CN114113600A - Application of urine alpha 1-antitrypsin and polypeptide fragment thereof in burn - Google Patents
Application of urine alpha 1-antitrypsin and polypeptide fragment thereof in burn Download PDFInfo
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- CN114113600A CN114113600A CN202010863339.XA CN202010863339A CN114113600A CN 114113600 A CN114113600 A CN 114113600A CN 202010863339 A CN202010863339 A CN 202010863339A CN 114113600 A CN114113600 A CN 114113600A
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- Prior art keywords
- antitrypsin
- burn
- alpha
- urine
- antibody
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- 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
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Abstract
The invention provides an application of urine Alpha 1-antitrypsin (Alpha-1-antitrypsin) and polypeptide fragments thereof, in particular to an application of urine Alpha 1-antitrypsin 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 alpha 1-antitrypsin and polypeptide fragments thereof are expressed in burn patients to be higher than healthy people (normal control group), and the content thereof is gradually increased along with the aggravation of burn. 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 alpha 1-antitrypsin and the polypeptide fragment thereof.
Description
Technical Field
The invention relates to new application of urine alpha 1-antitrypsin and polypeptide fragments thereof, in particular to application of urine alpha 1-antitrypsin 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.
Alpha 1-antitrypsin (Alpha-1-antitrypsin, Alpha 1-AT) is a single-chain glycoprotein that has the ability to cross the microvasculature and enter the tissue through the capillary endothelial space, is produced in the liver, and is mainly synthesized by the association of the hepatocyte synovial endoplasmic reticulum with the Golgi complex. Alpha 1-antitrypsin is mainly involved in negative regulation of plasma protease activity, inhibiting over-activation of protease, inhibiting synthesis and release of various inflammatory mediators, inhibiting activation of polymorphonuclear leukocytes, and playing a role in eliminating cytotoxic substances such as peroxides and free radicals, protecting tissue cells, and promoting regeneration and repair of damaged tissue cells. In the research, the content of the alpha 1-antitrypsin protein in urine of a burn patient is up-regulated compared with that of a healthy human group, and is in a certain correlation with the burn degree, and the more serious the burn degree is, the higher the content of the protein in the urine 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 alpha 1-antitrypsin 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 urinary alpha 1-antitrypsin is shown in SEQ ID NO.1 (MPSSVSWGIL LLAGLCCLVP VSLAEDPQGD AAQKTDTSHH DQDHPTFNKI)
TPNLAEFAFS LYRQLAHQSN STNIFFSPVS IATAFAMLSL GTKADTHDEI
LEGLNFNLTE IPEAQIHEGF QELLRTLNQP DSQLQLTTGN GLFLSEGLKL
VDKFLEDVKK LYHSEAFTVN FGDTEEAKKQ INDYVEKGTQ GKIVDLVKEL
DRDTVFALVN YIFFKGKWER PFEVKDTEEE DFHVDQVTTV KVPMMKRLGM
FNIQHCKKLS SWVLLMKYLG NATAIFFLPD EGKLQHLENE LTHDIITKFL
ENEDRRSASL HLPKLSITGT YDLKSVLGQL GITKVFSNGA DLSGVTEEAP
LKLSKAVHKA VLTIDEKGTE AAGAMFLEAI PMSIPPEVKF NKPFVFLMIE
QNTKSPLFMG KVVNPTQK); 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 test kit for alpha 1-antitrypsin in urine of a burn patient and polypeptide fragments thereof.
Preferably, the kit comprises 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 to alpha 1-antitrypsin and polypeptide fragments thereof.
Preferably, the detection method comprises methods such as mass spectrometry for directly detecting alpha 1-antitrypsin and polypeptide fragments thereof and related kits thereof.
Preferably, the detection method comprises related nucleic acid detection methods for directly detecting alpha 1-antitrypsin 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 an alpha 1-antitrypsin standard, a humanized tag antibody standard; preferably, the quality control product comprises: alpha 1-antitrypsin quality control substance and humanized label antibody quality control substance; 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 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 inventors identified the differential polypeptide with statistical significance, and searched the database to obtain the differential protein alpha 1-antitrypsin.
Compared with healthy people, the alpha 1-antitrypsin and the polypeptide fragment thereof are proved to be highly expressed in urine of burn patients and increase along with the aggravation of the burn degree through research, and have better consistency with clinical diagnosis. Therefore, the detection of urine alpha 1-antitrypsin and polypeptide fragments 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 sample, and utilizes the urine sample to detect the urine alpha 1-antitrypsin 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 urine alpha 1-antitrypsin and its polypeptide fragments in burn and healthy control groups.
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.
Compared with healthy people, the alpha 1-antitrypsin is highly expressed in urine of burn patients, the content of the alpha 1-antitrypsin in urine of healthy control groups and burn groups is shown in figure 1, and the expression of the alpha 1-antitrypsin in urine of the normal control groups and the burn groups is remarkably different and is increased 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 alpha 1-antitrypsin and polypeptide fragment thereof in burn
<130> 1
<140> 20Pα1-AT-CN
<141> 2020-08-03
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 418
<212> PRT
<213> Human Urine
<400> 1
Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys
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Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala
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Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn
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Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln
50 55 60
Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser
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Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr
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His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro
100 105 110
Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn
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Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu
130 135 140
Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys
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Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu
165 170 175
Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys
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Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu
195 200 205
Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val
210 215 220
Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val
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Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys
245 250 255
Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala
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Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu
275 280 285
Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp
290 295 300
Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr
305 310 315 320
Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe
325 330 335
Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys
340 345 350
Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly
355 360 365
Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile
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Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu
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Gln Lys
Claims (9)
1. The urine alpha 1-antitrypsin and its polypeptide fragment are used in preparing preparations for burn diagnosis, differential diagnosis, burn area and degree evaluation, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research, etc.
2. The use of claim 1, wherein the urinary α 1-antitrypsin has an amino acid sequence 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 alpha 1-antitrypsin and polypeptide fragments thereof from urine of a patient with burn injury.
4. 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 antibodies or antibody fragments capable of specifically binding to α 1-antitrypsin and polypeptide fragments thereof.
5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for directly detecting alpha 1-antitrypsin and polypeptide fragments thereof.
6. The use of claim 3, wherein the detection method comprises a method for directly detecting alpha 1-antitrypsin and polypeptide fragments thereof or nucleic acid related thereto, and a kit related thereto.
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 alpha 1-antitrypsin standard, a humanized tag antibody standard; preferably, the quality control product comprises: alpha 1-antitrypsin control substance and humanized label antibody quality control substance; 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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