Acetylated SR-A polypeptide and application thereof in preparation of rheumatoid arthritis diagnostic product

Abstract

The invention discloses an acetylated SR-A polypeptide and application of the acetylated SR-A polypeptide in preparation of a rheumatoid arthritis (RA) diagnostic product, and the acetylated polypeptide is an acetylated ornithine SR-A polypeptide (AOSP) and / or an acetylated lysine SR-A polypeptide (ALSP). The acetylated polypeptide can be used as a detection antigen for detecting an anti-acetylated SR-A polypeptide antibody in a biological sample of an RA patient, and further can be used for preparing an RA diagnosis product, especially has better diagnosis efficiency in serum-negative RA, and an effective new method is provided for early diagnosis of RA, especially diagnosis of serum-negative RA.

Description

Technical Field

[0001] The present invention relates to the technical field of disease diagnosis, and in particular to an acetylated SR-A polypeptide and an application thereof in the preparation of a rheumatoid arthritis diagnosis product. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by erosive arthritis. Early diagnosis and precise treatment of RA are crucial. Early intervention can slow down joint destruction in most patients to prevent irreversible deformity or disability. The clinical diagnosis of RA mainly relies on medical history, physical examination and laboratory biomarkers. Currently, the indicators commonly used in clinical diagnosis of RA are rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP), but their diagnostic sensitivity is limited. About 1 / 3 of RA patients still cannot be diagnosed in time due to lack of diagnostic markers, resulting in delayed treatment.

[0003] Scavenger receptor A (SR-A) is a pattern recognition receptor that is mainly expressed on the surface of macrophages and dendritic cells and plays an important role in innate and adaptive immunity. Our research group previously found through a large-scale multicenter study that SR-A has a good diagnostic value in RA, with a sensitivity of 66% and a specificity of 91%. More importantly, the positive rate of SR-A in RA patients with negative serum anti-CCP antibodies is 49.72%, in RA patients with negative RF, and in RA patients with negative anti-CCP antibodies and negative RF, it is 42.58% (Hu F, et al. Scavenger receptor-A is a biomarker and effector of rheumatoid arthritis: A large-scale multicenter study. Nat Commun. 2020 Apr 20;11(1):1911.), which has a good supplementary diagnostic value.

[0004] Protein post-translational modifications, especially citrullination and acetylation, play a vital role in the pathogenesis of RA. Citrullination is a specific and important phenomenon in RA, which has been widely studied and is of great significance in the pathogenic mechanism of RA autoantigens. Citrullination can enhance the immunogenicity of antigenic peptides and enhance their binding to HLA-DRB1 molecules containing shared epitopes, thereby more significantly activating autoreactive T cells and B cells and inducing the occurrence of autoimmune reactions.

[0005] In addition to citrullination modification, the role of acetylation modification in the pathogenesis of RA has also been gradually recognized. Acetylation modification refers to the transfer of the acetyl group of acetyl-CoA to the N-terminus or lysine residue of the protein under the action of acetyltransferase, which is one of the most important modification methods to change the function of the protein. Acetylation modification can change the molecular characteristics of proteins or polypeptides, and may produce new antigenic epitopes, thereby inducing the production of anti-acetylated protein antibodies (anti-acetylated protein antibody, AAPA). Although the structures of acetylated and citrullinated polypeptides are very similar, the antibody spectra of AAPA and anti-citrullinated protein antibodies in RA are not the same.

[0006] Therefore, we hope to discover new diagnostic markers for RA from different perspectives, to make up for the clinically difficult diagnosis of seronegative RA, and to further improve the accuracy of RA diagnosis. Summary of the invention

[0007] The purpose of the present invention is to provide a new acetylated SR-A polypeptide, which can be used as a detection antigen to detect anti-acetylated SR-A polypeptide antibodies in biological samples of rheumatoid arthritis patients, and can further be used to prepare rheumatoid arthritis diagnostic products.

[0008] The technical solution of the present invention is described in detail as follows: In a first aspect, the present invention provides an acetylated SR-A polypeptide, which is an acetylated ornithine SR-A polypeptide (acetylated ornithine SR-A peptide, AOSP) and / or an acetylated lysine SR-A polypeptide (acetylatedlysine SR-A peptide, ALSP), and the amino acid sequence is as follows: AOSP: KGD-Orn(Ac-)-GAIGFPGS-Orn(Ac-)-GLPGYAG- Orn(Ac-) (SEQ ID NO: 1); ALSP: KGD-K(Ac-)-GAIGFPGS-K(Ac-)-GLPGYAG-K(Ac-) (SEQ ID NO: 2).

[0009] In a second aspect, the present invention provides the use of the above-mentioned acetylated SR-A polypeptide in the preparation of a product for the diagnosis of rheumatoid arthritis, wherein the product is used to detect the content of anti-acetylated SR-A polypeptide antibodies. The anti-acetylated SR-A polypeptide antibodies are anti-AOSP polypeptide antibodies and / or anti-ALSP polypeptide antibodies.

[0010] Compared with normal healthy people, patients with Sjögren's syndrome, patients with systemic lupus erythematosus and patients with osteoarthritis, the content of anti-acetylated SR-A polypeptide antibodies in biological samples of patients with rheumatoid arthritis was significantly increased.

[0011] In the above application, the rheumatoid arthritis patient includes any of the following: a patient who is positive for anti-CCP antibodies and positive for RF; a patient who is negative for anti-CCP antibodies (positive for RF or negative for RF); a patient who is negative for RF (positive or negative for anti-CCP antibodies); a patient who is negative for anti-CCP antibodies and negative for RF.

[0012] In the above application, the biological sample is blood, plasma, serum or tissue fluid.

[0013] In the above application, the diagnostic product is an ELISA detection kit.

[0014] In a third aspect, the present invention provides a rheumatoid arthritis diagnostic kit, comprising: the above-mentioned acetylated SR-A polypeptide, an ELISA plate, an ELISA coating solution, a washing solution, a blocking solution, a sample diluent, an enzyme-labeled secondary antibody, a chromogenic substrate and a stop solution.

[0015] The present invention has the following beneficial effects: The acetylated SR-A polypeptide provided by the present invention can be used as a detection antigen to effectively detect the levels of anti-AOSP antibodies and / or anti-ALSP antibodies in biological samples of patients. The levels of these two antibodies in biological samples of RA patients are significantly higher than those of healthy controls and other disease controls. It has good diagnostic efficacy as an RA biomarker for diagnosing rheumatoid arthritis, especially in anti-CCP antibody-negative RA, RF-negative RA, and anti-CCP antibody-negative and RF-negative RA, and provides an effective new method for the early diagnosis of RA, especially the diagnosis of seronegative RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The statistical results of the expression levels of anti-AOSP antibodies in the serum of the RA group, different disease control groups and the healthy control group in Example 3, where anti-AOSP represents anti-AOSP antibodies, RA: rheumatoid arthritis, SS: Sjögren's syndrome, SLE: systemic lupus erythematosus, OA: osteoarthritis, and HC: healthy normal person.

[0017] Figure 2 This is the ROC curve of the anti-AOSP antibody in the diagnosis of RA in Example 3.

[0018] Figure 3 The statistical results of the expression levels of anti-AOSP antibodies in serum of different types of seronegative RA in Example 3, in which anti-AOSP represents anti-AOSP antibodies, and anti-CCP represents - RA: anti-CCP antibody negative rheumatoid arthritis, RF - RA: RF negative rheumatoid arthritis, (anti-CCP + RF) - RA: rheumatoid arthritis with negative anti-CCP antibodies and negative RF, HC: healthy normal subjects.

[0019] Figure 4 The statistical results of the expression levels of anti-ALSP antibodies in the serum of the RA group, different disease control groups and the healthy control group in Example 3, where anti-ALSP represents anti-ALSP antibodies, RA: rheumatoid arthritis, SLE: systemic lupus erythematosus, SS: Sjögren's syndrome, OA: osteoarthritis, and HC: healthy normal person.

[0020] Figure 5 This is the ROC curve of the anti-ALSP antibody in the diagnosis of RA in Example 3.

[0021] Figure 6 The statistical results of the expression levels of anti-ALSP antibodies in serum of different types of seronegative RA in Example 3. In the figure, anti-ALSP represents anti-ALSP antibodies, and anti-CCP - RA: anti-CCP antibody negative rheumatoid arthritis, RF - RA: RF negative rheumatoid arthritis, (anti-CCP + RF) - RA: rheumatoid arthritis with negative anti-CCP antibodies and negative RF, HC: healthy normal subjects.

[0022] Figure 1 , 3 , 4 , 6 Statistical results were analyzed using the Kruskal-Wallis H test for differences among multiple groups, and the Dunnet t test for multiple comparisons. p <0.05 was considered statistically significant, * p <0.05,**p <0.01,*** p <0.001. DETAILED DESCRIPTION

[0023] In order to make the technical personnel in this field better understand the present application scheme, the present application will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the described embodiments are only embodiments of a part of the present application, rather than all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work should belong to the scope of protection of this application. The instruments and reagents used in the embodiments are all derived from commercial channels unless otherwise specified.

[0024] Example 1 Preparation of acetylated SR-A polypeptide The amino acid sequence of human SR-A protein was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and the predicted potential antigenic epitopes of SR-A were obtained through the B cell epitope prediction tool in the IEDB database (http: / / www.iedb.org / ). At the same time, the SR-A amino acid sequence was compared with the RA autoantigenic epitopes in the IEDB database by BLAST sequence alignment, and the B cell antigenic epitopes of SR-A were determined in combination with the predicted potential antigenic epitopes.

[0025] The acetylated ornithine SR-A polypeptide (AOSP) was synthesized by solid phase peptide synthesis, and the sequence was KGD-Orn(Ac-)-GAIGFPGS-Orn(Ac-)-GLPGYAG-Orn(Ac-); the acetylated lysine SR-A polypeptide (ALSP) was synthesized, and the sequence was KGD-K(Ac-)-GAIGFPGS-K(Ac-)-GLPGYAG-K(Ac-). The above polypeptides were synthesized by Shanghai Nuoyou Biotechnology Co., Ltd.

[0026] Example 2 Acetylated SR-A polypeptide was used for detecting the levels of anti-AOSP antibodies and anti-ALSP antibodies in patient serum by ELISA method.

[0027] Based on the method of enzyme-linked immunosorbent assay (ELISA), an ELISA diagnostic kit was prepared. The kit components include: ELISA plate, acetylated SR-A peptide, ELISA coating solution, washing solution 0.05% PBST, blocking solution 3% BSA-PBST, sample diluent 1% BSA-PBST, goat anti-human enzyme-labeled secondary antibody, color substrate TMB and stop solution sulfuric acid. The components in the kit are all volume percentages, the solvent of the goat anti-human enzyme-labeled secondary antibody is glycerol, and the solvents of the other reagents are water.

[0028] (1) Expose the 96-well ELISA plate to ultraviolet light overnight to enhance adsorption.

[0029] (2) Peptide dissolution: Take out the synthesized acetylated SR-A peptide (AOSP or ALSP), return to room temperature, centrifuge at 12,000 rpm for 2 min, and dissolve the peptide to 1 mg / mL in sterile PBS.

[0030] (3) Coating: Dilute the peptide to 10 µg / mL with pre-diluted ELISA coating solution (1×), add 100 µL to each well, seal the plate with sealing film, and place it horizontally in a 4°C refrigerator overnight.

[0031] (4) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0032] (5) Blocking: Add 200 µL of 3% BSA-PBST to each well and incubate at room temperature for 2 h.

[0033] (6) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0034] (7) Dilute the serum with sample diluent (1% BSA-PBST) at a ratio of 1:100, mix well, add 100 µL to each well, and incubate at 37°C for 1 h.

[0035] (8) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0036] (9) Dilute goat anti-human IgG antibody at 1:15,000 in 1% BSA-PBST as diluent, add 100 µL to each well, and incubate at 37°C for 0.5 h.

[0037] (10) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0038] (11) Add 100 µL of TMB substrate to each well and react at room temperature in the dark for 5-20 min.

[0039] (12) Add 100 µL of 2 M sulfuric acid solution to each well to terminate the reaction and read the absorbance (optical density, OD) at 450 nm / 570 nm on a microplate reader.

[0040] (13) Each plate has a standard positive serum and a blank well. Ten RA sera with OD>1.5 were selected and mixed in the same volume to form the standard positive serum.

[0041] (14) The results are expressed in AU values, which are calculated as follows: AU = [(OD 肽 -OD 非特异背景 ) 待测血清 / (OD 肽 -OD 非特异背景 ) 阳性血清 ]×100.

[0042] Example 3 Diagnostic value of anti-acetylated SR-A polypeptide antibodies in RA This study first included 475 patients and healthy controls, including 150 patients with rheumatoid arthritis (RA), 75 patients with Sjögren's syndrome (SS), 75 patients with systemic lupus erythematosus (SLE), 75 patients with osteoarthritis (OA), and 100 healthy controls, and collected their sera to detect the levels of corresponding anti-ALSP antibodies. RA patients met the 2010 ACR and EULAR diagnostic criteria for RA, and SLE, SS, and OA patients met their respective diagnostic or classification criteria. All sera were collected by the Department of Rheumatology and Immunology, Peking University People's Hospital from 2017 to 2020. This study was approved by the Research Ethics Committee of Peking University People's Hospital.

[0043] To further explore the diagnostic value of anti-AOSP and anti-ALSP antibodies in seronegative RA, based on the above, sera from seronegative RA patients were further collected, and the levels of anti-AOSP and anti-ALSP antibodies were detected in the sera of 140 patients with anti-CCP antibody-negative RA, 155 patients with RF-negative RA, and 107 patients with anti-CCP antibody-negative and RF-negative RA.

[0044] 1. Diagnostic value of anti-AOSP antibodies in seronegative RA In order to explore whether there are anti-AOSP antibodies in RA, we first detected the levels of anti-AOSP antibodies in the serum of 475 cases of RA patients, disease control groups (SLE, SS, OA) and healthy controls by the ELISA method in Example 2. For the results, please refer to Figure 1 , showing that the level of anti-AOSP antibodies in the serum of RA patients was higher than that of disease controls and healthy controls.

[0045] We further analyzed the receiver operator characteristic (ROC) curve of anti-AOSP antibody, and the results are as follows Figure 2As shown, the area under the ROC curve (AUC) of anti-AOSP antibodies was 0.641, and the 95% confidence interval was 0.586-0.697, which was greater than 0.5, indicating that anti-AOSP antibodies in serum have clinical significance in the diagnosis of RA.

[0046] To further study the diagnostic value of anti-AOSP antibodies in RA, we set the AU value at the maximum value of the Youden index as the cut-off value (AU value = 25.46), and calculated that the sensitivity of anti-AOSP antibodies in RA was 32.67%, the specificity was 91.08%, the positive predictive value was 62.82%, and the negative predictive value was 74.56%, indicating that anti-AOSP antibodies have certain clinical diagnostic value in RA, see Table 1.

[0047] Table 1 Diagnostic value of anti-AOSP antibodies in RA Next, we further collected sera from seronegative RA patients and measured anti-AOSP antibody levels in sera from 140 anti-CCP antibody-negative RA patients, 155 RF-negative RA patients, and 107 anti-CCP antibody-negative and RF-negative RA patients. The results are shown in Figure 3 Compared with the healthy control (HC) group, the anti-AOSP antibody levels in the serum of patients with different types of seronegative RA were significantly increased. The positive rate of anti-AOSP antibody in anti-CCP antibody-negative RA was 34.28%, the positive rate in RF-negative RA was 35.48%, and the positive rate in anti-CCP antibody-negative and RF-negative RA was 34.58%, which has a good diagnostic value, see Table 2.

[0048] Table 2 Diagnostic value of anti-AOSP antibodies in seronegative RA In summary, anti-AOSP antibodies are potential biomarkers for RA and have good diagnostic efficacy in seronegative RA, providing an effective new method for the early diagnosis of RA, especially seronegative RA.

[0049] 2. Diagnostic value of anti-ALSP antibodies in seronegative RA In order to explore whether anti-ALSP antibodies exist in RA, we first detected the levels of anti-ALSP antibodies in the serum of 475 patients with RA, disease control groups (SLE, SS, OA) and healthy controls using the ELISA method in Example 2. Figure 4 , showing that the level of anti-ALSP antibodies in the serum of RA patients was higher than that in disease controls and healthy controls.

[0050] We further analyzed the ROC curve of anti-ALSP antibody and the results were as follows Figure 5 As shown, the AUC of anti-ALSP antibody was 0.636, and the 95% confidence interval was 0.580-0.692, which was greater than 0.5, indicating that anti-ALSP antibodies in serum have clinical significance in the diagnosis of RA.

[0051] To further study the diagnostic value of anti-ALSP antibodies in RA, we set the maximum value of the Youden index as the cut-off value (cut-off value = 23.73), and calculated that the sensitivity of anti-ALSP antibodies in RA was 29.33%, the specificity was 93.54%, the positive predictive value was 67.69%, and the negative predictive value was 74.15%, indicating that anti-ALSP antibodies have certain clinical diagnostic value in RA, see Table 3.

[0052] Table 3 Diagnostic value of anti-ALSP antibodies in RA Next, we analyzed the diagnostic value of anti-ALSP antibody levels in the sera of 140 anti-CCP antibody-negative RA patients, 155 RF-negative RA patients, and 107 anti-CCP antibody-negative and RF-negative RA patients. Figure 6 Compared with the healthy control group, the anti-ALSP antibody levels in the serum of patients with different types of seronegative RA were significantly increased. The positive rate of anti-ALSP antibody in anti-CCP antibody-negative RA was 20.71%, the positive rate in RF-negative RA was 20.65%, and the positive rate in anti-CCP antibody-negative and RF-negative RA was 19.63%, which has a good diagnostic value, see Table 4.

[0053] Table 4 Diagnostic value of anti-ALSP antibodies in seronegative RA In summary, anti-ALSP antibodies are potential biomarkers for RA and have good diagnostic efficacy in seronegative RA, providing an effective new method for the early diagnosis of RA, especially seronegative RA.

[0054] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. Acetylated SR-A polypeptide, characterized in that It is acetylated ornithine SR-A polypeptide (AOSP) and / or acetylated lysine SR-A polypeptide (ALSP), and the amino acid sequence is as follows: AOSP: KGD-Orn(Ac-)-GAIGFPGS-Orn(Ac-)-GLPGYAG- Orn(Ac-); ALSP: KGD-K(Ac-)-GAIGFPGS-K(Ac-)-GLPGYAG-K(Ac-).

2. Use of the acetylated SR-A polypeptide according to claim 1 in the preparation of a diagnostic product for rheumatoid arthritis, characterized in that: The product is used to detect the content of anti-acetylated polypeptide antibodies.

3. The use according to claim 2, characterized in that: Compared with normal healthy people, patients with Sjögren's syndrome, systemic lupus erythematosus and osteoarthritis, the content of anti-acetylated peptide antibodies in biological samples of patients with rheumatoid arthritis is significantly increased.

4. The use according to claim 3, characterized in that: The rheumatoid arthritis patients include any of the following: patients who are positive for anti-CCP antibodies and positive for RF; patients who are negative for anti-CCP antibodies; patients who are negative for RF; and patients who are negative for anti-CCP antibodies and negative for RF.

5. The use according to claim 2, characterized in that: The biological sample is blood, plasma, serum or tissue fluid.

6. The use according to claim 2, characterized in that: The diagnostic product is an ELISA detection kit.

7. A rheumatoid arthritis diagnostic kit, characterized in that: include: The acetylated SR-A polypeptide according to claim 1, an ELISA plate, an ELISA coating solution, a washing solution, a blocking solution, a sample diluent, an enzyme-labeled secondary antibody, a chromogenic substrate and a stop solution.

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