CN113092756A

CN113092756A - Application of urine prothrombin and polypeptide fragment thereof in allergic diseases

Info

Publication number: CN113092756A
Application number: CN201911334180.6A
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 an application of urine Prothrombin (Prothrombin) protein and a polypeptide fragment thereof, in particular to an application of urine Prothrombin and a polypeptide fragment thereof in preparing 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 prothrombin and the polypeptide fragment thereof in an 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 prothrombin and the polypeptide fragment thereof.

Description

Application of urine prothrombin and polypeptide fragment thereof in allergic diseases

Technical Field

The invention relates to new application of urine prothrombin and a polypeptide fragment thereof, in particular to application of urine prothrombin and a polypeptide fragment 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.

Prothrombin, also known as factor II, is synthesized by the liver and is a vitamin K dependent zymogen. Prothrombin plays a central role in the coagulation mechanism. Activated factor x is activated to form thrombin in the presence of activated factor v and a phospholipid surface provided by platelets or other cells. Thrombin is a proteolytic enzyme that hydrolyzes a variety of coagulation factors, converting fibrinogen into fibrin. Studies have shown that the coagulation and fibrinolysis systems are involved in the pathogenesis of allergic diseases, suggesting that the urine of patients with allergic diseases contains a reduced amount of fibrinogen compared to healthy individuals.

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 prothrombin and polypeptide fragments thereof in preparing a preparation for detecting allergic diseases.

Preferably, the amino acid sequence of the urine prothrombin is shown in SEQ ID NO.1 (MAHVRGLQLP GCLALAALCS LVHSQHVFLA PQQARSLLQR VRRANTFLEE VRKGNLEREC VEETCSYEEA FEALESSTAT DVFWAKYTAC ETARTPRDKL AACLEGNCAE GLGTNYRGHV NITRSGIECQ LWRSRYPHKP EINSTTHPGA DLQENFCRNP DSSTTGPWCY TTDPTVRRQE CSIPVCGQDQ VTVAMTPRSE GSSVNLSPPL EQCVPDRGQQ YQGRLAVTTH GLPCLAWASA QAKALSKHQD FNSAVQLVEN FCRNPDGDEE GVWCYVAGKP GDFGYCDLNY CEEAVEEETG DGLDEDSDRA IEGRTATSEY QTFFNPRTFG SGEADCGLRP LFEKKSLEDK TERELLESYI DGRIVEGSDA EIGMSPWQVM LFRKSPQELL CGASLISDRW VLTAAHCLLY PPWDKNFTEN DLLVRIGKHS RTRYERNIEK ISMLEKIYIH PRYNWRENLD RDIALMKLKK PVAFSDYIHP VCLPDRETAA SLLQAGYKGR VTGWGNLKET WTANVGKGQP SVLQVVNLPI VERPVCKDST RIRITDNMFC AGYKPDEGKR GDACEGDSGG PFVMKSPFNN RWYQMGIVSW GEGCDRDGKY GFYTHVFRLK KWIQKVIDQF GE); 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 prothrombin and polypeptide fragments thereof are from urine exosomes.

Preferably, the urine prothrombin and polypeptide fragments thereof are low expressed in patients with allergic diseases.

Preferably, the preparation is a kit for detecting the urine prothrombin and the polypeptide fragments thereof of patients with allergic diseases.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding prothrombin 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 standard comprises a prothrombin standard, a humanized tag antibody standard; preferably, the quality control product comprises: prothrombin control products and humanized label antibody quality control products; 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, differential expression prothrombin is obtained through database retrieval, and the prothrombin is low in expression in urine of allergic diseases compared with a healthy human group.

The research proves that compared with healthy people, the prothrombin 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 detection of the urine prothrombin and the polypeptide fragment thereof can be used for detection or auxiliary diagnosis 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 prothrombin 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 distribution of prothrombin in urine of healthy persons and patients with allergic disease.

FIG. 2 is a scattergram of prothrombin expression in urine samples of healthy persons and patients with allergic disease.

FIG. 3 is a ROC curve of prothrombin expression in urine of patients with allergic diseases.

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. 8 of each targetThe same positions 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) 2790.4 was retrieved in the database and identified as prothrombin.

Compared with healthy people, the prothrombin is low expressed in urine of patients with allergic diseases, and as shown in fig. 2, the expression of prothrombin in 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 2790.4 in urine of 60 patients with allergic diseases and 60 healthy people, establishes a ROC curve with an area of 0.698 below the curve, and reflects that the sensitivity and specificity of prothrombin are good 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> application of urine prothrombin and polypeptide fragment thereof in allergic diseases

<130> 19PProthrombin-CN

<140> 19PProthrombin-CN

<141> 2019-12-06

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 622

<212> PRT

<213> Human,Urine

<400> 1

Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu

35 40 45

Glu Glu Val Arg Lys Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr

50 55 60

Cys Ser Tyr Glu Glu Ala Phe Glu Ala Leu Glu Ser Ser Thr Ala Thr

65 70 75 80

Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro

85 90 95

Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu

100 105 110

Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile Glu

115 120 125

Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn Ser

130 135 140

Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro

145 150 155 160

Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val

165 170 175

Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr

180 185 190

Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro

195 200 205

Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg

210 215 220

Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala

225 230 235 240

Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln

245 250 255

Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val

260 265 270

Trp Cys Tyr Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys Asp Leu

275 280 285

Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp

290 295 300

Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr

305 310 315 320

Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys

325 330 335

Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu

340 345 350

Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser

355 360 365

Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys

370 375 380

Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp

385 390 395 400

Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn

405 410 415

Phe Thr Glu Asn Asp Leu Leu Val Arg Ile Gly Lys His Ser Arg Thr

420 425 430

Arg Tyr Glu Arg Asn Ile Glu Lys Ile Ser Met Leu Glu Lys Ile Tyr

435 440 445

Ile His Pro Arg Tyr Asn Trp Arg Glu Asn Leu Asp Arg Asp Ile Ala

450 455 460

Leu Met Lys Leu Lys Lys Pro Val Ala Phe Ser Asp Tyr Ile His Pro

465 470 475 480

Val Cys Leu Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gln Ala Gly

485 490 495

Tyr Lys Gly Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp Thr

500 505 510

Ala Asn Val Gly Lys Gly Gln Pro Ser Val Leu Gln Val Val Asn Leu

515 520 525

Pro Ile Val Glu Arg Pro Val Cys Lys Asp Ser Thr Arg Ile Arg Ile

530 535 540

Thr Asp Asn Met Phe Cys Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg

545 550 555 560

Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser

565 570 575

Pro Phe Asn Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly Glu

580 585 590

Gly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr Thr His Val Phe Arg

595 600 605

Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln Phe Gly Glu

610 615 620

Claims

1. Application of urine prothrombin and polypeptide fragments thereof in preparing preparations for detecting allergic diseases.

2. The use of claim 1, wherein the amino acid sequence of the urine prothrombin 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 of claim 1, wherein the prothrombin and polypeptide fragments thereof are from urine.

4. The use of claim 1, wherein the urine prothrombin and polypeptide fragments thereof are low expressed in patients with allergic disease.

5. The use of claim 1, wherein the preparation is a kit for detecting prothrombin and its polypeptide fragments in urine of patients with allergic diseases.

6. The use of claim 5, wherein the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding prothrombin 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 standard comprises a prothrombin standard, a humanized tag antibody standard; preferably, the quality control product comprises: prothrombin control products and humanized label antibody quality control products; 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.