Carbamylated polypeptide and application thereof in preparation of rheumatoid arthritis diagnostic kit

Abstract

The invention discloses a carbamylated polypeptide and application thereof in preparation of a rheumatoid arthritis diagnostic kit. The invention provides a cyclization carbamylation polypeptide as shown in SEQ ID NO. 1 and SEQ ID NO. 2 or a combination of the cyclization carbamylation polypeptide and the combination of the cyclization carbamylation polypeptide and the combination. Tests prove that the cyclized carbamylation polypeptide antigens as shown in SEQ ID NO.1 and SEQ ID NO.2 independently have high diagnostic value for diagnosing the negative RA of the anti-cyclic citrullinated peptide antibody, and respectively have application prospects of being developed into diagnostic kits for the negative RA of the anti-cyclic citrullinated peptide antibody; the combination of SEQ ID NO.1 and SEQ ID NO.2 further improves the diagnosis value of the anti-cyclic citrullinated peptide protein antibody negative RA, and has the application prospect of being developed into the anti-cyclic citrullinated peptide protein antibody negative RA diagnosis kit in a combined manner.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, relates to polypeptides and clinical applications thereof, and specifically relates to aminoformylated polypeptides and applications thereof in preparing a rheumatoid arthritis diagnostic kit. Background Art

[0002] Rheumatoid arthritis (RA) is an inflammatory synovitis disease that often occurs in small joints such as the hands and feet. Its disease characteristics are mainly chronic inflammatory synovitis. It is an autoimmune disease. If it is not treated in time, the disease will continue to develop, causing local inflammation, systemic joint deformities, and involvement of extra-articular organs, thus affecting daily life. Early diagnosis and early treatment can effectively improve prognosis and reduce the risk of disability.

[0003] In recent years, various studies have shown that RA patients have a variety of autoantibodies, such as rheumatoid factor (RF), anti-keratin antibodies (AKA), anti-perinuclear factor (APF) and anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies). RA is a heterogeneous disease, with some patients being positive for RF and / or anti-CCP antibodies (referred to as serum positive), and others being negative for RF and anti-CCP antibodies (referred to as serum negative).

[0004] How to strengthen the diagnosis of RA patients with negative anti-CCP antibodies has great clinical value. The applicant has discovered a polypeptide marker with high diagnostic efficacy for RA patients with negative anti-CCP antibodies, and proposed the present invention based on this discovery. Summary of the invention

[0005] The object of the present invention is to provide a carbamylated polypeptide and use thereof in preparing a rheumatoid arthritis diagnostic kit.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The use of the cyclized aminobamylated polypeptides of the sequences shown in SEQ ID NO.1 and / or SEQ ID NO.2 in combination or alone for preparing a diagnostic kit for rheumatoid arthritis:

[0008]

[0009] Wherein, X in each sequence represents carbamylated lysine, and its connection mode with the adjacent amino acid in the above polypeptide is shown in the following formula:

[0010]

[0011] The rheumatoid arthritis is anti-cyclic citrullinated peptide antibody-negative rheumatoid arthritis.

[0012] A cyclized carbamylated polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2:

[0013]

[0014] Wherein, X in each sequence represents carbamylated lysine, and its connection mode with the adjacent amino acid in the above polypeptide is shown in the following formula:

[0015]

[0016] A polypeptide combination, which is a combination of the polypeptides shown in the following sequences SEQ ID NO.1 and SEQ ID NO.2:

[0017]

[0018] Wherein, X in each sequence represents carbamylated lysine, and its connection mode with the adjacent amino acid in the above polypeptide is shown in the following formula:

[0019]

[0020] Beneficial effects:

[0021] The present invention provides a cyclic aminobamylated polypeptide shown in SEQ ID NO.1, SEQ ID NO.2 or a combination thereof. Experiments have shown that the cyclic aminobamylated polypeptide antigens shown in SEQ ID NO.1 and SEQ ID NO.2 have high diagnostic value for diagnosing anti-cyclic citrullinated peptide antibody-negative RA alone, and have application prospects for developing anti-cyclic citrullinated peptide antibody-negative RA diagnostic kits; the diagnostic value of SEQ ID NO.1 combined with SEQ ID NO.2 for diagnosing anti-cyclic citrullinated peptide protein antibody-negative RA is further improved, and has application prospects for jointly developing anti-cyclic citrullinated peptide antibody-negative RA diagnostic kits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The numerical distribution and mean of the ELISA test results of SEQ ID NO.1 polypeptide antigen in anti-cyclic citrullinated peptide antibody negative RA and HC samples and the ROC curve for distinguishing anti-cyclic citrullinated peptide antibody negative RA and HC samples;

[0023] Figure 2 The numerical distribution and mean of the ELISA test results of SEQ ID NO.2 polypeptide antigen in anti-cyclic citrullinated peptide antibody negative RA and HC samples and the ROC curve for distinguishing anti-cyclic citrullinated peptide antibody negative RA and HC samples;

[0024] Figure 3ROC curve of the combined diagnosis of SEQ ID NO.1 and SEQ ID NO.2 polypeptide antigens to distinguish between anti-cyclic citrullinated peptide antibody-negative RA and HC samples. DETAILED DESCRIPTION

[0025] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0026] Example 1: Discovery and synthesis of carbamylated polypeptides

[0027] 1. Experimental Materials

[0028] 1. Sample source

[0029] Plasma and serum were collected from rheumatoid arthritis subjects (RA) and healthy subjects (HC) who signed informed consent forms. Plasma should be freshly collected and separated. Plasma must be separated and collected within 6 hours of blood collection. The collected whole blood was placed in EDTA anticoagulant tubes, and the plasma was transferred to disposable sterile microcentrifuge tubes free of RNase.

[0030] The method for processing whole blood samples into serum is as follows: collect 2 ml of venous blood, put it in a 5 ml clean centrifuge tube, and anticoagulate it; after standing for 30 minutes, centrifuge it at 3500 rpm for 10 minutes, and take the supernatant; if the sample is not used immediately, it must be stored below -20°C.

[0031] 2. Instruments and reagents

[0032] Phosphatase inhibitors, trypsin, iodoacetamide, C18 desalting column, TMT labeling reagent, ziptip desalting column, liquid phase tandem Q Exactive HF-X mass spectrometer, Eppendorf pipette, vacuum centrifugal concentrator, etc.

[0033] 2. Experimental Methods

[0034] 1. Protein extraction

[0035] The plasma samples were taken out of the -80°C freezer, and 4 volumes of lysis buffer (8 M urea, 1% protease inhibitor and 1% phosphatase inhibitor) were added, and ultrasonic lysis was performed. The samples were centrifuged at 20000 g for 10 min at 4°C, and the supernatant was transferred to a new tube, and the protein concentration was determined using a BCA kit.

[0036] 2. Trypsin digestion

[0037] Take 100 μg of protein solution, add TCEP to a final concentration of 10 mM, and reduce at 56°C for 60 min. Then add iodoacetamide to a final concentration of 20 mM, and incubate at room temperature in the dark for 30 min. Finally, dilute the urea concentration of the sample to less than 2 M. Add pancreatin at a mass ratio of 1:50 (pancreatin: protein), and enzymolysis at 37°C overnight.

[0038] 3. Peptide desalting

[0039] Take 10mg C18 desalting column (waters), add methanol to activate twice, 1mL each time, and after the methanol is basically dripped dry, add 0.1% trifluoroacetic acid (TFA) 3 times, 1mL each time to balance the column. Dissolve the peptide sample with 0.1% TFA to a volume of 300μL-1mL, and adjust the pH to 2-3 with 10% TFA. Repeat the loading of the peptide solution 5-6 times to make it fully hang on the column, and then wash it with 0.1% TFA 3 times, 1mL each time to remove salt ions. Elute the peptide hanging on the column with 80% acetonitrile solution and vacuum concentrate and dry.

[0040] 4. TMT technology mark

[0041] Before labeling the sample, take out the TMT labeling reagent and restore it to room temperature. For each labeling reagent of 0.8 mg, add 42 μL of anhydrous acetonitrile (mass spectrometry grade) and vortex thoroughly to dissolve. Take out 10 μg of protein from each desalted peptide sample and transfer it to a new microcentrifuge tube to prepare the labeled TMT reagent. Add 4 μL of different TMT labeling reagent solutions to each protein sample, vortex lightly, centrifuge instantly, and shake at room temperature for 1 hour. After labeling, add 1 μL of 5% hydroxylamine to each sample mixture, shake at room temperature for 2 hours to quench. After quenching the reaction, combine all samples of the same group of labels into a new microcentrifuge tube, vortex to mix, and place in a vacuum centrifuge concentrator for concentration and drying.

[0042] 5. Desalting of mixed peptide samples

[0043] Take the ziptip desalting column and desalt it with a 20μL Eppendorf pipette. First, activate the ziptip with 100% acetonitrile and 50% acetonitrile solution for 3 times, and then balance it with 0.1% formic acid for 5-7 times. After classification, the concentrated and dried peptides are dissolved with 20μL 0.1% formic acid, and the pH is adjusted to 3 with 10% formic acid. The ziptip is repeatedly and slowly loaded and pumped 30-40 times, and washed with 0.1% formic acid 10 times to remove salt ions. Finally, the peptides on the ziptip column are eluted with 20μL 80% acetonitrile, concentrated and dried, and prepared for mass spectrometry detection.

[0044] 6. Liquid phase separation

[0045] After the peptides were dissolved in liquid chromatography mobile phase A, they were separated using the EASY-nLC 1200 ultra-high performance liquid phase system. Mobile phase A was 98% water / 2% ACN solution containing 0.1% formic acid; mobile phase B was 80% acetonitrile / 20% aqueous solution containing 0.1% formic acid. Liquid phase gradient settings: 0-3min, 8% to 8% B; 3-20min, 8% to 12% B; 20-80min, 12% to 25% B; 80-85min, 25% to 95% B; 85-90min, 95% to 100% B; the flow rate was maintained at 330nL / min.

[0046] 7. Mass spectrometry

[0047] The peptides were analyzed by LC-MS / MS using an EASY-nLC 1200 nanoliter liquid phase tandem Q Exactive HF-X mass spectrometer. Mass spectrometry data were collected in a data-dependent mode in positive ion mode, with a primary scan resolution of 70,000, a scan range of 350 m / z to 1800 m / z, an automatic gain control (AGC) upper limit of 3e6, and a maximum injection time of 20 ms. The secondary mass spectrometry resolution was 35,000, the AGC upper limit was 1e5, the maximum injection time was 100 ms, and a 0.4 Da precursor ion screening window (Isolation window) was used to screen the top 20 precursor ions in intensity and enter the HCD collision cell for fragmentation, with a normalized fragmentation energy of 30%. The dynamic exclusion time was 50 s for the parent ions with charge states of 1, 8, and uncharged.

[0048] 8. Retrieval of raw mass spectrometry data

[0049] The raw data files obtained from Q Exactive HF-X were searched using Maxquant 1.6.1.0 software. Search parameter settings: the database was SwissProt human database (updated in July 2019), a reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cleavage method was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum length of the peptide was set to 6 amino acid residues; the maximum number of peptide modifications was set to 5; the mass error tolerance of the primary precursor ion of the First search and Main search was set to 20ppm and 4.5ppm, respectively, and the mass error tolerance of the secondary fragment ion was 0.02Da. The carbamidomethylation of cysteine ​​(Carbamidomethyl_C) was set as a fixed modification, and the oxygen of methionine (Oxidation_M), citrullination of arginine (Citrullination_R), and carbamylation of lysine (Carbamylation_K) were set as dynamic modifications. TMT-6plex was used for secondary ion quantification, and peptides with a false discovery rate (FDR) less than 1% were screened for analysis.

[0050] 9. ELISA to verify the antibody level in serum

[0051] The detected self-peptide is synthesized in vitro, and the cyclized peptide of the self-peptide is synthesized (the cyclized peptide has higher stability), and the antigenicity of the self-peptide and its cyclized peptide is verified by ELISA. The cyclized peptide is a peptide in which the amino acids at the N-terminus and C-terminus of the self-peptide are replaced with cysteine, and the cysteines at both ends form disulfide bonds to cyclize the peptide.

[0052] 3. Experimental Results

[0053] The detected self-peptide sequences and their cyclized peptides are shown below:

[0054] 1. Peptide 653 (65-3):

[0055] Self-peptide sequence:

[0056] GLDVGWKSRXGQGLTGPTLLPG

[0057] Cyclized peptide sequence:

[0058]

[0059] 2. Peptide 654 (65-4)

[0060] Self-peptide sequence:

[0061] LRDNDKTRYMGXGVSKAVEHI

[0062] Cyclized peptide sequence:

[0063]

[0064] In the sequences SEQ ID NO.1 and SEQ ID NO.2, X represents carbamylated lysine, and the connection mode between X and the adjacent amino acid in the above polypeptide is shown in the following formula:

[0065]

[0066] Example 2: Diagnostic efficacy of carbamylated peptides for rheumatoid arthritis

[0067] 1. Experimental Materials

[0068] 1. Research subjects and groups

[0069] Rheumatoid arthritis subjects with negative anti-cyclic citrullinated peptide antibodies who were admitted to West China Hospital from March 2022 to July 2024 were selected as the RA group, and healthy subjects in the same period were selected as the HC group. The inclusion and exclusion criteria were as follows.

[0070] Inclusion criteria: patients who met the RA classification criteria revised by the American College of Rheumatology in 1987 or the new classification criteria for RA by ACR / EULAR in 2010 and whose serum anti-cyclic citrullinated peptide antibody test was negative.

[0071] Exclusion criteria:

[0072] Among them, the research subjects excluded patients with other autoimmune diseases (such as systemic lupus erythematosus), patients with systemic metabolic diseases (such as hypertension, diabetes), and patients who did not meet the RA classification criteria revised by the American College of Rheumatology in 1987 or the new classification criteria for RA by ACR / EULAR in 2010.

[0073] 2. Instruments and reagents

[0074] Highly adsorbable polystyrene 96-well plate (705071-Greiner Bio-One), TMB (3,3',5,5'-tetramethylbenzidine), HRP-coupled goat anti-human IgG secondary antibody, bovine serum albumin (BSA), phosphate buffer PBS (PH=7.2), phosphate Tween buffer PBST (PBS+0.5% Tween 20), blocking solution (PBST+1% BSA), sample diluent (PBST+0.5% BSA), self-peptide and cyclized polypeptide thereof (sequence as shown in Example 1).

[0075] 2. Experimental Methods

[0076] 1. ELISA method to determine the antibody level of the corresponding peptide in serum

[0077] (1) Peptide coating

[0078] Use phosphate buffered saline to dilute the peptide to 0.25μg / uL, add 100μL of peptide dilution to each well, and coat at 37 degrees Celsius for 2h. After coating, wash the plate 3 times with PBST, 200μL each time, let it stand for 2min after adding washing solution each time, shake it gently intermittently, shake off the liquid, and pat it dry on paper.

[0079] (2) Closed

[0080] Add 100 μL of blocking solution to each well. Incubate at 37°C for 2 hours to block the sites on the plate that are not coupled to peptides and reduce nonspecific recognition in subsequent operations. After blocking, invert the plate to dry, and wash 3 times with PBST, using the same washing method as before.

[0081] (3) Serum incubation

[0082] Dilute the anti-cyclic citrullinated peptide antibody negative RA and healthy human serum at 1:100 with sample diluent, gently mix, add 100 μL to each well, and incubate at 37°C for 30 minutes. After incubation, wash three times with PBST, using the same washing method as before.

[0083] (4) Incubation with secondary antibody

[0084] HRP-conjugated goat anti-human secondary antibody was diluted 1:10000 with PBST, 100 μL was added to each well, and incubated at 37 degrees Celsius for 30 minutes. After incubation, wash three times with PBST, using the same washing method as before.

[0085] (5) Color development and termination

[0086] The TMB color developing solution was balanced to room temperature, and 100 μL was added to each well. After addition, it was allowed to stand at room temperature in the dark for 10-30 min. The solution turned blue, and when the blue color no longer deepened, 2M H2SO4 was added to terminate the reaction, and the solution turned yellow.

[0087] (6) Reading

[0088] Within 30 minutes after termination, the absorbance at 450 nm was measured using an enzyme-labeled instrument.

[0089] (7) Data analysis

[0090] OD value of sample = test result - OD value of blank well. Standardize the sample according to the test result of the standard on each plate to eliminate the error between plates.

[0091] The cutoff value is AVERAGE (healthy people) + 2*SD (healthy people).

[0092] 2. Data processing method

[0093] When evaluating the diagnostic efficacy of peptides for RA, the OD values ​​of anti-cyclic citrullinated peptide antibody-negative RA and healthy samples were first tested by Mann-Whitney U test and plotted to obtain the positive rate of the peptide ELISA test results in anti-cyclic citrullinated peptide antibody-negative RA and whether there was a significant difference between the two groups. Secondly, the ROC curve of peptide diagnosis to distinguish anti-cyclic citrullinated peptide antibody-negative RA and HC was established based on the ELISA test results of the peptide, and the area under the curve (AUC) and 95% confidence interval were calculated. For the drawing of the combined diagnosis ROC curve, the disease state was first used as the dependent variable, and the ELISA test results of the two peptides were used as covariates, and binary logistic regression was performed. The combined measurement result is the test result of the cyclic aminocarbamylated peptide antigen shown in SEQ ID NO.1 and SEQ ID NO.2 multiplied by their respective regression coefficients, and then the sum of the test values.

[0094] 3. Experimental Results

[0095] The number of samples tested by single or combined ELISA of cyclic peptides, the positive rate in anti-cyclic citrullinated peptide antibody-negative RA or HC, and the AUC value for diagnostic differentiation between anti-cyclic citrullinated peptide antibody-negative RA and HC are shown in the following table.

[0096] Figure 1 , 2 The left column shows the numerical distribution and mean comparison of the single cyclized peptide ELISA test results in anti-cyclic citrullinated peptide antibody RA and HC samples. Figure 1 , 2 The right column shows the ROC curve of a single cyclized peptide for distinguishing anti-cyclic citrullinated peptide antibody-negative RA and HC samples. Figure 3 ROC curve of the combined diagnosis of SEQ ID NO.1 and SEQ ID NO.2 cyclized peptides for distinguishing anti-cyclic citrullinated peptide antibody-negative RA and HC samples.

[0097]

[0098] Those skilled in the art know that the area under the ROC curve AUC indicates the accuracy of the diagnostic test. The area under the ROC curve AUC has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. When AUC is 0.5, it means no diagnostic significance; when AUC is between 0.5 and 0.7, it means that the diagnostic accuracy is low; when AUC is between 0.7 and 0.9, it means that the diagnostic accuracy is medium; when AUC>0.9, it means that the diagnosis has a high accuracy.

[0099] In summary, the above test results show that the cyclized aminobamylated polypeptide antigens shown in SEQ ID NO.1 and SEQ ID NO.2 have high diagnostic values ​​for diagnosing anti-cyclic citrullinated peptide antibody-negative RA alone, and have application prospects for developing into diagnostic kits for anti-cyclic citrullinated peptide antibody-negative RA respectively; the diagnostic value of SEQ ID NO.1 combined with SEQ ID NO.2 for diagnosing anti-cyclic citrullinated peptide protein antibody-negative RA is further improved, and has the application prospect of being jointly developed into a diagnostic kit for anti-cyclic citrullinated peptide antibody-negative RA.

[0100] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Use of the following cyclized aminoformylated polypeptides represented by SEQ ID NO.1 and / or SEQ ID NO.2 in combination or alone for preparing a diagnostic kit for rheumatoid arthritis: SEQ ID NO.1: SEQ ID NO.2: in, In each sequence, X represents carbamylated lysine, and its connection mode with the adjacent amino acid in the above polypeptide is shown in the following formula: The rheumatoid arthritis is anti-cyclic citrullinated peptide antibody-negative rheumatoid arthritis.

2. A cyclized aminoformylated polypeptide, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2: SEQ ID NO.1: SEQ ID NO.2: in, In each sequence, X represents carbamylated lysine, and its connection mode with the adjacent amino acid in the above polypeptide is shown in the following formula:

3. A polypeptide combination, which is a combination of the polypeptides shown in the following sequences SEQ ID NO.1 and SEQ ID NO.2: SEQ ID NO.1: SEQ ID NO.2: in, In each sequence, X represents carbamylated lysine, and its connection mode with the adjacent amino acid in the above polypeptide is shown in the following formula:

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