Polypeptide for detecting rheumatoid arthritis diagnostic marker and application thereof

Abstract

The invention provides a polypeptide for detecting a diagnostic marker of rheumatoid arthritis and application of the polypeptide. The rheumatoid arthritis diagnostic marker is an anti-bispecific protein phosphatase 11 antibody, the amino acid sequence of polypeptide of the antibody is shown as SEQ ID NO.1, and the sequence of a gene for coding the protein is shown as SEQ ID NO.2. The polypeptide is applied to preparation of a reagent for diagnosing rheumatoid arthritis. By detecting the rheumatoid arthritis diagnostic marker, the kit can be effectively used for detecting the rheumatoid arthritis, the detection rate is increased, and a basis is provided for preventing and treating the rheumatoid arthritis as soon as possible.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically relates to a polypeptide for detecting a diagnostic marker of rheumatoid arthritis and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease that mainly causes inflammatory reactions in the synovium of multiple small joints throughout the body, which in turn causes symptoms such as morning stiffness, swelling, pain, and dysfunction in the joints. The global prevalence of RA is 0.2% to 1%, and most patients are women. Existing tests are all clinical tests, such as morning stiffness in or around the joints, lasting at least 1 hour; at least 3 joint soft tissues or effusions at the same time, swelling in at least one joint area in the wrist, metacarpophalangeal, and proximal interphalangeal joints; symmetrical arthritis; rheumatoid nodules; positive serum RF; osteoporosis and joint space narrowing on X-rays. Rheumatoid arthritis can be diagnosed if 4 of the above seven items are met. However, the X-ray of RA patients with a course of less than 6 months may be normal. Therefore, the sensitivity of existing RA diagnosis is not high, and a considerable number of early RA have not been identified. But at the same time, it is particularly important that new RA cases may benefit from early effective intervention to avoid progression to chronic, erosive RA, which can further lead to disability, affect long-term quality of life, and increase disease mortality. Therefore, for rheumatoid arthritis, early diagnosis and treatment are the key to preventing irreversible joint destruction. It is very important to determine the diagnostic markers for rheumatoid arthritis and improve their detection rate. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides polypeptides for detecting diagnostic markers of rheumatoid arthritis and applications thereof.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The polypeptide for detecting the diagnostic marker of rheumatoid arthritis provided by the present invention is a dual specificity protein phosphatase 11 (DUSP11) protein and an antibody against the dual specificity protein phosphatase 11 of the diagnostic marker of rheumatoid arthritis.

[0006] The present invention also provides the use of the polypeptide in preparing a reagent for diagnosing rheumatoid arthritis. Furthermore, the amino acid sequence of the polypeptide, i.e., bispecific protein phosphatase 11, is shown in SEQ ID NO.1, and the gene sequence encoding the polypeptide is shown in SEQ ID NO.2.

[0007] Wherein, the diagnosis comprises: determining the level of antibodies against dual-specific protein phosphatase 11 in a biological sample obtained from a patient presenting with rheumatoid arthritis; optionally,

[0008] The level of antibodies in the biological sample is compared to control data, wherein the detectability of antibodies reactive to dual specificity protein phosphatase 11 in the sample relative to the control data indicates a likelihood of having ACPA-negative rheumatoid arthritis.

[0009] Wherein, the biological sample is a serum sample.

[0010] The level of anti-dual specific protein phosphatase 11 antibodies is measured by the following steps, comprising:

[0011] S1, contacting a serum sample from a patient with the polypeptide, i.e., dual-specific protein phosphatase 11;

[0012] S2, formation of an antibody-protein complex between the antibodies present in the serum sample and the dual-specific protein phosphatase 11;

[0013] S3, washing to remove any unbound antibody;

[0014] S4, adding a detection antibody that is labeled and reactive to the antibody from the serum sample;

[0015] S5, washing to remove any unbound labeled detection antibody; and

[0016] S6. Converting the marker of the detection antibody into a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-duplicate specific protein phosphatase 11 antibodies in the patient.

[0017] Wherein, the bispecific protein phosphatase 11 is coated on a solid support.

[0018] Wherein, the support is a porous plate, fluorescent microspheres or colloidal gold.

[0019] Wherein, the detection antibody is labeled by covalently linking to an enzyme, a marker having a fluorescent compound or a metal, or a marker having a chemiluminescent compound.

[0020] Furthermore, the detection antibody is an anti-human IgG antibody labeled with PE-Cy5.

[0021] The beneficial effects of the present invention are:

[0022] The polypeptides for detecting diagnostic markers for rheumatoid arthritis provided by the present invention and their applications can greatly improve the detection rate of rheumatoid arthritis by screening polypeptides as antigens and detecting anti-polypeptide antibodies in the biological sample to be tested, i.e., serum. Through detection on a protein chip, the risk of rheumatoid arthritis can be successfully predicted and prevented in advance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a bar graph showing the distribution of the results of using a protein chip coated with the recombinant protein 1 of the dual-specific protein phosphatase 11 to detect DUSP11 antibodies in different sample groups in Example 3.

[0024] Figure 2 This is a bar graph showing the distribution of the results of using a protein chip coated with the recombinant protein 2 of the dual-specific protein phosphatase 11 to detect DUSP11 antibodies in different sample groups in Example 3. DETAILED DESCRIPTION

[0025] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

[0026] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are all analytically pure or above.

[0027] The samples used in the present invention are 647 serum samples provided by the rheumatology and immunology department of a medical institution, including: 350 serum samples from patients diagnosed with RA, with an average age ± standard deviation of 45.2 ± 12.5; 9 serum samples from osteoarthritis, with an average age ± standard deviation of 67.2 ± 16.6; 48 serum samples from systemic lupus erythematosus, with an average age ± standard deviation of 36.8 ± 12.4; 10 serum samples from ANCA-associated vasculitis, with an average age ± standard deviation of 46.9 ± 16.3; 28 serum samples from Behcet's disease, with an average age ± standard deviation of The mean age ± standard deviation was 54.2±20.7; 39 sera from ankylosing spondylitis, with a mean age ± standard deviation of 38.2±15.1; 21 sera from Sjögren's syndrome, with a mean age ± standard deviation of 52.7±13.2; 132 sera from healthy subjects, with a mean age ± standard deviation of 37.5±12.1; 10 sera from Takayasu arteritis, with a mean age ± standard deviation of 38.4±13.5; Among them, the diagnosis of RA was based on the criteria established by ACR / EULAR in 2010, and the other patients also met their respective diagnostic and / or classification criteria.

[0028] All sera were collected by the Department of Rheumatology and Immunology of medical institutions from 2006 to 2014. All disease sera were from clinically diagnosed patients. Patients with controversial diagnoses had their diagnoses confirmed after consultation with at least three chief physicians. All rheumatoid arthritis sera were tested for serum corresponding antibodies, including ANA 3 items: DNA-IF (immunofluorescence method), ANA-IF (immunofluorescence method) and ds-DNA (ELISA method), anti-CCP antibody, i.e. ACPA test (>25IU / ml defined as positive), RF test, AKA and APF test, MCV test, GPI test. All RA patients with negative anti-CCP antibodies and / or anti-AKA, APF, MCV antibodies met the diagnostic criteria for RA synovitis by ultrasound or MRI.

[0029] Example 1 Preparation of DUSP11 Antigen

[0030] 1.1 Construction of recombinant plasmid

[0031] The dual-specific protein phosphatase 11 protein (DUSP11) was selected, and its nucleotide sequence and amino acid sequence are shown in GNnBank sequence number: NM_003584.3. The gene sequence was modified according to the preferred codons of Escherichia coli. After a large number of experimental studies, the expression efficiency was increased, and the exogenous protein expression efficiency was more than 70%. The secondary structure of the recombinant protein was screened by bioinformatics, and the optimized gene sequence was obtained as shown in SEQ ID NO.2. SEQ ID NO.2: CCGGAACGCTGGAAAGATT ATCTGCCGGTGGGCCAGCGCATGCCGGGCACCCGCTTTATTGCGTTTAAAGTGCCGCTGCAGAAAAGCTTTGAAAAAAAACTGGCGCCGGAAGAATGCTTTAGCCCGCTGGATCTGTTTAACAAAATTCGCGA ACAGAACGAAGAACTGGGCCTGATTATTGATCTGACCTATACCCAGCGCTATTATAAACCGGAAGATCTGCCGGAAACCGTGCCGTATCTGAAAATTTTTACCGTGGGCCATCAGGTGCCGGATGATGAAAACCA TTTTTAAATTTAAACATGCGGTGAACGGCTTTCTGAAAGAAAACAAAGATAACGATAAACTGATTGGGCTGCATTGCACCCATGGCCTGAACCGCACCGGCTATCTGATTTGCCGCTATCTGATTGATGTGGAA GGCGTGCGCCCGGATGATGCGATTGAACTGTTTAACCGCTGCCGCGGCCATTGCCTGGAACGCCAGAACTATATTGAAGATCTGCAGAACGGCCCGATTCGCAAAAACTGGAACAGCAGC, and its corresponding amino acid sequence is SEQ ID NO.1: PERWKDYLPVGQRMPGTRFIAFKVPLQ KSFEKKLAPEECFSPLDLFNKIREQNEELGLIIDLTYTQRYYKPEDLPETVPYLKIFTVGHQVPDDETIFKFKHAVNGFLKENKDNDKLIGVHCTHGLNRTGYLICRYLIDVEGVRPDDAIELFNRCRGHCLERQNYIEDLQNGPIRKNWNSS. Shanghai Bioengineering Co., Ltd. was commissioned to synthesize the gene sequence (SEQ ID NO.1), and the gene was cloned into the expression vector pNT30a to obtain the recombinant plasmid.Experimental studies have shown that if a non-optimized gene sequence is used for recombinant expression, a recombinant protein can be obtained.

[0032] 1.2 Expression and identification of recombinant proteins

[0033] The recombinant plasmids 1 and 2 with correct sequencing were transformed into Escherichia coli BL21 respectively, and the single colony with correct identification was inoculated into LB liquid culture medium containing ampicillin (concentration of 10 μg / mL), and cultured at 37°C overnight with shaking. The next day, it was inoculated into a new LB liquid culture medium containing ampicillin at a ratio of 1:50 and cultured with shaking until OD600 reached 0.7, then the bacterial solution temperature was cooled to 22°C, IPTG was added to make its final concentration 0.75 mmol / L, and induced expression was performed at 22°C; 1 mL of bacterial solution was drawn after 6 hours, and lysed by ultrasound for 10 minutes, the bacterial solution was collected, boiled in a water bath for 10 minutes, centrifuged at 4°C and 10000 r / min for 5 minutes, placed on ice, and the supernatant and precipitate were drawn for 12% SDS-PAGE electrophoresis detection; after the electrophoresis, the gel was stained with Coomassie Brilliant Blue for 90 minutes, and the protein induced expression was observed after decolorization for 2 hours, and the target protein appeared in the precipitate, which was a protein of about 22KD in size.

[0034] 1.3 Purification of recombinant protein

[0035] 2 mL of induced expression bacterial solution was taken separately, centrifuged at 4°C, 12000 r / min for 30 minutes, the cell pellets were collected, washed three times with 0.01M pH7.4 PBS, suspended in pH8.0 lysis buffer (Tris-Nacl), ultrasonicated for 15 minutes, centrifuged at 4°C, 12000 r / min for 30 minutes, the pellet was suspended in 8 mol / L urea at pH8.0, shaken on ice for 2 hours to dissolve inclusion bodies, centrifuged at 12000 r / min for 30 minutes, the supernatant was filtered through a 0.22 μm filter membrane and purified using a His Trap TMHP (histidine) affinity adsorption column purification system, the purity was identified by SDS-PAGE, and finally the purified protein was dialyzed into 0.01 mol / L, pH7.4 PBS, concentrated by PNG-20000, and a purified recombinant protein with a molecular weight of about 22 KD was obtained, recorded as protein 1.

[0036] Example 2 Selection and preparation of dual-specific protein phosphatase 11 protein

[0037] For the selection of dual-specific protein phosphatase 11 protein, the inventors of the present invention conducted multiple experiments and selected the amino acid sequence as shown in SEQ ID NO.3 as an example, SEQ ID NO.3: AKKKGGN HIPERWKDYLPVGQRMPGTRFIAFKVPLQKSFEKKLAPEECFSPLDLFNKIREQNEELGLIIDLTYTQRYYKPEDLPETVPYLKIFTVGHQVPDDETIFKFKHAVNGFLKENKDNDKLIGVHCTHGLNRTGYLICRYLIDVEGVRPDDAIELFNRCRGHCLERQNYIEDLQNGPIRK, modified the gene sequence according to the preferred codons of Escherichia coli, screened the secondary structure of the recombinant protein by bioinformatics, and verified by multiple experiments. The final determined gene sequence is shown in SEQ ID NO.4: GCGAAAAAAAAAGGCGGCAACC.

[0038] According to the sequence shown in SEQ ID NO.4, Shanghai Yingjun Company was commissioned to synthesize, and the expression vector PGEX-4T-2 was connected to obtain the connection product, i.e., the recombinant plasmid, which was transformed into DH5α competent cells; the recombinant plasmid was identified by PCR and double enzyme digestion, and the positive clones were screened and sent to Shanghai Jun Company for sequencing, and the recombinant plasmid with correct sequencing was transformed into Escherichia coli BL21, and the single colony with correct identification was inoculated into LB liquid culture medium containing ampicillin, and cultured at 37°C with shaking overnight, and the next day it was inoculated into a new LB liquid culture medium containing ampicillin at a ratio of 1:100 and cultured with shaking until OD600 reached 0.8, and then the bacterial solution temperature was cooled to 22°C, and IPTG with a final concentration of 0.8mmol / L was added to induce expression at 22°C; and 1mL of bacterial solution was drawn at 1h, 2h, 3h, 4h, 5h, and 6h, respectively, and ultrasonically lysed, the bacterial solution was collected, boiled at 100°C for 10min, centrifuged at 4°C and 12000r / min for 3min, placed on ice, and the supernatant was drawn for SDS-PA GE electrophoresis detection; after the electrophoresis, the gel was stained with Coomassie Brilliant Blue for 80 minutes, and then decolorized for 2 hours to observe the protein induction expression; the target protein with a molecular weight of about 23KD was expressed from 1 hour after induction, and the yield was the highest at 6 hours.

[0039] 2 ml of bacterial solution induced for 6 hours of expression was taken and centrifuged at 12000 r / min for 30 minutes, the cell pellet was collected, washed three times with 0.01 M pH 7.4 PBS, suspended in pH 8.0 lysis buffer (Tris-NaCl), sonicated, and centrifuged. 10 μl of supernatant and precipitate were taken for SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue R-250 after electrophoresis. The specific target protein band was checked after decolorization with decolorizing solution. The results showed that the recombinant protein was expressed in the form of inclusion bodies. The cell precipitates (mainly containing some cell debris and inclusion bodies) were collected after ultrasonic centrifugation, suspended in 8 mol / L urea at pH 8.0, and shaken on ice for 2 hours to dissolve the inclusion bodies. The supernatant was centrifuged at 12000r / min for 30 minutes, and the supernatant was filtered through a 0.22μm filter membrane and purified using a His Trap TMHP (histidine) affinity adsorption column purification system. The purity was identified by SDS-PAGE. Finally, the purified protein was dialyzed into 0.03mol / L, pH 7.4 PBS and concentrated with PEG-20000 to obtain purified recombinant DUSP11 protein 2 with a molecular weight of approximately 23KD.

[0040] The N-termini of the recombinant proteins of the dual-specific protein phosphatase 11 obtained above, namely, DUSP11 protein 1 and DUSP11 protein 2, have glutathione S-transferase (GST) tags. The recombinant proteins of the dual-specific protein phosphatase 11 obtained above, DUSP11 protein 1 and DUSP11 protein 2, are coated on the chip using conventional methods. The chip quality is verified by hybridization with mouse anti-GST monoclonal antibody. When the correlation of the fluorescence signal values ​​of every two repeated protein spots on the chip reaches more than 97%, it is considered that the repeatability of every two spots on the chip is good.

[0041] 238 RA sera and 197 control sera (38 SLE sera, 9 Takayasu arteritis sera, 18 Behcet's disease sera, and 132 healthy human sera) were selected for hybridization with a protein chip to form an antibody-protein complex between the antibodies present in the serum samples and DUSP11; then washed to remove any unbound antibodies; added anti-human IgG antibodies labeled with PE-Cy5 for reaction, and after the reaction, the serum was detected with anti-human IgG antibodies labeled with PE-Cy5, and identified by signal acquisition and data analysis. The reaction of the dual-specific protein phosphatase 11 protein antibody in the serum with the dual-specific protein phosphatase 11 protein antigen probe was detected with anti-human IgG antibodies labeled with PE-Cy5. Specifically, the antibody level against dual-specific protein phosphatase 11 was measured by the following steps:

[0042] S1, contacting a serum sample from a patient with a bispecific protein phosphatase 11 coated on a chip;

[0043] S2, formation of an antibody-protein complex between the antibodies present in the serum sample and the dual-specific protein phosphatase 11;

[0044] S3, washing to remove any unbound antibody;

[0045] S4, adding a labeled detection antibody reactive to the antibody from the serum sample, i.e., PE-Cy5 labeled anti-human IgG antibody reaction;

[0046] S5, washing to remove any unbound detection antibody;

[0047] S6. Convert the marker of the detection antibody into a detectable fluorescent signal.

[0048] Scan and obtain the fluorescence signal graph of each chip, and drag the graph and the template file of the chip, i.e., the gail file, into the GenePix Pro 6.0 software for one-to-one correspondence. Then convert the signal information of all probes on each chip collected by the GenePix Pro 6.0 software and import it into an Excel table. The foreground signal intensity (F635median) of each probe point minus the surrounding background signal intensity (B635median) is used as the signal value of the point.

[0049] First, the data of ACPA-negative RA patients and healthy controls and disease controls were subjected to chi-square test or Fisher's exact test. As diagnostic marker proteins, parameters such as T score and p value can be obtained; for each protein, 1000 different cutoff values ​​were selected, and sensitivity and specificity can be calculated according to each cutoff value. The ROC curve was drawn with these 1000 points (1-specificity, sensitivity), and the AUC was calculated. The cutoff value corresponding to the point with the highest sum of sensitivity and specificity was the optimal cutoff. The results are shown in Table 1. In the results of hybridization reaction with large sample serum, the sensitivity of protein antigen immune reaction with ACPA-negative RA serum was greater than 25%, and it also had specificity that was different from healthy controls and disease controls. DUSP11 protein 1, the cutoff value was 2.0944, and its sensitivity was 64.30%, DUSP11 protein 2, the cutoff value was 2.2490, and the sensitivity was 26.50%.

[0050] Table 1 Cutoff values ​​and corresponding AUCs of protein 1 and protein 2 in different RAs

[0051] cutoff value Sensitivity Specificity AUC Protein 1 2.0944 0.6430 0.9090 0.852 Protein 2 2.2490 0.2650 0.9540 0.653

[0052] Example 3

[0053] External serum samples (60 RA sera, 10 SLE sera, 10 Takayasu arteritis sera, 10 Behcet's disease sera, and 30 healthy human sera) were used to verify the DUSP11 protein 1 and DUSP11 protein 2 obtained above. The protein chip prepared according to protein 1 and protein 2 in Example 2 was used for detection to obtain fluorescence values. The results were judged based on the cutoff value. Figure 1 and Figure 2. Through calculation, the accuracy of the test results obtained for DUSP11 protein 1 is 0.7833, and the accuracy of DUSP11 protein 2 is 0.6083, which is higher and can avoid missed detection. It can be seen that the polypeptide obtained by the optimized sequence, that is, the gene sequence of DUSP11 protein 1, can be used to detect diagnostic markers for rheumatoid arthritis and for the detection of rheumatoid arthritis.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide for detecting a diagnostic marker for rheumatoid arthritis, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and the gene sequence encoding the protein is shown in SEQ ID NO.

2.

2. Use of the polypeptide according to claim 1 in the preparation of a reagent for diagnosing rheumatoid arthritis.

3. The use according to claim 2, characterized in that: The risk of developing rheumatoid arthritis is determined by detecting the level of antibodies corresponding to the polypeptide in the biological sample.

4. The use according to claim 3, characterized in that: Detecting the level of antibodies corresponding to the polypeptide in a biological sample comprises the following steps: S1. contacting a serum sample from a patient with a polypeptide; S2, formation of an antibody-protein complex between the antibody and the polypeptide present in the serum sample; S3, washing to remove unbound antibodies; S4, adding a detection antibody that is labeled and reactive to the antibody from the serum sample; S5, washing to remove any unbound labeled detection antibody; S6. Converting the marker of the detection antibody into a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-polypeptide antibodies in the patient.

5. The use according to claim 4, characterized in that: The polypeptide is coated on a solid support.

6. The use according to claim 4, characterized in that: The support is a porous plate, fluorescent microspheres or colloidal gold.

7. The use according to claim 4, characterized in that: The detection antibody is an anti-human IgG antibody labeled with PE-Cy5.

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