muscle weakness. Dermatomyositis (DM) is the most common subtype of inflammatory myopathy. There are various specific autoantibodies in the sera of IIM patients, including two major categories: myositis-specific autoantibodies (MSAs) and myositis-related antibodies (MAAs). Anti-transcription intermediary factor 1-γ (TIF1-γ) antibody is a novel autoantibody specific to DM and belongs to one of the MSAs antibodies. Research indicates that the sensitivity of anti-TIF1-γ antibody in diagnosing DM complicated with tumors is 50.0% - 71.4%, and the specificity is 89.0% - 91.3%. The TIF1-γ antibody can be used as a reliable biomarker for monitoring secondary malignancies in adult DM patients.

[0003] Dermatomyositis is mainly treated with hormones and immunosuppressants. Hormones can improve the inflammatory state of patients and inhibit the immune disorder. In addition, using a large dose of hormones at the initial stage of the disease can also inhibit the overactive immune state of patients. Some patients are prone to recurrence during the process of reducing the dosage of hormones and need to be treated with immunosuppressants. The choice of immunosuppressants is determined according to the severity of muscle, skin, or lung involvement. If patients experience gastrointestinal symptoms such as acid reflux, nausea, and vomiting when using hormones and immunosuppressants, some drugs that prevent excessive gastric acid secretion or protect the gastric mucosa can also be used for treatment.

[0004] 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 malignancies. However, there are few types of antibodies available for detection currently, making it difficult to meet the detection requirements. Therefore, developing anti-TIF1-γ antibodies with high affinity has important application value in the diagnosis and detection of idiopathic inflammatory myopathy. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide an anti-TIF1-γ monoclonal antibody and its application. The present invention constructs a phage human antibody library using PBMC from patients positive for TIF1-γ antibody, and screens out a human-derived anti-TIF1-γ monoclonal antibody that binds to TIF1-γ by specifically binding to TIF1-γ. By specifically binding to TIF1-γ, the antibody can quantitatively detect the level of anti-TIF1-γ autoantibody in DM patients, thereby monitoring the disease activity and being used for the clinical diagnosis of DM patients.

[0006] To achieve the object of the present invention, the following technical solutions are adopted:

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

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

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

[0010] Monoclonal antibodies are the largest and fastest-growing group of therapeutic proteins. Currently, more than 500 therapeutic antibodies and their derivatives are in clinical trials, with a focus on cancer treatment and autoimmune diseases. The present invention isolated PBMC from patients positive for TIF1-γ antibody, extracted RNA and performed quality control on the RNA. The qualified RNA was reverse transcribed into cDNA using RT-PCR technology, and all antibody VH and VL gene fragments were amplified. The in vitro amplified VK and Vλ gene fragments were cloned into the pATA-scFv-2 vector to construct an antibody combinatorial library.

[0011] The antibody gene combinatorial library is inserted immediately downstream of the leader sequence of gene III (g3) or gene VIII (g8) of the phage-encoded membrane protein. Through superinfection with helper phage, the polypeptide expressed by the exogenous antibody gene can be displayed at the N-terminus of the phage coat proteins pIII or pVIII in the form of a fusion protein. Each phage particle encodes and presents a different antibody, and it contains billions of individual clones. In these antibody libraries, the genes encoding those antibodies that can bind to the antigen are enriched for affinity in vitro against the antigen - gentle elution - phage amplification, and the above enrichment and screening process is continued to be repeated until a phage library of antibodies 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 the ELISA method, 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 having at least 90% identity with the sequence shown in SEQ ID NO:8.

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

[0014] Preferably, the heavy chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO:8, and the light chain amino acid sequence of the monoclonal antibody is as 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 containing the nucleic acid molecule according to the second aspect.

[0017] In a fourth aspect, the present invention provides a host cell containing at least one copy of the expression vector according to the third aspect; or the nucleic acid molecule according to the second aspect is integrated into the chromosome of the host cell.

[0018] In a fifth aspect, the present invention provides a composition for detecting TIF1-γ in a sample, the composition comprising the anti-TIF1-γ monoclonal antibody according to 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 according to the first aspect or the composition for detecting TIF1-γ in a sample according to the fifth aspect.

[0020] In a 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] Through three rounds of screening of the antibody phage library of the present invention, clones with an antigen group greater than 3 times that of the control group were defined as positive clones, and these monoclonal antibodies were subjected to sequencing analysis. After excluding incorrect antibody sequences and repeated antibody sequences, and combining the antigen-antibody specific binding ability reflected by the ELISA experiment, the TIF1-γ-R7P1-C2 antibody was finally obtained. The antibody has high activity, good stability, and strong specificity, can quantitatively detect the level of anti-TIF1-γ autoantibody in DM patients, and has important application value in the detection and diagnosis of idiopathic inflammatory myopathy. Description of the Drawings

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

[0024] Figure 2 It is the PCR agarose gel electrophoresis pattern of monoclonal bacteria of the KH phage library.

[0025] Figure 3 It is the PCR agarose gel electrophoresis pattern of monoclonal bacteria of the λH phage library.

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

[0027] Figure 5 It is the OD value of the TIF1-γ-R7P1-C2 antibody under different dilution concentrations. Detailed Embodiments

[0028] 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.

[0029] For those technical or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in the field or in accordance with the product specifications. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0030] Example 1

[0031] Construction method of human-derived ScFv phage display library

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

[0033] Table 1

[0034] 1. Library construction

[0035] 1.1 Assembly of heavy chain variable region (VH) and light chain variable region (VL)

[0036] The RNA of TIF1-γ antibody-positive patients was reverse transcribed into cDNA using the HiScript® III First Strand cDNA Synthesis Kit (+gDNA Clear), and the VH and VL fragments of DNA were amplified. The PCR reaction conditions and steps are shown in Table 2.

[0037] Table 2

[0038] Among them, the three steps of denaturation, annealing, and extension (1) are 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 phage display library of light chain variable region

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

[0063] The VK and Vλ gene fragments obtained by in vitro amplification were cloned into the pATA-scFv-2 vector through cloning technology to form a VK library and a Vλ library.

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

[0065] Table 3

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

[0067] Table 4

[0068] Incubate the above ligation system at 16 °C overnight and inactivate it 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 the electroporation cuvette (0.1 cm gap) and the microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube for each transformation reaction).

[0072] 1.2.4.3 Take out the electroporation competent cells from the -80 °C refrigerator and place them on ice until they are completely melted (10 - 15 minutes). After the cells are thawed, gently mix them. Put 50 μL of the cells into a frozen microcentrifuge tube on ice.

[0073] 1.2.4.4 Carefully add 3 μL of the DNA mixture to the frozen electroporation cuvette without generating bubbles. Quickly flick the tube downwards with the wrist to make the cells sediment at the bottom.

[0074] 1.2.4.5 Perform electroporation at 600 Ω, 10 μF, and 1.8 kV. Immediately add 1 mL of pre-warmed SOC medium to each tube within 10 seconds of the pulse. Shake at 37 °C and 250 rpm for 1 hour.

[0075] 1.2.4.6 Collect all the electroporated media. Serially dilute 10 μL of the culture into 90 μL of SOC medium and spread it on 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 volume plated.

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

[0077] 1.3.1 Digest the 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] Incubate the above ligation reaction system overnight at 16 °C and inactivate it 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 Pre-warm 4 mL of SOC medium (Sigma, S1797) at 37 °C. Place the electroporation cuvette (0.2 cm gap) and microcentrifuge tubes on ice (one cuvette and one microcentrifuge tube for each transformation reaction).

[0085] 1.3.3.3 Take out the electroporation competent cells from the -80 °C refrigerator and place them on ice until they are completely melted (10 - 15 minutes). After the cells are thawed, gently mix them.

[0086] 1.3.3.4 Carefully add 6 μL of the DNA mixture to the chilled electroporation cuvette without generating bubbles. Quickly flick the tube downwards with the wrist to deposit the cells at the bottom.

[0087] 1.3.3.5 Perform electroporation at 600 Ω, 10 μF and 2.5 kV. Immediately add 2 mL of pre-warmed SOC medium to each tube within 10 seconds of the pulse. Shake at 37 °C and 250 rpm for 1 hour.

[0088] 1.3.3.6 Collect all the electroporated media. Serially 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 plated.

[0089] 1.4 Library evaluation

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

[0091] Table 7

[0092] Among them, the three steps of denaturation, annealing, and extension (1) were repeated 30 times. The primer sequences are as follows: Forward primer (F) for pATA-scFv-2 vector identification: agcggataacaatttcacacagga (SEQ ID NO:29). Reverse primer (R) for pATA-scFv-2 vector identification: gcccccttattagcgtttgccatc (SEQ ID NO:30). The results of agarose gel electrophoresis after PCR are as Figures 1 - 3 shown. Figure 1 It is the agarose gel electrophoresis map of monoclonal bacteria PCR of the VL phage library; among them, lane M: DL2000, lanes 1-16 are pATA-VK, and lanes 17-32 are pATA-Vλ. Figure 2 It is the agarose gel electrophoresis map of monoclonal bacteria PCR of the KH phage library; among them, lane M: DL2000, lanes 1-48 are pATA-scFv-KH. Figure 3 It is the agarose gel electrophoresis map of monoclonal bacteria PCR of the λH phage library; among them, lane M: DL2000, lanes 1-48 are pATA-scFv-λH.

[0093] 1.4.2 Sequencing: Positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing quality control results are shown in Tables 8 and 9. Table 8 is the analysis of IGLV germline genes, and Table 9 is the analysis of IGHV germline genes. It can be seen from the table that the diversity of the heavy and light chains of the library is relatively comprehensive, covering most of the main 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 the TIF1-γ gene was recombined into the expression vector plasmid pET28b to obtain the TIF1-γ-pET28b expression vector; the cloning sites are Nde1 / Xho1, and the amino acid sequence of TIF1-γ is as shown in SEQ ID NO:31. The gene sequence is as shown in SEQ ID NO:32.

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

[0099] Example 2

[0100] Preparation of Monoclonal Antibody Specifically Binding to TIF1-γ

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

[0102] Table 10

[0103] 1 First Round of Panning

[0104] 1.1 Immunotube Solid-Phase Panning

[0105] 1.1.1 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).

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

[0109] 1.1.5 Incubation: 1×10 12 pfu phage library (or the amplified phages from the previous round) was 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. The pre-incubated library was then 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 of 0.05% PBST.

[0111] 1.1.7 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) to neutralize the solution to pH = 7.0 - 8.0.

[0112] 1.1.8 After diluting the eluted phage, infect the exponentially growing Escherichia coli TG1, plate and measure the titer.

[0113] 1.2 Amplification of the eluted phage

[0114] 1.2.1 Pipette the eluted phage and add it to the exponentially growing Escherichia coli TG1 bacterial solution. After standing at 37°C for 30 minutes, culture at 220 rpm for 30 minutes - 1 hour.

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

[0116] 1.2.3 Add the helper phage to the bacterial solution. After standing at 37°C for 30 minutes, culture at 220 rpm for 45 minutes - 1 hour.

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

[0118] 1.2.5 The next day, centrifuge the bacterial solution at 4°C, 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.

[0119] 1.2.6 Centrifuge at 4°C, 8000 rpm for 30 minutes, discard the supernatant; resuspend the pellet with 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 the exponentially growing TG1, plate and measure the titer.

[0121] 2 Second to eighth round of panning

[0122] Use the amplified phage for the second round of panning, and the steps are the same as 1.1 and 1.2. Repeat the panning for multiple rounds, and 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 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.

[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 phages after each round of amplification with PBS, with the dilution factor increasing by 3 times. The initial concentration is 1×1012 pfu / mL. Add 100 μL of the diluted amplified phages to each well; incubate at 30°C for 1 hour and wash 4 - 6 times with 300 μL of PBST.

[0129] 3.5 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.

[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 Read the values using an ELISA reader at 450 nm - 620 nm. Table 12 shows the results of polyclonal phage ELISA.

[0132] Table 12

[0133] 4 Monoclonal phage ELISA screening

[0134] 4.1 Select an appropriate round, dilute the eluted phages to a suitable concentration, infect logarithmic-phase TG1 cells, and plate them.

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

[0136] 4.3 Add helper phages to the medium in the 96-deep well plate, let it stand at 37°C for 30 minutes; shake culture at 37°C and 250 rpm for 45 minutes - 1 hour.

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

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

[0139] 4.6 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.

[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 with the blocking solution 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 an ELISA reader at 450 nm - 620 nm, and sequence the highly specific clones.

[0146] 5 Secondary verification ELISA of positive clones

[0147] After sequencing the positive clones obtained by monoclonal screening, remove the double-peak sequences and repetitive sequences to obtain the final positive clones. The positive clones are subjected to secondary ELISA detection and verification according to steps 4.2 - 4.12 of step 4 to ensure the authenticity of the positive results. Table 13 shows the results of 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 CDR1 of the heavy chain variable region is as shown in SEQ ID NO:1: GYTFTGYY.

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

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

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

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

[0156] The CDR3 of the light chain variable region is as 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 as 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 as shown in SEQ ID NO:7:

[0160] SYELTQPLSVSAAPGTTATFTCGGDNIGNKRVHWYQQRPGQAPVLVVSDDYDRPSGIPERFSGSHSGEAAILTISGVEAGDEADYYCQLWDDDSDHVVFGGGTQLTVL.

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

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

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

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

[0165] Example 3

[0166] ELISA detection of OD values under different dilution concentrations of the antibody

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

[0168] 1. Coating: 100 μL / well of TIF1-γ protein (4 μg / mL) is used to coat the ELISA plate and incubated 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. Incubation with the positive antibody: The TIF1-γ-R7P1-C2 antibody is serially diluted, and 100 μL of the diluted antibody solution is added to each well and incubated 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. Incubation with the secondary antibody: Dilute 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.

[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 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.

[0176] 9. Reading the plate: Use an ELISA reader to read the values at 450 nm - 620 nm, as Figure 5 shown.

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

[0178] In summary, a high-affinity antibody named TIF1-γ-R7P1-C2 was screened in the present invention. 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 diagnosis and detection of dermatomyositis.

[0179] 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 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 comprises a sequence that is at least 90% identical to the sequence shown in SEQ ID NO:

8.

3. The anti-TIF1-γ monoclonal antibody according to claim 1, characterized in that 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.

4. 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.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-TIF1-γ monoclonal antibody according to any one of claims 1 to 4.

6. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that The host cell contains at least one copy of the expression vector of claim 6; or the nucleic acid molecule of claim 5 is integrated into the chromosome of the host cell.

8. A composition for detecting TIF1-γ in a sample, characterized in that: The composition comprises the anti-TIF1-γ monoclonal antibody according to any one of claims 1 to 4.

9. A kit for detecting TIF1-γ in a sample, characterized in that: The kit comprises the anti-TIF1-γ monoclonal antibody according to any one of claims 1 to 4 or the composition for detecting TIF1-γ in a sample according to claim 8.

10. Use of the anti-TIF1-γ monoclonal antibody according to any one of claims 1 to 4, the composition for detecting TIF1-γ in a sample according to claim 8, or the kit for detecting TIF1-γ in a sample according to claim 9 in the preparation of a product for diagnosing and / or detecting idiopathic inflammatory myopathy.

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