CN112904012B

CN112904012B - Application of urine IGKC protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Info

Publication number: CN112904012B
Application number: CN201911228349.XA
Authority: CN
Inventors: 张曼; 胡智颖
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2023-01-17
Anticipated expiration: 2039-12-04
Also published as: CN112904012A

Abstract

The invention provides an application of a urine Immunoglobulin kappa constant region (IGKC) and a polypeptide fragment thereof, in particular to an application of urine IGKC protein and a polypeptide fragment thereof in preparing a preparation for monitoring normal pregnancy or gestational diabetes. The research proves that compared with a normal women group (normal control) with normal childbearing age, the expression of the IGKC and the polypeptide fragment thereof is increased in normal pregnancy and gestational diabetes groups, and the IGKC 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 IGKC protein and the polypeptide fragment thereof.

Description

Application of urine IGKC protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Technical Field

The invention relates to new application of urine immunoglobulin kappa constant region (IGKC) protein and a polypeptide fragment thereof, in particular to application of urine IGKC protein and a polypeptide fragment thereof in monitoring normal pregnancy or gestational diabetes.

Background

In the course of pregnancy, in order to meet the requirements of fetal growth and development, the reproductive, blood circulation, urinary, respiratory, digestive and endocrine systems of pregnant women undergo a series of changes, in particular the endocrine system, the secretion of gonadotropic hormone is reduced, and the secretion of prolactin, thyroid stimulating hormone, adrenocorticotropic hormone, glucocorticoid, aldosterone, thyroid stimulating hormone and the like is increased. In the middle and late gestation period, part of pregnant women have reduced sensitivity to insulin, and have decomposition and antagonism on insulin, so that insulin activity is reduced, and the pregnant women cannot normally compensate for the physiological change to cause Gestational Diabetes Mellitus (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 immunoglobulin kappa constant region is a membrane-bound or secreted glycoprotein produced by B lymphocytes as part of an immunoglobulin. In the recognition phase of humoral immunity, membrane-bound immunoglobulins act as receptors, triggering clonal expansion of B lymphocytes and differentiation into plasma cell-secreting immunoglobulins upon specific antigen binding. The secreted immunoglobulins mediate the effector phase of humoral immunity, thereby eliminating bound antigen. In order to maintain the progress of pregnancy, there is a significant change in maternal immune function: the shift of immune function from Th1 to Th2 during pregnancy, suppression of cellular immune function and predominance of humoral immune function, are beneficial for maintaining pregnancy. Compared with normal pregnant women, the levels of IgA, igE, igM and IgE of GDM patients who can well control blood sugar through diet control have no obvious change, but the IgE of the GDM patients who need to be subjected to an insulin treatment group is obviously increased and the IgG is obviously reduced, which indicates that the GDM patients have humoral immunity regulation disorder, and the heavier the disease degree is, the more obvious the function damage is. Although there is an increase and decrease in the level of immunoglobulin in humoral immunity in GDM patients, this experiment shows that the secretion of immunoglobulin light chain kappa type in GDM patients is increased as a whole, compared to normal pregnant women.

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 can be quickly excreted into urine after entering blood, and the probability that the proteins and polypeptides are detected in the urine is much higher than that in the blood; before urine is collected, a possible protein degradation process in urine is completed, so that urine protein can be kept stable for a long 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 immunoglobulin kappa constant region (IGKC) protein and polypeptide fragments thereof in preparing preparations for monitoring normal pregnancy or gestational diabetes.

Preferably, the amino acid sequence of the urinary IGKC is shown in SEQ ID NO.1 (RTVAAPSVFI FPPSDEQLKS GTASVVCLN NFYPREADVQ WKVDNALQSG NSQEVTEQD SKDSTYLSS TLTLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC); 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 IGKC protein and polypeptide fragments thereof are from urine.

Preferably, the urine IGKC protein and the polypeptide fragment thereof are highly expressed in patients with normal pregnancy or gestational diabetes.

Preferably, the preparation is a urine IGKC 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 IGKC 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 an IGKC protein standard and a humanized tag antibody standard; preferably, the quality control product comprises: IGKC protein control product and humanized label antibody quality control product; preferably, the solid support comprises: particles, 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, putting 1 mu l of sample on an Anchorchip target plate, ionizing the sample, performing mass spectrometry, and collecting data in the range of 1000-10000Da to obtain a mass spectrometry polypeptide diagram consisting of protein peaks with different mass-to-charge ratios. And (3) analyzing all mass spectrograms of normal reproductive age women, normal pregnant women and a pregnant diabetes group by using BioExplorer analysis software, and screening the 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 IGKC protein is obtained through database retrieval, and compared with a normal reproductive group, the IGKC protein is highly expressed in urine of normal pregnant women and a pregnant diabetes group.

Compared with the normal childbearing group, the IGKC protein and the polypeptide fragment thereof are proved by research to be highly expressed in urine of normal pregnant women and pregnant diabetes patients, and have better consistency with fasting blood glucose and glycosylated hemoglobin. Therefore, the detection of urine IGKC 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 sample, and utilizes the urine sample to detect the urine IGKC 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 IGKC protein in urine from normal women of childbearing age, normal pregnant women and gestational diabetic patients.

FIG. 2 is a scattergram of IGKC protein expression in urine samples from normal fertile women, normal pregnant women and gestational diabetic patients.

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

FIG. 4 is a comparison of the uniformity of IGKC protein expression in the middle of gestation with Fasting Plasma Glucose (FPG) in gestation diabetic patients.

FIG. 5 is a comparison of the uniformity of expression of IGKC protein in the middle of gestation in gestation diabetic patients with glycated hemoglobin (HbA 1C%).

FIG. 6 is a ROC curve of urine IGKC 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-stage 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 a file 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;

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

acute and chronic infection, tumor, cardiovascular disease;

previous presence of impaired 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. Ionizing the sample by irradiation of a nitrogen laser, performing mass spectrometry, and collecting 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. 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 with mass to charge ratio (m/z) 1500.7 were statistically different between the 3 groups (P < 0.05), 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 acetonitrile solution (2% acetonitrile) and liquid phase B was 0.1% formic acid acetonitrile solution (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 as 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 database was searched as 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 card value, percolator algorithm, FDR < 1%. The differentially expressed polypeptide peak with a mass to charge ratio (m/z) of 1500.7 was retrieved in the database and identified as an IGKC protein.

Compared with the normal control group, the IGKC protein is highly expressed in urine of pregnant women (N) with normal pregnancy and patients with Gestational Diabetes (GDM) groups, as shown in fig. 2, the IGKC protein has significant differences in urine of three groups of women with normal gestational age, women with normal pregnancy and patients with gestational diabetes, and also has significant differences in two groups of women with normal pregnancy and patients with gestational diabetes.

Further dividing Gestational Diabetes Mellitus (GDM) 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 figure 3, the peak values of the TBC1D5a protein in the GDM-M polypeptide are higher than the peak value of the GDM-L polypeptide, and the difference is not significant.

The IGKC protein is respectively subjected to trend analysis with Fasting Plasma Glucose (FPG) and glycosylated hemoglobin (HbA 1C%) results detected when OGTT is performed in the middle gestation period of a GDM patient, and the trend is consistent with the variation trend of FPG and HbA 1C%. As shown in fig. 4 and 5.

Further, the inventors detected the peak of 1500.7 polypeptide in urine of 60 gestational diabetes patients and 31 normal pregnant women, and established an ROC curve with an area of 0.677 under the curve, as shown in FIG. 6, which shows that the sensitivity and specificity of the reaction IGKC 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

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Claims

1. The application of urine immunoglobulin kappa constant region protein IGKC in preparing a preparation for monitoring normal pregnancy of women or diabetes in pregnancy of women is disclosed, wherein the amino acid sequence of the urine IGKC protein is shown as SEQ ID No. 1.

2. The use of claim 1, wherein the urinary IGKC protein is highly expressed in a normal pregnant woman or a pregnant diabetic patient.

3. The use of claim 1, wherein the preparation is a urine IGKC protein detection kit for a normal pregnant woman or a pregnant diabetic patient.

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 an IGKC 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 an IGKC protein standard, a humanized tag antibody standard; the quality control product comprises: IGKC protein control product and humanized label antibody quality control product; the solid phase carrier comprises: particles, 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.