CN114252620A - Application of urine neutrophil gelatinase-associated lipocalin and polypeptide fragment thereof in allergic diseases - Google Patents

Application of urine neutrophil gelatinase-associated lipocalin and polypeptide fragment thereof in allergic diseases Download PDF

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CN114252620A
CN114252620A CN202010996137.2A CN202010996137A CN114252620A CN 114252620 A CN114252620 A CN 114252620A CN 202010996137 A CN202010996137 A CN 202010996137A CN 114252620 A CN114252620 A CN 114252620A
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neutrophil gelatinase
associated lipocalin
urine
antibody
polypeptide fragments
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张曼
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Abstract

The invention provides application of urine neutrophil gelatinase-associated lipocalin (NGAL) and polypeptide fragments thereof, in particular to application of urine neutrophil gelatinase-associated lipocalin 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 invention proves that the expression of urine neutrophil gelatinase-associated lipocalin and polypeptide fragments 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 preservation of the urine specimen, and utilizes the urine specimen to detect the urine neutrophil gelatinase-associated lipocalin and the polypeptide fragments thereof.

Description

Application of urine neutrophil gelatinase-associated lipocalin and polypeptide fragment thereof in allergic diseases
Technical Field
The invention relates to a new application of urine neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof, in particular to an application of urine neutrophil gelatinase-associated lipocalin 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.
Neutrophil gelatinase-associated lipocalin (NGAL) is a secreted protein of small relative molecular mass found in activated neutrophils. Under normal physiological conditions, NGAL exhibits low levels of expression in tissues such as liver, kidney, stomach, intestine, pancreas, bronchi and prostate. Research shows that NGAL is involved in biological processes such as inflammation, immunoreaction, cell differentiation and apoptosis of organisms. The content of NGAL in urine of patients with allergic diseases in the research is up-regulated compared with that of healthy people, and the content of NGAL 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 neutrophil gelatinase-associated lipocalin 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 neutrophil gelatinase-associated lipocalin is shown in SEQ ID No.1 (MPLGLLWLGL ALLGALHAQA QDSTSDLIPA PPLSKVPLQQ NFQDNQFQGK WYVVGLAGNA ILREDKDPQK MYATIYELKE DKSYNVTSVL FRKKKCDYWI RTFVPGCQPG EFTLGNIKSY PGLTSYLVRV VSTNYNQHAM VFFKKVSQNR EYFKITLYGR TKELTSELKE NFIRFSKSLG LPENHIVFPV PIDQCIDG); 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 kit for detecting the urine neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof of patients with allergic diseases.
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 neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof.
Preferably, the detection method comprises a mass spectrometry method for directly detecting the neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof and a related kit thereof.
Preferably, the detection method comprises related nucleic acid detection methods for directly detecting neutrophil gelatinase-associated lipocalin 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 neutrophil gelatinase-associated lipocalin standard, a humanized tag antibody standard; preferably, the quality control product comprises: a quality control product of neutrophil gelatinase-associated lipocalin and a quality control product of humanized label antibody; 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 neutrophil gelatinase-associated lipocalin.
Compared with healthy people, the neutrophil gelatinase-associated lipocalin 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 detection of the urine neutrophil gelatinase-associated lipocalin 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 preservation of the urine specimen, and utilizes the urine specimen to detect the urine neutrophil gelatinase-associated lipocalin and the polypeptide fragments 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 neutrophil gelatinase-associated lipocalin 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 neutrophil gelatinase-associated lipocalin is highly expressed in urine of allergic disease patients, the content of the neutrophil gelatinase-associated lipocalin in urine of healthy control groups and allergic disease groups is shown in figure 1, and the expression of the neutrophil gelatinase-associated lipocalin in urine of normal control groups and 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 neutrophil gelatinase-associated lipocalin and polymorphic fragments thereof in allergic diseases
<130> 1
<140> 20PNGAL-CN
<141> 2020-08-21
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 198
<212> PRT
<213> Human Urine
<400> 1
Met Pro Leu Gly Leu Leu Trp Leu Gly Leu Ala Leu Leu Gly Ala Leu
1 5 10 15
His Ala Gln Ala Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro
20 25 30
Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln
35 40 45
Gly Lys Trp Tyr Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu
50 55 60
Asp Lys Asp Pro Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu
65 70 75 80
Asp Lys Ser Tyr Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys
85 90 95
Asp Tyr Trp Ile Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe
100 105 110
Thr Leu Gly Asn Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val
115 120 125
Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys
130 135 140
Lys Val Ser Gln Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg
145 150 155 160
Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser
165 170 175
Lys Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile
180 185 190
Asp Gln Cys Ile Asp Gly
195

Claims (9)

1. The application of urine neutrophil gelatinase-associated lipocalin and polypeptide fragments 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 according to claim 1, wherein the amino acid sequence of the urinary neutrophil gelatinase-associated lipocalin is 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 according to claim 1, wherein the preparation is a kit for detecting neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof in urine of patients with allergic diseases.
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 neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof.
5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for direct detection of neutrophil gelatinase-associated lipocalin and polypeptide fragments thereof.
6. The use of claim 3, wherein the detection method comprises a method for directly detecting neutrophil gelatinase-associated lipocalin and its polypeptide fragments 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 a neutrophil gelatinase-associated lipocalin standard, a humanized tag antibody standard; preferably, the quality control product comprises: a neutrophil gelatinase-associated lipocalin control product and a 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.
CN202010996137.2A 2020-09-21 2020-09-21 Application of urine neutrophil gelatinase-associated lipocalin and polypeptide fragment thereof in allergic diseases Pending CN114252620A (en)

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