CN113777315A - Application of uroubiquitin-like protein ISG15 and polypeptide fragment thereof in burn - Google Patents
Application of uroubiquitin-like protein ISG15 and polypeptide fragment thereof in burn Download PDFInfo
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- CN113777315A CN113777315A CN202010519796.7A CN202010519796A CN113777315A CN 113777315 A CN113777315 A CN 113777315A CN 202010519796 A CN202010519796 A CN 202010519796A CN 113777315 A CN113777315 A CN 113777315A
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Abstract
The invention provides application of urine Ubiquitin-like protein ISG15 (Ubiquitin-like protein ISG15) and polypeptide fragments thereof, in particular application of urine Ubiquitin-like protein ISG15 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. 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 invention proves that urine ubiquitin-like protein ISG15 and the polypeptide fragment thereof are expressed in burn patients and are down-regulated compared with healthy people (normal control group). Can be used for auxiliary diagnosis and disease condition monitoring of burn patients. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient preservation of the urine specimen, and utilizes the urine specimen to detect the urine ubiquitin-like protein ISG15 and the polypeptide fragment thereof.
Description
Technical Field
The invention relates to a new application of urine ubiquitin-like protein ISG15 and a polypeptide fragment thereof, in particular to an application of urine ubiquitin-like protein ISG15 and a polypeptide fragment 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.
Ubiquitin-like protein ISG15 (Ubiquitin-like protein ISG15, ISG15) was the first identified Ubiquitin-like protein family member. Ubiquitin-like proteins are a class of small molecule proteins that are similar in sequence and structure to ubiquitin and, like ubiquitin, can be covalently modified by covalent binding to intracellular proteins. Stimulation by both virus and type I interferon strongly induces expression of ISGl5 protein, which plays an important regulatory role in antiviral innate immune response. ISGl5 can also modulate intracellular activity through covalent modification of various proteins within the cell. In the study, the ubiquitin-like protein ISG15 in urine of burn patients is expressed and reduced in expression compared with healthy people, and the protein content in urine of burn patients 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 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 ubiquitin-like protein ISG15 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 uroubiquitin-like protein ISG15 is shown in SEQ ID NO.1 (MGWDLTVKML AGNEFQVSLS SSMSVSELKA QITQKIGVHA FQQRLAVHPS GVALQDRVPL ASQGLGPGST VLLVVDKCDE PLSILVRNNK GRSSTYEVRL TQTVAHLKQQ VSGLEGVQDD LFWLTFEGKP LEDQLPLGEY GLKPLSTVFM NLRLRGGGTE PGGRS); 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 uroubiquitin-like protein ISG15 and polypeptide fragments thereof of burn 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 ubiquitin-like protein ISG15 and a polypeptide fragment thereof.
Preferably, the detection method comprises methods such as mass spectrometry for directly detecting ubiquitin-like protein ISG15 and polypeptide fragments thereof and related kits thereof.
Preferably, the detection method comprises related nucleic acid detection methods for directly detecting the ubiquitin-like protein ISG15 and the polypeptide fragment thereof and related kits 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 a ubiquitin-like protein ISG15 standard, a humanized tag antibody standard; preferably, the quality control product comprises: quality control products of ubiquitin-like protein ISG15 and humanized label antibody; 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 non-labeled quantitative calculation on data obtained in the mass spectrum of the burn group and the normal control group by adopting a Labelfree algorithm in a Maxquant algorithm. 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 searches by using a database to obtain the differential protein ubiquitin-like protein ISG 15.
Compared with healthy people, the ubiquitin-like protein ISG15 and the polypeptide fragment thereof are low in expression in urine of burn patients and have better consistency with clinical diagnosis. Therefore, the detection of the uroubiquitin-like protein ISG15 and the polypeptide fragment thereof can be used for auxiliary diagnosis or disease monitoring of burns.
The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient preservation of the urine specimen, and utilizes the urine specimen to detect the urine ubiquitin-like protein ISG15 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 ubiquitin-like protein ISG15 and its polypeptide fragments in burn group and healthy control group.
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 database used at this time 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.
Compared with healthy people, the ubiquitin-like protein ISG15 is low expressed in urine of burn patients, and as shown in figure 1, the ubiquitin-like protein ISG15 has significant difference in expression in urine of normal control group and burn group.
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 ubiquitin-like protein ISG15 and polypeptide fragment thereof in burn
<130> 1
<140> 20PISG15
<141> 2020-05-10
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 165
<212> PRT
<213> Human Urine
<400> 1
Met Gly Trp Asp Leu Thr Val Lys Met Leu Ala Gly Asn Glu Phe Gln
1 5 10 15
Val Ser Leu Ser Ser Ser Met Ser Val Ser Glu Leu Lys Ala Gln Ile
20 25 30
Thr Gln Lys Ile Gly Val His Ala Phe Gln Gln Arg Leu Ala Val His
35 40 45
Pro Ser Gly Val Ala Leu Gln Asp Arg Val Pro Leu Ala Ser Gln Gly
50 55 60
Leu Gly Pro Gly Ser Thr Val Leu Leu Val Val Asp Lys Cys Asp Glu
65 70 75 80
Pro Leu Ser Ile Leu Val Arg Asn Asn Lys Gly Arg Ser Ser Thr Tyr
85 90 95
Glu Val Arg Leu Thr Gln Thr Val Ala His Leu Lys Gln Gln Val Ser
100 105 110
Gly Leu Glu Gly Val Gln Asp Asp Leu Phe Trp Leu Thr Phe Glu Gly
115 120 125
Lys Pro Leu Glu Asp Gln Leu Pro Leu Gly Glu Tyr Gly Leu Lys Pro
130 135 140
Leu Ser Thr Val Phe Met Asn Leu Arg Leu Arg Gly Gly Gly Thr Glu
145 150 155 160
Pro Gly Gly Arg Ser
165
Claims (9)
1. Application of uroubiquitin-like protein ISG15 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.
2. The use according to claim 1, wherein the amino acid sequence of uroubiquitin-like protein ISG15 is 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 of claim 1, wherein the preparation is a kit for detecting uroubiquitin-like protein ISG15 and polypeptide fragments thereof in burn patients.
4. The use according to claim 3, wherein the kit comprises one or more of an immunization method of antigen-antibody reaction and a kit thereof such as aptamer antibody or antibody fragment capable of specifically binding ubiquitin-like protein ISG15 and polypeptide fragment thereof.
5. The use of claim 3, wherein the detection method comprises a mass spectrometry method for directly detecting the ubiquitin-like protein ISG15 and its polypeptide fragment and its related kit.
6. The use of claim 3, wherein the detection method comprises a method for directly detecting ubiquitin-like protein ISG15 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 ubiquitin-like protein ISG15 standard, a humanized tag antibody standard; preferably, the quality control product comprises: ubiquitin-like protein ISG15 quality 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.
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CN202010519796.7A CN113777315A (en) | 2020-06-09 | 2020-06-09 | Application of uroubiquitin-like protein ISG15 and polypeptide fragment thereof in burn |
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