阅读说明：本技术 尿液蛋白质z及其多肽片段在烧伤中的应用 (Application of urine protein Z 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蛋白质Z(Vitamin K-dependent protein Z)及其多肽片段的应用,具体为尿液蛋白质Z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,尿液蛋白质Z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蛋白质Z及其多肽片段。(The invention provides application of urine protein Z (Vitamin K-dependent protein Z) and polypeptide fragments thereof, in particular to application of urine protein Z 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 present invention proves that urine protein Z and its polypeptide fragment are expressed in burn patients down-regulated and reduced with the degree of burn, 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 sample, and utilizes the urine sample to detect the urine protein Z and the polypeptide fragment thereof.)

1. The urine protein Z 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 protein Z 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 of claim 1, wherein the preparation is a kit for detecting protein Z 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 protein Z and polypeptide fragments thereof.

5. The use according to claim 3, wherein the detection method comprises mass spectrometry and related kits for directly detecting protein Z and its polypeptide fragments.

6. The use of claim 3, wherein the detection method comprises a method for directly detecting related nucleic acid of protein Z and its polypeptide fragment and 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 protein Z standard, a humanized tag antibody standard; preferably, the quality control product comprises: protein Z control, humanized label antibody quality control; 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 new application of urine protein Z and polypeptide fragments thereof, in particular to application of urine protein Z 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.

Protein Z (PZ) is a vitamin K-dependent plasma glycoprotein belonging to the family of serine protease inhibitors. It can be used as an auxiliary factor of protein Z-dependent protease inhibitor (ZPI), and can improve ZPI activity of blood coagulation factor FXa and enhance anticoagulation effect. Since the structure of PZ is similar to other vitamin K dependent coagulation factors in many aspects, it has been a hot target for researchers at home and abroad to study bleeding and thrombotic diseases. Research shows that the protein Z is an important plasma protein related to diseases such as thrombosis, tumor, inflammation, obstetrics and gynecology. Protein Z in urine of burn patients in the research is expressed and reduced in comparison with healthy people, and has a certain correlation with the burn degree, and the more serious the burn degree is, the lower the protein content is.

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 protein Z 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 protein Z is shown in SEQ ID NO.1 (MAGCVPLLQG LVLVLALHRV EPSVFLPASK ANDVLVRWKR AGSYLLEELF EGNLEKECYE EICVYEEARE VFENEVVTDE FWRRYKGGSP CISQPCLHNG SCQDSIWGYT CTCSPGYEGS NCELAKNECH PERTDGCQHF CLPGQESYTC SCAQGYRLGE DHKQCVPHDQ CACGVLTSEK RAPDLQDLPW QVKLTNSEGK DFCGGVIIRE NFVLTTAKCS LLHRNITVKT YFNRTSQDPL MIKITHVHVH MRYDADAGEN DLSLLELEWP IQCPGAGLPV CTPEKDFAEH LLIPRTRGLL SGWARNGTDL GNSLTTRPVT LVEGEECGQV LNVTVTTRTY CERSSVAAMH WMDGSVVTRE HRGSWFLTGV LGSQPVGGQA HMVLVTKVSR YSLWFKQIMN); 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 protein Z 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 protein Z and polypeptide fragments thereof.

Preferably, the detection method comprises methods such as mass spectrometry for directly detecting the protein Z and polypeptide fragments thereof and related kits thereof.

Preferably, the detection method comprises related nucleic acid detection methods for directly detecting the protein Z 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 protein Z standard, a humanized tag antibody standard; preferably, the quality control product comprises: protein Z 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 inventors identified the differential polypeptide with statistical significance, and searched the database to obtain the differential protein Z.

Compared with healthy people, the protein Z and the polypeptide fragment thereof are low in expression in urine of burn patients, are reduced along with the aggravation of the burn degree, and have better consistency with clinical diagnosis. Therefore, the urine protein Z 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 protein Z 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 protein Z and its polypeptide fragments in burn and healthy control groups.

FIG. 2 is a schematic representation of the involvement of protein Z 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 this time is a Unit _ Home database generatedThe mass spectrum original file is processed by adopting MaxQuant software, and the retrieval parameter setting is shown in table 1.

Compared with healthy people, the protein Z is low expressed in urine of burn patients as shown in figure 1, the main biological processes involved in the protein Z are shown in figure 2, and the expression of the protein Z 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

<110> Zhang Man

<120> application of urine protein Z and polypeptide fragment thereof in burn

<130> 1

<140> 20PPROZ-CN

<141> 2020-05-03

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 400

<212> PRT

<213> Human Urine

<400> 1

Met Ala Gly Cys Val Pro Leu Leu Gln Gly Leu Val Leu Val Leu Ala

1 5 10 15

Leu His Arg Val Glu Pro Ser Val Phe Leu Pro Ala Ser Lys Ala Asn

20 25 30

Asp Val Leu Val Arg Trp Lys Arg Ala Gly Ser Tyr Leu Leu Glu Glu

35 40 45

Leu Phe Glu Gly Asn Leu Glu Lys Glu Cys Tyr Glu Glu Ile Cys Val

50 55 60

Tyr Glu Glu Ala Arg Glu Val Phe Glu Asn Glu Val Val Thr Asp Glu

65 70 75 80

Phe Trp Arg Arg Tyr Lys Gly Gly Ser Pro Cys Ile Ser Gln Pro Cys

85 90 95

Leu His Asn Gly Ser Cys Gln Asp Ser Ile Trp Gly Tyr Thr Cys Thr

100 105 110

Cys Ser Pro Gly Tyr Glu Gly Ser Asn Cys Glu Leu Ala Lys Asn Glu

115 120 125

Cys His Pro Glu Arg Thr Asp Gly Cys Gln His Phe Cys Leu Pro Gly

130 135 140

Gln Glu Ser Tyr Thr Cys Ser Cys Ala Gln Gly Tyr Arg Leu Gly Glu

145 150 155 160

Asp His Lys Gln Cys Val Pro His Asp Gln Cys Ala Cys Gly Val Leu

165 170 175

Thr Ser Glu Lys Arg Ala Pro Asp Leu Gln Asp Leu Pro Trp Gln Val

180 185 190

Lys Leu Thr Asn Ser Glu Gly Lys Asp Phe Cys Gly Gly Val Ile Ile

195 200 205

Arg Glu Asn Phe Val Leu Thr Thr Ala Lys Cys Ser Leu Leu His Arg

210 215 220

Asn Ile Thr Val Lys Thr Tyr Phe Asn Arg Thr Ser Gln Asp Pro Leu

225 230 235 240

Met Ile Lys Ile Thr His Val His Val His Met Arg Tyr Asp Ala Asp

245 250 255

Ala Gly Glu Asn Asp Leu Ser Leu Leu Glu Leu Glu Trp Pro Ile Gln

260 265 270

Cys Pro Gly Ala Gly Leu Pro Val Cys Thr Pro Glu Lys Asp Phe Ala

275 280 285

Glu His Leu Leu Ile Pro Arg Thr Arg Gly Leu Leu Ser Gly Trp Ala

290 295 300

Arg Asn Gly Thr Asp Leu Gly Asn Ser Leu Thr Thr Arg Pro Val Thr

305 310 315 320

Leu Val Glu Gly Glu Glu Cys Gly Gln Val Leu Asn Val Thr Val Thr

325 330 335

Thr Arg Thr Tyr Cys Glu Arg Ser Ser Val Ala Ala Met His Trp Met

340 345 350

Asp Gly Ser Val Val Thr Arg Glu His Arg Gly Ser Trp Phe Leu Thr

355 360 365

Gly Val Leu Gly Ser Gln Pro Val Gly Gly Gln Ala His Met Val Leu

370 375 380

Val Thr Lys Val Ser Arg Tyr Ser Leu Trp Phe Lys Gln Ile Met Asn

385 390 395 400