CN114252618A - Application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragment thereof in allergic diseases - Google Patents

Application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragment thereof in allergic diseases Download PDF

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CN114252618A
CN114252618A CN202010996099.0A CN202010996099A CN114252618A CN 114252618 A CN114252618 A CN 114252618A CN 202010996099 A CN202010996099 A CN 202010996099A CN 114252618 A CN114252618 A CN 114252618A
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microglobulin
trypsin inhibitor
alpha
urine
inhibitor precursor
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张曼
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract

The invention provides application of urine alpha-1-microglobulin/trypsin inhibitor precursor (Protein AMBP) and polypeptide fragments thereof, in particular application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof in preparation of preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like. The research proves that the expression of urine alpha-1-microglobulin/trypsin inhibitor precursor and the polypeptide fragment thereof is increased in patients with allergic diseases compared with healthy people (normal control group). Can be used for various purposes of detecting patients with allergic diseases. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine sample, and utilizes the urine sample to detect the urine alpha-1-microglobulin/trypsin inhibitor precursor and the polypeptide fragment thereof.

Description

Application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragment thereof in allergic diseases
Technical Field
The invention relates to a new application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof, in particular to an application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof in preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Background
Allergic diseases are also called allergic diseases, and typical allergic diseases comprise allergic rhinitis, bronchial asthma, allergic dermatitis, eczema, allergic conjunctivitis, anaphylactic shock and the like. According to the statistics of the world health organization, the number of allergic people is huge in the world, and about one third of people suffer from allergic diseases. In recent years, due to rapid change of life style of people, remarkable improvement of industrialization degree and remarkable aggravation of air pollution, the incidence rate of allergic diseases rises year by year, serious allergic diseases even endanger life, and the problem becomes a global public health focus at present.
Currently, clinical auxiliary examination of allergic diseases mainly includes Skin Prick Test (SPT), serum sIgE detection and the like, but SPT patients have poor pain compliance and are easily affected by drugs, and the risk of systemic anaphylaxis can be increased, so that the SPT test is not suitable for severe allergic constitution and children and old people. The detection of sIgE is also not a non-invasive one, and often requires repeated blood drawing of the patient. In order to improve the life quality and compliance of patients with allergic diseases and relieve the pain of repeated blood collection and prick tests, the urine protein or polypeptide research is expected to realize the diagnosis of the allergic diseases assisted by painless, convenient, quick and easily repeated urine detection and also lay a foundation for the further research of the urine polypeptide detection kit.
Alpha-1-microglobulin/trypsin inhibitor precursor (AMBP) is a glycoprotein consisting of 352 amino acids. AMBP is produced in the liver, synthesized and secreted by hepatocytes, and is detectable in both blood and urine. AMBP participates in a plurality of biological processes, and researches show that the AMBP plays an important role in physiological and pathological processes such as tissue injury, immunoregulation, inflammatory reaction, cell oxidation resistance and the like. In the research, the AMBP content in urine of patients with allergic diseases is up-regulated compared with that in healthy people, and the content in urine is increased.
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 patients with allergic diseases in blood collection for many times, the experiment is expected to realize the diagnosis and disease monitoring of patients with allergic diseases assisted by painless, convenient, quick and easily repeated urine detection through the research of urine protein or polypeptide on the basis of the methodology of the earlier stage, and also lays a foundation for the further research of urine polypeptide detection kits.
Disclosure of Invention
The invention aims to provide application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof in preparation of preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
Preferably, the amino acid sequence of the urine alpha-1-microglobulin/trypsin inhibitor precursor is shown in SEQ ID No.1 (MRSLGALLLL LSACLAVSAG PVPTPPDNIQ VQENFNISRI YGKWYNLAIG STCPWLKKIM DRMTVSTLVL GEGATEAEIS MTSTRWRKGV CEETSGAYEK TDTDGKFLYH KSKWNITMES YVVHTNYDEY AIFLTKKFSR HHGPTITAKL YGRAPQLRET LLQDFRVVAQ GVGIPEDSIF TMADRGECVP GEQEPEPILI PRVRRAVLPQ EEEGSGGGQL VTEVTKKEDS CQLGYSAGPC MGMTSRYFYN GTSMACETFQ YGGCMGNGNN FVTEKECLQT CRTVAACNLP IVRGPCRAFI QLWAFDAVKG KCVLFPYGGC QGNGNKFYSE KECREYCGVP GDGDEELLRF SN); 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 urine alpha-1-microglobulin/trypsin inhibitor precursor and a polypeptide fragment detection kit thereof for patients with allergic diseases.
Preferably, the kit comprises an immunological method of antigen-antibody reaction and kits thereof such as one or more of an aptamer antibody or antibody fragment capable of specifically binding to alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof.
Preferably, the detection method comprises methods such as mass spectrometry for directly detecting alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof and related kits.
Preferably, the detection method comprises related nucleic acid detection methods for directly detecting alpha-1-microglobulin/trypsin inhibitor precursor 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-microglobulin/trypsin inhibitor precursor standard, a humanized tag antibody standard; preferably, the quality control product comprises: alpha-1-microglobulin/trypsin inhibitor precursor quality 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.
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 allergic disease 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 quantitative calculation on data obtained in the mass spectrum of the allergic disease 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 alpha-1-microglobulin/trypsin inhibitor precursor.
Compared with healthy people, the alpha-1-microglobulin/trypsin inhibitor precursor and the polypeptide fragment thereof are high in expression in urine of patients with allergic diseases and have better consistency with clinical diagnosis. Therefore, the method for detecting the urine alpha-1-microglobulin/trypsin inhibitor precursor and the polypeptide fragment thereof can be used for auxiliary diagnosis or disease condition monitoring of allergic diseases.
The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine sample, and utilizes the urine sample to detect the urine alpha-1-microglobulin/trypsin inhibitor precursor 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-microglobulin/trypsin inhibitor precursor and its polypeptide fragments in allergic disease groups and healthy control groups.
Detailed Description
Example 1Collection and processing of urine specimens
Allergic disease patients are selected as an allergic disease group, and health examinees in the same period are selected as a 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.
Figure DEST_PATH_IMAGE001
Compared with healthy people, the alpha-1-microglobulin/trypsin inhibitor precursor is highly expressed in urine of allergic disease patients, the content of the alpha-1-microglobulin/trypsin inhibitor precursor in urine of healthy control groups and allergic disease groups is shown in figure 1, and the expression of the alpha-1-microglobulin/trypsin inhibitor precursor in urine of the normal control groups and the allergic disease groups is significantly different.
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-microglobulin/trypsin inhibitor precursor and polymorphic fragment thereof in allergic diseases
<130> 1
<140> 20PAMBP-CN
<141> 2020-08-21
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<170> SIPOSequenceListing 1.0
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<213> Human Urine
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Met Arg Ser Leu Gly Ala Leu Leu Leu Leu Leu Ser Ala Cys Leu Ala
1 5 10 15
Val Ser Ala Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Gln Val Gln
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Glu Asn Phe Asn Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Leu Ala
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Ile Gly Ser Thr Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr
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Val Ser Thr Leu Val Leu Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser
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Met Thr Ser Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thr Ser Gly
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Ala Tyr Glu Lys Thr Asp Thr Asp Gly Lys Phe Leu Tyr His Lys Ser
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Lys Trp Asn Ile Thr Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp
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Glu Tyr Ala Ile Phe Leu Thr Lys Lys Phe Ser Arg His His Gly Pro
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Thr Ile Thr Ala Lys Leu Tyr Gly Arg Ala Pro Gln Leu Arg Glu Thr
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Leu Leu Gln Asp Phe Arg Val Val Ala Gln Gly Val Gly Ile Pro Glu
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Asp Ser Ile Phe Thr Met Ala Asp Arg Gly Glu Cys Val Pro Gly Glu
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Gln Glu Pro Glu Pro Ile Leu Ile Pro Arg Val Arg Arg Ala Val Leu
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Pro Gln Glu Glu Glu Gly Ser Gly Gly Gly Gln Leu Val Thr Glu Val
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Thr Lys Lys Glu Asp Ser Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys
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Met Gly Met Thr Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys
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Glu Thr Phe Gln Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val
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Thr Glu Lys Glu Cys Leu Gln Thr Cys Arg Thr Val Ala Ala Cys Asn
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Leu Pro Ile Val Arg Gly Pro Cys Arg Ala Phe Ile Gln Leu Trp Ala
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Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys
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Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu Cys Arg Glu Tyr
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Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Leu Leu Arg Phe Ser Asn
340 345 350

Claims (9)

1. The application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragment thereof in preparing preparations for allergic disease diagnosis, differential diagnosis, disease degree judgment, treatment effect evaluation, monitoring, prognosis evaluation, mechanism research and the like.
2. The use of claim 1, wherein the amino acid sequence of said urinary α -1-microglobulin/trypsin inhibitor precursor is as set forth 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 alpha-1-microglobulin/trypsin inhibitor precursor and its polypeptide fragments in urine of patients with allergic diseases.
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 alpha-1-microglobulin/trypsin inhibitor precursors and polypeptide fragments thereof.
5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for direct detection of α -1-microglobulin/trypsin inhibitor precursor and polypeptide fragments thereof.
6. The use of claim 3, wherein the detection method comprises methods for directly detecting alpha-1-microglobulin/trypsin inhibitor precursor and its polypeptide fragment or its related nucleic acid detection, and related kits.
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-microglobulin/trypsin inhibitor precursor standard, a humanized tag antibody standard; preferably, the quality control product comprises: alpha-1-microglobulin/trypsin inhibitor precursor control and 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.
CN202010996099.0A 2020-09-21 2020-09-21 Application of urine alpha-1-microglobulin/trypsin inhibitor precursor and polypeptide fragment thereof in allergic diseases Pending CN114252618A (en)

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