CN113092772A

CN113092772A - Application of urine cadherin subgroup SA-2 and polypeptide fragment thereof in allergic diseases

Info

Publication number: CN113092772A
Application number: CN201911334101.1A
Authority: CN
Inventors: 张曼; 刘娜
Original assignee: Beijing Shijitan Hospital
Current assignee: Beijing Shijitan Hospital
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2021-07-09

Abstract

The invention provides application of urine cadherin subgroup SA-2 (Cohesin subbunit SA-2) protein and a polypeptide fragment thereof, and particularly relates to application of urine cadherin subgroup SA-2 and the polypeptide fragment thereof in preparation of a preparation for detecting allergic diseases. The allergic diseases are also called allergic diseases, and typical allergic diseases (allergic diseases) include allergic rhinitis, bronchial asthma, allergic dermatitis, eczema, allergic conjunctivitis, anaphylactic shock, and the like, and are hereinafter collectively referred to as allergic diseases. According to the invention, compared with a healthy human group (normal control), the expression of the urine mucin subgroup SA-2 and the polypeptide fragment thereof in the allergic disease patient group is reduced, and the method can be used for auxiliary diagnosis of the allergic disease patient. 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 mucoprotein subgroup SA-2 and the polypeptide fragment thereof.

Description

Application of urine cadherin subgroup SA-2 and polypeptide fragment thereof in allergic diseases

Technical Field

The invention relates to a new application of urine cadherin subgroup SA-2 and polypeptide fragments thereof, in particular to an application of urine cadherin subgroup SA-2 and polypeptide fragments thereof in monitoring allergic diseases.

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.

Zonulin is one of the major regulatory factors involved in sister chromatid separation during mid/late cell division transformation, and is a multi-protein complex composed of 4 core subunits, one of which is the SA2 subunit. SA2 plays an important role in relieving the cohesion complex of sister chromatids, repairing DNA double-strand breaks and regulating genes. Loss of expression of the SA2 protein may result in segregation of chromosomal abnormalities, inefficient repair of DNA double strand breaks, or aberrant transcriptional function. Studies have shown that SA2 loss is associated with the course of breast cancer, and this study shows that the urine of allergic disease patients has a reduced content of the subset of mucin SA-2 compared to healthy humans.

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 alleviate the pain of the patients with allergic diseases in the multiple blood sampling and prick test, the test is expected to be helpful for the diagnosis of the allergic diseases by detecting urine protein or polypeptide on the basis of the previous methodology.

Disclosure of Invention

The invention aims to provide application of urine cadherin subgroup SA-2 and polypeptide fragments thereof in preparing a preparation for detecting allergic diseases.

Preferably, the amino acid sequence of said urine mucin subgroup SA-2 is as shown in SEQ ID NO.1 (MIAAPEIPTD FNLLQESETH FSSDTDFEDI EGKNQKQGKG KTCKKGKKGP AEKGKGGNGG GKPPSGPNRM NGHHQQNGVE NMMLFEVVKM GKSAMQSVVD DWIESYKHDR DIALLDLINF FIQCSGCKGV VTAEMFRHMQ NSEIIRKMTE EFDEDSGDYP LTMAGPQWKK FKSSFCEFIG VLVRQCQYSI IYDEYMMDTV ISLLTGLSDS QVRAFRHTST LAAMKLMTAL VNVALNLSIN MDNTQRQYEA ERNKMIGKRA NERLELLLQK RKELQENQDE IENMMNAIFK GVFVHRYRDA IAEIRAICIE EIGIWMKMYS DAFLNDSYLK YVGWTMHDKQ GEVRLKCLTA LQGLYYNKEL NSKLELFTSR FKDRIVSMTL DKEYDVAVQA IKLLTLVLQS SEEVLTAEDC ENVYHLVYSA HRPVAVAAGE FLYKKLFSRR DPEEDGMMKR RGRQGPNANL VKTLVFFFLE SELHEHAAYL VDSMWDCATE LLKDWECMNS LLLEEPLSGE EALTDRQESA LIEIMLCTIR QAAECHPPVG RGTGKRVLTA KEKKTQLDDR TKITELFAVA LPQLLAKYSV DAEKVTNLLQ LPQYFDLEIY TTGRLEKHLD ALLRQIRNIV EKHTDTDVLE ACSKTYHALC NEEFTIFNRV DISRSQLIDE LADKFNRLLE DFLQEGEEPD EDDAYQVLST LKRITAFHNA HDLSKWDLFA CNYKLLKTGI ENGDMPEQIV IHALQCTHYV ILWQLAKITE SSSTKEDLLR LKKQMRVFCQ ICQHYLTNVN TTVKEQAFTI LCDILMIFSH QIMSGGRDML EPLVYTPDSS LQSELLSFIL DHVFIEQDDD NNSADGQQED EASKIEALHK RRNLLAAFCK LIVYTVVEMN TAADIFKQYM KYYNDYGDII KETMSKTRQI DKIQCAKTLI LSLQQLFNEM IQENGYNFDR SSSTFSGIKE LARRFALTFG LDQLKTREAI AMLHKDGIEF AFKEPNPQGE SHPPLNLAFL DILSEFSSKL LRQDKRTVYV YLEKFMTFQM SLRREDVWLP LMSYRNSLLA GGDDDTMSVI SGISSRGSTV RSKKSKPSTG KRKVVEGMQL SLTEESSSSD SMWLSREQTL HTPVMMQTPQ LTSTIMREPK RLRPEDSFMS VYPMQTEHHQ TPLDYNRRGT SLMEDDEEPI VEDVMMSSEG RIEDLNEGMD FDTMDIDLPP SKNRRERTEL KPDFFDPASI MDESVLGVSM F); 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, said subset of mucins SA-2 and polypeptide fragments thereof are from urinary exosomes.

Preferably, said urine mucin subgroup SA-2 and polypeptide fragments thereof are low expressed in patients with allergic diseases.

Preferably, the preparation is a urine cadherin subgroup SA-2 of patients with allergic diseases and a polypeptide fragment detection kit thereof.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to the subset of mucin SA-2 and polypeptide fragments thereof.

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.

Preferably, the standards include the mucin subgroup SA-2 standard, a humanized tag antibody standard; preferably, the quality control product comprises: a mucin subgroup SA-2 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.

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 allergic diseases, centrifuges the urine samples, takes supernate, and purifies and separates the urine samples by using weak cation exchange magnetic beads. Uniformly mixing 1 mu l of sample and 10 mu l of matrix, taking 1 mu l of point on an Anchorchip target plate, ionizing the sample, carrying out mass spectrometry, and collecting data within the range of 1000-10000Da to obtain a mass spectrometry polypeptide diagram consisting of protein peaks with different mass-to-charge ratios. All mass spectra of healthy persons and groups of allergic diseases were analyzed using the BioExplorer analysis software to screen for differential polypeptides. Then, the inventor carries out matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification on the differential polypeptide with statistical significance, and searches a database to obtain the differential expression mucin subgroup SA-2, wherein the mucin subgroup SA-2 is low in expression in urine of allergic diseases compared with a healthy human group.

Compared with healthy people, the invention proves that the mucoprotein subgroup SA-2 and the polypeptide fragment thereof have low expression in urine of patients with allergic diseases and better consistency with clinical diagnosis. Therefore, the urine mucin subgroup SA-2 and the polypeptide fragment thereof can be used for detecting or assisting in diagnosing 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 mucoprotein subgroup SA-2 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 peak intensity profile of the mucin subgroup SA-2 in urine of healthy humans and patients with allergic disease.

FIG. 2 is a scatter plot of the expression of the mucin subgroup SA-2 in urine samples from healthy humans and patients with allergic disease.

FIG. 3 is a ROC curve for expression of the mucin subgroup SA-2 in urine from patients with allergic disease.

Detailed Description

Example 1Collection and processing of urine specimens

60 patients with allergic diseases are collected in clinic diagnosis of the Beijing century jar hospital affiliated to the university of capital medical science, and the age of the patients is 23-58 years as a group of the allergic diseases (allergic diseases). 60 healthy examiners of the physical examination center of the Beijing century bed hospital affiliated to the university of capital medical science were collected as a control group. After urine collection, 400 Xg centrifugation for 5min, supernatant was collected and split charged and frozen at-80 ℃. A comparison of the clinical characteristics of these subjects is shown in table 1.

Table 1 general clinical data of patients in allergic disease (allergic disease) group and control group are compared.

Example 2Magnetic bead purification and urine polypeptide screening

And (3) enriching the polypeptide in the urine by using weak cation exchange magnetic beads, and analyzing the polypeptide spectrum in the urine by using a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Mu.l of the eluate was mixed with 10. mu.l of matrix (0.3% of. alpha. -cyano-4-hydroxycinnamic acid, HCCA) and 1. mu.l of the spot was applied to an Anchorchip target plate and dried at room temperature. The specimen is ionized by nitrogen laser irradiation and calibrated before further mass spectrometry of the specimen. After calibrating the instrument with the standard, the sample is detected, data in the range of 1-10kDa are collected, and a mass spectrogram consisting of protein peaks with different mass-to-charge ratios is obtained. For each MALDI target, a total of 400 laser shots were performed. The 8 different positions of each target point were irradiated 50 times each, and the average value represents one specimen. Thereby obtaining the polypeptide maps of all samples. Three biological replicates were performed per sample. All spectra obtained from the urine samples were analyzed using the BioExplorer software. The software removes the base line from the mass spectrogram and standardizes the mass spectrogram, the mass range is 1000-10000Da, the signal-to-noise ratio (S/N) is more than 5, the mass drift does not exceed 0.1 percent, and the mass spectrum is measured in the mass spectrum measuring device>Peaks detected in 80% of the samples were considered informative and were screened for differential polypeptides. The polypeptide peaks of mass to charge ratio (m/z) 1066.5 were statistically different between the 2 groups (m/z)P<0.01) and the distribution of the average peak intensities thereof is shown in fig. 1.

Example 3Identification and analysis of differential Polypeptides

From the sample tube, 20 ul was taken for LC-MS/MS analysis. An EASY-nLC1000 HPLC system was used for the separation. Liquid phase a was 0.1% formic acid in acetonitrile (2% acetonitrile) and liquid phase B was 0.1% formic acid in acetonitrile (98% acetonitrile). The chromatographic spray needle is PN: FS 360-20-10-N-20-C12. Samples were separated by capillary HPLC and analyzed by Q-exact mass spectrometer (Thermo). The ion source voltage is 3.5kv, the analysis time is 120min, and the scanning range of the parent ion is 300-2000 m/z. The mass-to-charge ratios of the polypeptides and polypeptide fragments were collected by collecting 20 fragments (MS 2scan, HCD) after each complete scan. At M/Z200, the MS1 resolution was 70,000, and the MS2 resolution was 17,500. Data analysis software Peaks8.5 (Bioinformatics Solutions Inc.) was used for the search. The search database was the ipi human database (v 3.45). The parent ion error was set to 10 ppm, the fragment ion error was set to 0.02 Da, and the digestion mode was non-digestion.

The differentially expressed polypeptide peak of mass to charge (m/z) 2197.0 was retrieved in the database and identified as the mucin subgroup SA-2.

Compared with healthy people, the expression of the mucin subgroup SA-2 in the urine of patients with allergic diseases is low, and as shown in FIG. 2, the expression of the mucin subgroup SA-2 in the urine of healthy people and patients with allergic diseases is significantly different.

Further, the inventor detects a polypeptide peak value with a mass-to-charge ratio (m/z) of 2197.0 in urine of 60 patients with allergic diseases and 60 healthy people, establishes a ROC curve with an area of 0.780 under the curve, and reflects that the sensitivity and specificity of the cadherin subgroup SA-2 are better as shown in FIG. 3.

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 (Beijing railway general Hospital)

<120> use of urine mucin subgroup SA-2 and polypeptide fragments thereof in allergic diseases

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<140> 19PSA2-CN

<141> 2019-12-06

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Claims

1. Application of urine cadherin subgroup SA-2 and polypeptide fragments thereof in preparing preparations for detecting allergic diseases.

2. The use according to claim 1, wherein the amino acid sequence of urine cadherin subgroup SA-2 is 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. Use according to claim 1, wherein the subset of mucins SA-2 and polypeptide fragments thereof is from urine.

4. The use according to claim 1, wherein said urine cadherin subgroup SA-2 and polypeptide fragments thereof is low expressed in patients with allergic disease.

5. The use according to claim 1, wherein the preparation is a kit for detecting urinary cadherin subgroup SA-2 and polypeptide fragments thereof of patients with allergic diseases.

6. The use according to claim 5, wherein the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to mucin subgroup SA-2 and polypeptide fragments thereof.

7. The use according to claim 5, 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.

8. The use of claim 7, wherein the standards comprise a mucin subgroup SA-2 standard, a humanized tag antibody standard; preferably, the quality control product comprises: a mucin subgroup SA-2 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.