阅读说明：本技术 尿液软骨间层蛋白及其多肽片段在烧伤中的应用 (Application of urine cartilage intermediate layer protein and polypeptide fragment thereof in burn ) 是由 张曼 王佶图 于 2020-06-05 设计创作，主要内容包括：本发明提供一种尿液软骨间层蛋白(Cartilage intermediate-layer protein)及其多肽片段的应用,具体为尿液软骨间层蛋白及其多肽片段在制备用于烧伤诊断、鉴别诊断、烧伤面积及程度评价、治疗效果评价、监测、预后评估及机理研究等制剂的应用。烧伤是日常生活中常见的重要创伤,每年每100万人中约有5000～100000人烧伤,据世界卫生组织统计,每年全球死于烧伤患者超过30万人,严重烧伤救治存活率仍处于较低水平。本发明通过研究证实,与健康人(正常对照组)相比,尿液软骨间层蛋白及其多肽片段在烧伤患者中表达下调,并随烧伤程度加重而降低。可用于烧伤患者的辅助诊断及病情监测。本发明发挥尿液标本获取无创、可大规模重复取样、保存方便的优势,利用尿液标本检测尿液软骨间层蛋白及其多肽片段。(The invention provides application of a urine Cartilage intermediate-layer protein (Cartilage intermediate-layer protein) and a polypeptide fragment thereof, in particular to application of the urine Cartilage intermediate-layer protein and the polypeptide fragment 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 cartilage intermediate layer protein and polypeptide fragments thereof are expressed in burn patients and are reduced along with the increase of the burn degree 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 cartilage interstratified protein and the polypeptide fragment thereof.)

1. The urine cartilage intermediate layer 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 according to claim 1, wherein the amino acid sequence of the urine chondroprotein 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 the cartilaginous interlayer protein and polypeptide fragments thereof in urine of a patient with burn.

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 the intercartilaginous protein and polypeptide fragments thereof.

5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for directly detecting the cartilage intermediate layer protein and its polypeptide fragments.

6. The use according to claim 3, wherein the detection method comprises a method for directly detecting the chondroprotein and the related nucleic acid of the polypeptide fragment thereof, and a related kit thereof.

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 an intercartilaginous protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: cartilage intermediate layer protein 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 phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide, cellulose, and carriers with similar functions.

Technical Field

The invention relates to a new application of urine cartilage intermediate layer protein and polypeptide fragments thereof, in particular to application of urine cartilage intermediate layer 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.

The Cartilage Intermediate Layer Protein (CILP) is a matrix component of human articular cartilage, and is named because the distribution is limited to cartilage tissues and is abundant in the cartilage Intermediate layer. However, the presence of this protein is now found in meniscus, tendons, ligaments, synovium and intervertebral discs and less research is currently available. According to the research, the expression of the cartilaginous interlayer protein in urine of a burn patient is reduced compared with that of a healthy human group, the expression is correlated with the burn degree to a certain extent, and the protein content is lower when the burn degree is more serious.

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 cartilage intermediate layer 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 chondroprotein is shown in SEQ ID NO.1 (MVGTKAWVFS FLVLEVTSVL GRQTMLTQSV RRVQPGKKNP SIFAKPADTL ESPGEWTTWF NIDYPGGKGD YERLDAIRFY YGDRVCARPL RLEARTTDWT PAGSTGQVVH GSPREGFWCL NREQRPGQNC SNYTVRFLCP PGSLRRDTER IWSPWSPWSK CSAACGQTGV QTRTRICLAE MVSLCSEASE EGQHCMGQDC TACDLTCPMG QVNADCDACM CQDFMLHGAV SLPGGAPASG AAIYLLTKTP KLLTQTDSDG RFRIPGLCPD GKSILKITKV KFAPIVLTMP KTSLKAATIK AEFVRAETPY MVMNPETKAR RAGQSVSLCC KATGKPRPDK YFWYHNDTLL DPSLYKHESK LVLRKLQQHQ AGEYFCKAQS DAGAVKSKVA QLIVIASDET PCNPVPESYL IRLPHDCFQN ATNSFYYDVG RCPVKTCAGQ QDNGIRCRDA VQNCCGISKT EEREIQCSGY TLPTKVAKEC SCQRCTETRS IVRGRVSAAD NGEPMRFGHV YMGNSRVSMT GYKGTFTLHV PQDTERLVLT FVDRLQKFVN TTKVLPFNKK GSAVFHEIKM LRRKKPITLE AMETNIIPLG EVVGEDPMAE LEIPSRSFYR QNGEPYIGKV KASVTFLDPR NISTATAAQT DLNFINDEGD TFPLRTYGMF SVDFRDEVTS EPLNAGKVKV HLDSTQVKMP EHISTVKLWS LNPDTGLWEE EGDFKFENQR RNKREDRTFL VGNLEIRERR LFNLDVPESR RCFVKVRAYR SERFLPSEQI QGVVISVINL EPRTGFLSNP RAWGRFDSVI TGPNGACVPA FCDDQSPDAY SAYVLASLAG EELQAVESSP KFNPNAIGVP QPYLNKLNYR RTDHEDPRVK KTAFQISMAK PRPNSAEESN GPIYAFENLR ACEEAPPSAA HFRFYQIEGD RYDYNTVPFN EDDPMSWTED YLAWWPKPME FRACYIKVKI VGPLEVNVRS RNMGGTHRQT VGKLYGIRDV RSTRDRDQPN VSAACLEFKC SGMLYDQDRV DRTLVKVIPQ GSCRRASVNP MLHEYLVNHL PLAVNNDTSE YTMLAPLDPL GHNYGIYTVT DQDPRTAKEI ALGRCFDGTS DGSSRIMKSN VGVALTFNCV ERQVGRQSAF QYLQSTPAQS PAAGTVQGRV PSRRQQRASR GGQRQGGVVA SLRFPRVAQQ PLIN); 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 cartilage intermediate layer protein and the polypeptide fragments thereof in urine of a burn patient.

Preferably, the kit includes 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 the chondroprotein and a polypeptide fragment thereof.

Preferably, the detection method comprises methods such as mass spectrometry for directly detecting the cartilage intermediate layer protein and the polypeptide fragment thereof and related kits thereof.

Preferably, the detection method comprises related nucleic acid detection and related kits for directly detecting the chondroprotein and polypeptide fragments 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 an intercartilaginous protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: cartilage interstratified protein quality control products and humanized label antibody quality control products; 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 patients with different burn degrees, centrifugates for 5min at 4000r/min, absorbs supernatant, determines 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 cartilaginous interlayer protein.

Compared with healthy people, the cartilage intermediate layer protein and the polypeptide fragment thereof are low in expression in urine of burn patients and are reduced along with the aggravation of the burn degree through research, and the cartilage intermediate layer protein and the polypeptide fragment thereof have better consistency with clinical diagnosis. Therefore, the detection of the urine cartilage intermediate layer 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 specimen, and utilizes the urine specimen to detect the urine cartilage interstratified 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 cartilage intermediate layer protein and its polypeptide fragments in burn group and healthy control group.

FIG. 2 is a schematic representation of the involvement of the intercartilaginous proteins in the main 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 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 expression of the cartilaginous interlayer protein in urine of a burn patient is low, as shown in figure 1, the main biological process involved in the production is shown in figure 2, and the expression of the cartilaginous interlayer protein in urine of a normal control group and a burn group is remarkably different and is reduced along with the aggravation of the burn degree.

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

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