Monoclonal antibody resisting aminoacyl-tRNA synthetase and application thereof

Abstract

The invention relates to the field of monoclonal antibodies, in particular to a monoclonal antibody resisting amino acyl-tRNA synthetase. The invention provides a monoclonal antibody for resisting amino acyl-tRNA synthetase. The amino acid sequences of three CDR regions of a heavy chain of the monoclonal antibody respectively have amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the amino acid sequences of the three CDR regions of the light chain of the gene respectively have the amino acid sequences shown as SEQ ID NO: 4, 5 and 6. The amino acyl-tRNA synthetase monoclonal antibody disclosed by the invention is obtained by utilizing a phage display technology. The monoclonal antibody has the advantages of strong specificity, high affinity, high detection sensitivity and no matrix effect, and solves the problems of interchangeability and consistency of quality control products. Through identification, when the monoclonal antibody is used for repeated detection for multiple times, the stability is good, and the accuracy is high.

Description

Technical Field

[0001] The present invention relates to the field of monoclonal antibodies, and particularly to monoclonal antibodies against aminoacyl-tRNA synthetase. Background Art

[0002] Autoimmunity is a reaction of the body's immune system against its own antigens, generating autoantibodies and autoreactive T cells. Under normal physiological conditions, autoimmunity can promptly remove senescent and dead self-cells to maintain the stability of the internal environment. Under certain pathological conditions, autoreactive T cells and autoantibodies that directly and indirectly damage self-tissues may be produced, causing lesions and dysfunctions in the corresponding organs and tissues, which is called an autoimmune disease. Common autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polymyositis / dermatomyositis, mixed connective tissue disease, hyperthyroidism, juvenile diabetes, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, and many kinds of skin diseases, chronic liver diseases, etc.

[0003] Anti-Jo-1 antibody is commonly found in polymyositis (PM), and the positive detection rate can reach 40% - 50%; in patients with overlap of PM and DM (dermatomyositis), the positive detection rate of anti-Jo-1 antibody is 25%; in patients with isolated DM, the detection rate of anti-Jo-1 antibody is less than 10%; in other autoimmune diseases, anti-Jo-1 antibody is negative, so anti-Jo-1 antibody is specific for the diagnosis of PM. In addition, in patients with overlap of PM and scleroderma, the positive detection rate of anti-Jo-1 antibody can be as high as 85%; in patients with overlap of progressive systemic sclerosis (PSS) and PM, the positive detection rate of anti-Jo-1 antibody is about 25%; in patients with PM accompanied by pulmonary interstitial fibrosis, the positive rate of anti-Jo-1 antibody can reach 60%. Jo-1 antibody is one of the histidyl-tRNA synthetase antibodies. This cytoplasmic enzyme catalyzes the esterification reaction of histidine and tRNA, and it exists in the form of a homodimer in the cytoplasm, and the subunit molecular weight is about 50 kD. The antigenic determinant site of Jo-1 is at the N-terminus, which is a region of about 32 amino acids with a helical structure. Anti-Jo-1 antibody is rarely detected in non-myositis patients. There is evidence that anti-Jo-1 antibody is related to MHC class II genes, and the antibody titer indicates that the body's response to produce Jo-1 is somehow related to the pathogenesis of myositis along with the disease activity degree.

[0004] Quality control samples are samples with known concentration contents, and are used to identify whether the measurement results of an instrument are accurate by measuring with the instrument. The JO-1 quality control sample will experience a period of storage and transportation before use. Even when used in accordance with the reagent storage conditions and expiration date regulations, there is a great risk of instability, which will cause trouble in the use of the supporting reagents. Therefore, providing a monoclonal anti-Jo-1 antibody with strong stability and high reactivity can solve the above problems. Summary of the Invention

[0005] In view of this, the present invention provides a monoclonal antibody against aminoacyl-tRNA synthetase. The present invention uses phage display technology to obtain the aminoacyl-tRNA synthetase monoclonal antibody of the present invention. This monoclonal antibody has strong specificity, high affinity, high detection sensitivity, and no matrix effect, and solves the interchangeability and consistency problems of quality control samples. After identification, repeated detection using this monoclonal antibody shows good stability and high accuracy.

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

[0007] The present invention provides a monoclonal antibody against aminoacyl-tRNA synthetase. (1) The amino acid sequences of the three CDR regions of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 1, 2, and 3; and

[0008] (2) The amino acid sequences of the three CDR regions of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 4, 5, and 6; or

[0009] (3) The amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences described in (1) or (2), and amino acid sequences that are functionally the same or similar to the amino acid sequences shown in (1) or (2); or

[0010] (4) Amino acid sequences having at least 80% homology with the sequences described in (1), (2), or (3).

[0011] In some embodiments of the present invention, the sequence of SEQ ID NO: 1 is: GFTFSSY W.

[0012] In some embodiments of the present invention, the sequence of SEQ ID NO: 2 is: IKQDGSE K.

[0013] In some embodiments of the present invention, the sequence of SEQ ID NO: 3 is: ARDLGSG WPHEY.

[0014] In some embodiments of the present invention, the sequence of SEQ ID NO: 4 is: QSISSY.

[0015] In some embodiments of the present invention, the sequence of SEQ ID NO:5 above is: AAS.

[0016] In some embodiments of the present invention, the sequence of SEQ ID NO:6 above is: QXSYSTP YT; wherein: X can be Q or H.

[0017] In some embodiments of the present invention, for the above monoclonal antibody, (5), its heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7 and / or SEQ ID NO:8; and

[0018] (6), its light chain variable region has the amino acid sequence shown in SEQ ID NO:9 and / or SEQ ID NO:10; or

[0019] (7), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (5) or (6), and having the same or similar function as the amino acid sequence shown in (5) or (6I); or

[0020] (8), an amino acid sequence having at least 80% homology with the sequence described in (5), (6) or (7).

[0021] In some embodiments of the present invention, the sequence of SEQ ID NO:7 above is: EVQLLES GGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGSGWP HEYWGQGTLVTVSS.

[0022] In some embodiments of the present invention, the sequence of SEQ ID NO:8 above is: QVQLVQS GGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGS EKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGSGWP HEYWGQGTLVTVSS.

[0023] In some embodiments of the present invention, the sequence of SEQ ID NO:9 above is: DIVMTQS PSSLSASIGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVDIK.

[0024] In some embodiments of the present invention, the sequence of SEQ ID NO:10 is: NIQLTQS PSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQHSYSTPYTFGQGTKVDIK.

[0025] In some embodiments of the present invention, in the above monoclonal antibody, the plurality is 2, 3, 4 or 5.

[0026] The present invention also provides a nucleic acid molecule encoding the above monoclonal antibody.

[0027] The present invention also provides an expression vector, comprising the above nucleic acid molecule.

[0028] The present invention also provides a host, which is transformed and / or transfected with the above expression vector.

[0029] The present invention also provides the use of the above monoclonal antibody, the above nucleic acid molecule, the above expression vector and / or the above host in any of the following;

[0030] (I), preparing a product for detecting aminoacyl-tRNA synthetase; and / or

[0031] (II), preparing a product for detecting the content of Jo-1 antibody; and / or

[0032] (III), preparing a product for detecting eosinophilic airway inflammation-related diseases; and / or

[0033] (IV), preparing a product for detecting autoimmune diseases.

[0034] The present invention also provides a drug, which is characterized by comprising the above monoclonal antibody, the above nucleic acid molecule, the above expression vector and / or the above host and a pharmaceutically acceptable excipient.

[0035] The present invention also provides a kit, comprising the above monoclonal antibody, the above nucleic acid molecule, the above expression vector and / or the above host and an acceptable adjuvant.

[0036] The present invention utilizes phage display technology to obtain the aminoacyl-tRNA synthetase monoclonal antibody of the present invention. This monoclonal antibody has strong specificity, high affinity, high detection sensitivity, no matrix effect, and solves the problems of interchangeability and consistency of quality control products. After identification, repeated detection using this monoclonal antibody shows good stability and high accuracy. Description of the Drawings

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art.

[0038] Figure 1 Show the SDS-PAGE electrophoresis pattern of recombinant antibody purification. Detailed implementation manners

[0039] The present invention discloses monoclonal antibodies against aminoacyl-tRNA synthetase.

[0040] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0041] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-limiting, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context otherwise.

[0042] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0043] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is only intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0044] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values and percentages used in this disclosure are modified by "about". Herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific numerical value or range.

[0045] The present invention provides monoclonal antibodies against aminoacyl-tRNA synthetase and their applications. The antibodies of the present invention are mainly used to detect the content of aminoacyl-tRNA synthetase by western blot.

[0046] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0047] The present invention provides a monoclonal antibody against aminoacyl-tRNA synthetase,

[0048] (1) The CDR1, CDR2, and CDR3 of its heavy chain sequentially have the amino acid sequences shown in SEQ ID No: 1, 2, and 3; and

[0049] (2) The CDR1, CDR2, and CDR3 of its light chain sequentially have the amino acid sequences shown in SEQ ID No: 4, 5, and 6; or

[0050] (3) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids on the basis of the amino acid sequence shown in (1) or (2); or

[0051] (4) A sequence having a homology of more than 80% with the amino acid sequence shown in any one of (1) to (3).

[0052] In some specific embodiments of the present invention, the monoclonal antibody,

[0053] (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID No: 7 or SEQ ID No: 8; and

[0054] (6) Its light chain variable region has the amino acid sequence shown in SEQ ID No: 9 or SEQ ID No: 10; or

[0055] (7) A sequence obtained by substituting, deleting, adding, and / or replacing one or more amino acids on the basis of the amino acid sequence shown in (5) or (6); or

[0056] (8) A sequence having a homology of more than 80% with the amino acid sequence shown in any one of (5) to (7).

[0057] The light chain of monoclonal antibody J233:

[0058] NIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSYSTPYTFGQG TKVDIK (shown in SEQ ID NO: 11);

[0059] Light chain QSISSY (as shown in SEQ ID NO: 12).........___AAS (as shown in SEQ ID NO: 13)............QHSYSTPYT (as shown in SEQ ID NO: 14).....................;

[0060] Heavy chain: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPG KGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDT AVYYCARDLGSGWPHEYWGQGTLVTVSS (as shown in SEQ ID NO: 15);

[0061] Heavy chain GFTFSSYW (as shown in SEQ ID NO: 16).......___IKQDGSEK (as shown in SEQ ID NO: 17).......ARDLGSGWPHEY (as shown in SEQ ID NO: 18)..................

[0062] The present invention also provides the use of the monoclonal antibody in the preparation of a reagent or kit for detecting aminoacyl - tRNA synthetase.

[0063] The present invention also provides the use of the monoclonal antibody in the preparation of a reagent and / or kit for diagnosing the content of Jo - 1 antibody.

[0064] The present invention also provides the use of the monoclonal antibody in the preparation of a reagent and / or kit for detecting eosinophilic airway inflammation - related diseases;

[0065] The autoimmune diseases detected include diseases such as polymyositis (PM) and dermatomyositis (DM).

[0066] Based on the above research, the present invention also provides a reagent, including the monoclonal antibody.

[0067] In Examples 1 - 5 of the present invention, the raw materials and reagents used can be purchased from the market.

[0068] The present invention will be further described below in conjunction with examples:

[0069] Example 1 Screening of human serum positive samples

[0070] Prepare an Anti-Jo-1 IgG detection kit, detect 20 human sera collected. After diluting 20 μL of the sample with 380 μL of diluent, take 10 μL, add 20 μL of magnetic beads and 90 μL of sample diluent, incubate for 15 min, wash magnetically, then add 100 μL of enzyme conjugate, incubate for 17 min, wash magnetically, add substrate to detect the signal value. The test results are shown in Table 1, and one positive sample 18 is detected. Isolate the lymphocytes of this person, and routinely extract the total RNA of lymphocytes by the Trizol method, and reverse transcribe to synthesize cDNA.

[0071] Table 1 Screening of positive samples

[0072]

[0073] Example 2 Splicing of scFv gene and construction of phage screening

[0074] 1. Splicing of scFv gene:

[0075] Amplify the light chain variable region and heavy chain variable region of the antibody by PCR respectively. The PCR reaction program is shown in Table 2: The PCR products are recovered by 1% agarose gel. The amplified light chain variable region and heavy chain variable region are spliced into scFv by the overlap-PCR method, and the products are recovered by 1% agarose gel and stored at -20 °C.

[0076] Table 2 PCR amplification program

[0077]

[0078]

[0079] 2. Construction and screening of phage single-chain antibody library:

[0080] The phagemid vector pcomb3XSS and the purified and recovered ScFv fragment are digested with SfiI to construct a recombinant plasmid. The recombinant plasmid is electrotransformed into TG1 competent cells to construct an immune single-chain antibody library, and a primary phage single-chain antibody library is prepared; the primary phage single-chain antibody library is enriched and screened for 3 rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; 558 monoclonal colonies are picked, the monoclonal phage supernatant is prepared, and the positive clones are identified by the Phage-ELISA method to obtain the positive sequences. After library construction, panning and screening, the titers of 10 phage supernatants all meet the requirements, as shown in Table 3.

[0081] Table 3 Reactivity of phage supernatant

[0082] Number Luminescence value Y201 50,446,292 Y216 57,744,461 Y218 51,714,830 Y226 58,224,202 Y232 55,083,209 Y233 48,395,436 Y249 48,627,015 Y253 53,386,107 Y266 55,618,318 Y273 62,397,770 Y274 69,184,845 Y280 50,257,598

[0083] Example 3 Expression of the recombinant monoclonal antibody of the present invention and antibody purification

[0084] 1. Construction of transient transfection cell line

[0085] Amplify the variable region of the heavy chain and the variable region of the light chain of the antibody by PCR respectively to obtain the antibody variable region gene. The heavy chain antibody gene and the light chain antibody gene are respectively homologous recombined with the RGFc-PCMV3 and RCL-PCMV3 vectors digested with KasI / BamHI to obtain the expression vector of the human full antibody sequence. After the recombined full antibody plasmid is transferred into competent DH5α cells, positive clones are selected for sequencing and plasmid extraction. The extracted plasmid is transfected into HEK293 cells. After 48 h of transfection, when the antibody is detected to be expressed by ELISA, feeding is added. After 7 d of transfection, the cell supernatant is collected to obtain the supernatant of the target protein.

[0086] 2. Purification of antibody SPA and SDS-PAGE identification column equilibration

[0087] Set the flow rate at 6.4 mL / min, change to the equilibration buffer 0.02 mol / L PBS pH 7.4 to rinse the chromatography column, set the flow rate at 3.8 mL / min, perform sample loading and purification. When 10 μL of the sample in the flow-through tube is added to 200 μL of the G250 staining solution and turns blue, put the flow-through tube into a 250 mL conical flask and start collecting the flow-through. Then equilibrate again: set the flow rate at 6.4 mL / min, and use the equilibration buffer 0.02 mol / L PBS pH 7.4 to rinse the chromatography column again until no protein flows through; dissociation: place 10 4 mL centrifuge tubes on the dissociation tube rack, add 200 μL of 1 mol / L Tris (pH 8.5) to each tube, set the constant flow pump at a flow rate of 6.4 mL / min, and dissociate the target protein with the dissociation buffer 0.2 mol / L Gly + 0.15 mol / L NaCl pH 2.7. When 10 μL of the sample from the flow-through tube is added to 200 μL of the G250 staining solution and turns blue, start manual collection, collect 4 mL in each tube, and stop collection when 10 μL of the sample from the flow-through tube is added to 200 μL of the G250 staining solution and is colorless. The collected protein is dialyzed into 10 mM PBS PH7.2 buffer, the concentration is adjusted to 2 mg / mL, and then detected by SDS-PAGE, as Figure 1 shown. It shows that the purity of the purified antibody is high.

[0088] Example 4 Screening of high-reactivity recombinant human Jo-1 antibody with Autoimmune Biotechnology Co., Ltd. kits

[0089] Prepare an Anti-Jo-1 IgG detection kit to detect the reactivity of recombinant antibodies. After diluting 20 μL of the sample with 380 μL of diluent, take 10 μL, add 20 μL of magnetic beads and 90 μL of sample diluent. After incubating for 15 min, wash with magnetic force, then add 100 μL of enzyme conjugate. After incubating for 17 min and washing with magnetic force, add the substrate to detect the signal value. The test results are shown in Table 4. After diluting the 4 purified antibodies 500-fold, they are all higher than 200 AU / mL, and the generality of the antibodies can be further evaluated.

[0090] Table 4 Reactivity of Recombinant Jo-1 Antibodies

[0091]

[0092] Example 5 Detection of Jo-1 Antibody Stability

[0093] Use a commercially available Anti-Jo-1 IgG magnetic particle luminescence kit to detect 4 recombinant antibodies and detect the stability of the antibodies. The experimental results are as follows: The stability of 266 is the strongest.

[0094] Table 5

[0095]

[0096] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A monoclonal antibody against aminoacyl-tRNA synthetase, characterized in that (1) The amino acid sequences of the three CDR regions of its heavy chain have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively; and (2) the amino acid sequences of the three CDR regions of its light chain have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively; or (3) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1) or (2), and having the same or similar function as the amino acid sequence described in (1) or (2); or (4) An amino acid sequence that is at least 80% homologous to the sequence described in (1), (2) or (3).

2. The monoclonal antibody according to claim 1, characterized in that (5) Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 8; and (6) whose light chain variable region has the amino acid sequence shown in SEQ ID NO: 9 and / or SEQ ID NO: 10; or (7), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (5) or (6), and having the same or similar function as the amino acid sequence described in (5) or (6); or (8) An amino acid sequence that is at least 80% homologous to the sequence described in (5), (6) or (7).

3. The monoclonal antibody according to claim 1 or 2, characterized in that The plurality is 2, 3, 4 or 5.

4. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that Comprising the nucleic acid molecule as described in claim 4.

6. A host, characterized in that Transformation and / or transfection is carried out using the expression vector according to claim 5.

7. Use of the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 and / or the host according to claim 6 in any of the following; (I), preparing a product for detecting aminoacyl-tRNA synthetases; and / or (II), preparing a product for detecting the content of Jo-1 antibodies; and / or (III) preparing products for detecting diseases associated with eosinophilic airway inflammation; and / or (IV) Preparation of products for detecting autoimmune diseases.

8. A drug, characterized in that The invention comprises the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 and / or the host according to claim 6, and a pharmaceutically acceptable excipient.

9. A kit comprising the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5 and / or the host according to claim 6 and an acceptable auxiliary agent.

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