Monoclonal antibody for identifying Profilin-1 and application thereof

Abstract

The invention relates to the field of biological medicine, and provides a monoclonal antibody for recognizing Profilin-1 and application of the monoclonal antibody. The antibody provided by the invention is selected from at least one of the following: (1) light chain CDR sequences as shown in SEQ ID NO: 5, 6 and 7, and heavy chain CDR sequences as shown in SEQ ID NO: 8, 9 and 10; (2) light chain CDR sequences as shown in SEQ ID NO: 11, 12 and 13, and heavy chain CDR sequences as shown in SEQ ID NO: 14, 15 and 16; or an amino acid sequence having at least 80% sequence identity compared with (1) or (2). In addition, the invention also discloses an enzyme immunoassay kit prepared by using the antibody, and the enzyme immunoassay kit has relatively high sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a monoclonal antibody for recognizing Profilin-1 and application thereof. Background Art

[0002] Profilin-1 is a protein involved in the reorganization of the actin skeleton in cells. It plays a role in multiple cellular functions, including cell movement, cell division, and cell signaling. Recent studies have found that in addition to its role in cell biology, Profilin-1 may also be related to the pathogenesis of depression.

[0003] In the pathological mechanism of depression, inflammatory response is an important factor. Studies have shown that there is an increase in inflammatory markers in the peripheral blood of patients with depression, such as C-reactive protein (CRP), which may be related to the severity of depressive symptoms. In addition, chronic low-grade inflammation may be associated with a certain subtype of depression and specific depressive symptoms, and there may be gender differences. As a protein, changes in the expression level of Profilin-1 may be related to inflammatory response.

[0004] In the biomarker research of depression, some researchers proposed that the composite effect of 6 biomarkers can reach a diagnostic sensitivity and specificity of 67% and 69% for major depressive disorder, including profilin-1. This suggests that profilin-1 may be a potential biomarker for the diagnosis of depression.

[0005] In addition, proteomics research on depression is also being explored, aiming to reveal the molecular mechanisms of depression by detecting changes in protein expression. These studies may help discover new biomarkers and provide a basis for early diagnosis and treatment of depression.

[0006] In summary, although the direct link between Profilin-1 and depression requires further research to clarify, existing studies have shown that it may play a role in the pathogenesis of depression and may serve as a potential biomarker for diagnosing depression. Future studies may reveal the specific mechanism of action of Profilin-1 in depression and how it can work with other biomarkers to help with the diagnosis and treatment of depression.

[0007] The immunoassay method based on the specific reaction between antigen and antibody is low-cost, fast, highly accurate and sensitive, and its core is the specific antibody. Currently, there is no diagnostic reagent raw material with high specificity for Profilin-1 in the industry.

[0008] Currently, there is an urgent need to develop an anti-Profilin-1 antibody with strong specificity and high sensitivity. Summary of the invention

[0009] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0010] In one aspect, the present invention provides an anti-Profilin-1 antibody or antigen-binding fragment, comprising: A CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: Light chain variable region CDR sequences: SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; Heavy chain variable region CDR sequences: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or Light chain variable region CDR sequences: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; Heavy chain variable region CDR sequences: SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. The present invention provides an anti-Profilin-1 antibody or antigen-binding fragment, and an ELISA kit prepared using the antibody or antigen-binding fragment has high sensitivity and specificity.

[0011] According to some embodiments of the present invention, the antibody or antigen-binding fragment includes: a light chain variable region CDR1 sequence shown in SEQ ID NO:5, a light chain variable region CDR2 sequence shown in SEQ ID NO:6, a light chain variable region CDR3 sequence shown in SEQ ID NO:7, a heavy chain variable region CDR1 sequence shown in SEQ ID NO:8, a heavy chain variable region CDR2 sequence shown in SEQ ID NO:9, and a heavy chain variable region CDR3 sequence shown in SEQ ID NO:10, or a light chain variable region CDR1 sequence shown in SEQ ID NO:11, a light chain variable region CDR2 sequence shown in SEQ ID NO:12, a light chain variable region CDR3 sequence shown in SEQ ID NO:13, a heavy chain variable region CDR1 sequence shown in SEQ ID NO:14, a heavy chain variable region CDR2 sequence shown in SEQ ID NO:15, and a heavy chain variable region CDR3 sequence shown in SEQ ID NO:16.

[0012] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the following: (a) having a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2; (b) having a light chain variable region as shown in SEQ ID NO:3 and a heavy chain variable region as shown in SEQ ID NO:4; An amino acid sequence having at least 80% sequence identity with (a) or (b).

[0013] (c) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO: 1, and having the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO: 2; (d) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region as shown in SEQ ID NO:3, and having the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region as shown in SEQ ID NO:4; Or an amino acid sequence having one or more conservative amino acid substitutions compared to (c) or (d).

[0014] According to some embodiments of the present invention, the antibody or antigen-binding fragment further comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian antibody.

[0015] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from at least one of a murine antibody, a rabbit antibody, a primate antibody or mutants thereof.

[0016] According to some embodiments of the present invention, the heavy chain constant region and the light chain constant region are derived from a murine antibody or a mutant thereof.

[0017] According to some embodiments of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody; According to some embodiments of the present invention, the monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody and a minimum recognition unit.

[0018] Another aspect of the present invention provides an antibody analog, which comprises the variable region or the CDR region in the variable region of the above-mentioned antibody or antigen-binding fragment.

[0019] In another aspect, the present invention provides an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding fragment.

[0020] According to some embodiments of the invention, the polynucleotide comprises: (e) having a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 17, a nucleic acid sequence having at least 80% sequence identity to (e); or (f) having a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 18, a nucleic acid sequence having at least 80% sequence identity to (f); or (g) having a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 19, a nucleic acid sequence having at least 80% sequence identity to (g); or (h) having a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 20, A nucleic acid sequence having at least 80% sequence identity with (h).

[0021] In another aspect, the present invention provides an expression vector comprising the above-mentioned polynucleotide.

[0022] In another aspect, the present invention provides a hybridoma cell, which can produce the above-mentioned anti-Profilin-1 antibody or antigen-binding fragment.

[0023] In yet another aspect, the present invention provides a method for preparing an anti-Profilin-1 antibody or antigen fragment, comprising culturing the above-mentioned recombinant cell or the above-mentioned hybridoma cell.

[0024] In yet another aspect, the present invention provides use of the above-mentioned antibody or antigen-binding fragment and the above-mentioned antibody analog in preparing a kit for detecting Profilin-1.

[0025] Another aspect of the present invention provides a kit, comprising at least one of the above-mentioned antibodies or antigen-binding fragments, antibody analogs, vectors, recombinant cells, and hybridoma cells.

[0026] Another aspect of the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment, antibody analog, polynucleotide, vector, recombinant cell, hybridoma cell.

[0027] Another aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment, comprising culturing the above-mentioned recombinant cell or hybridoma cell.

[0028] Another aspect of the present invention provides a medicine, including the above-mentioned antibody or antigen-binding fragment, antibody analog, polynucleotide, vector, recombinant cell, hybridoma cell, and composition.

[0029] Another aspect of the present invention provides a use of the above-mentioned antibody or antigen-binding fragment, antibody analog, polynucleotide, vector, recombinant cell, hybridoma cell, and composition in the preparation of a drug, wherein the drug is used to prevent Profilin-1-mediated related diseases; According to some embodiments of the present invention, the Profilin-1-mediated related diseases include depression, Alzheimer's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), etc.

[0030] The present invention provides a monoclonal antibody against Profilin-1, which has the characteristics of high affinity and specificity, and also provides an in vitro diagnostic test kit based on the antibody. The present invention solves the problem of relative shortage of antibody raw materials and test kits for Profilin-1 detection projects in the current market.

[0031] Additional aspects and advantages of the present disclosure will be given in part in the following description and in part will be obvious from the following description or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is a map of the pCDNA3.1-PFN1 plasmid that expresses Profilin-1 recombinant protein. Figure 2 SDS-PAGE was used to identify the expression of the target protein Profilin-1. Figure 3 This is the standard curve of the ELISA kit prepared using antibodies CF172 and AE132. Figure 4 The ELISA kit prepared with antibodies CF172 and AE132 was used to detect Profilin-1 in human serum, and the correlation between the concentration of Profilin-1 in serum and the score of the Self-Rating Anxiety Scale (SAS) was compared.

Claims

1. A monoclonal antibody or antigen-binding fragment that recognizes Profilin-1, characterized in that: include: A CDR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: Light chain variable region CDR sequences: SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; Heavy chain variable region CDR sequences: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or Light chain variable region CDR sequences: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; Heavy chain variable region CDR sequences: SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:

16.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that include: The light chain variable region CDR1 sequence shown in SEQ ID NO:5, the light chain variable region CDR2 sequence shown in SEQ ID NO:6, the light chain variable region CDR3 sequence shown in SEQ ID NO:7, the heavy chain variable region CDR1 sequence shown in SEQ ID NO:8, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:9, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:10, or the light chain variable region CDR1 sequence shown in SEQ ID NO:11, the light chain variable region CDR2 sequence shown in SEQ ID NO:12, the light chain variable region CDR3 sequence shown in SEQ ID NO:13, the heavy chain variable region CDR1 sequence shown in SEQ ID NO:14, the heavy chain variable region CDR2 sequence shown in SEQ ID NO:15, and the heavy chain variable region CDR3 sequence shown in SEQ ID NO:

16.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that Include at least one of the following: (a) having a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2; (b) having a light chain variable region as shown in SEQ ID NO:3 and a heavy chain variable region as shown in SEQ ID NO:4; An amino acid sequence having at least 80% sequence identity with (a) or (b).

4. The antibody or antigen-binding fragment according to claim 1, characterized in that Include at least one of the following: (c) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO:1, and having the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO:2; (d) having the CDR1, CDR2 and CDR3 sequences of the light chain variable region as shown in SEQ ID NO:3, and having the CDR1, CDR2 and CDR3 sequences of the heavy chain variable region as shown in SEQ ID NO:4; Or an amino acid sequence having one or more conservative amino acid substitutions compared to (c) or (d).

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that Further comprising at least one of a heavy chain constant region and a light chain constant region, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a mammalian antibody; Optionally, the heavy chain constant region and the light chain constant region are derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody or a mutant thereof; Optionally, the heavy chain constant region and the light chain constant region are derived from a murine antibody or a mutant thereof.

6. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment includes a monoclonal antibody or a polyclonal antibody; Optionally, the monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody, and a minimum recognition unit.

7. An antibody analog, characterized in that The antibody analog comprises the variable region or the CDR region in the variable region of the antibody or antigen-binding fragment according to any one of claims 1 to 6.

8. An isolated polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment according to any one of claims 1 to 6.

9. The polynucleotide according to claim 8, characterized in that The polynucleotide comprises: (e) having a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 17, a nucleic acid sequence having at least 80% sequence identity to (e); or (f) having a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 18, a nucleic acid sequence having at least 80% sequence identity to (f); or (g) having a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 19, a nucleic acid sequence having at least 80% sequence identity to (g); or (h) having a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 20, A nucleic acid sequence having at least 80% sequence identity with (h).

10. An expression vector, characterized in that: Comprising the polynucleotide according to claim 8 or 9.

11. A recombinant cell, characterized in that Comprising the expression vector according to claim 10.

12. A hybridoma cell, characterized in that: The hybridoma cell can produce the anti-Profilin-1 antibody or antigen-binding fragment according to any one of claims 1 to 6.

13. A method for preparing an anti-Profilin-1 antibody or antigen fragment, characterized in that: The method comprises culturing the recombinant cell according to claim 11 or the hybridoma cell according to claim 12.

14. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 6 and the antibody analog according to claim 7 in preparing a kit, characterized in that: The kit is used for detecting Profilin-1.

15. A kit, characterized in that: The invention comprises at least one of the antibody or antigen-binding fragment according to any one of claims 1 to 6, the antibody analog according to claim 7, the vector according to claim 10, the recombinant cell according to claim 11, and the hybridoma cell according to claim 12.

16. A composition, characterized in that The invention comprises the antibody or antigen-binding fragment according to any one of claims 1 to 6, the antibody analog according to claim 7, the polynucleotide according to claim 8 or 9, the vector according to claim 10, the recombinant cell according to claim 11, and the hybridoma cell according to claim 12.

17. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 6, characterized in that: The method comprises culturing the recombinant cell according to claim 11 and the hybridoma cell according to claim 12.

18. A drug, characterized in that The invention comprises the antibody or antigen-binding fragment according to any one of claims 1 to 6, the antibody analog according to claim 7, the polynucleotide according to claim 8 or 9, the vector according to claim 10, the recombinant cell according to claim 11, the hybridoma cell according to claim 12, and the composition according to claim 16.

19. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 6, the antibody analog according to claim 7, the polynucleotide according to claim 8 or 9, the vector according to claim 10, the recombinant cell according to claim 11, the hybridoma cell according to claim 12, or the composition according to claim 16 in the preparation of a medicament for preventing Profilin-1-mediated related diseases; Optionally, the Profilin-1-mediated related diseases include depression, Alzheimer's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), etc.

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