Acetylated SR-A polypeptide and its application in the preparation of diagnostic products for rheumatoid arthritis

Abstract

The present invention discloses an acetylated SR-A polypeptide and its application in the preparation of a diagnostic product for rheumatoid arthritis (RA). Among them, the acetylated polypeptide is acetylated ornithine SR-A polypeptide (AOSP) and / or acetylated lysine SR-A polypeptide (ALSP). This acetylated polypeptide can be used as a detection antigen to detect anti-acetylated SR-A polypeptide antibodies in biological samples of RA patients, and thus can be used to prepare a diagnostic product for RA. In particular, it has good diagnostic efficacy in seronegative RA, providing an effective new method for the early diagnosis of RA, especially for the diagnosis of seronegative RA.

Description

Technical Field

[0001] The present invention relates to the technical field of disease diagnosis, and particularly relates to an acetylated SR-A polypeptide and its application in the preparation of a rheumatoid arthritis diagnostic 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 commonly used clinical indicators for diagnosing RA are rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP), but the sensitivity of their diagnosis is limited. There are still about one-third of RA patients who are delayed in treatment because they cannot be diagnosed in time due to the lack of diagnostic markers.

[0003] Scavenger receptor-A (SR-A) is a pattern recognition receptor mainly expressed on the surface of macrophages and dendritic cells and plays an important role in innate immunity and adaptive immunity. Our research group previously found through a large-sample multicenter study that SR-A has 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 negative for serum anti-CCP antibodies is 49.72%, the positive rate in RF-negative RA patients is 39.13%, and the positive rate in RA patients negative for both anti-CCP antibodies and RF 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.), showing good complementary diagnostic value.

[0004] Post-translational modifications of proteins, especially citrullination and acetylation modifications, play crucial roles in the pathogenesis of RA. Citrullination modification is a specific and important phenomenon in RA and has been extensively studied, which is of great significance in the pathogenic mechanism of RA autoantigens. Citrullination modification can enhance the immunogenicity of antigen peptides and their binding affinity with HLA-DRB1 molecules containing shared epitopes, thus activating autoreactive T cells and B cells more significantly 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 coenzyme A to the N-terminus or lysine residues of proteins under the action of acetylase, which is one of the main modification methods to change protein functions. After acetylation modification, the molecular characteristics of proteins or polypeptides can be changed, and new antigenic epitopes may be generated, thus inducing the production of anti-acetylated protein antibodies (AAPA). Although the structures of acetylated polypeptides and citrullinated polypeptides are very similar, the antibody spectra of AAPA and anti-citrullinated protein antibodies are different in RA.

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

[0007] The object 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 for preparing rheumatoid arthritis diagnostic products.

[0008] The technical solution of the present invention is described in detail as follows:

[0009] In the first aspect, the present invention provides an acetylated SR-A polypeptide, which is an acetylated ornithine SR-A peptide (AOSP) and / or an acetylated lysine SR-A peptide (ALSP), and the amino acid sequences are as follows:

[0010] AOSP:

[0011] KGD-Orn(Ac-)-GAIGFPGS-Orn(Ac-)-GLPGYAG- Orn(Ac-) (SEQ ID NO:1);

[0012] ALSP:

[0013] KGD-K(Ac-)-GAIGFPGS-K(Ac-)-GLPGYAG-K(Ac-) (SEQ ID NO:2).

[0014] In a second aspect, the present invention provides the use of the above-mentioned acetylated SR-A polypeptide in the preparation of a diagnostic product for rheumatoid arthritis, and the product is used to detect the content of anti-acetylated SR-A polypeptide antibody. The anti-acetylated SR-A polypeptide antibody is an anti-AOSP polypeptide antibody and / or an anti-ALSP polypeptide antibody.

[0015] The content of anti-acetylated SR-A polypeptide antibody in the biological samples of patients with rheumatoid arthritis is significantly increased compared with that of normal healthy people, patients with Sjogren's syndrome, patients with systemic lupus erythematosus, and patients with osteoarthritis.

[0016] In the above application, the patients with rheumatoid arthritis include any one of the following: patients positive for anti-CCP antibody and positive for RF; patients negative for anti-CCP antibody (positive or negative for RF); patients negative for RF (positive or negative for anti-CCP antibody); patients negative for anti-CCP antibody and negative for RF.

[0017] In the above application, the biological sample is blood, plasma, serum or tissue fluid.

[0018] In the above application, the diagnostic product is an ELISA detection kit.

[0019] In a third aspect, the present invention provides a diagnostic kit for rheumatoid arthritis, comprising: the above-mentioned acetylated SR-A polypeptide, an enzyme-linked immunosorbent assay (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 termination solution.

[0020] The present invention has the following beneficial effects:

[0021] The acetylated SR-A polypeptide provided by the present invention can effectively detect the levels of anti-AOSP antibody and / or anti-ALSP antibody in the biological samples of patients as a detection antigen. The levels of these two antibodies in the biological samples of RA patients are significantly higher than those of healthy controls and other disease controls. It has good diagnostic efficacy in diagnosing rheumatoid arthritis as a RA biomarker, especially in anti-CCP antibody-negative RA, RF-negative RA, and anti-CCP antibody-negative and RF-negative RA, providing an effective new method for the early diagnosis of RA, especially for the diagnosis of seronegative RA. Description of the Drawings

[0022] Figure 1Statistical results of the expression levels of anti-AOSP antibodies in the sera of the RA group, different disease control groups, and healthy control group in Example 3. In the figure, anti-AOSP represents anti-AOSP antibody, RA: rheumatoid arthritis, SS: Sjogren's syndrome, SLE: systemic lupus erythematosus, OA: osteoarthritis, HC: healthy normal individuals.

[0023] Figure 2 ROC curve of anti-AOSP antibody in the diagnosis of RA in Example 3.

[0024] Figure 3 Statistical results of the expression levels of anti-AOSP antibodies in the sera of different types of seronegative RA in Example 3. In the figure, anti-AOSP represents anti-AOSP antibody, anti-CCP - RA: rheumatoid arthritis with negative anti-CCP antibody, RF - RA: rheumatoid arthritis with negative RF, (anti-CCP + RF) - RA: rheumatoid arthritis with negative anti-CCP antibody and negative RF, HC: healthy normal individuals.

[0025] Figure 4 Statistical results of the expression levels of anti-ALSP antibodies in the sera of the RA group, different disease control groups, and healthy control group in Example 3. In the figure, anti-ALSP represents anti-ALSP antibody, RA: rheumatoid arthritis, SLE: systemic lupus erythematosus, SS: Sjogren's syndrome, OA: osteoarthritis, HC: healthy normal individuals.

[0026] Figure 5 ROC curve of anti-ALSP antibody in the diagnosis of RA in Example 3.

[0027] Figure 6 Statistical results of the expression levels of anti-ALSP antibodies in the sera of different types of seronegative RA in Example 3. In the figure, anti-ALSP represents anti-ALSP antibody, anti-CCP - RA: rheumatoid arthritis with negative anti-CCP antibody, RF - RA: rheumatoid arthritis with negative RF, (anti-CCP + RF) - RA: rheumatoid arthritis with negative anti-CCP antibody and negative RF, HC: healthy normal individuals.

[0028] Figure 1 、 3 The statistical results of 3, 4, and 6 were analyzed for differences among multiple groups using the Kruskal-Wallis H test, and multiple comparisons were performed using the Dunnett t test. p P < 0.05 was considered statistically significant, * p P < 0.05, **p <0.01, *** p <0.001。 Detailed implementation manners

[0029] In order to enable those skilled in the art of the present technology to better understand the solution of this application, the following will clearly and completely describe this application in combination with the embodiments and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application. The instruments and reagents used in the embodiments are from commercial channels without special instructions.

[0030] Example 1 Preparation of acetylated SR-A polypeptide

[0031] Obtain the amino acid sequence of human SR-A protein in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and obtain the predicted potential antigenic epitopes of SR-A through the B cell epitope prediction tool in the IEDB database (http: / / www.iedb.org / ). At the same time, perform a BLAST sequence alignment of the SR-A amino acid sequence with the RA autoantigen epitopes in the IEDB database, and jointly determine the B cell antigenic epitopes of SR-A in combination with the above predicted potential antigenic epitopes.

[0032] Use solid-phase peptide synthesis to synthesize acetylated ornithine SR-A polypeptide (AOSP) with the sequence KGD-Orn(Ac-)-GAIGFPGS-Orn(Ac-)-GLPGYAG-Orn(Ac-); synthesize acetylated lysine SR-A polypeptide (ALSP) with the sequence KGD-K(Ac-)-GAIGFPGS-K(Ac-)-GLPGYAG-K(Ac-). The above polypeptides were synthesized by Shanghai Nuoyou Biotechnology Co., Ltd.

[0033] Example 2 Use acetylated SR-A polypeptides to detect the levels of anti-AOSP antibodies and anti-ALSP antibodies in the sera of patients by ELISA method.

[0034] Based on the Enzyme-linked immunosorbent assay (ELISA) method, an ELISA diagnostic kit is prepared. The components of the kit include: enzyme-linked immunosorbent assay plates, acetylated SR-A polypeptides, 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, chromogenic substrate TMB, and termination solution sulfuric acid. The percentages of each component in the kit are volume percentages. The solvent of the goat anti-human enzyme-labeled secondary antibody is glycerol, and the solvents of the other reagents are water.

[0035] (1) Pre-irradiate the 96-well enzyme-linked immunosorbent assay plates with ultraviolet light overnight to improve the adsorption capacity.

[0036] (2) Peptide dissolution: Take out the synthesized acetylated SR-A polypeptide (AOSP or ALSP), let it return to room temperature, centrifuge at 12000 rpm for 2 min, and dissolve the polypeptide in sterile PBS to 1 mg / mL.

[0037] (3) Plate coating: Dilute the polypeptide to 10 µg / mL with the pre-diluted ELISA coating solution (1×), add 100 µL to each well, seal the plate with a sealing film, and place it horizontally in a 4°C refrigerator overnight.

[0038] (4) Plate washing: Add 300 µL of 0.05% PBST to each well, let it stand for 2 min, and repeat 3 times.

[0039] (5) Blocking: Add 200 µL of 3% BSA-PBST to each well and let it stand at room temperature for 2 h.

[0040] (6) Plate washing: Add 300 µL of 0.05% PBST to each well, let it stand for 2 min, and repeat 3 times.

[0041] (7) Dilute the serum with the 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.

[0042] (8) Plate washing: Add 300 µL of 0.05% PBST to each well, let it stand for 2 min, and repeat 3 times.

[0043] (9) Dilute the goat anti-human IgG antibody with 1% BSA-PBST as the diluent at a ratio of 1:15000, mix well, add 100 µL to each well, and incubate at 37°C for 0.5 h.

[0044] (10) Plate washing: Add 300 µL of 0.05% PBST to each well, let it stand for 2 min, and repeat 3 times.

[0045] (11) Add 100 µL of TMB substrate to each well and react at room temperature in the dark for 5 - 20 min.

[0046] (12)Add 100 µL of 2 M sulfuric acid solution to each well to terminate the reaction, and read the absorbance value (optical density, OD value) at a wavelength of 450 nm / 570 nm on an enzyme-linked immunosorbent assay (ELISA) reader.

[0047] (13)Each plate has standard positive serum and blank wells. Select 10 cases of RA serum with OD > 1.5, mix them in the same volume, and this is the standard positive serum.

[0048] (14)The results are expressed in AU value, and its calculation method is:

[0049] 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.

[0050] Example 3 Diagnostic value of anti-acetylated SR-A polypeptide antibody in RA

[0051] This study first included 475 patients and healthy controls, including 150 patients with rheumatoid arthritis (RA), 75 patients with Sjogren's syndrome (SS), 75 patients with systemic lupus erythematosus (SLE), 75 patients with osteoarthritis (OA), and 100 healthy controls. Their sera were collected and the levels of corresponding anti-ALSP antibodies were detected. RA patients met the 2010 ACR and EULAR RA diagnostic criteria, and SLE, SS, and OA patients met their respective diagnostic or classification criteria. All sera were collected by the Rheumatology and Immunology Department of Peking University People's Hospital from 2017 to 2020. This study obtained the review and approval of the Research Ethics Committee of Peking University People's Hospital.

[0052] To further explore the diagnostic value of anti-AOSP antibody and anti-ALSP antibody in seronegative RA, on the basis of the above, sera from seronegative RA patients were further collected, and the levels of anti-AOSP antibody and anti-ALSP antibody were detected in the sera of 140 RA patients negative for anti-CCP antibody, 155 RA patients negative for RF, and 107 RA patients negative for both anti-CCP antibody and RF.

[0053] (I)Diagnostic value of anti-AOSP antibody in seronegative RA

[0054] To investigate whether there are anti-AOSP antibodies in RA, we first detected the levels of anti-AOSP antibodies in the sera of 475 cases in total, including RA patients, disease control groups (SLE, SS, OA) and healthy control groups, by the ELISA method of Example 2. The results are shown in Figure 1 , showing that the levels of anti-AOSP antibodies in the sera of RA patients are higher than those in the disease control and healthy control groups.

[0055] We further analyzed the receiver operator characteristic (ROC) curve of anti-AOSP antibodies. The results are shown in Figure 2 . The area under the ROC curve (AUC) of anti-AOSP antibodies is 0.641, and the 95% confidence interval is 0.586 - 0.697, which is greater than 0.5, indicating that anti-AOSP antibodies in the serum have clinical significance in the diagnosis of RA.

[0056] To further study the diagnostic value of anti-AOSP antibodies in RA, we set the AU value at the maximum Youden index as the Cut-off value (AU value = 25.46). The sensitivity of anti-AOSP antibodies in RA was calculated to be 32.67%, the specificity was 91.08%, the positive predictive value was 62.82%, and the negative predictive value was 74.56%, suggesting that anti-AOSP antibodies have certain clinical diagnostic value in RA. See Table 1.

[0057] Table 1 Diagnostic value of anti-AOSP antibodies in RA

[0058]

[0059] Next, we further collected the sera of seronegative RA patients. The levels of anti-AOSP antibodies were detected 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. The results are shown in Figure 3 . Compared with the healthy control (HC) group, the levels of anti-AOSP antibodies in the sera of different types of seronegative RA patients are significantly increased. The positive rate of anti-AOSP antibodies in anti-CCP antibody-negative RA is 34.28%, in RF-negative RA is 35.48%, and in anti-CCP antibody-negative and RF-negative RA is 34.58%, having good diagnostic value. See Table 2.

[0060] Table 2 Diagnostic value of anti-AOSP antibodies in seronegative RA

[0061]

[0062] In summary, anti-AOSP antibody is a potential biomarker for RA and has good diagnostic efficacy in seronegative RA, providing an effective new method for the early diagnosis of RA, especially for the diagnosis of seronegative RA.

[0063] (2) Diagnostic value of anti-ALSP antibody in seronegative RA

[0064] To explore whether there is anti-ALSP antibody in RA, we first detected the levels of anti-ALSP antibody in the sera of 475 cases including RA patients, disease control groups (SLE, SS, OA) and healthy control group by the ELISA method in Example 2. The results are as Figure 4 shown, indicating that the level of anti-ALSP antibody in the sera of RA patients is higher than that in disease controls and healthy controls.

[0065] We further analyzed the ROC curve of anti-ALSP antibody, and the results are as Figure 5 shown. The AUC of anti-ALSP antibody is 0.636, and the 95% confidence interval is 0.580 - 0.692, which is greater than 0.5, indicating that anti-ALSP antibody in serum has clinical significance in the diagnosis of RA.

[0066] To further study the diagnostic value of anti-ALSP antibody in RA, we set the value at the maximum Youden index as the cut-off value (cut-off value = 23.73), and calculated that the sensitivity of anti-ALSP antibody in RA is 29.33%, the specificity is 93.54%, the positive predictive value is 67.69%, and the negative predictive value is 74.15%, suggesting that anti-ALSP antibody has certain clinical diagnostic value in RA, see Table 3.

[0067] Table 3 Diagnostic value of anti-ALSP antibody in RA

[0068]

[0069] Next, we analyzed the diagnostic value of the level of anti-ALSP antibody in the sera of 140 RA patients negative for anti-CCP antibody, 155 RA patients negative for RF, and 107 RA patients negative for both anti-CCP antibody and RF. The results are as Figure 6 shown. Compared with the healthy control group, the levels of anti-ALSP antibody in the sera of different types of seronegative RA patients are all significantly increased. The positive rate of anti-ALSP antibody in RA patients negative for anti-CCP antibody is 20.71%, the positive rate in RA patients negative for RF is 20.65%, and the positive rate in RA patients negative for both anti-CCP antibody and RF is 19.63%, showing good diagnostic value, see Table 4.

[0070] Table 4 Diagnostic value of anti-ALSP antibody in seronegative RA

[0071]

[0072] In summary, the anti-ALSP antibody is a potential biomarker for RA and has good diagnostic efficacy in seronegative RA, providing an effective new method for the early diagnosis of RA, especially for the diagnosis of seronegative RA.

[0073] In this article, specific examples are used to elaborate on the inventive concept in detail. The description of the above embodiments is only for helping to understand the core idea of the present invention. It should be noted that for those of ordinary skill in the art, any obvious modifications, equivalent replacements or other improvements made without departing from the inventive concept shall be included within the protection scope of the present invention.

Claims

1. An acetylated SR-A polypeptide, characterized in that, It is an acetylated ornithine SR-A polypeptide (AOSP) and / or an 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 for detecting the content of anti-acetylated polypeptide antibody.

3. The application according to claim 2, wherein Compared with normal healthy people, patients with Sjogren's syndrome, patients with systemic lupus erythematosus and patients with osteoarthritis, the content of anti-acetylated polypeptide antibody in the biological samples of patients with rheumatoid arthritis increases significantly.

4. The application according to claim 3, characterized in that, The patients with rheumatoid arthritis include any one of the following: patients positive for anti-CCP antibody and positive for RF; patients negative for anti-CCP antibody; patients negative for RF; patients negative for anti-CCP antibody and negative for RF.

5. The application according to claim 3, characterized in that The biological sample is blood, plasma, serum or tissue fluid.

6. The application according to claim 2, wherein The diagnostic product is an ELISA detection kit.

7. A diagnostic kit for rheumatoid arthritis, characterized in that, It includes: The acetylated SR-A polypeptide described in claim 1, an enzyme-linked immunosorbent assay (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 termination solution.

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