CN117233396A - Application of urine extracellular matrix protein 1 and polypeptide fragment thereof in systemic lupus erythematosus - Google Patents
Application of urine extracellular matrix protein 1 and polypeptide fragment thereof in systemic lupus erythematosus Download PDFInfo
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- CN117233396A CN117233396A CN202210241035.9A CN202210241035A CN117233396A CN 117233396 A CN117233396 A CN 117233396A CN 202210241035 A CN202210241035 A CN 202210241035A CN 117233396 A CN117233396 A CN 117233396A
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- Prior art keywords
- pro
- leu
- extracellular matrix
- matrix protein
- glu
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- 102100031758 Extracellular matrix protein 1 Human genes 0.000 title claims abstract description 42
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 32
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 32
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- 239000012634 fragment Substances 0.000 title claims abstract description 25
- 201000000596 systemic lupus erythematosus Diseases 0.000 title abstract description 7
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides application of urine extracellular matrix protein 1 (Extracellular matrix protein 1) and polypeptide fragments thereof, in particular application of the urine extracellular matrix protein 1 and polypeptide fragments thereof in preparation of diagnosis, differential diagnosis, disease degree and activity judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like of systemic lupus erythematosus (Systemic lupus erythematosus, SLE). SLE is a diffuse connective tissue disease mediated by autoimmunity and highlighted by immune inflammation, most of which appear as hidden diseases and often involve multiple systems of the body. The invention proves that the expression of the urine extracellular matrix protein 1 and the polypeptide fragment thereof in SLE active patients is reduced through researches. Detection can be applied for various purposes of SLE patients. The invention plays the advantages of noninvasive acquisition of urine samples, large-scale repeated sampling and convenient preservation, and utilizes the urine samples to detect the extracellular matrix protein 1 and the polypeptide fragments thereof.
Description
Technical Field
The invention relates to a new application of urine extracellular matrix protein 1 and a polypeptide fragment thereof, in particular to an application of urine extracellular matrix protein 1 and a polypeptide fragment thereof in SLE diagnosis, differential diagnosis, disease degree and activity judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Background
Systemic lupus erythematosus (Systemic lupus erythematosus, SLE) is an autoimmune connective tissue disease that can involve multiple organs throughout the body, well developed in women of childbearing age. According to epidemiological statistical studies, SLE prevalence in China is 70/10 ten thousand, and women are up to 113/10 ten thousand. The cause of the disease is not clear, SLE is frequently a hidden onset disease, clinical manifestations are complex and various, and red spots distributed in the nose bridge and cheeks in butterfly form are changes of SLE characteristics. Skin lesions of SLE also include light sensitivity, hair loss, hand and foot palmar and periungual erythema, discoid erythema, reynolds phenomenon, and the like. In addition, SLE often develops in multiple systems, especially those involving vital visceral systems such as lupus nephritis, neuropsychiatric lupus, pericardial effusion, blood and digestive system damage. It can be seen that the method is particularly important for timely diagnosis, effective disease monitoring and targeted treatment of the patient.
Extracellular matrix protein 1 (Extracellular matrix protein, ECM 1) is a secreted protein involved in biological processes such as cell proliferation, angiogenesis, embryogenesis, skin differentiation and tumor formation. The ECM1 gene is located in the epidermal differentiation complex (Epidermal differentiation complex, EDC) region adjacent to human chromosome 1q21, and contains 10 exons, which are closely related to the keratinocyte differentiation process. The extracellular matrix protein 1 content of SLE active patients in urine is reduced in the study.
Compared with the common clinical blood sample, the urine can be completely and noninvasively collected continuously in a large amount; without steady state regulation, more kinds and larger amplitude of changes can be accumulated, and many pathophysiological changes of the body may be manifested in urine. Protein polypeptides with relatively small molecular weights such as hormones and cytokines can be excreted into urine quickly after entering blood, and the probability of being detected in the urine is much higher than that in the blood; prior to urine collection, the possible proteolytic processing of urine is completed, so that urine proteins remain stable for a longer period of time. In order to relieve the pain of patients with SLE caused by repeated blood sampling, the experiment is expected to realize the diagnosis and disease monitoring of SLE patients with painless, convenient, quick and easily repeated urine detection through urine protein or polypeptide research on the basis of early-stage methodology, and lays a foundation for further research of urine polypeptide detection kits.
Disclosure of Invention
The invention aims to provide an application of urine extracellular matrix protein 1 and polypeptide fragments thereof in preparing SLE diagnosis, differential diagnosis, disease degree and activity judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like
Preferably, the amino acid sequence of the urine extracellular matrix protein 1 comprises the amino acid sequence shown in SEQ ID NO.1 (MGTTARAALV LTYLAVASAA SEGGFTATGQ RQLRPEHFQE VGYAAPPSPP LSRSLPMDHP DSSQHGPPFE GQSQVQPPPS QEATPLQQEK LLPAQLPAEK EVGPPLPQEA VPLQKELPSL QHPNEQKEGT PAPFGDQSHP EPESWNAAQH CQQDRSQGGW GHRLDGFPPG RPSPDNLNQI CLPNRQHVVY GPWNLPQSSY SHLTRQGETL NFLEIGYSRC CHCRSHTNRL ECAKLVWEEA MSRFCEAEFS VKTRPHWCCT RQGEARFSCF QEEAPQPHYQ LRACPSHQPD ISSGLELPFP PGVPTLDNIK NICHLRRFRS VPRNLPATDP LQRELLALIQ LEREFQRCCR
QGNNHTCTWK AWEDTLDKYC DREYAVKTHH HLCCRHPPSP TRDECFARRA PYPNYDRDIL TIDIGRVTPN LMGHLCGNQR VLTKHKHIPG LIHNMTARCC DLPFPEQACC AEEEKLTFIN DLCGPRRNIW RDPALCCYLS PGDEQVNCFN INYLRNVALV SGDTENAKGQ GEQGSTGGTN ISSTSEPKEE), SEQ ID NO.2 (MGTTARAALV LTYLAVASAA SEGGFTATGQ RQLRPEHFQE VGYAAPPSPP LSRSLPMDHP DSSQHGPPFE GQSQVQPPPS QEATPLQQEK LLPAQLPAEK EVGPPLPQEA VPLQKELPSL QHPNEQKEGT PAPFGDQSHP EPESWNAAQH CQQDRSQGGW GHRLDGFPPG RPSPDNLNQI CLPNRQHVVY GPWNLPQSSY SHLTRQGETL NFLEIGYSRC CHCRSHTNRL ECAKLVWEDT LDKYCDREYA VKTHHHLCCR HPPSPTRDEC FARRAPYPNY DRDILTIDIG RVTPNLMGHL CGNQRVLTKH KHIPGLIHNM TARCCDLPFP EQACCAEEEK LTFINDLCGP RRNIWRDPAL CCYLSPGDEQ VNCFNINYLR NVALVSGDTE NAKGQGEQGS TGGTNISSTS EPKEE), SEQ ID NO.3 (MALPLRDRVK EATPLQQEKL LPAQLPAEKE VGPPLPQEAV PLQKELPSLQ HPNEQKEGTP APFGDQSHPE PESWNAAQHC QQDRSQGGWG HRLDGFPPGR PSPDNLNQIC LPNRQHVVYG PWNLPQSSYS HLTRQGETLN FLEIGYSRCC HCRSHTNRLE CAKLVVRLGS), SEQ ID NO.4 (MGTTARAALV LTYLAVASAA SEGGFTATGQ RQLRPEHFQE VGYAAPPSPP LSRSLPMDHP DSSQHGPPFE GQSGKEGRGP RPHSQPWLGE RVGCSHIPPS IVQPPPSQEA TPLQQEKLLP AQLPAEKEVG PPLPQEAVPL QKELPSLQHP NEQKEGTPAP FGDQSHPEPE SWNAAQHCQQ DRSQGGWGHR LDGFPPGRPS PDNLNQICLP NRQHVVYGPW NLPQSSYSHL TRQGETLNFL EIGYSRCCHC RSHTNRLECA KLVWEEAMSR FCEAEFSVKT RPHWCCTRQG EARFSCFQEE APQPHYQLRA CPSHQPDISS GLELPFPPGV PTLDNIKNIC HLRRFRSVPR NLPATDPLQR ELLALIQLER EFQRCCRQGN NHTCTWKAWE DTLDKYCDRE YAVKTHHHLC CRHPPSPTRD ECFARRAPYP NYDRDILTID IGRVTPNLMG HLCGNQRVLT KHKHIPGLIH NMTARCCDLP FPEQACCAEE EKLTFINDLC GPRRNIWRDP ALCCYLSPGD EQVNCFNINY LRNVALVSGD TENAKGQGEQ)
GSTGGTNISS TSEPKEE); or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO 1-4 and has the same function as the amino acid sequence shown in SEQ ID NO 1-4.
Preferably, the preparation is a detection kit for urine extracellular matrix protein 1 and polypeptide fragments thereof of SLE patients.
Preferably, the kit comprises one or more of an immunization method of antigen-antibody reaction and a kit thereof, such as an aptamer antibody or an antibody fragment capable of specifically binding to extracellular matrix protein 1 and a polypeptide fragment thereof.
Preferably, the detection method comprises a method of directly detecting mass spectrum of extracellular matrix protein 1 and polypeptide fragments thereof and the like and a related kit thereof.
Preferably, the detection method comprises methods such as related nucleic acid detection for directly detecting extracellular matrix protein 1 and polypeptide fragments thereof and related kits thereof.
Preferably, the kit further comprises a component selected from the group consisting of: solid phase carrier, diluent, reference substance, standard substance, quality control substance, detection antibody, secondary antibody diluent, luminous reagent, washing liquid, color developing liquid and stop solution.
Preferably, the standard comprises an extracellular matrix protein 1 standard, a humanized tag antibody standard; preferably, the quality control product comprises: extracellular matrix protein 1 quality control and humanized tag antibody quality control; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates or affinity membranes, and other carriers of equivalent function.
Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose and has similar functions.
The inventors first collected urine samples from healthy persons, SLE active patients (aSLE) and stationary phase patients (sSLE), centrifuged at 4000r/min for 5min, and the supernatant was aspirated, and the protein concentration was measured by the Bradford method for SDS-PAGE enzymatic hydrolysis. Label-free mass spectrometry of urine samples was performed by an orbitrapF type mass spectrometer. The data obtained in mass spectrometry for SLE group and normal control group were quantitatively calculated. Screening the differential polypeptide by taking the difference of protein expression amount over 1.5 times and the P <0.05 as a reference standard through statistical test. Then the inventor identifies the differential polypeptide with statistical significance, and searches by using a database to obtain the differential protein extracellular matrix protein 1. The inventor uses Western Blot immunoblotting test and parallel reaction monitoring technology (PRM) to verify extracellular matrix protein 1 and the content thereof in urine of SLE patients and healthy people.
The research proves that the extracellular matrix protein 1 and the polypeptide fragment thereof are expressed in urine of SLE active patients, and have better consistency with clinical diagnosis. Therefore, the detection of the extracellular matrix protein 1 of urine and the polypeptide fragment thereof can be used for the auxiliary diagnosis or disease monitoring of SLE.
The invention plays the advantages of noninvasive acquisition of urine samples, large-scale repeated sampling and convenient preservation, and utilizes the urine samples to detect the extracellular matrix protein 1 and the polypeptide fragments thereof.
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments, as illustrated in the accompanying drawings.
Drawings
FIG. 1 is a graph showing the content of extracellular matrix protein 1 and polypeptide fragments thereof in SLE patients and healthy control groups.
FIG. 2 is a graph showing Western Blot immunoblotting test results of extracellular matrix protein 1 of urine and polypeptide fragments thereof in SLE patients and healthy control groups.
FIG. 3 is a quantitative view of urine extracellular matrix protein 1 and polypeptide fragments thereof in a PRM of a SLE patient and a healthy control group.
Detailed Description
Example 1Urine specimen collection and processing
SLE patients were selected as SLE group, and contemporaneous healthy physical examination persons were selected as normal control group. 30ml of fresh morning urine samples of each group of study subjects were collected after admission, and the urine in the morning catheters was collected by the disabled and placed in a dry, clean container. Centrifuging the collected urine specimen at 4000r/min for 5min, sucking the supernatant, subpackaging 2ml per tube, and storing in a refrigerator at-80 ℃.
Example 2Mass spectrometry and screening of urine polypeptides
Urine sample protein is extracted and the concentration of the extracted protein is determined. Mass spectrometry of urine samples was performed by orbitrapf type mass spectrometry. The data obtained in the mass spectrum of the experimental group and the normal control group are quantitatively calculated. The comparison between groups adopts t-test for differential analysis, and the differential expression protein is screened by taking the difference of protein expression amount over 1.5 times and the P <0.05 as a reference standard through statistical test.
Example 3Identification and analysis of differential Polypeptides
The database used is a uniprot_homo database, the generated mass spectrum original file is processed by MaxQuant software, and the retrieval parameter setting is shown in table 1.
Example 4Verification of urine extracellular matrix protein 1
The results of Label-free mass spectrometry show that extracellular matrix protein 1 is low-expressed in urine of SLE active patients, the content of the extracellular matrix protein 1 in urine of SLE patients and healthy control groups is shown in figure 1, the extracellular matrix protein 1 in urine of SLE patients and healthy people is detected by using Western Blot immunoblotting test, the results of which are shown in figure 2, and the extracellular matrix protein 1 in urine of SLE patients and healthy people is detected by using Parallel Reaction Monitoring (PRM), and the results of which are shown in figure 3. The results were confirmed to be consistent, with significant differences in extracellular matrix protein 1 expression between groups.
Although the invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and the invention is defined in the following claims.
Sequence listing
<110> Bowman
<120> application of urine extracellular matrix protein 1 and polypeptide fragment thereof in systemic lupus erythematosus
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Claims (9)
1. The application of the urine extracellular matrix protein 1 and the polypeptide fragment thereof in preparing SLE diagnosis, differential diagnosis, disease degree and activity judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
2. The use according to claim 1, wherein the amino acid sequence of the urine extracellular matrix protein 1 comprises the amino acid sequence shown in SEQ ID No.1 to 4; or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO 1-4 and has the same function as the amino acid sequence shown in SEQ ID NO 1-4.
3. The use according to claim 1, wherein the formulation is a SLE patient urine extracellular matrix protein 1 and polypeptide fragment detection kit thereof.
4. The use according to claim 3, wherein the kit comprises one or more of an immunization method of an antigen-antibody reaction and a kit thereof such as an aptamer antibody or an antibody fragment capable of specifically binding to extracellular matrix protein 1 and a polypeptide fragment thereof.
5. The use according to claim 3, wherein the detection method comprises a method for directly detecting mass spectra of extracellular matrix protein 1 and polypeptide fragments thereof and the like and a kit related thereto.
6. The use according to claim 3, wherein the detection method comprises a method for directly detecting extracellular matrix protein 1 and polypeptide fragments thereof or related nucleic acid detection and the like and related kits thereof.
7. The use according to claim 3, wherein the kit further comprises a component selected from the group consisting of: solid phase carrier, diluent, reference substance, standard substance, quality control substance, detection antibody, secondary antibody diluent, luminous reagent, washing liquid, color developing liquid and stop solution.
8. The use of claim 7, wherein the standard comprises an extracellular matrix protein 1 standard, a humanized tag antibody standard; preferably, the quality control product comprises: extracellular matrix protein 1 control, humanized tag antibody quality control; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates or affinity membranes, and other carriers of equivalent function.
9. The use according to claim 8, wherein the solid support is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose and a support with similar function.
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| CN202210241035.9A CN117233396A (en) | 2022-03-13 | 2022-03-13 | Application of urine extracellular matrix protein 1 and polypeptide fragment thereof in systemic lupus erythematosus |
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| CN202210241035.9A CN117233396A (en) | 2022-03-13 | 2022-03-13 | Application of urine extracellular matrix protein 1 and polypeptide fragment thereof in systemic lupus erythematosus |
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