CN113759133A - Application of urinary angiotensinogen and polypeptide fragment thereof in burn - Google Patents
Application of urinary angiotensinogen and polypeptide fragment thereof in burn Download PDFInfo
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- CN113759133A CN113759133A CN202010504022.7A CN202010504022A CN113759133A CN 113759133 A CN113759133 A CN 113759133A CN 202010504022 A CN202010504022 A CN 202010504022A CN 113759133 A CN113759133 A CN 113759133A
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Abstract
The invention provides application of Angiotensinogen (Angiotensinogen) protein and polypeptide fragments thereof, in particular to application of Angiotensinogen 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 research proves that compared with healthy people (normal control group), the expression of the urinary angiotensinogen and the polypeptide fragment thereof in the burn patients is increased, and the increase degree is increased along with the increase of the burn degree. 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 storage of the urine specimen, and utilizes the urine specimen to detect the angiotensinogen and the polypeptide fragment thereof.
Description
Technical Field
The invention relates to new application of urinary angiotensinogen and polypeptide fragments thereof, in particular to application of the urinary angiotensinogen and the 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.
Angiotensinogen (AGT) is the only precursor substance of the renin-angiotensin system, containing 485 amino acid residues. Under the action of renin, 10 amino acids of the N-terminal cut off by AGT are equivalent to angiotensin I, other downstream angiotensin components are all converted from angiotensin I, the rest part is des (AngI) AGT, and the function is not clear at present. Glucocorticoid, insulin, estrogen, thyroid hormone and the like can regulate the synthesis of AGT, and the AGT is found to be related to the occurrence of hypertension, coronary heart disease, myocardial infarction, various kidney diseases and the like. In the study, the expression of angiotensinogen in urine of burn patients is up-regulated compared with that of healthy people, and the expression is in a certain correlation with the burn degree, and the protein content is higher 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 the urinary angiotensinogen and the polypeptide fragment 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.
Preferably, the amino acid sequence of the urinary angiotensinogen is shown as SEQ ID NO.1 (MRKRAPQSEM APAGVSLRAT ILCLLAWAGL AAGDRVYIHP FHLVIHNEST CEQLAKANAG KPKDPTFIPA PIQAKTSPVD EKALQDQLVL VAAKLDTEDK LRAAMVGMLA NFLGFRIYGM HSELWGVVHG ATVLSPTAVF GTLASLYLGA LDHTADRLQA ILGVPWKDKN CTSRLDAHKV LSALQAVQGL LVAQGRADSQ AQLLLSTVVG VFTAPGLHLK QPFVQGLALY TPVVLPRSLD FTELDVAAEK IDRFMQAVTG WKTGCSLMGA SVDSTLAFNT YVHFQGKMKG FSLLAEPQEF WVDNSTSVSV PMLSGMGTFQ HWSDIQDNFS VTQVPFTESA CLLLIQPHYA SDLDKVEGLT FQQNSLNWMK KLSPRTIHLT MPQLVLQGSY DLQDLLAQAE LPAILHTELN LQKLSNDRIR VGEVLNSIFF ELEADEREPT ESTQQLNKPE VLEVTLNRPF LFAVYDQSAT ALHFLGRVAN PLSTA); 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 burn patient urinary angiotensinogen and polypeptide fragments thereof.
Preferably, the kit comprises an antigen-antibody reactive immunization method and kits thereof such as one or more of an aptamer antibody or antibody fragment capable of specifically binding angiotensinogen and polypeptide fragments thereof.
Preferably, the detection method comprises methods such as mass spectrometry for directly detecting angiotensinogen and polypeptide fragments thereof and related kits thereof.
Preferably, the detection method comprises related nucleic acid detection methods for directly detecting angiotensinogen 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 angiotensinogen standard, a humanized tag antibody standard; preferably, the quality control product comprises: angiotensin antigen 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.
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 inventor identifies the differential polypeptide with statistical significance, and utilizes a database to search to obtain the differential protein angiotensinogen.
Compared with healthy people, the angiotensinogen and the polypeptide fragment thereof are highly expressed in urine of burn patients, are increased along with the aggravation of the burn degree, and have better consistency with clinical diagnosis. Therefore, the detection of the urinary angiotensinogen 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 storage of the urine specimen, and utilizes the urine specimen to detect the angiotensinogen 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 uroangiotensinogen and its polypeptide fragments in burn group and healthy control group.
Figure 2 is a schematic representation of the involvement of angiotensinogen 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, angiotensinogen is highly expressed in urine of burn patients as shown in figure 1, and the main biological processes involved in the angiotensinogen are shown in figure 2, and the expression of the angiotensinogen in urine of normal control groups and 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 urinary angiotensinogen and polypeptide fragment thereof in burn
<130> 1
<140> 20PAGT-CN
<141> 2020-05-03
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 485
<212> PRT
<213> Human Urine
<400> 1
Met Arg Lys Arg Ala Pro Gln Ser Glu Met Ala Pro Ala Gly Val Ser
1 5 10 15
Leu Arg Ala Thr Ile Leu Cys Leu Leu Ala Trp Ala Gly Leu Ala Ala
20 25 30
Gly Asp Arg Val Tyr Ile His Pro Phe His Leu Val Ile His Asn Glu
35 40 45
Ser Thr Cys Glu Gln Leu Ala Lys Ala Asn Ala Gly Lys Pro Lys Asp
50 55 60
Pro Thr Phe Ile Pro Ala Pro Ile Gln Ala Lys Thr Ser Pro Val Asp
65 70 75 80
Glu Lys Ala Leu Gln Asp Gln Leu Val Leu Val Ala Ala Lys Leu Asp
85 90 95
Thr Glu Asp Lys Leu Arg Ala Ala Met Val Gly Met Leu Ala Asn Phe
100 105 110
Leu Gly Phe Arg Ile Tyr Gly Met His Ser Glu Leu Trp Gly Val Val
115 120 125
His Gly Ala Thr Val Leu Ser Pro Thr Ala Val Phe Gly Thr Leu Ala
130 135 140
Ser Leu Tyr Leu Gly Ala Leu Asp His Thr Ala Asp Arg Leu Gln Ala
145 150 155 160
Ile Leu Gly Val Pro Trp Lys Asp Lys Asn Cys Thr Ser Arg Leu Asp
165 170 175
Ala His Lys Val Leu Ser Ala Leu Gln Ala Val Gln Gly Leu Leu Val
180 185 190
Ala Gln Gly Arg Ala Asp Ser Gln Ala Gln Leu Leu Leu Ser Thr Val
195 200 205
Val Gly Val Phe Thr Ala Pro Gly Leu His Leu Lys Gln Pro Phe Val
210 215 220
Gln Gly Leu Ala Leu Tyr Thr Pro Val Val Leu Pro Arg Ser Leu Asp
225 230 235 240
Phe Thr Glu Leu Asp Val Ala Ala Glu Lys Ile Asp Arg Phe Met Gln
245 250 255
Ala Val Thr Gly Trp Lys Thr Gly Cys Ser Leu Met Gly Ala Ser Val
260 265 270
Asp Ser Thr Leu Ala Phe Asn Thr Tyr Val His Phe Gln Gly Lys Met
275 280 285
Lys Gly Phe Ser Leu Leu Ala Glu Pro Gln Glu Phe Trp Val Asp Asn
290 295 300
Ser Thr Ser Val Ser Val Pro Met Leu Ser Gly Met Gly Thr Phe Gln
305 310 315 320
His Trp Ser Asp Ile Gln Asp Asn Phe Ser Val Thr Gln Val Pro Phe
325 330 335
Thr Glu Ser Ala Cys Leu Leu Leu Ile Gln Pro His Tyr Ala Ser Asp
340 345 350
Leu Asp Lys Val Glu Gly Leu Thr Phe Gln Gln Asn Ser Leu Asn Trp
355 360 365
Met Lys Lys Leu Ser Pro Arg Thr Ile His Leu Thr Met Pro Gln Leu
370 375 380
Val Leu Gln Gly Ser Tyr Asp Leu Gln Asp Leu Leu Ala Gln Ala Glu
385 390 395 400
Leu Pro Ala Ile Leu His Thr Glu Leu Asn Leu Gln Lys Leu Ser Asn
405 410 415
Asp Arg Ile Arg Val Gly Glu Val Leu Asn Ser Ile Phe Phe Glu Leu
420 425 430
Glu Ala Asp Glu Arg Glu Pro Thr Glu Ser Thr Gln Gln Leu Asn Lys
435 440 445
Pro Glu Val Leu Glu Val Thr Leu Asn Arg Pro Phe Leu Phe Ala Val
450 455 460
Tyr Asp Gln Ser Ala Thr Ala Leu His Phe Leu Gly Arg Val Ala Asn
465 470 475 480
Pro Leu Ser Thr Ala
485
Claims (9)
1. The application of the urinary angiotensinogen and the polypeptide fragment 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 said prouroangiotensinogen is represented by 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 according to claim 1, wherein the preparation is a test kit for the detection of angiotensinogen and its polypeptide fragments in urine of burn patients.
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 angiotensinogen and polypeptide fragments thereof.
5. The use according to claim 3, wherein the detection method comprises mass spectrometry and related kits for directly detecting angiotensinogen and its polypeptide fragments.
6. The use according to claim 3, wherein the detection method comprises a method of directly detecting related nucleic acid such as angiotensinogen and polypeptide fragments 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 angiotensinogen standard, a humanized tag antibody standard; preferably, the quality control product comprises: angiotensinogen 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.
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| CN202010504022.7A CN113759133A (en) | 2020-06-05 | 2020-06-05 | Application of urinary angiotensinogen and polypeptide fragment thereof in burn |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998033813A2 (en) * | 1997-02-04 | 1998-08-06 | University Of Southern California | Method for accelerating healing of thermal injuries |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998033813A2 (en) * | 1997-02-04 | 1998-08-06 | University Of Southern California | Method for accelerating healing of thermal injuries |
Non-Patent Citations (2)
| Title |
|---|
| ONISHI S 等: "Resistin forms a network with inflammatory cytokines and is associated with prognosis in major burns", BURNS, vol. 48, no. 7, 27 October 2021 (2021-10-27), pages 1680 - 1689 * |
| 赵乔妹;刘丹;雷婷;张曼;: "不同程度烧伤患者凝血功能变化及意义", 标记免疫分析与临床, no. 05, 25 May 2019 (2019-05-25), pages 36 - 40 * |
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