Bacterial polypeptide gfo and its use in the preparation of diagnostic products for rheumatoid arthritis

Abstract

The application discloses a bacterial polypeptide Gfo and application thereof in preparing a rheumatoid arthritis diagnosis product, and belongs to the technical field of rheumatoid arthritis diagnosis. The amino acid sequence of the bacterial polypeptide Gfo is LNSHGFLPETE. The diagnosis product prepared from the bacterial polypeptide Gfo can effectively detect that the level of an anti-bacterial polypeptide Gfo antibody in a biological sample of a rheumatoid arthritis patient is significantly higher than that of a healthy person and other rheumatism immune diseases similar to rheumatoid arthritis. Moreover, in patients negative to CCP antibody and RF, the level is still significantly increased. Therefore, the anti-bacterial polypeptide Gfo antibody is an important biomarker of rheumatoid arthritis, and provides an effective new method for diagnosis of RA, in particular, diagnosis of serum-negative RA.

Description

Technical Field

[0001] This invention relates to the field of rheumatoid arthritis diagnostic technology, specifically to the bacterial polypeptide Gfo and its application in the preparation of rheumatoid arthritis diagnostic products. Background Technology

[0002] Rheumatoid arthritis (RA) is a systemic, heterogeneous autoimmune disease characterized by destructive, chronic polyarthritis. It commonly affects peripheral small joints, is frequently accompanied by multi-system complications, and can ultimately lead to joint deformities and loss of function.

[0003] The clinical diagnosis of rheumatoid arthritis (RA) mainly relies on medical history, physical examination, and laboratory biomarkers. Currently, the commonly used indicators for RA diagnosis are rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). However, their diagnostic sensitivity is limited, and about one-third of RA patients still cannot be diagnosed in a timely manner due to the lack of diagnostic markers, thus delaying treatment. Therefore, discovering new diagnostic markers for RA to fill the gaps in the diagnosis of clinically challenging seronegative RA is an urgent clinical problem to be solved.

[0004] The tonsils are the first line of immune defense against microbial antigens, maintaining local and systemic immune homeostasis through continuous participation in immune responses. Disruption of their immune tolerance mechanism may lead to abnormal reactions to symbiotic microorganisms, thereby triggering autoimmune diseases. As a secondary lymphoid organ, the tonsils are more directly involved in immune regulation. The close interaction between the tonsillar flora and the host immune system may affect autoimmune responses, which is an important mechanism in the pathogenesis of rheumatoid arthritis (RA). Our previous research found that Bacteroides A1C1 was significantly elevated in the tonsillar flora of RA patients (Li J, et al. The aberrant tonsillar microbiota modulates autoimmune responses in rheumatoid arthritis. JCIInsight. 2024 Aug 20;9(18):e175916). Antibodies stimulated by Bacteroides may be closely related to the occurrence and development of RA and are potential diagnostic markers for RA. Although current research has shown that tonsillar flora imbalance is related to the development of RA, there is still a lack of microbial-related immunodiagnostic methods, especially for diagnostic markers for serologically negative RA patients. Diagnosis of RA is crucial, as early intervention can slow joint damage in most patients and prevent irreversible deformities or disabilities.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to address the ongoing need for novel serum biomarkers to better diagnose rheumatoid arthritis (RA), particularly RA with negative serum antibodies. We provide a bacterial polypeptide, Gfo, which can detect corresponding antibody levels in biological samples from RA patients, thereby improving the diagnosis of RA.

[0007] The technical solution of this invention is described in detail below:

[0008] In a first aspect, the present invention provides a bacterial polypeptide Gfo, the amino acid sequence of which is LNSHGFLPETE (SEQ ID NO:1), which is a polypeptide against Bacteroides A1C1 (Gfo / Idh / MocA family oxidoreductase, Gfo).

[0009] Optionally or preferably, the above-mentioned bacterial polypeptide Gfo has a carrier protein BSA coupled to the N-terminus of its amino acid sequence. The coupled amino acid sequence is as follows: BSA-C-LNSHGFLPETE (SEQ ID NO:2), which can increase immunogenicity.

[0010] Secondly, the present invention provides the application of the above-mentioned bacterial polypeptide Gfo in the preparation of a diagnostic product for rheumatoid arthritis, wherein the product is used to detect the content of antibacterial polypeptide Gfo antibody in a patient's biological sample.

[0011] Optionally or preferably, in the above applications, the content of antibacterial peptide Gfo antibody in biological samples from rheumatoid arthritis patients is significantly increased compared to healthy individuals, patients with Sjögren's syndrome, patients with systemic lupus erythematosus, and patients with osteoarthritis.

[0012] Optionally or preferably, in the above applications, the biological sample is serum, plasma, tissue fluid, or blood.

[0013] Optionally or preferably, in the above applications, the diagnostic product is an ELISA test kit.

[0014] Thirdly, the present invention provides a diagnostic kit for rheumatoid arthritis, comprising: the above-mentioned bacterial polypeptide Gfo, 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] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The bacterial polypeptide Gfo provided by this invention, as a detection antigen, can effectively detect antibacterial polypeptide Gfo antibodies in patient biosamples using an ELISA method. The level of antibacterial polypeptide Gfo antibodies in RA patient biosamples is significantly higher than that in healthy controls and other disease controls, and can be used as a biomarker for RA diagnosis. It exhibits particularly good diagnostic efficacy in anti-CCP antibody-negative RA, RF-negative RA, and anti-CCP antibody-negative and RF-negative RA, providing an effective new method for RA diagnosis, especially for the diagnosis of seronegative RA. Attached Figure Description

[0017] Figure 1 The levels of antibacterial peptide Gfo antibody in serum samples from different groups in this example are shown. HC represents the healthy normal group, OA represents the osteoarthritis group, SS represents the Sjögren's syndrome group, SLE represents the systemic lupus erythematosus group, and anti-CCP is also mentioned. + RA refers to the CCP antibody-positive rheumatoid arthritis group, anti-CCP - RA refers to the rheumatoid arthritis group that is negative for CCP antibodies.

[0018] Figure 2 The ROC curve of the antibacterial peptide Gfo antibody in the Total RA diagnosis is shown in the example.

[0019] Figure 3 In the examples, the antibacterial peptide Gfo antibody was used in anti-CCP. + ROC curve in RA diagnosis.

[0020] Figure 4 In the examples, the antibacterial peptide Gfo antibody was used in anti-CCP. - ROC curve in RA diagnosis.

[0021] Figure 5 The levels of antibacterial peptide Gfo antibody and anti-CCP in serum samples from different groups of RA patients in the examples are shown. - For the CCP antibody negative group, RF - The group was RF-negative (anti-CCP+RF). - The group was anti-CCP antibody negative and RF negative. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial channels.

[0023] Example 1 Synthesis of bacterial polypeptide Gfo

[0024] The amino acid sequence of Bacteroides A1C1 protein Gfo / Idh / MocA family oxidoreductase (GFO) was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). The potential antigenic epitopes of the bacterial protein were predicted using the B cell epitope prediction tool in the IEDB database (http: / / www.iedb.org / ). After analysis, a polypeptide Gfo was finally identified: LNSHGFLPETE (SEQ ID NO:1).

[0025] The bacterial polypeptide Gfo, with the sequence BSA-C-LNSHGFLPETE (SEQ ID NO:2), was synthesized using a solid-phase polypeptide synthesis method. The sequence contains a carrier protein BSA coupled to the N-terminus via a cysteine ​​residue.

[0026] Example 2: ELISA diagnostic product prepared from bacterial polypeptide Gfo for the diagnosis of RA

[0027] The ELISA kit includes: bacterial peptide Gfo, microplate, ELISA coating solution, washing solution (0.05% PBST), blocking solution (PBST containing 3% BSA), diluent (PBST containing 1% BSA), enzyme-labeled secondary antibody (goat anti-human IgG antibody), chromogenic substrate (TMB), and stop solution (sulfuric acid solution).

[0028] Detection method:

[0029] (1) Preheat the 96-well microplate to UV light overnight to improve adsorption capacity.

[0030] (2) Peptide lysing: Take out the synthesized bacterial peptide Gfo (sequence BSA-C-LNSHGFLPETE), bring it to room temperature, centrifuge at 12000 rpm for 2 min, and dissolve the peptide in sterile PBS to 1 mg / mL.

[0031] (3) Plate 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 the sealing membrane, and place it in a refrigerator at 4°C overnight.

[0032] (4) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0033] (5) Sealing: Add 200 µL of PBST containing 3% BSA to each well and let stand at room temperature for 2 h.

[0034] (6) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0035] (7) Dilute the serum with PBST containing 1% BSA at a ratio of 1:100, mix well and add 100 µL to each well, and incubate at 37°C for 1 h.

[0036] (8) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0037] (9) Dilute the goat anti-human IgG antibody at a ratio of 1:15000 with PBST containing 1% BSA as a diluent, mix well, add 100 µL to each well, and incubate at 37°C for 0.5 h.

[0038] (10) Washing: Add 300 µL of 0.05% PBST to each well, let stand for 2 min, and repeat 3 times.

[0039] (11) Add 100 µL of TMB substrate to each well and react at room temperature in the dark for 5-20 min.

[0040] (12) Add 100 µL of 2 M sulfuric acid solution to each well to terminate the reaction, and read the absorbance (optical density, OD value) at 450 nm / 570 nm wavelength on the microplate reader.

[0041] (13) Each plate contains standard positive serum and blank wells. Ten cases of RA serum with OD > 1.5 were selected and mixed in the same volume to obtain the standard positive serum.

[0042] (14) The result is expressed as an AU value, which is calculated as follows:

[0043] 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.

[0044] This study initially included 610 patients and healthy volunteers, comprising 280 patients with rheumatoid arthritis (RA), 68 with Sjögren's syndrome (SS), 72 with systemic lupus erythematosus (SLE), 69 with osteoarthritis (OA), and 121 healthy controls. RA patients met the 2010 ACR and EULAR diagnostic criteria for RA, while SLE, SS, and OA patients met their respective diagnostic or classification criteria. All serum samples were collected by the Department of Rheumatology and Immunology at Peking University People's Hospital from 2019 to 2023. This study was approved by the Research Ethics Committee of Peking University People's Hospital.

[0045] To further explore the diagnostic value of anti-Gfo antibodies in seronegative RA, serum samples were collected from patients with seronegative RA. The levels of anti-Gfo antibodies were detected in the serum of 108 patients with anti-CCP antibody-negative RA, 119 patients with RF-negative RA, and 100 patients with both anti-CCP antibody-negative and RF-negative RA.

[0046] To investigate the presence of the antibacterial peptide Gfo antibody in RA, we first detected the level of Gfo antibody in the serum of 610 individuals, including RA patients, disease controls (SLE, SS, OA), and healthy controls, using ELISA. The results showed that the level of anti-Gfo antibody in the serum of RA patients was higher than that in disease controls and healthy controls. (See [link to ELISA]). Figure 1 .

[0047] We further analyzed the receiver operating characteristic (ROC) curves of the antibacterial peptide Gfo antibody in RA, using healthy individuals and those with SLE, SS, and OA as controls. The area under the ROC curve (AUC) for the antibacterial peptide Gfo antibody was 0.719, with a 95% confidence interval of 0.679–0.760 (p < 0.001), indicating that serum antibacterial peptide Gfo antibody has clinical significance in the diagnosis of RA. (See [link to relevant documentation]). Figure 2 .

[0048] To further investigate the diagnostic value of antibacterial peptide Gfo antibody in RA, we set the AU value (mean + 3*SD) in healthy individuals as the cut-off value (AU value = 44.96). The calculated sensitivity of antibacterial peptide Gfo antibody in RA was 30.71%, the specificity was 95.15%, the positive predictive value was 84.31%, and the negative predictive value was 61.81% (see Table 1). This suggests that antibacterial peptide Gfo antibody has certain clinical diagnostic value in overall RA.

[0049] In anti-CCP-positive RA, with healthy individuals and those with SLE, SS, and OA as controls, the area under the ROC curve (AUC) of the antibacterial peptide Gfo antibody was 0.722, with a 95% confidence interval of 0.676–0.767 (p < 0.001). (See [link to relevant documentation]). Figure 3 The sensitivity of the antibacterial peptide Gfo antibody in anti-CCP-positive RA was 32.14%, the specificity was 95.15%, the positive predictive value was 79.75%, and the negative predictive value was 70.25% (see Table 1). This suggests that the antibacterial peptide Gfo antibody has certain clinical diagnostic value in anti-CCP-positive RA.

[0050] In anti-CCP-negative RA, with healthy individuals and those with SLE, SS, and OA as controls, the area under the ROC curve (AUC) of the antibacterial peptide Gfo antibody was 0.705, with a 95% confidence interval of 0.641–0.770 (p < 0.001). This indicates that serum antibacterial peptide Gfo antibody has clinical significance in the diagnosis of anti-CCP-negative RA. (See also...) Figure 4 The sensitivity of the bacterial peptide Gfo antibody in anti-CCP negative RA was 27.38%, the specificity was 95.15%, the positive predictive value was 58.97%, and the negative predictive value was 83.73% (see Table 1). This suggests that the antibacterial peptide Gfo antibody has certain clinical diagnostic value in anti-CCP negative RA.

[0051] Table 1. Diagnostic value of antibacterial peptide Gfo antibodies in RA

[0052] Group Number of examples Number of positive cases of antibacterial peptide Gfo antibody Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Total RA 280 86 30.71 95.15 84.31 61.81 <![CDATA[Anti-CCP + RA]]> 196 63 32.14 95.15 79.75 70.25 <![CDATA[Anti-CCP - RA]]> 84 23 27.38 95.15 58.97 83.73 SLE 72 6 8.33 - - - SS 68 7 10.26 - - - OA 69 2 2.90 - - - HC 121 1 0.83 - - -

[0053] To further elucidate the diagnostic value of antibacterial peptide Gfo antibody in autoantibody-negative RA, we conducted further studies in 108 patients with anti-CCP antibody-negative RA, 119 patients with RF-negative RA, and 100 patients with both anti-CCP antibody-negative and RF-negative RA. Figure 5 The results showed that the positive rate of antibacterial peptide Gfo antibody was 25.93% in anti-CCP antibody-negative RA, 23.53% in RF-negative RA, and 24% in both anti-CCP antibody-negative and RF-negative RA, indicating good diagnostic value (Table 2).

[0054] Table 2. Diagnostic value of antibacterial peptide Gfo antibody in serum-negative RA

[0055]

[0056] In summary, antibacterial peptide Gfo antibody is an important biomarker for RA and has good diagnostic efficacy in seronegative RA, providing an effective new method for the diagnosis of RA, especially seronegative RA.

[0057] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. A bacterial polypeptide Gfo, characterized in that, The amino acid sequence is LNSHGFLPETE.

2. The bacterial polypeptide Gfo according to claim 1, characterized in that, The N-terminus of the amino acid sequence is also coupled with the carrier protein BSA, and the coupled amino acid sequence is BSA-C-LNSHGFLPETE.

3. The application of the bacterial polypeptide Gfo described in claim 1 or 2 in the preparation of diagnostic products for rheumatoid arthritis, characterized in that, The product is used to detect the content of antibacterial peptide Gfo antibody in patient biological samples.

4. The application according to claim 3, characterized in that, Compared with healthy individuals, patients with Sjögren's syndrome, patients with systemic lupus erythematosus, and patients with osteoarthritis, the levels of antibacterial peptide Gfo antibodies were significantly increased in biological samples from patients with rheumatoid arthritis.

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

6. The application according to claim 3, characterized in that, The diagnostic product is an ELISA test kit.

7. A diagnostic kit for rheumatoid arthritis, characterized in that, include: The bacterial polypeptide Gfo, enzyme-labeled plate, ELISA coating solution, washing solution, blocking solution, sample dilution solution, enzyme-labeled secondary antibody, chromogenic substrate and stop solution as described in claim 1 or 2.

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