CN112881689B

CN112881689B - Application of urine blood coagulation factor IX protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Info

Publication number: CN112881689B
Application number: CN201911198521.1A
Authority: CN
Inventors: 张曼; 胡智颖
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2023-02-10
Anticipated expiration: 2039-11-29
Also published as: CN112881689A

Abstract

The invention provides an application of urine Coagulation Factor IX (Coagulation Factor IX) protein and a polypeptide fragment thereof, in particular to an application of the urine Coagulation Factor IX protein and the polypeptide fragment thereof in preparation of a preparation for monitoring normal pregnancy or gestational diabetes. The research proves that compared with a group of women with normal reproductive age (normal control), the expression of the blood coagulation factor IX and the polypeptide fragment thereof in a normal pregnancy and gestational diabetes group is increased, and the blood coagulation factor IX and the polypeptide fragment thereof can be used for monitoring and auxiliary diagnosis of patients with gestational state or gestational diabetes. 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 coagulation factor IX protein and the polypeptide fragment thereof.

Description

Application of urine blood coagulation factor IX protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Technical Field

The invention relates to new application of urine blood coagulation factor IX protein and polypeptide fragments thereof, in particular to application of the urine blood coagulation factor IX protein and the polypeptide fragments thereof in monitoring normal pregnancy or gestational diabetes.

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 of adverse pregnancy ending of gestational diabetes women such as gestational hypertension, abortion, cesarean section, premature delivery, newborn hypoglycemia, and neonatal hyperbilirubinemia 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 the gestational diabetes patients, relieve the pain of blood sampling for multiple times of gestational blood sugar detection, and formulate more refined and personalized monitoring indexes aiming at different gestational periods, and hope to realize painless, convenient, rapid 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 also lay a foundation for the further research of the urine polypeptide detection kit.

The blood coagulation factor IX is a molecule playing an important role in a blood coagulation mechanism, participates in the activation process of prothrombin endogenous, changes various hormone levels in a gestation body, particularly obviously increases the estrogen level, and the estrogen can promote the generation of the blood coagulation factor and fibrin of an organism to increase and reduce the antithrombin level. Compared with healthy pregnant women, the expression levels of the coagulation factors VIII and IXa and the activity of vWF of the GDM pregnant women are obviously increased. The risk of the arteriovenous thrombus stroke of the GDM patient is high, and the blood coagulation factor IX is one of the important reasons of the thrombus stroke. Similarly, the fibrinogen level of the GDM patient is higher than that of a healthy pregnant woman, so that the hypercoagulable state of the blood of the GDM patient is more obvious, and the hypercoagulable state of the blood of the pregnant woman during pregnancy is caused, and the slight hypercoagulability of the blood has positive effects on maintaining the integrity of fetal membranes, quickly stopping bleeding after delivery and regenerating endometrium, and is a physiological protection mechanism. The results of the polypeptide detected in the urine are completely consistent with the increase of the coagulation factor IX and the increase of the content of plasma fibrinogen in the change of maternal blood systems during pregnancy, and the blood is in a high coagulation state.

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 coagulation factor IX protein and polypeptide fragments thereof in preparation of preparations for monitoring normal pregnancy or gestational diabetes.

Preferably, the amino acid sequence of the urine coagulation factor IX is shown in SEQ ID No.1 (MQRVNMIMAE SPGLITICLL GYLLSAECTV FLDHENANKI LNRPKRYNSG KLEEFVQGNL ERECMEEKCS FEEAREVFEN TERTTEFWKQ YYDGDGCESN PCLNGGSCKD DINSYECWCP FQFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVL HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL T); 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 coagulation factor IX protein and polypeptide fragments thereof are from urine.

Preferably, the urine coagulation factor IX protein and polypeptide fragments thereof are highly expressed in patients with normal pregnancy or gestational diabetes.

Preferably, the preparation is a blood coagulation factor IX protein and polypeptide fragment detection kit for patients with normal pregnancy or gestational diabetes.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to factor IX 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 the second antibody diluent.

Preferably, the standard comprises a coagulation factor IX protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: a blood coagulation factor IX protein control product and a 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 first and second electrodes are formed of a metal, 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 sample to be spotted on an Anchorchip target plate, carrying out mass spectrometry after ionizing the sample, collecting data within the range of 1000-10000Da, and obtaining a mass spectrum 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, obtains the blood coagulation factor IX protein with differential expression by database retrieval, and the blood coagulation factor IX protein is highly expressed in urine of normal pregnant women and gestational diabetes patients compared with the normal child-bearing group.

Compared with the normal reproductive age group, the blood coagulation factor IX protein and the polypeptide fragment thereof are highly expressed in the urine of normal pregnant women and pregnant diabetics, and have better consistency with fasting blood glucose and glycosylated hemoglobin. Therefore, the urine blood coagulation factor IX protein and the polypeptide fragment thereof can be used for monitoring or auxiliary diagnosis of normal pregnancy or gestational diabetes.

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 coagulation factor IX 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 profile of coagulation factor IX protein in urine from women of normal reproductive age, women of normal pregnancy and gestational diabetic patients.

FIG. 2 is a scatter plot of the expression of coagulation factor IX protein in urine samples from women of normal child bearing age, women of normal pregnancy and gestational diabetes.

FIG. 3 is a scattergram of the expression of coagulation factor IX protein in the GDM-M and GDM-L groups.

FIG. 4 is a comparison of the uniformity of coagulation factor IX protein expression in the mid-gestation period with Fasting Plasma Glucose (FPG) in gestational diabetic patients.

FIG. 5 is a comparison of the uniformity of the expression of coagulation factor IX protein and glycated hemoglobin (HbA 1C%) in mid-gestation of gestational diabetic patients.

FIG. 6 is a ROC curve of urine coagulation factor IX protein expression in urine of 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 construction of files are carried out in our hospital from the early pregnancy (8-12 weeks) by diabetics in the gestational period and normal pregnant women, and the data are complete;

the pregnancy carries 75g Oral Glucose Tolerance Test (OGTT) 24-28 weeks, all of which conform to the pregnancy in the ninth edition "gynaecology and obstetricsStandard for diagnosis of diabetes during pregnancy; exclusion criteria:

acute and chronic infectious, tumor, cardiovascular disease;

impaired glucose tolerance has existed in the past;

severe liver and kidney dysfunction;

those with mental 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 calibrating the instrument with the standard, 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. Ionizing the sample by nitrogen laser, analyzing mass spectrum, collecting data within 1000-10000Da range, and obtaining mass spectrogram comprising 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 peak of mass-to-charge ratio (m/z) 1079.2 has statistical differences between 3 groups (P < 0.05), and the distribution of the average peak area 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). 100% A solution was equilibrated on a 100X 100mm BEHnonaACquisity column with a flow rate of 400nl/min. The chromatographic spray needle is PN: FS360-20-10-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 300-2000m/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 17,500 with a resolution of 70,000,MS2. A search was performed using the data analysis software PD (Proteome discover 1.4, thermo). The search database is Mascot. The parent ion error was set to 20ppm, the fragment ion error was set to 1Da, the digestion system was non-digestion, and the modification was Oxidation (M) of methionine. Data calorie value, percolator algorithm, FDR is less than or equal to 1 percent. The differentially expressed polypeptide peak of mass-to-charge ratio (m/z) 1079.2 was retrieved in the database and identified as a coagulation factor IX protein.

Compared with the normal control group, the blood coagulation factor IX protein is highly expressed in the urine of the pregnant woman (N) with normal pregnancy and the diabetic (GDM) with pregnancy, as shown in figure 2, the expression of the blood coagulation factor IX protein in the urine of three groups of women with normal gestational age, women with normal pregnancy and diabetic patients with pregnancy is significantly different, and the difference is also significant in two groups of the women with normal pregnancy and diabetic patients with pregnancy.

Further, gestational Diabetes Mellitus (GDM) is divided into a middle gestational period (GDM-M, 13-27 weekend) and a late gestational period (GDM-L, 28 weeks and later) according to gestational weeks, as shown in fig. 3, the peak values of the blood coagulation factor IX protein in the GDM-M polypeptide are higher than those of the GDM-L polypeptide, and the difference is not significant.

Trend analysis is carried out on the coagulation factor IX protein and Fasting Plasma Glucose (FPG) and glycosylated hemoglobin (HbA 1C%) results detected when OGTT is performed in the middle gestation period of a GDM patient respectively, and the trend analysis is consistent with the change trend of FPG and HbA 1C%. As shown in fig. 4 and 5.

Further, the inventor detects the polypeptide peak value of 1079.2 in urine of 60 gestational diabetes patients and 31 normal pregnant women, establishes a ROC curve, the area under the curve is 0.631, and as shown in FIG. 6, the sensitivity and specificity of the reaction coagulation factor IX protein are good.

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 coagulation factor IX protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

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Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala

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Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly

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Claims

1. The application of urine coagulation factor IX protein in preparing a preparation for monitoring normal pregnancy of women is disclosed, wherein the amino acid sequence of the urine coagulation factor IX protein is shown as SEQ ID No. 1.

2. The use of claim 1, wherein said urinary coagulation factor IX protein is highly expressed in a normally pregnant woman.

3. The use of claim 1, wherein the formulation is a urine coagulation factor IX protein assay kit for a normal pregnant woman.

4. The use of claim 3, wherein the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to factor IX protein.

5. 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.

6. The use of claim 5, wherein the standard comprises a coagulation factor IX protein standard, a humanized tag antibody standard; the quality control product comprises: a blood coagulation factor IX protein quality control product and a humanized label antibody quality control product; the solid phase carrier comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

7. The use of claim 6, wherein the solid phase carrier is selected from the group consisting of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.