CN112924682A

CN112924682A - Application of urine fibrinogen alpha chain protein and polypeptide fragment thereof in normal pregnancy

Info

Publication number: CN112924682A
Application number: CN201911233918.XA
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: CN112924682B

Abstract

The invention provides an application of a urine Fibrinogen alpha chain (FGA) protein and a polypeptide fragment thereof, in particular to an application of a urine FGA protein and a polypeptide fragment thereof in preparation of a preparation for monitoring normal pregnancy. The research proves that compared with a normal women group with the reproductive age (normal control), the FGA protein and the polypeptide fragment thereof have higher expression in a normal pregnancy group and can be used for monitoring the pregnancy state. 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 FGA protein and the polypeptide fragment thereof.

Description

Application of urine fibrinogen alpha chain protein and polypeptide fragment thereof in normal pregnancy

Technical Field

The invention relates to a new application of urine fibrinogen alpha chain (FGA) protein and a polypeptide fragment thereof, in particular to an application of urine FGA protein and a polypeptide fragment 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.

Fibrinogen is converted to fibrin monomers by the action of thrombin. The in vivo various hormone levels change during pregnancy, especially the estrogen level is obviously increased, the estrogen can promote the protogenesis of body blood coagulation factors and fibrin to increase, the antithrombin level is reduced, which leads to the high coagulation state of the pregnant woman blood during pregnancy, the slight high coagulation of the blood has positive effects on maintaining the integrity of fetal membranes, rapidly stopping bleeding after delivery and regenerating endometrium, and the physiological protection mechanism is realized. The results of the polypeptide detected in the urine are completely consistent with the increase of the fibrinogen content in the plasma in the change of the maternal blood system during the gestation period, 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 sampling for multiple times in blood sugar detection of pregnant women, the experiment hopes to realize the monitoring and understanding of the pregnancy state through the detection of urine protein or polypeptide on the basis of the exploration of the methodology in the early stage.

Disclosure of Invention

The invention aims to provide application of a urinary fibrinogen alpha chain (FGA) protein and a polypeptide fragment thereof in preparation of a preparation for monitoring normal pregnancy.

Preferably, the amino acid sequence of said urine FGA is shown in SEQ ID No.1 (MFSMRIVCLV LSVVGTAWTA DSGEGDFLAE GGGVRGPRVV ERHQSACKDS DWPFCSDEDW NYKCPSGCRM KGLIDEVNQD FTNRINKLKN SLFEYQKNNK DSHSLTTNIM EILRGDFSSA NNRDNTYNRV SEDLRSRIEV LKRKVIEKVQ HIQLLQKNVR AQLVDMKRLE VDIDIKIRSC RGSCSRALAR EVDLKDYEDQ QKQLEQVIAK DLLPSRDRQH LPLIKMKPVP DLVPGNFKSQ LQKVPPEWKA LTDMPQMRME LERPGGNEIT RGGSTSYGTG SETESPRNPS SAGSWNSGSS GPGSTGNRNP GSSGTGGTAT WKPGSSGPGS TGSWNSGSSG TGSTGNQNPG SPRPGSTGTW NPGSSERGSA GHWTSESSVS GSTGQWHSES GSFRPDSPGS GNARPNNPDW GTFEEVSGNV SPGTRREYHT EKLVTSKGDK ELRTGKEKVT SGSTTTTRRS CSKTVTKTVI GPDGHKEVTK EVVTSEDGSD CPEAMDLGTL SGIGTLDGFR HRHPDEAAFF DTASTGKTFP GFFSPMLGEF VSETESRGSE SGIFTNTKES SSHHPGIAEF PSRGKSSSYS KQFTSSTSYN RGDSTFESKS YKMADEAGSE ADHEGTHSTK RGHAKSRPVR DCDDVLQTHP SGTQSGIFNI KLPGSSKIFS VYCDQETSLG GWLLIQQRMD GSLNFNRTWQ DYKRGFGSLN DEGEGEFWLG NDYLHLLTQR GSVLRVELED WAGNEAYAEY HFRVGSEAEG YALQVSSYEG TAGDALIEGS VEEGAEYTSH NNMQFSTFDR DADQWEENCA EVYGGGWWYN NCQAANLNGI YYPGGSYDPR NNSPYEIENG VVWVSFRGAD YSLRAVRMKI RPLVTQ); 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 FGA protein and polypeptide fragments thereof are from urine.

Preferably, the urinary FGA protein and polypeptide fragments thereof are highly expressed in normal pregnant women.

Preferably, the preparation is a detection kit for the urine FGA protein of a normal pregnant woman and a polypeptide fragment thereof.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to FGA 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 an FGA protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: FGA 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, obtains the FGA protein with differential expression by database retrieval, and compared with a normal child-bearing group, the FGA protein is highly expressed in urine of normal pregnant women and pregnant diabetic patients.

Compared with the normal group of child bearing age, the FGA protein and the polypeptide fragment thereof are proved by research to be highly expressed in the urine of normal pregnant women and patients with gestational diabetes. Therefore, the detection of the urine FGA protein and the polypeptide fragment thereof can be used for monitoring or auxiliary diagnosis of normal pregnancy.

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 FGA 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 graph of the peak area distribution of FGA protein in urine of women of normal childbearing age, women of normal pregnancy and gestational diabetic patients.

FIG. 2 is a scattergram of FGA protein expression in urine samples from women of normal child bearing age, women of normal pregnancy and gestational diabetes.

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;

existed in the pastImpaired glucose tolerance;

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 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) 2661.8 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) 2661.8 was retrieved in the database and identified as an FGA protein.

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

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 urinary fibrinogen alpha chain (FGA) protein and polypeptide fragments thereof in the preparation of a formulation for monitoring normal pregnancy.

2. The use according to claim 1, wherein the amino acid sequence of said urinary FGA protein 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. The use according to claim 1, wherein the FGA protein and polypeptide fragments thereof are derived from urine.

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

5. The use of claim 1, wherein the agent is a kit for detecting FGA protein and its polypeptide fragments in urine of normal pregnant women.

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 FGA 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 FGA protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: FGA 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.