CN114252617A - Application of urine macrophage colony stimulating factor-1 and polypeptide fragment thereof in allergic diseases - Google Patents

Application of urine macrophage colony stimulating factor-1 and polypeptide fragment thereof in allergic diseases Download PDF

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CN114252617A
CN114252617A CN202010996093.3A CN202010996093A CN114252617A CN 114252617 A CN114252617 A CN 114252617A CN 202010996093 A CN202010996093 A CN 202010996093A CN 114252617 A CN114252617 A CN 114252617A
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stimulating factor
macrophage colony
colony stimulating
urine
antibody
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张曼
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

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Abstract

The invention provides application of urine Macrophage colony stimulating factor-1 (macrophagal colony-stimulating factor 1) and polypeptide fragments thereof, in particular to application of the urine Macrophage colony stimulating factor-1 and the polypeptide fragments thereof in preparation of preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like. The research proves that the expression of the urinary macrophage colony stimulating factor-1 and the polypeptide fragment thereof is reduced in patients with allergic diseases compared with healthy people (normal control group). Can be used for various purposes of detecting patients with allergic diseases. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine specimen, and utilizes the urine specimen to detect the urine macrophage colony stimulating factor-1 and the polypeptide fragment thereof.

Description

Application of urine macrophage colony stimulating factor-1 and polypeptide fragment thereof in allergic diseases
Technical Field
The invention relates to a new application of urine macrophage colony stimulating factor-1 and a polypeptide fragment thereof, in particular to an application of the urine macrophage colony stimulating factor-1 and the polypeptide fragment thereof in preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Background
Allergic diseases are also called allergic diseases, and typical allergic diseases comprise allergic rhinitis, bronchial asthma, allergic dermatitis, eczema, allergic conjunctivitis, anaphylactic shock and the like. According to the statistics of the world health organization, the number of allergic people is huge in the world, and about one third of people suffer from allergic diseases. In recent years, due to rapid change of life style of people, remarkable improvement of industrialization degree and remarkable aggravation of air pollution, the incidence rate of allergic diseases rises year by year, serious allergic diseases even endanger life, and the problem becomes a global public health focus at present.
Currently, clinical auxiliary examination of allergic diseases mainly includes Skin Prick Test (SPT), serum sIgE detection and the like, but SPT patients have poor pain compliance and are easily affected by drugs, and the risk of systemic anaphylaxis can be increased, so that the SPT test is not suitable for severe allergic constitution and children and old people. The detection of sIgE is also not a non-invasive one, and often requires repeated blood drawing of the patient. In order to improve the life quality and compliance of patients with allergic diseases and relieve the pain of repeated blood collection and prick tests, the urine protein or polypeptide research is expected to realize the diagnosis of the allergic diseases assisted by painless, convenient, quick and easily repeated urine detection and also lay a foundation for the further research of the urine polypeptide detection kit.
Macrophage colony-stimulating factor-1 (CSF-1) has mitotic promoting effect, and can regulate proliferation and differentiation of blood precursor cell, granulocyte and Macrophage. CSF-1 interacts with genetic factors and plays a key role in the movement and reproduction of cells. In the research, the CSF-1 content in urine of patients with allergic diseases is reduced in expression compared with that in healthy people, and the content in urine is reduced.
Compared with the common clinical blood sample, the urine can be collected in a non-invasive, continuous and large amount; without homeostatic regulation, more various and larger changes can be accumulated, and many pathophysiological changes of the body can be reflected in urine. Some protein polypeptides with relatively small molecular weight, such as hormones and cytokines, are excreted into urine quickly after entering blood, and the probability that the proteins and polypeptides are detected in urine is much higher than that in blood; before urine is collected, a possible protein degradation process in urine is completed, so that urine protein can be kept stable for a longer time. In order to relieve the pain of patients with allergic diseases in blood collection for many times, the experiment is expected to realize the diagnosis and disease monitoring of patients with allergic diseases assisted by painless, convenient, quick and easily repeated urine detection through the research of urine protein or polypeptide on the basis of the methodology of the earlier stage, and also lays a foundation for the further research of urine polypeptide detection kits.
Disclosure of Invention
The invention aims to provide application of urine macrophage colony stimulating factor-1 and polypeptide fragments thereof in preparation of preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Preferably, the amino acid sequence of the urine macrophage colony stimulating factor-1 is shown as SEQ ID NO.1
(MTAPGAAGRC PPTTWLGSLL LLVCLLASRS ITEEVSEYCS HMIGSGHLQS
LQRLIDSQME TSCQITFEFV DQEQLKDPVC YLKKAFLLVQ DIMEDTMRFR
DNTPNAIAIV QLQELSLRLK SCFTKDYEEH DKACVRTFYE TPLQLLEKVK
NVFNETKNLL DKDWNIFSKN CNNSFAECSS QDVVTKPDCN CLYPKAIPSS
DPASVSPHQP LAPSMAPVAG LTWEDSEGTE GSSLLPGEQP LHTVDPGSAK
QRPPRSTCQS FEPPETPVVK DSTIGGSPQP RPSVGAFNPG MEDILDSAMG
TNWVPEEASG EASEIPVPQG TELSPSRPGG GSMQTEPARP SNFLSASSPL
PASAKGQQPA DVTGTALPRV GPVRPTGQDW NHTPQKTDHP SALLRDPPEP
GSPRISSLRP QGLSNPSTLS AQPQLSRSHS SGSVLPLGEL EGRRSTRDRR
SPAEPEGGPA SEGAARPLPR FNSVPLTDTG HERQSEGSFS PQLQESVFHL
LVPSVILVLL AVGGLLFYRW RRRSHQEPQR ADSPLEQPEG SPLTQDDRQV
ELPV); or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.
Preferably, the preparation is a urine macrophage colony stimulating factor-1 and a polypeptide fragment detection kit thereof for patients with allergic diseases.
Preferably, the kit includes one or more of an immunological method of antigen antibody reaction and kits thereof such as aptamer antibodies or antibody fragments capable of specifically binding macrophage colony stimulating factor-1 and polypeptide fragments thereof.
Preferably, the detection method comprises methods such as mass spectrometry for directly detecting macrophage colony stimulating factor-1 and polypeptide fragments thereof and related kits.
Preferably, the detection method comprises a related nucleic acid detection method for directly detecting macrophage colony stimulating factor-1 and polypeptide fragments thereof and a related kit thereof.
Preferably, the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and the second antibody diluent.
Preferably, the standard comprises macrophage colony stimulating factor-1 standard, humanized label antibody standard; preferably, the quality control product comprises: macrophage colony stimulating factor-1 quality control product and humanized label antibody quality control product; preferably, the solid support comprises: particles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, micro-porous plates or affinity membranes and other carriers with the same functions.
Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose, and has similar functions.
The inventor firstly collects urine samples of healthy people and allergic disease patients, centrifugates for 5min at 4000r/min, absorbs supernatant, measures the concentration of extracted protein by a Bradford method, and carries out SDS-PAGE enzymolysis. The Label-free mass spectrometry of the urine samples was performed by the OrbitrapFasion type mass spectrometer. And performing quantitative calculation on data obtained in the mass spectrum of the allergic disease group and the normal control group. The differential polypeptide is screened by using the difference of protein expression amount more than 1.5 times and P <0.05 as a reference standard through statistical test. Then, the inventor identifies the differential polypeptide with statistical significance, and utilizes a database to search to obtain the differential protein macrophage colony stimulating factor-1.
Compared with healthy people, the macrophage colony stimulating factor-1 and the polypeptide fragment thereof are low in expression in urine of patients with allergic diseases and have better consistency with clinical diagnosis. Therefore, the urine macrophage colony stimulating factor-1 and the polypeptide fragment thereof can be used for auxiliary diagnosis or disease condition monitoring of allergic diseases.
The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine specimen, and utilizes the urine specimen to detect the urine macrophage colony stimulating factor-1 and the polypeptide fragment thereof.
In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
FIG. 1 is a graph of the content of urinary macrophage colony stimulating factor-1 and its polypeptide fragments in allergic disease groups and healthy control groups.
Detailed Description
Example 1Collection and processing of urine specimens
Allergic disease patients are selected as an allergic disease group, and health examinees in the same period are selected as a normal control group. 30ml samples of fresh morning urine were collected from each group of subjects after admission, and those who failed to urinate normally collected their morning urine from their catheters and placed in dry, clean containers. Centrifuging the collected urine specimen at 4000r/min for 5min, sucking supernatant, subpackaging 2ml per tube, and storing in a refrigerator at-80 ℃.
Example 2Mass spectrometry and screening of urine polypeptides
Extracting protein from urine sample, and determining the concentration of extracted protein. Mass spectrometry of urine samples was performed by orbitrapfuision type mass spectrometry. And performing quantitative calculation on data obtained in the mass spectrum of the experimental group and the normal control group. The comparison among groups adopts t-test to carry out differential analysis, and differential expression proteins are screened by using the difference of protein expression quantity more than 1.5 times and taking the statistical test that P <0.05 as a reference standard.
Example 3Identification and analysis of differential Polypeptides
The used database is a Unit _ Homo database, the generated mass spectrum original file is processed by MaxQuant software, and the retrieval parameter setting is shown in Table 1.
Figure DEST_PATH_IMAGE002
Compared with healthy people, the expression of the macrophage colony stimulating factor-1 in the urine of the allergic disease patients is low, the content of the macrophage colony stimulating factor-1 in the urine of the healthy control group and the allergic disease groups is shown in figure 1, and the expression of the macrophage colony stimulating factor-1 in the urine of the normal control group and the allergic disease groups is obviously different.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Zhang Man
<120> application of urine macrophage colony stimulating factor-1 and polymorphic fragment thereof in allergic diseases
<130> 1
<140> 20PCSF-1-CN
<141> 2020-08-21
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 554
<212> PRT
<213> Human Urine
<400> 1
Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu
1 5 10 15
Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr
20 25 30
Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu
35 40 45
Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln
50 55 60
Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys
65 70 75 80
Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr
85 90 95
Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu
100 105 110
Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu
115 120 125
Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln
130 135 140
Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu
145 150 155 160
Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala
165 170 175
Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu
180 185 190
Tyr Pro Lys Ala Ile Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His
195 200 205
Gln Pro Leu Ala Pro Ser Met Ala Pro Val Ala Gly Leu Thr Trp Glu
210 215 220
Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro
225 230 235 240
Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln Arg Pro Pro Arg Ser
245 250 255
Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser
260 265 270
Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser Val Gly Ala Phe Asn
275 280 285
Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met Gly Thr Asn Trp Val
290 295 300
Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile Pro Val Pro Gln Gly
305 310 315 320
Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gln Thr Glu
325 330 335
Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala
340 345 350
Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu Pro
355 360 365
Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn His Thr Pro
370 375 380
Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro
385 390 395 400
Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln Gly Leu Ser Asn Pro
405 410 415
Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Arg Ser His Ser Ser Gly
420 425 430
Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp
435 440 445
Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala
450 455 460
Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly
465 470 475 480
His Glu Arg Gln Ser Glu Gly Ser Phe Ser Pro Gln Leu Gln Glu Ser
485 490 495
Val Phe His Leu Leu Val Pro Ser Val Ile Leu Val Leu Leu Ala Val
500 505 510
Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro
515 520 525
Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr
530 535 540
Gln Asp Asp Arg Gln Val Glu Leu Pro Val
545 550

Claims (9)

1. The application of the urine macrophage colony stimulating factor-1 and the polypeptide fragment thereof in preparing preparations for allergic disease diagnosis, differential diagnosis, disease degree 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 said urinary macrophage colony stimulating factor-1 is as shown in SEQ ID No. 1; or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.
3. The use according to claim 1, wherein the preparation is a kit for detecting macrophage colony stimulating factor-1 and polypeptide fragments thereof in urine of patients with allergic diseases.
4. The use according to claim 3, wherein the kit comprises one or more of an immunological method of antigen antibody reaction and kits thereof such as aptamer antibody or antibody fragment capable of specifically binding macrophage colony stimulating factor-1 and polypeptide fragments thereof.
5. The use according to claim 3, wherein the detection method comprises a method for directly detecting mass spectra of macrophage colony stimulating factor-1 and polypeptide fragments thereof and a related kit thereof.
6. The use of claim 3, wherein the detection method comprises a method for directly detecting macrophage colony stimulating factor-1 and its polypeptide fragment or its related nucleic acid detection, and its related kit.
7. The use according to claim 3, wherein the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and the second antibody diluent.
8. The use of claim 7, wherein the standard comprises a macrophage colony stimulating factor-1 standard, a humanized tag antibody standard; preferably, the quality control product comprises: macrophage colony stimulating factor-1 control product and humanized label antibody quality control product; preferably, the solid support comprises: particles, microspheres, glass slides, test strips, plastic beads, liquid phase chips, micro-porous plates or affinity membranes and other carriers with the same functions.
9. The use of claim 8, wherein the solid support is made of a material selected from the group consisting of polyvinyl chloride
Any one of ethylene, polystyrene, polyacrylamide and cellulose, and carriers with similar functions.
CN202010996093.3A 2020-09-21 2020-09-21 Application of urine macrophage colony stimulating factor-1 and polypeptide fragment thereof in allergic diseases Pending CN114252617A (en)

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