CN114252607A

CN114252607A - Application of urine V-ATPASE subunit S1 and polypeptide fragment thereof in allergic diseases

Info

Publication number: CN114252607A
Application number: CN202011012765.9A
Authority: CN
Inventors: 张曼; 刘娜
Original assignee: Beijing Shijitan Hospital
Current assignee: Beijing Shijitan Hospital
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2022-03-29

Abstract

The invention provides application of urine V-ATPASE subunit S1 (V-type proton ATPase subunit S1) and a polypeptide fragment thereof, in particular application of urine V-ATPASE subunit S1 and a polypeptide fragment 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 urine V-ATPASE subunit S1 and the polypeptide fragment thereof have reduced expression in allergic disease patients 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 specimen, and utilizes the urine specimen to detect the urine V-ATPASE subunit S1 and the polypeptide fragment thereof.

Description

Application of urine V-ATPASE subunit S1 and polypeptide fragment thereof in allergic diseases

Technical Field

The invention relates to new application of urine V-ATPASE subunit S1 and a polypeptide fragment thereof, in particular to application of urine V-ATPASE subunit S1 and a polypeptide fragment 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.

The V-ATPase subunit S1 (V-type proton ATPase subbunit S1, V-ATPase subbunit S1) is involved in a plurality of biological processes such as cellular iron plasma homeostasis, transferrin transport, iron ion metabolism and ion transmembrane transport metabolism. V-ATPase is a complex composed of multiple subunits, mainly composed of two domains, the peripherally located V1 domain and the transmembrane V0 domain, which are widely present in eukaryotic cells. In the research, the content of V-ATPase subunit S1 in urine of patients with allergic diseases is reduced compared with that in healthy people, and the content in urine is reduced.

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 V-ATPASE subunit S1 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 V-ATPASE subunit S1 is shown in SEQ ID NO.1 (MMAAMATARV RMGPRCAQAL WRMPWLPVFL SLAAAAAAAA AEQQVPLVLW SSDRDLWAPA ADTHEGHITS DLQLSTYLDP ALELGPRNVL LFLQDKLSIE DFTAYGGVFG NKQDSAFSNL ENALDLAPSS LVLPAVDWYA VSTLTTYLQE KLGASPLHVD LATLRELKLN ASLPALLLIR LPYTASSGLM APREVLTGND EVIGQVLSTL KSEDVPYTAA LTAVRPSRVA RDVAVVAGGL GRQLLQKQPV SPVIHPPVSY NDTAPRILFW AQNFSVAYKD QWEDLTPLTF GVQELNLTGS FWNDSFARLS LTYERLFGTT VTFKFILANR LYPVSARHWF TMERLEVHSN GSVAYFNASQ VTGPSIYSFH CEYVSSLSKK GSLLVARTQP SPWQMMLQDF QIQAFNVMGE QFSYASDCAS FFSPGIWMGL LTSLFMLFIF TYGLHMILSL KTMDRFDDHK GPTISLTQIV); 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 V-ATPASE subunit S1 and polypeptide fragments thereof in urine of patients with allergic diseases.

Preferably, the kit includes one or more of an immunological method of antigen-antibody reaction and kits thereof such as an aptamer antibody or antibody fragment capable of specifically binding to V-ATPASE subunit S1 and polypeptide fragments thereof.

Preferably, the detection method comprises methods such as mass spectrometry for directly detecting the V-ATPASE subunit S1 and the polypeptide fragment thereof and related kits thereof.

Preferably, the detection method comprises related nucleic acid detection methods for directly detecting the V-ATPASE subunit S1 and the polypeptide fragment 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 V-ATPASE subunit S1 standard, a humanized tag antibody standard; preferably, the quality control product comprises: V-ATPASE subunit S1 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 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 searches a database to obtain the differential protein V-ATPASE subunit S1.

Compared with healthy people, the V-ATPASE subunit S1 and the polypeptide fragment thereof are low in expression in urine of patients with allergic diseases and have better consistency with clinical diagnosis. Therefore, the urine V-ATPASE subunit S1 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 specimen, and utilizes the urine specimen to detect the urine V-ATPASE subunit S1 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 V-ATPASE subunit S1 and its polypeptide fragment in allergic disease group and healthy control group.

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.

TABLE 1 Max Quant software search parameter Table

Compared with healthy people, the V-ATPASE subunit S1 is low in expression in urine of patients with allergic diseases, the content of the V-ATPASE subunit S1 in urine of healthy control groups and the urine of allergic diseases is shown in figure 1, and the expression of the V-ATPASE subunit S1 in urine of normal control groups and the urine of allergic diseases 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> Beijing century bed Hospital affiliated to capital medical university

<120> application of urine V-ATPase subunit S1 and polymorphic fragment thereof in allergic diseases

<130> 1

<140> 20PV-ATPASE SUBUNIT S1-CN

<141> 2020-09-24

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 470

<212> PRT

<213> Human Urine

<400> 1

Met Met Ala Ala Met Ala Thr Ala Arg Val Arg Met Gly Pro Arg Cys

1 5 10 15

Ala Gln Ala Leu Trp Arg Met Pro Trp Leu Pro Val Phe Leu Ser Leu

20 25 30

Ala Ala Ala Ala Ala Ala Ala Ala Ala Glu Gln Gln Val Pro Leu Val

35 40 45

Leu Trp Ser Ser Asp Arg Asp Leu Trp Ala Pro Ala Ala Asp Thr His

50 55 60

Glu Gly His Ile Thr Ser Asp Leu Gln Leu Ser Thr Tyr Leu Asp Pro

65 70 75 80

Ala Leu Glu Leu Gly Pro Arg Asn Val Leu Leu Phe Leu Gln Asp Lys

85 90 95

Leu Ser Ile Glu Asp Phe Thr Ala Tyr Gly Gly Val Phe Gly Asn Lys

100 105 110

Gln Asp Ser Ala Phe Ser Asn Leu Glu Asn Ala Leu Asp Leu Ala Pro

115 120 125

Ser Ser Leu Val Leu Pro Ala Val Asp Trp Tyr Ala Val Ser Thr Leu

130 135 140

Thr Thr Tyr Leu Gln Glu Lys Leu Gly Ala Ser Pro Leu His Val Asp

145 150 155 160

Leu Ala Thr Leu Arg Glu Leu Lys Leu Asn Ala Ser Leu Pro Ala Leu

165 170 175

Leu Leu Ile Arg Leu Pro Tyr Thr Ala Ser Ser Gly Leu Met Ala Pro

180 185 190

Arg Glu Val Leu Thr Gly Asn Asp Glu Val Ile Gly Gln Val Leu Ser

195 200 205

Thr Leu Lys Ser Glu Asp Val Pro Tyr Thr Ala Ala Leu Thr Ala Val

210 215 220

Arg Pro Ser Arg Val Ala Arg Asp Val Ala Val Val Ala Gly Gly Leu

225 230 235 240

Gly Arg Gln Leu Leu Gln Lys Gln Pro Val Ser Pro Val Ile His Pro

245 250 255

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

260 265 270

Asn Phe Ser Val Ala Tyr Lys Asp Gln Trp Glu Asp Leu Thr Pro Leu

275 280 285

Thr Phe Gly Val Gln Glu Leu Asn Leu Thr Gly Ser Phe Trp Asn Asp

290 295 300

Ser Phe Ala Arg Leu Ser Leu Thr Tyr Glu Arg Leu Phe Gly Thr Thr

305 310 315 320

Val Thr Phe Lys Phe Ile Leu Ala Asn Arg Leu Tyr Pro Val Ser Ala

325 330 335

Arg His Trp Phe Thr Met Glu Arg Leu Glu Val His Ser Asn Gly Ser

340 345 350

Val Ala Tyr Phe Asn Ala Ser Gln Val Thr Gly Pro Ser Ile Tyr Ser

355 360 365

Phe His Cys Glu Tyr Val Ser Ser Leu Ser Lys Lys Gly Ser Leu Leu

370 375 380

Val Ala Arg Thr Gln Pro Ser Pro Trp Gln Met Met Leu Gln Asp Phe

385 390 395 400

Gln Ile Gln Ala Phe Asn Val Met Gly Glu Gln Phe Ser Tyr Ala Ser

405 410 415

Asp Cys Ala Ser Phe Phe Ser Pro Gly Ile Trp Met Gly Leu Leu Thr

420 425 430

Ser Leu Phe Met Leu Phe Ile Phe Thr Tyr Gly Leu His Met Ile Leu

435 440 445

Ser Leu Lys Thr Met Asp Arg Phe Asp Asp His Lys Gly Pro Thr Ile

450 455 460

Ser Leu Thr Gln Ile Val

465 470

Claims

1. The urine V-ATPASE subunit S1 and the polypeptide fragment thereof are applied to the preparation of 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 V-ATPASE subunit S1 is 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 according to claim 1, wherein the agent is a detection kit for V-ATPASE subunit S1 and its polypeptide fragments in urine of patients with allergic diseases.

4. Use according to claim 3, wherein 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 V-ATPASE subunit S1 and polypeptide fragments thereof.

5. The use of claim 3, wherein the detection method comprises mass spectrometry and related kits for directly detecting V-ATPASE subunit S1 and its polypeptide fragment.

6. The use of claim 3, wherein the detection method comprises methods for directly detecting V-ATPASE subunit S1 and its polypeptide fragment or its related nucleic acid detection, and its 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 V-ATPASE subunit S1 standard, a humanized tag antibody standard; preferably, the quality control product comprises: V-ATPASE subunit S1 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.

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.