CN-121779530-B - Citrullinated T200C polypeptide and application thereof in preparation of rheumatoid arthritis diagnostic products

Abstract

The invention discloses citrullinated T200C polypeptide and application thereof in preparing rheumatoid arthritis diagnostic products, and belongs to the technical field of disease diagnosis. The amino acid sequence of citrullinated T200C polypeptide is GSM-Cit-GA-Cit-Cit-EPEEPEGAV. Studies have found that anti-citrullinated T200C polypeptide antibodies are significantly higher in RA patients than in healthy people and other rheumatic immune patients who are easily confused with RA. The citrullinated T200C polypeptide provided by the invention is used as a detection antigen, and detection of the anti-citrullinated T200C polypeptide antibody in a patient body can effectively identify RA, and has good diagnostic value particularly for anti-CCP antibody and/or RF negative RA, and ESR and/or CRP normal RA.

Inventors

• HU FANLEI
• LI ZHANGUO
• XIONG YUEJIAO
• CAO LULU

Assignees

• 北京大学人民医院

Dates

Publication Date

20260508

Application Date

20260309

Claims (9)

1. 1. Citrullinated T200C polypeptide characterized by the amino acid sequence as follows: GSM-Cit-GA-Cit-Cit-EPEEPEGAV。
2. 2. the citrullinated T200C polypeptide according to claim 1, wherein the N-terminal amino acid sequence is further coupled to the carrier protein BSA via a cysteine residue, the coupled amino acid sequence being as follows: BSA-C-GSM-Cit-GA-Cit-Cit-EPEEPEGAV。
3. 3. Use of a citrullinated T200C polypeptide according to claim 1 or 2 for the preparation of a diagnostic product for rheumatoid arthritis, wherein said product is used for detecting the anti-citrullinated T200C polypeptide antibody content in a biological sample of a patient.
4. 4. The use according to claim 3, wherein the level of anti-citrullinated T200C polypeptide antibodies in the biological sample of rheumatoid arthritis patients is significantly increased relative to normal healthy people, patients with sjogren's syndrome, patients with systemic lupus erythematosus and patients with osteoarthritis.
5. 5. The use according to claim 4, wherein the rheumatoid arthritis patients comprise anti-CCP antibody negative rheumatoid arthritis patients and/or RF negative rheumatoid arthritis patients.
6. 6. The use according to claim 4, wherein the rheumatoid arthritis patients comprise rheumatoid arthritis patients with normal erythrocyte sedimentation rate and/or rheumatoid arthritis patients with normal C-reactive protein.
7. 7. The use according to claim 3, wherein the biological sample is serum, plasma or joint fluid.
8. 8. The use according to claim 3, wherein the diagnostic product is an ELISA detection kit.
9. 9. A rheumatoid arthritis diagnosis kit is characterized by comprising the citrullinated T200C polypeptide, an ELISA plate, an ELISA coating liquid, a washing liquid, a sealing liquid, a diluting liquid, an ELISA secondary antibody, a chromogenic substrate and a stop liquid according to claim 1 or 2.

Description

Citrullinated T200C polypeptide and application thereof in preparation of rheumatoid arthritis diagnostic products Technical Field The invention relates to the technical field of disease diagnosis, in particular to application of citrullinated T200C polypeptide in preparation of rheumatoid arthritis diagnosis products. Background Rheumatoid arthritis (rheumatoid arthritis, RA) is a systemic inflammatory disease characterized by progressive joint damage. As a clinically common autoimmune disease, RA is a global disease, affecting about 0.5% -1% of the population worldwide, and only 500 ten thousand RA patients exist in China. Early diagnosis and treatment are critical to improve prognosis in RA patients. Current diagnosis of RA depends on comprehensive assessment of clinical symptoms, signs, laboratory and radiological indicators. Among them, rheumatoid factor (rheumatoid factor, RF) and anti-cyclic citrulline polypeptide (anti-cyclic citrullinated peptide antibodies, anti-CCP) antibodies are the most commonly used laboratory diagnostic indicators in clinic, but the diagnostic sensitivity is limited, and more than 30% of patients are seronegative (RF and anti-CCP negative). For seronegative RA patients, clinical diagnosis is mostly dependent on the evidence of symmetric polyarthritis, morning stiffness, rising inflammatory indexes, synovitis, bone marrow edema or early bone erosion and the like, which are shown by imaging examination, and the requirement on clinical experience is high, so that early patients are easy to misdiagnose or miss diagnosis, delay treatment and even cause disability. Thus, the discovery of new diagnostic markers for RA, particularly those that are effective in seronegative RA patients, is a clinical problem that is currently in need of resolution. Disclosure of Invention The invention aims to provide a novel antigen peptide, namely citrullinated T200C polypeptide (CTP for short), and an adaptive antibody, namely anti-citrullinated T200C polypeptide antibody (anti-CTP for short), which is used as a novel serological marker, has a good diagnosis effect on RA, and can be particularly used as a diagnosis supplement of seronegative RA. The technical scheme of the invention is as follows: in a first aspect, the present invention provides a citrullinated T200C polypeptide, abbreviated CTP, having the amino acid sequence: GSM-Cit-GA-Cit-Cit-EPEEPEGAV (SEQ ID NO: 1), citrulline is at positions 4, 7 and 8 in the sequence. The autoantigens in RA and health/disease control are enriched in the early stage of the subject group by an antibody affinity chromatography technology, mass spectrometry is carried out, and the result of the mass spectrometry is subjected to bioinformatics analysis, so that T200C is obviously enriched in serum of RA patients. T200C (Transmembrane protein C, transmembrane protein 200C) is a transmembrane protein, and the current academy has little research on its function. Next, we identified the B cell epitope of the T200C protein, and screened out peptide fragments with higher scores and containing arginine, and synthesized the polypeptide sequence that citrullinated all of its arginine, i.e. SEQ ID No. 1 described above. We found that Enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) using this citrullinated polypeptide as antigen has significant diagnostic value for RA, especially seronegative RA. Alternatively or preferably, the N-terminal of the amino acid sequence of the CTP is further coupled with carrier protein BSA through cysteine residues, so that the immunogenicity can be improved, and the coupled amino acid sequence is as follows: BSA-C-GSM-Cit-GA-Cit-Cit-EPEEPEGAV(SEQ ID NO:2)。 The product is used for detecting the content of anti-CTP antibodies in a biological sample of a patient. The anti-CTP antibody content in biological samples of RA patients was significantly increased relative to normal healthy people (healthy control, HC), sj (Sj) gren's syndrome, SS) patients, systemic lupus erythematosus (systemic lupus erythematosus, SLE) patients, and Osteoarthritis (OA) patients. The RA patients include anti-CCP antibody negative RA patients and/or RF negative RA patients, RA patients with normal Erythrocyte Sedimentation Rate (ESR) and/or C-reactive protein (CRP). The biological sample is serum, plasma or joint fluid. The diagnostic product is an ELISA detection kit. In a second aspect, the invention provides a RA diagnostic kit, which comprises the citrullinated T200C polypeptide, an ELISA plate, ELISA coating liquid, washing liquid, sealing liquid, diluent, enzyme-labeled secondary antibody, chromogenic substrate and stop solution, wherein the amino acid sequence of the citrullinated T200C polypeptide is GSM-Cit-GA-Cit-EPEEPEGAV or BSA-C-GSM-Cit-GA-Cit-EPEEPEGAV. Compared with the prior art, the invention has the following beneficial effects: Through a large number of research and screening works, the citrullinated T200C polypeptide,