CN111868073B - Specific polypeptide related to rheumatoid arthritis and application thereof - Google Patents
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Abstract
Provides a specific polypeptide related to rheumatoid arthritis and application thereof. The specific polypeptide comprises citrulline, wherein at least one side of the citrulline is connected with citrulline-like amino acids, and interval nitrogen-based amino acids are also arranged between the citrulline and the citrulline-like amino acids. The provided specific polypeptide related to rheumatoid arthritis has higher sensitivity and specificity in identifying autoimmune antibodies of patients with rheumatoid arthritis, can be directly used for detecting RA autoimmune antibodies, can also be used for immunizing animals, and can be used for producing polyclonal antibodies or monoclonal antibodies for detecting citrullinated protein, and can be used for producing identification kits for citrullinated protein in serum, joint fluid and body fluid of rheumatoid arthritis.
Description
Technical Field
The invention relates to a related technology for detecting rheumatoid arthritis, in particular to a polypeptide design of a Multi-citrulline Similar amino acid repetitive structure (MCSM) and application thereof in development of a detection kit related to rheumatoid arthritis.
Background
Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease, and national adopts RA classification standard revised by the American College of Rheumatology (ACR) in 1987, the specificity of which is 89% and the sensitivity of which is 91% -94%, but the standard is easy to miss diagnosis for some early or atypical patients. Rheumatoid Factor (RF) is an antibody against an antigenic determinant of the Fc fragment of human or animal IgG molecules, and is an autoantibody targeting denatured IgG. RF was originally discovered by Rose et al (1984) in the serum of patients with Rheumatoid Arthritis (RA), can appear in early stage of disease, is convenient to detect, has high sensitivity (about 80%), but has low specificity, the RF positive rate can reach 5% in normal people, and the RF positive rate also appears in other autoimmune diseases in large quantity. In addition, anti-CCP antibodies can appear early in arthritis and are associated with destruction of bone joints, but the sensitivity is 60% and the specificity is 90%. Clinical experiments at present, the most common detection indexes for detecting RA diseases comprise an anti-cyclic citrulline antibody (anti-CCP antibody) and a Rheumatoid Factor (RF), and also an anti-keratin antibody (AKA), an anti-filaggrin antibody (anti-filaggrin antibody, AFA), an anti-nuclear factor (APF), an anti-vimentin antibody and the like, so that important bases are provided for early clinical diagnosis of RA, guidance of treatment and prediction of prognosis.
Polypeptides synthesized from the cDNA sequence of filaggrin by Schellekens and Girbal Neuhause et al in 1998 confirmed that citrulline residues are an essential component of the recognition epitope for rheumatoid arthritis-specific anti-filaggrin antibodies. By analyzing the amino acid sequences of filggrin of each sequence number in the gene library, a peptide chain of about 20 amino acid residues with citrulline instead of arginine is synthesized. And a certain test shows that citrulline is a main constitutive antigenic determinant component recognized by the anti-filaggrin related antibody in the serum of the RA patient. In 2000, Schellekens replaced two serines in a citrullinated peptide chain consisting of 19 amino acid residues with cysteines, forming disulfide bonds with a similar structure to β -turns, and synthesized Cyclic Citrullinated Peptides (CCPs). The cyclic citrullinated peptide is used as an antigen matrix to detect the anti-cyclic citrullinated peptide antibody in the serum of RA patients by an ELISA method, and the sensitivity and specificity are obviously improved. This is a second generation CCP product. The synthesized cyclic citrullinated peptide not only increases the stability of the antigen, but also improves the sensitivity and specificity of CCP detection. CCP commercial detection kit is marketed in 2002 and is rapidly and widely applied to clinical detection, and is still the most common gold standard in RA detection. There are at least six common kits available today, respectively from Axis-Shield (US), Euro-Diagnostica (the Netherlands), Euroimmun (Germany), Inova (US), Phadia (Sweden/Germany), Abbott (US). The CCP2 detection kit is a plurality of polypeptides optimized by a large amount of screening on the basis of CCP1, the sequences are not published, the detection sensitivity is 66.7-77.7%, and the specificity is 86.1-98.8%. In recent years, CCP third generation detection reagent CCP3.1(Inova, US) can detect the serum antibody IgG and IgA levels of patients, CCP3 peptide sequence is not disclosed, and CCP3.1 has lower specificity than CCP2 in the aspect of RA diagnosis application. Anti-CarP (Anti-Carbamylated protein) antibodies are novel RA-specific detection antibodies identified in recent years, recognizing a different type of post-translational modification of proteins compared to CCP antibodies, and Anti-carbamoylation antibodies were present in the serum of 43% of RA patients.
Since the focus of research has been on CCP-antibody positive RA patients, CN1602426A discloses a method for detecting autoantibodies from patients suffering from rheumatoid arthritis, comprising contacting said autoantibodies with a peptide unit comprising an XG motif and a peptide unit comprising an XnonG motif. CN101918432A discloses a three-dimensional matrix of selected synthetic peptide mimic sequences preferentially recognized by autoimmune antibodies detected in patients with rheumatoid arthritis, which are capable of improving the specificity and sensitivity of detection of these autoantibodies in patients before symptoms occur, patients showing symptoms of rheumatoid arthritis, and patients diagnosed as positive for rheumatoid arthritis. CN101957365A discloses a kit for detecting a CCP and IgG bispecific antibody, which can detect a natural bispecific antibody existing in the serum of a rheumatoid arthritis patient, and is convenient to use, simple and easy to implement. CN102323402A discloses a CCP antibody in-vitro detection kit and a preparation method thereof, and the kit can be applied to the auxiliary diagnosis of rheumatoid arthritis. CN102796173A discloses an epitope of rheumatoid arthritis and application thereof, the epitope is only combined with IgG in serum of a patient, but not reacted with serum of healthy people, and can be used for preparing a medicament for diagnosing rheumatoid arthritis.
However, about 30 percent of patients with rheumatoid arthritis still have no clear detection index in clinic, and the patients are called patients with anti-citrulline negative (CCP-) rheumatoid arthritis. The citrulline detection reagent generally targets a single protein molecule and has good specificity on a certain subtype of RA. A series of researches show that a patient with positive resistance to citrulline (CCP +) has obviously different clinical manifestations from a patient with negative resistance to citrulline (CCP-) RA, and the lack of clinical diagnosis information of the patient with CCP-rheumatoid arthritis is partially limited by the fact that the existing detection method system cannot detect specific indications carried by the CCP-patient, and the CCP-patient can be an RA patient subtype containing a specific citrulline marker. Combining multiple detection of multiple targets is a very meaningful attempt, and the autoantibody types of RA patients can be subdivided, so that basis is better provided for clinical diagnosis and treatment.
CN106950365A discloses an ACPA negative RA diagnostic marker and application thereof, in particular to application of deoxyco-protein Dioxygenase (DOHH) or fragments thereof in preparing a reagent for diagnosing anti-citrullinated polypeptide antibody negative rheumatoid arthritis diseases. CN106950366A discloses an ACPA negative RA diagnostic marker and application thereof, in particular to application of Pentaxin related protein 3, namely PTX3 or fragments thereof in preparation of a reagent for diagnosing anti-citrullinated polypeptide antibody negative rheumatoid arthritis diseases. However, the diagnostic specificity of these negative rheumatoid arthritis was only 90%.
Early diagnosis and early treatment are the key to protect joint function and prevent joint destruction. Currently, a fraction of early patients cannot be diagnosed. The internationally accepted CCP method detects RA with up to 70% sensitivity and fails to diagnose patients at an early stage. Kasapcopurr et al, university of Turkey Italian, reported that, unlike adult Rheumatoid Arthritis (RA), the detection rate of serum anti-Cyclic Citrullinated Peptide (CCP) antibodies in Juvenile Idiopathic Arthritis (JIA) patients was very low. The investigators performed anti-CCP assays for 122 children with JIA and 27 adult RA patients. The results showed that only 3 of the polyarticular JIA children were positive for anti-CCP antibody, and these children were girls, had erosive joint lesions, and were also positive for Rheumatoid Factor (RF); while adult RA patients have a positive rate of up to 70% for anti-CCP antibodies.
RA autoantigen appears first in the body of an RA patient, reaches a certain reaction degree through the accumulation of the autoantigen, and stimulates T cells and memory cells in the body of the RA patient to generate corresponding autoantibodies. Antibodies against citrullinated autoantigens are considered to have high specificity in rheumatoid arthritis patients(1). Citrulline is a non-protein amino acid, and in nature, none of the trnas corresponds to citrulline, which is produced in vivo by the enzyme PADI. The PADI enzymes are classified into 5 types, of which PADI2 type and PADI4 type are found in the synovial fluid of patients with rheumatoid arthritis. Citrullination has several functions in the normal physiology of the body, such as citrullinated serine proteins are involved in the formation of the stratum corneum of the skin; citrullination of histones in the nucleus is associated with gene expression regulation; citrullinated proteins are also elevated in conditions of inflammation, trauma, apoptosis, aging(4,5,6,7)。
Among all the arginines in the protein, some are easily citrullinated, some are not easy, some are faster and some are slower(2)Structure of proteinArginine is located in the 3-dimensional protein folding structure and is related to the amino acid sequence around arginine. Citrullination changes the polarity of the amino acid side chains and thus also brings about antigenic changes(3). Although the change from arginine to citrulline has little effect on the change in molecular weight of proteins, there is a great difference in the recognition of antibody antigens due to the change in charge. In the population with HLA-DR4 gene, the affinity between citrullinated polypeptide and MHC molecules of HLA-DR4 is greatly improved compared to the corresponding arginylated polypeptide. The studies on CCP have revealed that the CCP autoantibodies are mostly associated with high affinity polypeptides of HLA-DR 4. However, no citrullinated antigen corresponding to the citrullinated antigen in vivo has been found. In the detection of citrullinated antigens, citrullination is found in many proteins, such as structural proteins, filaggrin, collagen; a gene regulatory proteome protein; intracellular protein-patterning proteins, enolase; extracellular protein fibrinogen, and the like.
Despite the presence of citrullinated antigens in RA serum, specific disease-causing autoantigens have not been identified to date. The existence of citrullinated antigen is not specific to rheumatoid arthritis, and the increase of citrullinated antigen is reported in other diseases, such as ankylosing spondylitis, psoriatic arthritis, non-degenerative spondyloarthropathy, multiple myeloma with damaged joints, osteoarthritis, gout, pseudogout, chondrocalcinosis and the like(8，9，10)In animal models, deposition of citrullinated protein in joint tissue correlates with severity of arthritis, but the concentration of citrullinated protein in the joints of rheumatoid arthritis patients does not correlate well with disease severity; in contrast, citrullinated proteins are sometimes found in some other diseases, such as in plaques of scleroderma(11)(ii) a Hippocampal region of alzheimer's disease; in the renal tubules of obstructive renal disease(12，13)(ii) a Therefore, the identification of citrullinated protein specific to rheumatoid arthritis is directly related to the specificity of detection.
Other rheumatoid arthritis-associated antigens are also human cartilage glycoprotein (HCgp-39), produced by chondrocytes, synovial fibroblasts, macrophages and neutrophils, and involved in tissue remodeling and degradation of the extracellular matrix. HCgp-39 is highly expressed in articular cartilage and synovium of RA patients. Studies have shown that HCgp-39 polypeptides are recognized by Peripheral Blood Mononuclear Cells (PBMCs) of RA patients, causing PBMCs to proliferate; and can induce BALB/c mice to generate chronic invasive arthritis.
Type II Collagen (CII) can stimulate recall response of T cells of RA patients to cause hyperplasia, and the CII and proteins of certain bacteria or germs (such as mycobacterium tuberculosis, EB virus and the like) are known to have common epitope QK/RRAA, and can be possibly subjected to molecular simulation or fuzzy recognition to cause diseases. A lot of researches in recent years show that the amino acid sequences at the 261-273 sites of CII are the main antigen epitopes of CII.
The aggrecan (aggrecan) is the major macromolecular proteoglycan component that makes up the cartilage matrix. The degradation of the polymeric hormones in patients with RA is accelerated, possibly leading to destruction of the articular cartilage. Experiments prove that the polymin can activate peripheral blood T cells of RA patients to induce autoimmune response. It is believed that the major T cell epitope for the induction of arthritis by polymin is present in the G1 globular domain of the antigen. Wherein the amino acids at the 280-292 position of the main peptide epitope can induce IgG2 type antibody reaction besides inducing T cell expansion and Interferon (IFN) -gamma secretion.
Glycoprotein P68, a molecular chaperone protein, also known as immunoglobulin binding protein (BiP). The experiment proves that P68 can stimulate the proliferation of peripheral blood and synovial T cells of RA patients. The P68 antibody had a sensitivity of 64% and a specificity of 99% for RA diagnosis.
P205, cartilage connexin (LP), CH65, osteopontin (osteopontin) and the like all can be used as autoantigens of joint parts to participate in RA immunopathological process.
Calpain (calpastatin), an antibody (ACAST-C27) aiming at 27-site amino acid epitope at C end of calpain can be seen in early RA which is seronegative, and may have certain significance for early diagnosis of RA.
Glucose 6-phosphate isomerase (GPI) is reported in the literature to be an important enzyme in glycolysis and gluconeogenesis processes, and high-titer GPI antibodies and soluble GPI molecules exist in synovial fluid and plasma of RA patients. Citrullination of arginine (R) residues in amino acids 306-324 (SHQESTRCRSRGRSGRSGS) of the filaggrin provides a major epitope recognized by AFA of RA patients, wherein the citrulline residue at the central position is crucial in the recognition process of the filaggrin by RA. It is believed that these peptides may also be one of the sources of citrullinated antigens, possibly featuring citrullination sites.
The existing RA antigen design mainly selects specific antigen regions from natural proteins, so that the efficiency is low, and the result is not satisfactory.
Reference documents:
1.van Boekel MA,Vossenaar ER,van den Hoogen FH,van Venrooij WJ:Autoantibody systems in rheumatoid arthritis:specificity,sensitivity and diagnostic value.Arthritis Res.4(2),87–93(2002).
2，Ryo
Akari Suzuki&Kazuhiko Yamamoto：Citrulline and anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis.Future Rheumatol.(2006)1(2),249–258.
3.Curis E,Nicolis I,Moinard C et al.:Almost all about citrulline in mammals.Amino Acids 29(3),177–205(2005).
4.van Stipdonk MJ,Willems AA,Amor S et al.:T-cells discriminate between differentially phosphorylated forms ofαB-crystallin,a major central nervous system myelin antigen.Int.Immunol.10(7),943–950(1998).
5.Rathmell JC,Thompson CB:The central effectors of cell death in the immune system.Ann.Rev.Immunol.17,781–828(1999).
6.Piacentini M,Colizzi V:Tissue transglutaminase:apoptosis versus autoimmunity.Immunol.Today 20(3),130–134(1999).
7.Hershko A,Ciechanover A:The ubiquitin system.Ann.Rev.Biochem.67,425–479(1998).
8.Vossenaar ER,Smeets TJ,Kraan MC,Raats JM,van Venrooij WJ,Tak PP:The presence of citrullinated proteins is not specific for rheumatoid synovial tissue.Arthritis Rheum.50(11),3485–3494(2004).
9.Chapuy-Regaud S,Sebbag M,Baeten D et al.:Fibrin deimination in synovial tissue is not specific for rheumatoid arthritis but commonly occurs during synovitides.J.Immunol.174(8),5057–5064(2005).
10.Kruithof E,Baeten D,De Rycke L et al.:Synovial histopathology of psoriatic arthritis,both oligo-and polyarticular,resembles spondyloarthropathy more than it does rheumatoid arthritis.Arthritis Res.Ther.7(3),R569–R580(2005).
11.Nicholas AP,Sambandam T,Echols JD,Tourtellotte WW:Increased citrullinated glial fibrillary acidic protein in secondary progressive multiple sclerosis.J.Comp.Neurol.473(1),128–136(2004).
12.Ishigami A,Ohsawa T,Hiratsuka M et al.:Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer’s disease.J.Neurosci.Res.80(1),120–128(2005).
13.Feng D,Imasawa T,Nagano T et al.:Citrullination preferentially proceeds in glomerular Bowman’s capsule and increases in obstructive nephropathy.Kidney Int.68(1),84–95(2005).
disclosure of Invention
The invention aims to overcome the limitations of the prior art and provide a brand-new artificially designed specific polypeptide related to rheumatoid arthritis and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a specific polypeptide associated with rheumatoid arthritis or cyclic peptides and aggregates thereof, said specific polypeptide having the following characteristics:
comprises 1-3 citrulline and 2-5 citrulline-like amino acids;
0-2 spaced amino acids are independently arranged between citrullines or between citrulline and citrulline-like amino acids;
the citrulline-like amino acid is independently selected from arginine, glutamine or asparagine.
In some examples, the specific polypeptide includes a citrulline core (Cit) -X- (Cit), X being a spacer amino acid, such as: (Cit) -G- (Cit), (Cit) -S- (Cit), (Cit) -T- (Cit). Experimental data show that this can bring about better results.
In some examples, the specific polypeptide has a citrulline-like amino acid profile on both the left and right sides of the citrulline core; further, the citrulline-like amino acids are spaced apart, and in particular, there is only one amino acid between two adjacent citrulline-like amino acids.
In some examples, the total number of citrullines in the specific polypeptide is 1-2. If 3 citrullines are present, 4-5 spacer amino acids exist between the 3 rd citrulline and the first two citrullines.
The specific polypeptides and cyclized peptides thereof can be further multimerized in the form of monomers, dimers, trimers, and multimers. The aggregates include aggregates of specific polypeptides, as well as aggregates of cyclized peptides of the specific polypeptides. The type of the spacer amino acid is not particularly limited, and may be various natural amino acids other than citrulline-like amino acids.
In some examples, the specific polypeptide has a total length of 10 to 40 amino acids, preferably 16 to 30 amino acids. This facilitates synthesis and has strong antigenicity.
In some examples, the spacer amino acid directly attached to the C-terminus of citrulline is selected from glycine, serine, threonine, alanine, and proline. The spacer amino acids have smaller side chains, smaller volume or easy folding, have smaller display influence on citrulline side chains, and are more favorable for obtaining specific polypeptides with better effect. Proline has a turning effect and may further provide the effect of a partial cyclic peptide.
In some examples, the specific polypeptide is at least one of the following polypeptides, wherein Cit refers to citrulline:
P01：HQDQG(Cit)S(Cit)NRAA
P02：DCDGQW(Cit)GP(Cit)SVE(Cit)HQSAKCK
P03：KCKQFRN(Cit)G(Cit)SPRAADCD
P05：SHQEST(Cit)G(Cit)SRG(Cit)SG(Cit)SGS
P06：HQDNQ(Cit)S(Cit)QNAA
P07：HQGQG(Cit)G(Cit)SQGQGRA
P08：HNGNG(Cit)G(Cit)SNGRGNA
P10：HANN(Cit)T(Cit)SNNGA
P11：RSQFNW(Cit)S(Cit)SRPR
P12：HQFRN(Cit)Q(Cit)RSRNHA
P13：HQYNFQW(Cit)S(Cit)GRPR
P14：GRFQM(Cit)H(Cit)RLIRH
P15：RSQFNF(Cit)P(Cit)SRPR
P16：KAAN(Cit)NNDAL(Cit)QAKQ
P17：HQYNFQW(Cit)S(Cit)GRP(Cit)NQAA
P18：DCDGQV(Cit)S(Cit)GRP(Cit)NQAAKCK
P19：HQMREL(Cit)S(Cit)GQVDQL(Cit)NDKAR。
in a second aspect of the present invention, there is provided:
use of a specific polypeptide, or cyclic peptides and aggregates thereof, the sequence of which is as described above in relation to the first aspect of the invention, for the preparation of an antigen for differentiating citrullinated autoantibodies.
In some examples, the citrullinated autoantibody is from rheumatoid arthritis serum, joint fluid, or body fluid.
In a third aspect of the present invention, there is provided:
a method of inducing citrullinated autoantibodies comprising the act of immunizing an animal with a specific polypeptide, or a cyclic peptide and aggregate thereof, the sequence of said specific polypeptide being as described in the first aspect of the invention.
In a fourth aspect of the present invention, there is provided:
the use of a specific polypeptide according to the first aspect of the invention or cyclic peptides and aggregates thereof, said use comprising:
inducing to generate monoclonal antibody;
for the evaluation of antibody titers;
the antibody is a specific citrullinated autoantibody for detecting rheumatoid arthritis.
In a fifth aspect of the present invention, there is provided:
a kit comprising at least one specific polypeptide, or a cyclic peptide and aggregates thereof, the sequence of said specific polypeptide being as described in the first aspect of the invention.
In some kit examples, the specific polypeptide or at least one of a cyclic peptide and an aggregate thereof; and/or
At least one detection antibody directed against fibrinogen, collagen, vimentin, enolase, human IgG, human IgM or human IgA.
The diagnostic accuracy of the kit can be further improved by using the kit in combination with other detection antibodies.
In some examples of kits, the kit is used for: detecting citrullinated autoantibodies and evaluating the disease condition of rheumatoid arthritis.
The invention has the beneficial effects that:
the specific polypeptide related to rheumatoid arthritis has higher sensitivity and specificity in identifying autoimmune antibodies of patients with rheumatoid arthritis, can be directly used for detecting RA autoimmune antibodies, can also be used for immunizing animals, and can be used for producing polyclonal antibodies or monoclonal antibodies for detecting citrullinated protein, and is used for producing identification kits for citrullinated protein in serum, joint fluid and body fluid of rheumatoid arthritis.
The specific polypeptide related to rheumatoid arthritis of the invention can also be cyclized. These specific polypeptides and their cyclized peptides can be further multimerized in the form of monomers, dimers, trimers, and multimers.
Drawings
FIG. 1 is a side chain group comparison of citrulline and similar amino acids;
FIG. 2 is a schematic diagram of a specific polypeptide of the present invention related to rheumatoid arthritis;
FIG. 3 is a graph showing the standard curve for CCP calibrator assay with different polypeptide coatings;
FIG. 4 shows the results of detection of RA-associated specific antibodies in the sera of 20 patients with RA, with different polypeptide coatings;
FIG. 5 shows the results of the detection of serum from 20 RA patients by different MCSM mabs in combination with different secondary antibodies.
Detailed Description
The inventor researches and discovers that glutamine and asparagine have certain similarity with citrulline in view of the side chain structure of citrulline as shown in figure 1. The invention discovers that around 1-3 citrullines, 3-5 amino acids similar to citrulline side chains are arranged on one side of the citrulline or form a special structure around the citrulline, and the special structure is named as a citrulline-like side chain repeating structure; the interval amino acid is also arranged between the citrulline and the side chain amino acid similar to the citrulline and consists of 0 to 2 amino acids. The pattern of its repeating structure is shown in figure 2. The repeat structure has higher sensitivity and specificity in the recognition of autoimmune antibodies in rheumatoid arthritis patients.
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1: design and Synthesis of Polycitrulline-like amino acid repeat (Multi-Cirtrulin silicon Motif, MCSM) polypeptide:
the designed polypeptide sequence is as follows:
P01：HQDQG(Cit)S(Cit)NRAA(SEQ ID NO.：1)
P02：DCDGQW(Cit)GP(Cit)SVE(Cit)HQSAKCK(SEQ ID NO.：2)
P03：KCKQFRN(Cit)G(Cit)SPRAADCD(SEQ ID NO.：3)
P04：HQCHQESTRGRSRGRCGRSGS(SEQ ID NO.：4)
P05：SHQEST(Cit)G(Cit)SRG(Cit)SG(Cit)SGS(SEQ ID NO.：5)
P06：HQDNQ(Cit)S(Cit)QNAA(SEQ ID NO.：6)
P07：HQGQG(Cit)G(Cit)SQGQGRA(SEQ ID NO.：7)
P08：HNGNG(Cit)G(Cit)SNGRGNA(SEQ ID NO.：8)
P10：HANN(Cit)T(Cit)SNNGA(SEQ ID NO.：9)
P11：RSQFNW(Cit)S(CIT)SRPR(SEQ ID NO.：10)
P12：HQFRN(Cit)Q(Cit)RSRNHA(SEQ ID NO.：11)
P13：HQYNFQW(Cit)S(Cit)GRPR(SEQ ID NO.：12)
P14：GRFQM(Cit)H(Cit)RLIRH(SEQ ID NO.：13)
P15：RSQFNF(Cit)P(Cit)SRPR(SEQ ID NO.：14)
P16：KAAN(Cit)NNDAL(Cit)QAKQ(SEQ ID NO.：15)
P17：HQYNFQW(Cit)S(Cit)GRP(Cit)NQAA(SEQ ID NO.：16)
P18：DCDGQV(Cit)S(Cit)GRP(Cit)NQAAKCK(SEQ ID NO.：17)
P19：HQMREL(Cit)S(Cit)GQVDQL(Cit)NDKAR(SEQ ID NO.：18)。
the polypeptide thus designed was synthesized by peptide chemical synthesis. For the purpose of subsequent detection, biotinylation may be added to the synthesis; cysteine at both ends can be added to form a cyclic polypeptide for the stability of the peptide chain and the improvement of the detection sensitivity.
Example 2 comparison of different citrullination and MSCM Polypeptides to detect differences in anti-citrullinated antibodies in human serum
Experimental materials:
enzyme label plate: NUNC446469
Streptavidin: hangzhou Niulongrc-SA
Coating buffer solution: PBS, pH7.4
Washing liquid: PBS + Triton X-100, pH7.4
Sealing liquid: PBS + 1% BSA, pH7.4
Sample diluent: PBS + 0.1% Triton X-100+ 1% BSA
Enzyme-labeled antibody: HRP-labeled mouse anti-human IgG (for 1:15000)
Enzyme-labeled antibody diluent: PBS + 1% BSA + 0.1% Triton X-100, pH7.4
Different biological polypeptides:
p04 (non citrullination)
P05 (citrullinated, no MSCM characteristic conformation)
P11 (citrullinated, MSCM-characterized conformation)
P18 (citrullinated, MSCM-characterized conformation)
European Mongolia CCP
Sample preparation:
healthy control: 20 examples of
RA, omon CCP +: 20 examples of
Calibration products: calibration product of European Mongolian CCP kit.
The experimental method comprises the following steps:
preparation of SA-coated ELISA plate
1) Diluting SA to 10ug/ml with PBS, adding 100 ul/hole into the bottom of a blank enzyme label plate, and incubating at 2-8 ℃ and 200rpm overnight;
2) discarding the liquid and patting to dry;
3) coating different biotinylated polypeptides;
4) respectively diluting different biotinylated polypeptides to 5ug/ml, adding 100 ul/hole into the bottom of a blank enzyme label plate, and incubating at room temperature and 200rpm for 1 hour;
5) discarding the liquid and patting to dry;
6) washing for 1 time: adding washing solution 200 ul/hole, standing for 2min, discarding solution, and drying;
7) and (3) sealing: adding the blocking solution into 200 ul/hole, and incubating at room temperature and 200rpm for 3 hours;
8) discarding the liquid and patting to dry; obtaining the SA-coated ELISA plate.
The SA coated ELISA plate can be directly used within one week and is preserved at the temperature of 2-8 ℃; drying is needed for long-term storage: inversely placing the mixture in an air-blast drying oven at 37 ℃ for drying for 2 hours; and (3) packaging the ELISA plate by using an aluminum foil bag and a drying agent, and storing at 2-8 ℃.
Test sample
1) Diluting the sample by 100 times with a sample diluent, and adding 100 ul/hole into a coated enzyme label plate;
2) incubation at room temperature at 200rpm for 1 hour;
3) washing for 3 times;
4) enzyme-labeled antibody (HRP-labeled mouse anti-human IgG, 1:15000) was added at 100 ul/well, and incubated at 200rpm for 30 minutes at room temperature;
5) washing for 3 times;
6) developing for 15min, and stopping;
7) reading by a microplate reader: OD450, 630 nm.
Plate distribution:
| 1 | 2 | 3 | 4 | 5 | 6 | ||
| A | calibration article 1 | RA01 | RA09 | RA17 | N05 | N13 | |
| B | Calibration article 2 | RA02 | RA10 | RA18 | N06 | N14 | |
| C | Calibration article 3 | RA03 | RA11 | RA19 | N07 | N15 | |
| D | Calibration article 4 | RA04 | RA12 | RA20 | N08 | N16 | |
| | Calibration article | 5 | RA05 | RA13 | N01 | N09 | N17 |
| F | BLANK | RA06 | RA14 | N02 | N10 | N18 | |
| G | BLANK | RA07 | RA15 | N03 | N11 | N19 | |
| H | BLANK | RA08 | RA16 | N04 | N12 | N20 |
The experimental results are as follows:
plate 1: p04 coated ELISA plate
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | 0.049 | 0.058 | 0.060 | 0.058 | 0.057 | 0.055 |
| B | 0.050 | 0.058 | 0.057 | 0.060 | 0.055 | 0.055 |
| C | 0.051 | 0.057 | 0.059 | 0.057 | 0.057 | 0.059 |
| D | 0.050 | 0.057 | 0.064 | 0.057 | 0.060 | 0.053 |
| E | 0.052 | 0.060 | 0.062 | 0.058 | 0.054 | 0.071 |
| F | 0.051 | 0.068 | 0.062 | 0.057 | 0.089 | 0.056 |
| G | 0.051 | 0.059 | 0.056 | 0.080 | 0.058 | 0.062 |
| H | 0.049 | 0.068 | 0.058 | 0.057 | 0.057 | 0.061 |
Plate 2: p05 coated ELISA plate
Plate 3: european Mongolia CCP
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | 0.104 | 1.914 | 2.565 | 2.639 | 0.062 | 0.059 |
| B | 0.274 | 1.178 | 2.626 | 2.111 | 0.062 | 0.063 |
| C | 0.740 | 2.202 | 2.742 | 1.854 | 0.063 | 0.068 |
| D | 2.121 | 2.824 | 2.668 | 2.792 | 0.066 | 0.057 |
| E | 2.677 | 2.873 | 2.860 | 0.072 | 0.058 | 0.078 |
| F | 0.055 | 2.848 | 2.340 | 0.093 | 0.061 | 0.059 |
| G | 0.053 | 2.820 | 1.084 | 0.093 | 0.059 | 0.062 |
| H | 0.053 | 2.987 | 0.995 | 0.066 | 0.063 | 0.064 |
Plate 4: coated p11 ELISA plate
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | 0.052 | 1.175 | 0.731 | 2.231 | 0.067 | 0.059 |
| B | 0.069 | 2.153 | 2.498 | 0.653 | 0.058 | 0.089 |
| C | 0.085 | 2.203 | 1.677 | 1.464 | 0.058 | 0.079 |
| D | 0.209 | 1.308 | 2.738 | 1.939 | 0.067 | 0.057 |
| E | 0.466 | 1.590 | 2.293 | 0.080 | 0.059 | 0.080 |
| F | 0.055 | 2.298 | 2.305 | 0.085 | 0.082 | 0.080 |
| G | 0.051 | 1.248 | 2.714 | 0.136 | 0.063 | 0.098 |
| H | 0.050 | 2.622 | 1.045 | 0.070 | 0.061 | 0.067 |
Plate 5: coated p18 ELISA plate
| 1 | 2 | 3 | 4 | 5 | 6 | |
| A | 0.065 | 0.338 | 1.779 | 1.468 | 0.062 | 0.057 |
| B | 0.092 | 0.461 | 0.507 | 0.377 | 0.055 | 0.056 |
| C | 0.197 | 0.069 | 0.293 | 0.187 | 0.056 | 0.061 |
| D | 0.762 | 2.431 | 1.760 | 1.813 | 0.062 | 0.062 |
| E | 1.514 | 1.385 | 0.763 | 0.059 | 0.056 | 0.066 |
| F | 0.052 | 1.089 | 0.362 | 0.061 | 0.058 | 0.055 |
| G | 0.054 | 2.439 | 0.228 | 0.082 | 0.058 | 0.063 |
| H | 0.051 | 2.939 | 0.061 | 0.066 | 0.058 | 0.058 |
The results for panels 1-5 are summarized in the following table:
FIG. 3 is a graph of a standard curve generated using a polypeptide-coated assay CCP calibrator (data for P04 and P05 are overlaid); FIG. 4 shows the results of detection of RA-associated specific antibodies in 20 patients with RA and normal human serum, with different polypeptide coatings.
The experimental results show that:
1) the P04 polypeptide without citrulline can not distinguish the serum of healthy people from the serum of RA patients at all, and the detection rate is 0;
2) polypeptide P05 with citrullination and without MSCM characteristic conformation even having 4 citrullination sites can not well distinguish the serum of healthy people from the serum of RA patients, and the detection rate of 20 RA sera is only 6;
3) the polypeptide P11 with citrullination and MSCM characteristic conformation can well distinguish serum of healthy people from RA patient, 20 healthy people are tested to be negative, 20 RA are tested to be positive, and the detection rate is completely consistent with that of an European Mongolian CCP kit;
4) polypeptide P18 with citrullination and a partial MSCM signature conformation was found negative in 20 healthy persons, and 17 positive in 20 RA persons, slightly lower in comparison with the Euromeng CCP kit.
Example 3: cross-linking the synthesized polypeptide with BSA, purifying, immunizing rabbit, and preparing polyclonal antibody
1) Coupling the synthesized polypeptide to carrier protein BSA or OVA by using an EDC method or a glutaraldehyde crosslinking method;
2) two (about 2Kg) healthy 6 week-sized new zealand white rabbits were selected;
3) mixing 1ml Freund's complete adjuvant and 1ml antigen, and making into milk white;
4) the primary immunization is 400ug of antigen, and the subsequent immunization is 100ug each time; back subcutaneous injection;
5) immunizing once every two weeks for 4-5 times in total, and finally collecting blood after verifying that the concentration of the antibody in serum reaches the standard;
6) antibodies in serum were purified using ProteinA/G affinity fillers.
The purified polyclonal antibody can be matched with one or more antibodies aiming at vimentin, fibrinogen, type II collagen and enolase to form a kit for detecting the specific antigen of the rheumatoid arthritis.
Example 4: after synthetic polypeptide and BSA are cross-linked and purified, the mouse is immunized, and monoclonal antibodies are screened
1) Coupling the synthesized polypeptide to carrier protein BSA or OVA by using an EDC method or a glutaraldehyde crosslinking method; the adjuvant can adopt Freund complete adjuvant, and can also adopt novel immunologic adjuvant;
2) selecting 6-8 weeks old BALB/C mice, immunizing the backs for 4-5 times at intervals of 2-3 weeks each time; 10 days after the last immunization, blood is taken to verify that the titer reaches the standard, and three days before spleen fusion is taken for strengthening the immunization; 50ug of each mouse was used for the first immunization, and 25ug of each mouse was used each time later;
3) selecting splenic cells of a mouse with the highest serum titer to fuse with mouse myeloma cells SP 20; electric fusion, chemical fusion, viral fusion can be used;
4) limiting dilution of the fused cells, and cloning in a 96-well plate;
5) HAT screening hybridoma cells; detecting positive cells of antigen detection, and carrying out expanded culture;
6) freezing and storing the cells by liquid nitrogen;
7) preparing an antibody by an ascites method or a hybridoma cell culture method;
8) antibodies were purified using proteinA/G affinity packing.
The purified monoclonal antibody can be matched with one or more antibodies aiming at vimentin, fibrinogen, type II collagen and enolase, and can be used for preparing a kit for detecting the specific antigen of the rheumatoid arthritis.
Example 5: assembly of kit for detecting autoantigen
1) And main reagents:
(1) polyclonal or monoclonal anti-MCSM antibodies;
(2) coating buffer solution: 0.1M to 0.5M carbonic acid buffer solution, and the pH value is 9.0 to 9.6;
(3) sealing liquid: 1-5% BSA (or 5% skimmed milk powder), 0.05-0.5M PBS buffer, 0.01-0.5% thimerosal, and pH 6.0-8.5;
(4) sample diluent: 1-5% BSA, 0.05-0.5M Tri-HCL buffer solution, 0.01-0.5% thimerosal, and pH 6.0-8.5;
(5) negative and positive control dilution: 1-5% BSA, 0.05-0.5M PBS buffer solution, 0.01-0.5% thimerosal, pH 6.0-8.5;
(6) washing liquid: 0.1 to 0.5 percent of Tween-20, 0.05 to 0.5M of PBS buffer solution, 0.01 to 0.5 percent of thimerosal and pH of 6.0 to 8.5;
(7) enzyme-labeled antibody diluent: 1-5% BSA, 0.05-0.5M PBS buffer solution, 0.01-0.5% thimerosal, pH 6.0-8.5;
(8) color development buffer: 0.01M to 1M phosphate-citric acid buffer solution, and the pH value is 3.0 to 5.0;
(9) color development liquid: TMB;
(10) stopping liquid: sulfuric acid.
2) Preparation of working solution for reaction plate
Coating the produced polyclonal antibody or monoclonal antibody resisting citrullinated peptide with a special structure at the screened optimal concentration, abandoning waste liquid after staying overnight (16-20 h), patting dry, and adding confining liquid for sealing; and after drying, putting the reaction plate into an incubator at 37 ℃ for drying for 1-3 h, and then putting the reaction plate into an aluminum foil bag with a drying agent for sealing and packaging.
Biotinylation anti-wavy protein, anti-fibrinogen antibody, anti-collagen antibody, anti-enolase antibody or antibody aiming at other antigens related to rheumatoid arthritis are mixed singly or in a certain proportion to prepare primary anti-working solution according to the optimal working concentration.
The enzyme-labeled streptavidin can be obtained by ordinary purchasing screening.
3) Negative and positive control preparation
Serum and normal human serum of patients diagnosed with rheumatoid arthritis are collected from hospitals, treated and diluted by a negative and positive reference substance diluent according to an appropriate ratio (1: 3 to 1: 100, preferably 1: 20).
The constructed kit comprises the following aspects: 96-well enzyme-linked immunosorbent assay plate, negative control, positive control, critical control, sample diluent, washing liquid, enzyme-labeled antibody, developing liquid, stop solution, sealing plate membrane and instructions.
Example 6: method of using kit
1) Pretreatment: placing the components of the kit at room temperature for balancing for 0.5-1 h, diluting the sample (1: 3 to 1: 100) by using a sample diluent, wherein the dilution can be determined according to specific experiments;
2) sample adding: adding diluted samples to be tested and 100 mul/hole of negative and positive control in the corresponding holes of the ELISA plate, and sealing the plate with a sealing plate film;
3) and (3) incubation: incubating the sample-added ELISA plate in a 200-280 rpm oscillator room temperature for 30-60 min;
4) washing: drying the incubated enzyme label plate by using a waste liquid, and washing for 4-6 times by using a washing liquid;
5) adding an anti-or enzyme-labeled antibody into an enzyme-labeled plate according to a ratio of 100 mu l/hole, and sealing the plate by using a sealing plate film;
6) and (3) incubation: incubating the sample-added ELISA plate in a 200-280 rpm oscillator room temperature for 30-60 min;
7) washing: drying the incubated enzyme label plate by using a waste liquid, and washing for 4-6 times by using a washing liquid;
8) the antibody detection kit directly enters the next step, the antigen detection kit is added with enzyme-labeled streptavidin for incubation, and the antibody detection kit enters the next step after being washed;
9) color development: diluting and uniformly mixing the substrate A and the substrate B according to the ratio of 1:10, adding the substrate A and the substrate B into an enzyme label plate according to the ratio of 100 mu l/hole, and incubating for 10-15 min at the room temperature of a 400rpm oscillator;
10) measuring: adding the stop solution into the ELISA plate according to 100 mul/hole, lightly beating and uniformly mixing, setting the wavelengths of 450nm and 630nm of an ELISA reader to measure the OD value of each hole by double wavelengths, and comparing with a standard substance or a reference substance to calculate a result.
Example 7: comparison of differences in detection of RA-specific antigens in human serum by different anti-MSCM monoclonal antibodies
Experimental materials:
enzyme label plate: NUNC446469
Coating antibody: several murine monoclonal antibodies against MSCM polypeptide G4, G6, G9, G11 (obtained by a series of steps including immunization of mice, cell fusion, antibody screening, etc. in example 4).
Coating buffer solution: CBS, pH9.0-9.6
Washing liquid: PBS + 0.05% Triton X-100, pH7.4
Sealing liquid: PBS + 3% BSA, pH7.4
Sample diluent: PBS + 0.1% Triton X-100+ 1% BSA
A first antibody:
goat anti-human fibrinogen polyclonal antibody
Sheep anti-human vimentin polyclonal antibody
Enzyme-labeled dimer:
HRP-labeled rabbit anti-sheep IgG
Enzyme-labeled antibody diluent: PBS + 1% BSA + 0.1% Triton X-100, pH7.4
Sample preparation:
serum of healthy controls: 20 cases (N01-N20)
Clinically confirmed RA patient sera: 20 cases (RA 01-RA 20)
The experimental method comprises the following steps:
preparation of different MSCM-resistant monoclonal antibody coated enzyme label plate
1) Diluting the coated antibody to 1-5 ug/ml by CBS, adding 100 ul/hole into the bottom of a blank enzyme label plate, and incubating overnight at 2-8 ℃ and 200 rpm;
2) discarding the liquid and patting to dry;
3) washing for 1 time: adding washing solution 200 ul/hole, standing for 2min, discarding solution, and drying;
4) and (3) sealing: adding the blocking solution into 200 ul/hole, and incubating at room temperature and 200rpm for 3 hours;
5) discarding the liquid and patting to dry;
6) the product can be directly used within one week and stored at 2-8 ℃;
7) drying is needed for long-term storage: inversely placing the mixture in an air blast drying oven at 37 ℃ for drying for 1 to 3 hours; and (3) packaging the ELISA plate by using an aluminum foil bag and a drying agent, and storing at 2-8 ℃.
Test sample
1) Diluting the sample with a sample diluent, and adding 100 ul/hole of the sample into a coated enzyme label plate;
2) incubation at room temperature at 200rpm for 1 hour;
3) washing for 3 times;
4) primary antibody (goat anti-human fibrinogen is expressed as 1: 1000 dilution; sheep anti-human vimentin was expressed as 1: 2500 dilution) and enzyme-labeled secondary antibody (same as RACP enzyme-labeled secondary antibody dilution 1: 2500) according to the following steps: 1, mixing, adding an enzyme label plate at 100 ul/hole, and incubating for 30 minutes at room temperature and 200 rpm;
5) washing for 5 times;
6) developing for 15min, and stopping;
7) reading by a microplate reader: OD450, 630 nm.
Plate distribution:
results of the experiment
Coating: g4
Coating: g6
Coating: g9
Coating: g11
And (4) analyzing results:
FIG. 5 shows the results of the detection of serum from 20 RA patients by different MCSM mabs in combination with different secondary antibodies.
And (4) conclusion:
1) the detection rate of different MSCM monoclonal antibodies to RA patients is different,
2) different secondary antibodies also had an effect on the results of the assay
3) The detection rate of the anti-fibrinogen secondary antibody for G4 is 16/20 (80%); the detection rate of G4 with the anti-vimentin secondary antibody is 15/20 (75%); cutoff is set to OD450-630 ≥ 0.2.
4) The detection rate of the anti-fibrinogen secondary antibody for G6 is 18/20 (90%); the detection rate of G6 using the second anti-vimentin antibody is 17/20 (85%);
5) the detection rate of the anti-fibrinogen secondary antibody for G9 is 17/20 (85%); the detection rate of G9 using the second anti-vimentin antibody is 17/20 (85%);
6) the detection rate of the anti-fibrinogen secondary antibody for G11 is 18/20 (90%); the detection rate of G11 with the anti-vimentin secondary antibody was 18/20 (90%).
Example 8: detection amount of amplification sample of MCSM polypeptide autoantibody rheumatoid arthritis diagnostic kit
Coating with streptavidin, combining with artificially synthesized biotinylated MSCM mixed polypeptide (cyclized P11, P07, P18), sealing, and oven drying. Sample sera were purified by 1: 100, adding 100ul of sample into the air of the reaction plate according to each hole, selecting the other holes, adding a standard substance or a reference substance, reacting for 1h at room temperature, adding an HRP-labeled anti-human IgG antibody after washing, reacting for 30min at room temperature, adding a TMB substrate after washing, reacting for 15min at room temperature, adding a stop solution to stop the reaction, detecting the light absorption values at 450nm and 630nm by using a spectrophotometer, and comparing with the standard substance or the reference substance to calculate the result.
Number and type of samples: the results of a total of 350 clinical sera, 214 normal patients and 136 RA patients are shown in table 1.
Table 1: statistical table of clinical diagnosis results of MCSM autoantibody detection kit Vs rheumatoid arthritis
As can be seen from the results in table 1, the kit for detecting autoantibodies using citrulline-like multimeric structured peptide (MSCM polypeptide) has a positive coincidence rate of 81.6% compared to the results of clinical judgment; the negative coincidence rate is 97.7%; the overall consistency was 91.4%. The sensitivity of the kit is 81 percent, the specificity is 91 percent, and the kit can be used for assisting a doctor to make judgment.
The combination of different polypeptides may improve the sensitivity and specificity of detection.
Example 9: detection quantity of amplified sample of rheumatoid arthritis autoantigen rheumatoid arthritis diagnosis kit
Coating an enzyme label plate with 1-5 ug/ml anti-MSCM antibody (the combination of G11 and G6, the proportion of 1: 1), diluting sample serum by 10-50 times, adding 100ul of the diluted sample serum into each hole, adding a standard substance or a reference substance into the rest holes, reacting at room temperature for 1h, washing, adding a biotin-labeled composite primary antibody (the combination of an anti-fibrinogen antibody and an anti-vimentin antibody, the proportion of 1: 1), reacting at room temperature for 1h, washing, adding diluted enzyme-labeled streptavidin, reacting at room temperature for 30min, washing, adding a TMB substrate, reacting at room temperature for 15min, adding a stop solution to stop the reaction, detecting the light absorption values at 450nm and 630nm by using a spectrophotometer, and comparing the light absorption values with the standard substance or the reference substance to calculate the result.
Number and type of samples: a total of 350 clinical sera, 214 normal patients and 136 RA patients. The results of the experiment are shown in table 2.
TABLE 2 statistical table of clinical diagnosis results of MCSM specific antigen detection kit Vs
As can be seen from the results in Table 2, the sensitivity of the diagnosis kit for rheumatoid arthritis autoantigen prepared by immunizing animals with citrulline-like multimeric structure peptides (MSCM polypeptides) to produce antibodies is 86% and the specificity is 96% compared with the results of clinical judgment, and the diagnosis kit for rheumatoid arthritis autoantigen is superior to other detection kits in both sensitivity and specificity and can also be used for assisting doctors in making judgment.
The combination of different coating antibodies and the combination of different recognition antibodies can further improve the sensitivity and specificity of detection.
The present invention is illustrated in detail by the examples described above, but the present invention is not limited to the details described above, i.e., it is not intended that the present invention be implemented by relying on the details described above. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Reid Biotechnology Ltd, Guangzhou City
<120> specific polypeptide related to rheumatoid arthritis and application thereof
<130> CCP
<160> 18
<170> PatentIn version 3.5
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<220>
<221> misc_feature
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His Gln Asp Gln Gly Xaa Ser Xaa Asn Arg Ala Ala
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Asp Cys Asp Gly Gln Trp Xaa Gly Pro Xaa Ser Val Glu Xaa His Gln
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Ser Ala Lys Cys Lys
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His Gln Cys His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Arg Cys
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Ser His Gln Glu Ser Thr Xaa Gly Xaa Ser Arg Gly Xaa Ser Gly Xaa
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His Gln Asp Asn Gln Xaa Ser Xaa Gln Asn Ala Ala
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His Gln Gly Gln Gly Xaa Gly Xaa Ser Gln Gly Gln Gly Arg Ala
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<213> Artificial sequence
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His Asn Gly Asn Gly Xaa Gly Xaa Ser Asn Gly Arg Gly Asn Ala
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His Ala Asn Asn Xaa Thr Xaa Ser Asn Asn Gly Ala
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Arg Ser Gln Phe Asn Trp Xaa Ser Xaa Ser Arg Pro Arg
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<222> (8)..(8)
<223> Xaa means citrulline
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His Gln Phe Arg Asn Xaa Gln Xaa Arg Ser Arg Asn His Ala
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<221> misc_feature
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<221> misc_feature
<222> (10)..(10)
<223> Xaa means citrulline
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His Gln Tyr Asn Phe Gln Trp Xaa Ser Xaa Gly Arg Pro Arg
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<210> 13
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Gly Arg Phe Gln Met Xaa His Xaa Arg Leu Ile Arg His
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<222> (9)..(9)
<223> Xaa means citrulline
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Arg Ser Gln Phe Asn Phe Xaa Pro Xaa Ser Arg Pro Arg
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<223> Xaa means citrulline
<400> 15
Lys Ala Ala Asn Xaa Asn Asn Asp Ala Leu Xaa Gln Ala Lys Gln
1 5 10 15
<210> 16
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<221> misc_feature
<222> (8)..(8)
<223> Xaa means citrulline
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa means citrulline
<220>
<221> misc_feature
<222> (14)..(14)
<223> Xaa means citrulline
<400> 16
His Gln Tyr Asn Phe Gln Trp Xaa Ser Xaa Gly Arg Pro Xaa Asn Gln
1 5 10 15
Ala Ala
<210> 17
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa means citrulline
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa means citrulline
<220>
<221> misc_feature
<222> (13)..(13)
<223> Xaa means citrulline
<400> 17
Asp Cys Asp Gly Gln Val Xaa Ser Xaa Gly Arg Pro Xaa Asn Gln Ala
1 5 10 15
Ala Lys Cys Lys
20
<210> 18
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<221> misc_feature
<222> (7)..(7)
<223> Xaa means citrulline
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa means citrulline
<220>
<221> misc_feature
<222> (16)..(16)
<223> Xaa means citrulline
<400> 18
His Gln Met Arg Glu Leu Xaa Ser Xaa Gly Gln Val Asp Gln Leu Xaa
1 5 10 15
Asn Asp Lys Ala Arg
20
Claims (7)
1. A specific polypeptide associated with rheumatoid arthritis, or cyclic peptides and aggregates thereof, said specific polypeptide being at least one of the following polypeptides, wherein Cit refers to citrulline:
P11：RSQFNW(Cit)S(Cit)SRPR
P18：DCDGQV(Cit)S(Cit)GRP(Cit)NQAAKCK。
2. use of a specific polypeptide, or cyclic peptides and aggregates thereof, the sequence of which is as defined in claim 1, for the preparation of an antigen for differentiating citrullinated autoantibodies.
3. Use according to claim 2, characterized in that: the citrullinated autoantibodies are derived from rheumatoid arthritis serum, joint fluid or body fluid.
4. A method of inducing citrullinated antibodies comprising the act of immunizing an animal with a specific polypeptide or cyclic peptide and aggregate thereof, wherein: the sequence of the specific polypeptide is as defined in claim 1.
5. Use of a specific polypeptide having the sequence according to claim 1, or a cyclic peptide and aggregate thereof, comprising:
inducing to generate monoclonal antibody;
for the evaluation of antibody titers;
the antibody is a specific citrullinated autoantibody for detecting rheumatoid arthritis.
6. A kit comprising at least one specific polypeptide or cyclic peptides and aggregates thereof, characterized in that: the sequence of the specific polypeptide is as defined in claim 1; the kit is used for: detecting specific citrullinated autoantibodies against rheumatoid arthritis, and evaluating the disease condition of the rheumatoid arthritis.
7. The kit of claim 6, further comprising:
antibodies induced by at least one of the specific polypeptides or cyclic peptides and aggregates thereof; and/or
At least one detection antibody that is anti-human IgG, human IgM or human IgA.
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| CN115458049B (en) * | 2022-06-29 | 2023-07-25 | 四川大学 | Method and device for predicting universal anti-citrullinated polypeptide antibody epitope based on bidirectional circulating neural network |
| CN116162134B (en) * | 2022-12-29 | 2023-12-01 | 旦生(北京)医学科技有限责任公司 | Polypeptide or polypeptide composition |
| CN117990903A (en) * | 2024-04-07 | 2024-05-07 | 北京大学人民医院 | Application of anti-PCOLCE antibody in preparation of rheumatoid arthritis supplementary diagnostic product |
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| WO2004087747A2 (en) * | 2003-03-31 | 2004-10-14 | Universita' Di Pisa | Citrullinated synthetic peptides and uses thereof |
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| FI20050814A0 (en) * | 2005-08-11 | 2005-08-11 | Procollagen Oy | Procedure for observing autoantibodies formed in rheumatoid arthritis |
| CN101726588B (en) * | 2008-10-15 | 2013-02-13 | 上海荣盛生物药业有限公司 | Composition for externally detecting rheumatoid arthritis antibody and application thereof |
| US20110014632A1 (en) * | 2008-12-08 | 2011-01-20 | Nanosphere, Inc. | Assays for clinical assessments of rheumatoid arthritis |
| US20140227303A1 (en) * | 2011-06-29 | 2014-08-14 | Viracor-Ibt Laboratories | Methods for diagnosing and monitoring diseases or conditions using disease modified biomolecules and measurement of a functional immune response |
| US9347941B2 (en) * | 2012-01-13 | 2016-05-24 | Catholic University Industry-Academic Cooperation Foundation | Diagnosis kit for rheumatoid arthritis |
| CN110161247A (en) * | 2018-02-11 | 2019-08-23 | 北京科美生物技术有限公司 | Detect homogeneous immunological detection reagent box and its application of cyclic citrullinated peptid |
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