CN112924681A

CN112924681A - Application of urine KRT10 protein and polypeptide fragment thereof in normal pregnancy

Info

Publication number: CN112924681A
Application number: CN201911230688.1A
Authority: CN
Inventors: 张曼; 胡智颖
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2021-06-08
Anticipated expiration: 2039-12-05
Also published as: CN112924681B

Abstract

The invention provides application of urine KRT10 protein and a polypeptide fragment thereof, in particular to application of urine KRT10 protein and a polypeptide fragment thereof in preparing a preparation for monitoring normal pregnancy, wherein the urine KRT is a type I cytoskeletal 10 protein (KRT 10) and the polypeptide fragment thereof is also called cytokeratin 10. According to the invention, compared with a normal women group with a normal reproductive age (a normal control), the KRT10 protein and the KRT10 polypeptide fragment are expressed in a normal pregnancy group to be increased, so that the KRT10 protein can be used for monitoring the pregnancy state. 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 KRT10 protein and the polypeptide fragment thereof in the urine.

Description

Application of urine KRT10 protein and polypeptide fragment thereof in normal pregnancy

Technical Field

The invention relates to a new application of urine keratin I-type cytoskeleton 10 (KRT10, also called cytokeratin 10) protein and polypeptide fragments thereof, in particular to an application of urine KRT10 protein and polypeptide fragments thereof in monitoring normal pregnancy.

Background

In the process of pregnancy, in order to meet the requirements of fetal growth and development, a series of changes occur in the reproductive, blood circulation, urinary, respiratory, digestive and endocrine systems of pregnant women, especially in the endocrine system, the secretion of gonadotropin is reduced, and the secretion of prolactin, thyroid stimulating hormone, adrenocorticotropic hormone, glucocorticoid, aldosterone, thyroid hormone and the like is increased. In the middle and late gestation period, part of pregnant women have reduced insulin sensitivity, and have decomposition and antagonism on insulin, so that insulin activity is reduced, and the pregnant women can not normally compensate the physiological change to cause Gestational Diabetes (GDM). If the blood sugar level is not well controlled, the incidence rate of adverse pregnancy fatalities of gestational diabetes women in gestational period hypertension diseases, abortion, cesarean section, premature delivery, neonatal hypoglycemia, neonatal hyperbilirubinemia and the like is obviously higher than that of normal pregnant and lying-in women. For the patients with gestational diabetes who are diagnosed, the later blood sugar control standard and control requirement are stricter, and the monitoring is more frequent. The method aims to improve the life quality and medical compliance of patients in gestation period, relieve the pain of blood sampling for multiple times in blood sugar detection in gestation period, formulate more refined and personalized monitoring indexes aiming at different gestation periods, expect to realize painless, convenient, quick and easily repeated urine detection or monitoring of the in vivo sugar level and sugar metabolism condition through the research of urine protein or polypeptide, and lay a foundation for the further research of urine polypeptide detection kits.

Keratin is the major protein constituting the epidermis, the fur follicle, of the hide, and more than 20 keratin peptide chains have been found. Keratin type I cytoskeleton 10 (KRT 10) plays a role in establishing the plantar skin epidermal barrier. Considering that during normal pregnancy, as weight of pregnant women and fetuses increases, sole load increases, KRT10 secretion in serum increases to maintain the protective property of the plantar dermoepidermal barrier, and KRT10 increase can be detected in urine of pregnant women during pregnancy.

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 blood glucose detection of GDM patients in blood sampling for multiple times, the experiment hopes to realize monitoring and understanding of the glucose level and the glucose metabolism level in GDM patients through detection of urine protein or polypeptide on the basis of the previous methodology exploration.

Disclosure of Invention

The invention aims to provide application of urine keratin I type cytoskeletal 10 (KRT10, also called as cytoskeletal protein 10) protein and polypeptide fragments thereof in preparing a preparation for monitoring normal pregnancy.

Preferably, the amino acid sequence of the urine KRT10 is shown in SEQ ID NO.1 (MSVRYSSSKH YSSSRSGGGG GGGGCGGGGG VSSLRISSSK GSLGGGFSSG GFSGGSFSRG SSGGGCFGGS SGGYGGLGGF GGGSFRGSYG SSSFGGSYGG IFGGGSFGGG SFGGGSFGGG GFGGGGFGGG FGGGFGGDGG LLSGNEKVTM QNLNDRLASY LDKVRALEES NYELEGKIKE WYEKHGNSHQ GEPRDYSKYY KTIDDLKNQI LNLTTDNANI LLQIDNARLA ADDFRLKYEN EVALRQSVEA DINGLRRVLD ELTLTKADLE MQIESLTEEL AYLKKNHEEE MKDLRNVSTG DVNVEMNAAP GVDLTQLLNN MRSQYEQLAE QNRKDAEAWF NEKSKELTTE IDNNIEQISS YKSEITELRR NVQALEIELQ SQLALKQSLE ASLAETEGRY CVQLSQIQAQ ISALEEQLQQ IRAETECQNT EYQQLLDIKI RLENEIQTYR SLLEGEGSSG GGGRGGGSFG GGYGGGSSGG GSSGGGHGGG HGGSSGGGYG GGSSGGGSSG GGYGGGSSSG GHGGSSSGGY GGGSSGGGGG GYGGGSSGGG SSSGGGYGGG SSSGGHKSSS SGSVGESSSK GPRY); 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 KRT10 protein and polypeptide fragments thereof are derived from urine.

Preferably, the KRT10 protein and its polypeptide fragments are highly expressed in normal pregnant women.

Preferably, the preparation is a kit for detecting KRT10 protein and polypeptide fragments thereof in urine of normal pregnant women.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding KRT10 protein 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 KRT10 protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: KRT10 protein control product, and humanized label antibody quality control product; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.

The inventor firstly collects urine samples of women of normal childbearing age, women of normal pregnancy and patients with gestational diabetes, takes supernatant after centrifugation, and utilizes weak cation exchange magnetic beads to purify and separate the urine samples. 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 spectrograms of normal fertile age women, normal pregnant women and gestational diabetes groups were analyzed using the BioExplorer analysis software to screen 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 KRT10 protein with differential expression, and the KRT10 protein is highly expressed in the urine of normal pregnant women and patients with gestational diabetes compared with the normal gestational age group.

According to the invention, compared with a normal child group, KRT10 protein and the polypeptide fragment thereof are highly expressed in urine of normal pregnant women and patients with gestational diabetes. Therefore, the detection of urine IGKC protein and the polypeptide fragment thereof can be used for monitoring normal pregnancy or auxiliary diagnosis.

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 KRT10 protein 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 peak area distribution diagram of KRT10 protein in urine of women of normal childbearing age, women of normal pregnancy and patients with gestational diabetes.

FIG. 2 is a scattergram of KRT10 protein expressed in urine samples from women of normal child-bearing age, women of normal pregnancy, and gestational diabetes patients.

Detailed Description

Example 1Collection and processing of urine specimens

60 gestational diabetes patients (GDM) are collected in obstetrical outpatient service and inpatient clinical diagnosis of Beijing century bed hospital affiliated to capital medical university, wherein 30 gestational middle (GDM-M, 13-27 weekend) samples and 30 gestational late (GDM-L, 28 weeks and later) samples are collected; 31 normal pregnant women (N) in routine obstetrical examination in the outpatient clinic; 31 pregnant women (T) with normal health examination, namely clean mid-stream urine specimens of a control group, are all 15-49 years old. After urine collection, 400 Xg centrifugation for 5min, supernatant was collected and split charged and frozen at-80 ℃. Inclusion criteria were:

the first pregnancy test and the first establishment of files are carried out in our hospital from the early pregnancy (8-12 weeks) of patients with gestational diabetes and normal pregnant women, and the data are complete;

75g Oral Glucose Tolerance Test (OGTT) is carried out at 24-28 weeks of pregnancy, and the results all accord with the diagnosis standard of gestational diabetes in ninth edition 'gynaecology obstetrics'; exclusion criteria:

acute and chronic infectious, tumor, cardiovascular disease;

previous presence of impaired glucose tolerance;

severe liver and kidney dysfunction;

combined extractMental and intellectual disabilities. A comparison of the clinical characteristics of these subjects is shown in Table 1-1.

TABLE 1-1 women of Normal gestational age (T), pregnant women of Normal pregnancy (N) and patients with Gestational Diabetes Mellitus (GDM)

Note: Alb/Cr indicates a urine microalbumin to creatinine ratio < 30.

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). After calibration of the instrument with standards, 1. 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 (Autoflex MALDI TOF, Bruker-Dalton) target plate and dried at room temperature. And ionizing the sample by irradiating the nitrogen laser, and then carrying out mass spectrometry, and acquiring data in the range of 1000-10000Da to obtain a mass spectrogram consisting of protein peaks with different mass-to-charge ratios. For each MALDI crystallisation spot, a total of 400 laser shots (50 shots at each of the 8 different positions of each crystallisation spot) were made, and the average represented a sample, thereby obtaining a polypeptide profile for all samples. Three biological replicates were performed per sample. The mass spectrograms of normal pregnant women (T), normal pregnant women (N) and Gestational Diabetes Mellitus (GDM) groups were analyzed using BioExplorer analysis software to screen differential polypeptides. The polypeptide peaks of mass to charge ratio (m/z) 1089.2 were statistically different (P < 0.05) between the 3 groups, and the distribution of the mean peak areas is shown in fig. 1.

Example 4Identification and analysis of differential Polypeptides

From the sample tube, 20ul 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 100% A solution was equilibrated on a 100X 100mm BEHnanoACquisity column at a flow rate of 400 nl/min. 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 PD (Proteome scanner 1.4, thermo) was used for the search. The search database is Mascot. The parent ion error was set to 20ppm, the fragment ion error was set to 1Da, the digestion mode was non-digestion, and the modification was oxidation (M) of methionine. Data card value, Percolator algorithm, FDR < 1%. The differentially expressed polypeptide peak of mass to charge (m/z) 1089.2 was retrieved in the database and identified as the KRT10 protein.

Compared with the normal control group, the KRT10 protein is highly expressed in the urine of the patients with normal pregnant women (N) and Gestational Diabetes (GDM) groups, as shown in figure 2, the KRT10 protein has significant difference in the expression in the urine of three groups of women with normal gestational age, women with normal pregnancy and patients with gestational diabetes, but has no significant difference in the urine of two groups of women with normal pregnancy and patients with gestational diabetes.

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

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Claims

1. Use of urine keratin type I cytoskeletal 10 (KRT10, also known as cytokeratin 10) protein and polypeptide fragments thereof in the preparation of a formulation for monitoring normal pregnancy.

2. The use of claim 1, wherein the amino acid sequence of the KRT10 protein in urine 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 of claim 1, wherein said KRT10 protein and polypeptide fragments thereof are derived from urine.

4. The use of claim 1, wherein said urinary KRT10 protein and polypeptide fragments thereof are highly expressed in normal pregnant women.

5. The use of claim 1, wherein the preparation is KRT10 protein and its polypeptide fragment detection kit from urine of normal pregnant women.

6. The use of claim 5, wherein the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding KRT10 protein 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 KRT10 protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: KRT10 protein control product, and humanized label antibody quality control product; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

9. The use of claim 8, wherein the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.