CN113804891A - Application of urine C-reactive protein and polypeptide fragment thereof in burn - Google Patents

Application of urine C-reactive protein and polypeptide fragment thereof in burn Download PDF

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CN113804891A
CN113804891A CN202010544887.6A CN202010544887A CN113804891A CN 113804891 A CN113804891 A CN 113804891A CN 202010544887 A CN202010544887 A CN 202010544887A CN 113804891 A CN113804891 A CN 113804891A
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urine
reactive protein
burn
antibody
polypeptide fragments
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张曼
王佶图
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Beijing Shijitan Hospital
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    • 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
    • G01N33/6893—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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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
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Abstract

The invention provides an application of urine C-reactive protein (C-reactive protein) and polypeptide fragments thereof, in particular to an application of urine C-reactive protein and polypeptide fragments thereof in preparing preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like. The burn is an important common wound in daily life, about 5000-100000 people burn in every 100 million people every year, according to the statistics of the world health organization, more than 30 million people die of burn patients all year around the world, and the survival rate of the serious burn treatment is still at a lower level. The research proves that the expression of urine C-reactive protein and polypeptide fragments thereof is increased in the burn patients compared with healthy people (normal control group). Can be used for various purposes of detecting burn patients. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient preservation of the urine sample, and utilizes the urine sample to detect the urine C-reactive protein and the polypeptide fragment thereof.

Description

Application of urine C-reactive protein and polypeptide fragment thereof in burn
Technical Field
The invention relates to new application of urine C-reactive protein and polypeptide fragments thereof, in particular to application of urine C-reactive protein and polypeptide fragments thereof in burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Background
Burn refers to the injury of skin, tissue and even deep viscera of human body caused by chemical substances such as flame, high-temperature gas, scorching solid or liquid, radioactive rays, electric energy, strong acid and strong alkali, etc., and is a systemic comprehensive disease. After burn, a large amount of reactions such as necrosis, infection, shock, blood coagulation dysfunction and the like of wound tissues can cause a series of pathophysiological changes of organisms. Burns of different degrees have different influences on human bodies, serious burns can damage the environment in the human bodies, the burn patients have pathophysiological changes of complexity of various systems, and relevant detection indexes can correspondingly change along with the difference of the severity of the burns. Timely detection of changes in these indices can provide valuable reference for clinicians in many areas, such as disease diagnosis, disease judgment, treatment selection, and patient prognosis assessment.
However, patients with severe burns have poor skin integrity, and clinical use of hematological tests as invasive tests on the skin in such patients has presented difficulties, and repeated blood draw tests can also exacerbate patient pain. Urine as ultrafiltrate of blood contains abundant biological information, and the collection process has the advantages of non-invasive and convenient operation, and the like, which is particularly obvious in the detection of burn patients. The biomarker which is helpful for burn diagnosis and reflects disease change is searched in urine, so that the life quality and compliance of burn patients can be improved, the pain of blood collection for many times is relieved, and a reference basis which is favorable for disease diagnosis and treatment is better provided for clinicians.
C-reactive protein (CRP) is a sensitive acute time-phase reaction protein synthesized by liver, when the organism is infected or has tissue damage, CRP can activate complement and strengthen phagocytosis of phagocyte to play an opsonization role, thereby eliminating pathogenic microorganism and damaged, necrotic and apoptotic tissue cells invading the organism and playing an important protective role in the natural immune process of the organism. It is not affected by erythrocyte, hemoglobin, body immunity, lipid, antibiotic, age and sex, and is a good index for reaction infection. In the research, the expression of C-reactive protein in the urine of the burn patient is up-regulated compared with that of a healthy human group, and the content of the protein in the urine of the burn patient is increased.
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 multiple blood sampling of burn patients, the experiment is expected to realize the diagnosis and disease monitoring of the burn patients by painless, convenient, quick and easily repeated urine detection through the research of urine protein or polypeptide on the basis of the methodology exploration of the early stage, and also lays a foundation for the further research of the urine polypeptide detection kit.
Disclosure of Invention
The invention aims to provide application of urine C-reactive protein and polypeptide fragments thereof in preparation of preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Preferably, the amino acid sequence of the urine C-reactive protein is shown in SEQ ID NO.1 (MEKLLCFLVL TSLSHAFGQT DMSRKAFVFP KESDTSYVSL KAPLTKPLKA FTVCLHFYTE LSSTRGYSIF SYATKRQDNE ILIFWSKDIG YSFTVGGSEI LFEVPEVTVA PVHICTSWES ASGIVEFWVD GKPRVRKSLK KGYTVGAEAS IILGQEQDSF GGNFEGSQSL VGDIGNVNMW DFVLSPDEIN TIYLGGPFSP NVLNWRALKY EVQGEVFTKP QLWP); 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 detection kit for the C-reactive protein in urine of a burn patient and polypeptide fragments thereof.
Preferably, the kit comprises one or more of an immunization method for antigen-antibody reaction and kits thereof such as aptamer antibodies or antibody fragments capable of specifically binding to C-reactive protein and polypeptide fragments thereof.
Preferably, the detection method comprises methods such as mass spectrometry for directly detecting the C-reactive protein and the polypeptide fragment thereof and related kits thereof.
Preferably, the detection method comprises related nucleic acid detection methods for directly detecting C-reactive protein and polypeptide fragments thereof and related kits.
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 a C-reactive protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: c reaction protein quality control material and humanized label antibody quality control material; 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 burn 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 burn 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 C reactive protein.
Compared with healthy people, the C-reactive protein and the polypeptide fragment thereof are highly expressed in urine of burn patients and have better consistency with clinical diagnosis. Therefore, the urine C-reactive protein and the polypeptide fragment thereof can be used for auxiliary diagnosis or disease condition monitoring of the burn.
The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient preservation of the urine sample, and utilizes the urine sample to detect the urine C-reactive protein 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 showing the content of urine C-reactive protein and its polypeptide fragments in burn and healthy control groups.
FIG. 2 is a schematic diagram of the participation of C-reactive protein in major biological processes.
Detailed Description
Example 1Collection and processing of urine specimens
Burn patients were selected as the burn group, and contemporary physical examiners were selected as the 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 640026DEST_PATH_IMAGE001
Compared with healthy people, the C-reactive protein is highly expressed in urine of burn patients as shown in figure 1, the main biological processes involved in the C-reactive protein are shown in figure 2, and the expression of the C-reactive protein in urine of normal control groups and the burn groups is remarkably 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> urine C-reactive protein and application of polypeptide fragment thereof in burn
<130> 1
<140> 20PCRP
<141> 2020-05-10
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 224
<212> PRT
<213> Human Urine
<400> 1
Met Glu Lys Leu Leu Cys Phe Leu Val Leu Thr Ser Leu Ser His Ala
1 5 10 15
Phe Gly Gln Thr Asp Met Ser Arg Lys Ala Phe Val Phe Pro Lys Glu
20 25 30
Ser Asp Thr Ser Tyr Val Ser Leu Lys Ala Pro Leu Thr Lys Pro Leu
35 40 45
Lys Ala Phe Thr Val Cys Leu His Phe Tyr Thr Glu Leu Ser Ser Thr
50 55 60
Arg Gly Tyr Ser Ile Phe Ser Tyr Ala Thr Lys Arg Gln Asp Asn Glu
65 70 75 80
Ile Leu Ile Phe Trp Ser Lys Asp Ile Gly Tyr Ser Phe Thr Val Gly
85 90 95
Gly Ser Glu Ile Leu Phe Glu Val Pro Glu Val Thr Val Ala Pro Val
100 105 110
His Ile Cys Thr Ser Trp Glu Ser Ala Ser Gly Ile Val Glu Phe Trp
115 120 125
Val Asp Gly Lys Pro Arg Val Arg Lys Ser Leu Lys Lys Gly Tyr Thr
130 135 140
Val Gly Ala Glu Ala Ser Ile Ile Leu Gly Gln Glu Gln Asp Ser Phe
145 150 155 160
Gly Gly Asn Phe Glu Gly Ser Gln Ser Leu Val Gly Asp Ile Gly Asn
165 170 175
Val Asn Met Trp Asp Phe Val Leu Ser Pro Asp Glu Ile Asn Thr Ile
180 185 190
Tyr Leu Gly Gly Pro Phe Ser Pro Asn Val Leu Asn Trp Arg Ala Leu
195 200 205
Lys Tyr Glu Val Gln Gly Glu Val Phe Thr Lys Pro Gln Leu Trp Pro
210 215 220

Claims (9)

1. The urine C-reactive protein and the polypeptide fragment thereof are applied to the preparation of preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
2. The use of claim 1, wherein the urine C-reactive protein has an amino acid sequence as set forth 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 of claim 1, wherein the preparation is a test kit for the detection of C-reactive protein in urine of burn patients and polypeptide fragments thereof.
4. Use according to claim 3, wherein the kit comprises one or more of an immunization method for antigen-antibody reaction and kits thereof such as aptamer antibodies or antibody fragments capable of specifically binding to C-reactive protein and polypeptide fragments thereof.
5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for direct detection of C-reactive protein and its polypeptide fragments.
6. The use of claim 3, wherein the detection method comprises a method of directly detecting C-reactive protein and its polypeptide fragments 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 C-reactive protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: c reaction protein control product, 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 phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, and carriers with similar functions.
CN202010544887.6A 2020-06-15 2020-06-15 Application of urine C-reactive protein and polypeptide fragment thereof in burn Pending CN113804891A (en)

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