CN112904014B

CN112904014B - Application of urine TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Info

Publication number: CN112904014B
Application number: CN201911229413.6A
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: 2023-01-13
Anticipated expiration: 2039-12-04
Also published as: CN112904014A

Abstract

The invention provides application of human urine TBC1 domain family member 5 subtype a (TBC 1 domain family member 5 isofomm a, TBC 1D5a) and polypeptide fragments thereof, in particular application of urine TBC1D5a protein and polypeptide fragments thereof in preparation of preparations for monitoring normal pregnancy or gestational diabetes. The research proves that compared with a normal women group (normal control) with the normal reproductive age, the expression of the TBC1D5a and the polypeptide fragment thereof in normal pregnancy and gestational diabetes groups is increased, and the TBC1D5a and the polypeptide fragment thereof can be used for monitoring and auxiliary diagnosis of patients with gestational states or gestational diabetes. The invention exerts the advantages of non-invasive urine specimen acquisition, large-scale repeated sampling and convenient preservation, and utilizes the urine specimen to detect the urine TBC1D5a protein and the polypeptide fragment thereof.

Description

Application of urine TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Technical Field

The invention relates to a novel application of urine TBC1 domain family member 5 subtype a (TBC 1D5 a) protein and a polypeptide fragment thereof, in particular to an application of urine TBC1D5a 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 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 with gestational diabetes, relieve the pain of blood sampling for multiple times in gestational blood sugar detection, formulate more refined and personalized monitoring indexes aiming at different gestational periods, and expect to realize painless, convenient, quick and easily repeated urine detection or monitoring of the in vivo sugar level and sugar metabolism conditions through the research of urine protein or polypeptide, thereby laying a foundation for the further research of urine polypeptide detection kits.

Human TBC1 domain family member subtype 5a (TBC 1D5 a) protein is one of the members of the TBC family protein. Most of TBC protein is a down-regulated molecule of a specific Rab protein, and can selectively promote the hydrolysis and inactivation of the specific Rab protein. Through combining with RAB7 to form functional Rab-GAP, ATG9 transport regulation, autophagy regulation and reverse transport complex regulation function are exerted. Autophagy generally maintains the metabolism of stressed cells by promoting intracellular catabolism and nutrient cycling. For stresses requiring increased glycolytic demand, the core autophagy mechanism also promotes glucose uptake and glycolytic flux by promoting cell surface expression of the glucose transporter GLUT1/SLC2 A1. During metabolic stress, the LC3+ autophagy compartment binds and sequesters the rabgap protein TBC1D5 away from its inhibitory interaction with the reverse transcriptase complex, thereby recruiting reverse transcriptase to the endosomal membrane and GLUT1 plasma membrane translocation. In contrast, in autophagy deficient cells, TBC1D 5's inhibitory interaction with reverse transcriptase remains unchanged, resulting in the incorrect entry of glut1 into the lysosomal internal compartment. In addition, deletion of TBC1D5 in autophagy-deficient cells rescues recruitment of reverse transcriptase to the plasma membrane and circulation to the GLUT1 surface. Thus, during metabolic stress, TBC1D5 shuttles to autophagosomes to facilitate reverse transcription dependent GLUT1 transport. However, the mechanism of physiological changes of TBC1D5 in pregnancy is not reported in the literature.

Compared with the common clinical blood sample, the urine can be collected in a large amount in a completely noninvasive, continuous and continuous way; without homeostatic regulation, more kinds of changes and larger range 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 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 human TBC1 domain family member 5 subtype a (TBC 1D5 a) protein and polypeptide fragments thereof in preparation of preparations for monitoring normal pregnancy or gestational diabetes.

<xnotran> , TBC1D5a SEQ ID NO.1 (MYHSLSETRH PLQPEEQEVG IDPLSSYSNK SGGDSNKNGR RTSSTLDSEG TFNSYRKEWE ELFVNNNYLA TIRQKGINGQ LRSSRFRSIC WKLFLCVLPQ DKSQWISRIE ELRAWYSNIK EIHITNPRKV VGQQDLMINN PLSQDEGSLW NKFFQDKELR SMIEQDVKRT FPEMQFFQQE NVRKILTDVL FCYARENEQL LYKQGMHELL APIVFVLHCD HQAFLHASES AQPSEEMKTV LNPEYLEHDA YAVFSQLMET AEPWFSTFEH DGQKGKETLM TPIPFARPQD LGPTIAIVTK VNQIQDHLLK KHDIELYMHL NRLEIAPQIY GLRWVRLLFG REFPLQDLLV VWDALFADGL SLGLVDYIFV AMLLYIRDAL ISSNYQTCLG LLMHYPFIGD VHSLILKALF LRDPKRNPRP VTYQFHPNLD YYKARGADLM NKSRTNAKGA PLNINKVSNS LINFGRKLIS PAMAPGSAGG PVPGGNSSSS SSVVIPTRTS AEAPSHHLQQ QQQQQRLMKS ESMPVQLNKG DVVTGSDAQV SVPVQTLTDL QGLSSKNISS SPSVESLPGG REFTGSPPSS ATKKDSFFSN ISRSRSHSKT MGRKESEEEL EAQISFLQGQ LNDLDAMCKY CAKVMDTHLV NIQDVILQEN LEKEDQILVS LAGLKQIKDI LKGSLRFNQS QLEAEENEQI TIADNHYCSS GQGQGRGQGQ SVQMSGAIKQ ASSETPGCTD RGNSDDFILI SKDDDGSSAR GSFSGQAQPL RTLRSTSGKS QAPVCSPLVF SDPLMGPASA SSSNPSSSPD DDSSKDSGFT IVSPLDI); </xnotran> 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 TBC1D5a protein and polypeptide fragments thereof are from urine.

Preferably, the urine TBC1D5a protein and polypeptide fragments thereof are highly expressed in normal gestation or gestation diabetes patients.

Preferably, the preparation is a TBC1D5a protein and polypeptide fragment detection kit for urine of 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 TBC1D5a 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 TBC1D5a protein standard, a humanized tag antibody standard; preferably, the quality control product comprises: TBC1D5a 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 specimens of normal women of childbearing age, normal pregnant women and pregnant diabetics, centrifugates the urine specimens, takes supernate, and utilizes weak cation exchange magnetic beads to purify and separate the urine specimens. 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, obtains the TBC1D5a protein with differential expression by searching a database, and compared with a normal fertile age group, the TBC1D5a protein is highly expressed in urine of normal pregnant women and pregnant diabetic groups.

Compared with the normal childbearing group, the TBC1D5a protein and the polypeptide fragment thereof are high in expression 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 the urine TBC1D5a 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 non-invasive urine specimen acquisition, large-scale repeated sampling and convenient preservation, and utilizes the urine specimen to detect the urine coagulation TBC1D5a 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 distribution graph of peak area of TBC1D5a protein in urine of women of normal child bearing age, women of normal pregnancy and patients with gestational diabetes.

FIG. 2 is a scattergram of TBC1D5a protein expressed in urine samples from women of normal child bearing age, women of normal pregnancy and gestational diabetic patients.

FIG. 3 is a scatter plot of expression of TBC1D5a protein in the GDM-M and GDM-L groups.

FIG. 4 is a comparison of the uniformity of TBC1D5a protein expression in the mid-gestation period of gestation diabetes patients compared to Fasting Plasma Glucose (FPG).

FIG. 5 is a comparison of the uniformity of expression of TBC1D5a protein and glycated hemoglobin (HbA 1C%) in mid-gestation of a gestational diabetic patient.

Fig. 6 is a ROC curve of urine TBC1D5a protein expressed in urine of gestational diabetes patients.

Detailed Description

Example 1Collection and processing of urine specimens

60 gestational diabetes mellitus patients (GDM) are collected in an obstetrical clinic and inpatient clinic diagnosis department outpatient service of Beijing century bed hospital affiliated to capital medical university, wherein 30 gestational mid-term (GDM-M, 13-27 weekends) samples and 30 gestational late-term (GDM-L, 28 weeks and beyond) 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 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 with mental and intellectual disabilities. A comparison of the clinical characteristics of these subjects is shown in Table 1-1.

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 point, a total of 400 laser shots (50 shots for each of the 8 different positions of each crystallisation point) were made, and the average value represented a sample, thereby obtaining a polypeptide map for all samples. Three biological replicates per sample were performed. 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) 1290.6 was statistically different between the 3 groups (P < 0.05) with the distribution of the average peak areas 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 the solution was equilibrated to a 100X 100mm BEHnanoACquisity 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 card value, percolator algorithm, FDR < 1%. The differentially expressed polypeptide peak of mass to charge (m/z) 1290.6 was retrieved in the database and identified as TBC1D5a protein.

Compared with the normal control group, TBC1D5a protein is highly expressed in urine of normal pregnant women (N) and Gestational Diabetes (GDM) patients, as shown in fig. 2, TBC1D5a protein has significant differences in expression in urine of three groups of normal pregnant women, normal pregnant women and gestational diabetes patients, and also in two groups of normal pregnant women and gestational diabetes patients.

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 value of the TBC1D5a protein in the GDM-M polypeptide is higher than that of the GDM-L polypeptide, and the difference is not significant.

Trend analysis is carried out on TBC1D5a protein and Fasting Plasma Glucose (FPG) and glycosylated hemoglobin (HbA 1C%) results detected when OGTT is performed in the middle of gestation of a GDM patient, and the trend is relatively consistent with the change trend of FPG and HbA 1C%. As shown in fig. 4 and 5.

Further, the inventor detects 1290.6 polypeptide peak in urine of 60 gestational diabetes patients and 31 normal pregnant women, establishes a ROC curve, the area under the curve is 0.661, as shown in FIG. 6, the sensitivity and specificity of the reaction TBC1D5a 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 TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

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Claims

1. The application of urine human TBC1 domain family member 5 subtype a protein TBC1D5a in the preparation of a preparation for monitoring normal pregnancy of women or diabetes in women pregnancy is disclosed, wherein the amino acid sequence of the urine TBC1D5a protein is shown as SEQ ID NO. 1.

2. The use of claim 1, wherein the urinary TBC1D5a protein is highly expressed in a normal pregnant woman or a pregnant diabetic patient.

3. The use of claim 1, wherein the formulation is a TBC1D5a protein assay kit from urine of 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 TBC1D5a 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 TBC1D5a protein standard, a humanized tagged antibody standard; the quality control product comprises: TBC1D5a 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.