Use of tctp protein in the preparation of non-invasive preterm birth diagnostic products

Abstract

The application discloses application of TCTP protein in preparation of noninvasive premature birth diagnosis products and relates to the technical field of biological medicine. The application can estimate the premature birth risk by detecting the TCTP protein content or the TCTP gene expression quantity in peripheral blood of a pregnant woman, the method only needs 0.5ml peripheral blood of the mother, is safe for the mother and infant, and has the advantages of simplicity, high sensitivity, low cost and simple operation. In 126 sample examples in the embodiment of the application, the premature birth detection rate of the method is 91%, and in the control group, the false positive rate is 8.6% (5 examples / 58 examples), so the method has high accuracy.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to application of TCTP protein in preparation of non-invasive premature birth diagnosis products. BACKGROUND

[0002] Premature birth accounts for 35.4% of the causes of global neonatal death, is the primary cause of neonatal death and birth defects, and is the main cause of death of children under the age of five. Premature birth is essentially related to abnormal initiation of delivery, so it is crucial to analyze the causes and mechanisms of delivery initiation. Predicting premature birth can strengthen prenatal care, reduce the incidence of premature birth, and improve the growth and development of premature infants.

[0003] At present, the methods for evaluating the risk of premature birth mainly include imaging examination, fetal fibronectin (fFN) detection and inflammatory marker detection. Among them, the imaging examination mainly measures the cervical length through transvaginal ultrasound to evaluate the cervical function state to evaluate the risk of premature birth, but the positive rate of predicting premature birth is low. Biochemical marker detection, such as fetal fibronectin (fFN) detection and inflammatory marker detection, has insufficient sensitivity and specificity.

[0004] Therefore, it is urgent in clinical practice to find non-invasive detection biomarkers for predicting and early diagnosing premature birth. For example, looking for molecular markers in maternal peripheral blood proteins, cfRNA, cfDNA and exosome RNA to reflect the pregnancy state, determine the gestational age and estimate the risk of premature birth. SUMMARY

[0005] Therefore, the application provides application of TCTP protein in preparation of non-invasive premature birth diagnosis products.

[0006] In order to achieve the above purpose, the application adopts the following technical scheme:

[0007] The application of the product for detecting TCTP protein in preparation of non-invasive premature birth diagnosis reagent, wherein the amino acid sequence of the TCTP protein is shown as SEQ ID NO. 3.

[0008] Preferably, the product is a kit.

[0009] Another object of the application is to provide application of the product for detecting TCTP gene expression in preparation of non-invasive premature birth diagnosis reagent, wherein the amino acid sequence coded by the TCTP gene is shown as SEQ ID NO. 3.

[0010] Preferably, the product is a probe set or a kit.

[0011] More preferably, the kit further comprises reagents for extracting total RNA from peripheral blood, and / or reagents for reverse transcription of cDNA from total RNA, and / or reagents for quantitative PCR of cDNA.

[0012] Advantages:

[0013] 1. The present application can estimate the risk of premature birth by detecting the content of TCTP protein or the expression amount of TCTP gene in the peripheral blood of pregnant women, which only needs 0.5 ml of peripheral blood of the mother, is safe for mother and infant, and has the advantages of simple method, high sensitivity, low cost and simple operation.

[0014] 2. In the 126 sample examples of the present application, the detection rate of premature birth by the method is 91%, and the false positive rate in the control group is 8.6% (5 / 58), so the method has high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0016] Figure 1 For the identification of cell subtypes in lung tissue of different gestational periods and adult mice; Epi-AT1, type I alveolar epithelial cells; Epi-AT2, type II alveolar epithelial cells; Ciliated cells, ciliated epithelial cells; Club cells, ciliated cells; Differentiation cells, differentiation cells; Fibroblast, fibroblast; Endothelial cell, endothelial cell; Lymphocyte, lymphocyte; Myeloid cell, myeloid cell.

[0017] Figure 2 For identifying molecular markers of cell subtypes in mouse lung tissue.

[0018] Figure 3 For the expression and distribution of Epi-AT1-Hopx + cells in lung tissue of different gestational periods and adult mice; E15.5: E15.5 fetal lung; E18.5: E18.5 fetal lung; Labor: fetal lung at delivery; Adult-8w: adult lung tissue. DAPI (blue) is a nuclear marker, and PDPN (green) and Hopx (red) are type I lung epithelial cell markers.

[0019] Figure 4 The proportion of different subgroups of alveolar epithelial cells in different gestational stages, neonatal mice and adult mice.

[0020] Figure 5 The proportion of different subgroups of alveolar epithelial cells in different developmental stages (E12.5, i.e. 12.5-day pregnant fetal mice, E18.5, i.e. 12.5-day pregnant fetal mice, Neonatal, i.e. 0.5-day-old neonatal mice, Adult, i.e. 8-week-old adult mice), wherein the left graph is the percentage proportion and the right graph is the actual proportion.

[0021] Figure 6 The distribution of up-regulated genes in E18.5 mouse fetal lung epithelial cells.

[0022] Figure 7 The expression distribution of TCTP gene in different subgroups of fetal lung epithelial cells in different developmental stages.

[0023] Figure 8 The transcription level and protein expression level of TCTP gene in lung tissues of fetal mice in different gestational stages.

[0024] Figure 9 The expression level of TCTP gene in mRNA and encoded protein in the fetal lung of preterm mice (RU-PTL) and control mice (TM-PTNL) in the same gestational stage.

[0025] Figure 10 The standard curve of TCTP protein determination (r>0.99).

[0026] Figure 11 The TCTP protein in the serum of preterm group mothers was significantly increased, P<0.001. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0028] Example 1

[0029] 1. Single-cell transcriptome sequencing results from lung tissues of mid-pregnancy (E12.5) (GSM4504959), late-pregnancy (E18.5) (GSM5343154, GSM5343155), neonatal mice (GSM4647247, GSM4647248), and adult mice (GSM4647254, GSM4647255) in public databases were analyzed using the Seurat software package in R. The analysis workflow included data standardization, differential gene identification, and dimensionality reduction clustering. Cell types were visualized using the DimPlot function in the Seurat package (see Appendix). Figure 1 Molecular markers for identifying cell types were visualized using ggplot2 software (see appendix). Figure 2 The combined results indicate that the type I alveolar epithelial cell subset Epi-AT1-Hopx is specifically present in fetal lung tissue during late pregnancy (E18.5).

[0030] 2. Lungs from pregnant mice (E15.5, E18.5, at parturition, and adult mice) were collected for immunofluorescence staining. The specific method was as follows:

[0031] (1) Sampling and paraffin sectioning: After treating the lungs with 4% paraformaldehyde for 24 hours, rinsed with PBS for 10 minutes, and then embedded in paraffin, the paraffin solidified overnight and was sectioned using a microtome. The lungs were then observed under a microscope to select a suitable field of view.

[0032] (2) Dewaxing: Place the paraffin sections on the slide rack with the same orientation and bake them in a constant temperature oven at 55°C for 30 minutes; at the same time, put the first dewaxing solution tank into the constant temperature oven at 55°C; put the paraffin sections and slide rack into the first dewaxing solution tank, and then take them out of the constant temperature oven and place them at room temperature. After 5 minutes, take out the sections and immerse them in the second dewaxing solution tank at room temperature. Then, put the paraffin sections into the tank in the order of dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3, or you can choose a gradient concentration of ethanol, for 5 minutes in each tank; rinse the sections with running water for 5 minutes.

[0033] (3) Staining: Completely cover the sample with 5% blank goat serum. The slides should be placed in a humidified chamber. For cell culture plates, the wells can be sealed directly and incubated in a 37°C constant temperature and humidity incubator for 30 min. Dilute the primary antibody 1:1000 and incubate the slides in the primary antibody dilution buffer at 4°C overnight. Recover the primary antibody and wash once with TBST for 5 min. Dilute the secondary antibody 1:5000. Incubate at room temperature in the dark for 1 h, and wash once with TBST buffer for 5 min. Add DAPI working solution to the sample and incubate at room temperature in the dark for 10 min. Remove the DAPI working solution and wash once with TBST buffer for 5 min. Wash three times with TBS buffer for 5 min each time. After adding anti-fluorescence attenuation mounting medium, observe and acquire images under a fluorescence microscope (see Appendix). Figure 3 ).

[0034] Immunofluorescence staining showed that the content of Epi-AT1-Hopx cells was significantly increased in the fetal lungs during late pregnancy (18.5 dpc), peaking at delivery, while this cell subset almost completely disappeared in the lung tissue of adult fetal rats. This result further suggests that this cell subset undergoes dramatic dynamic changes during fetal lung development and may be related to the initiation of labor.

[0035] 3. Regarding the appendix Figure 1 Further cluster analysis of lung epithelial cells in the lungs using the Seurat software package further confirmed the specific distribution of the Epi-AT1-Hopx cell subset in the fetal lungs during late pregnancy (see Appendix). Figures 4-5 To analyze the transition process of this cell subpopulation, we performed differential gene analysis on the sequencing results of mouse fetal lungs at various stages. We found that in the E18.5 fetal lungs, a series of genes related to protein translation and synthesis were significantly upregulated (see Appendix). Figure 6 The TCTP gene is specifically highly expressed in the E18.5 fetal lung Epi-AT1-Hopx cell subset (see Appendix). Figure 7 ).

[0036] 4. Collect mouse lungs at E12.5, E15.5, E18.5 and at the onset of labor, and analyze the mRNA expression of the TCTP gene and the expression level of the encoded TCTP protein in mouse fetal lung tissue using Realtime qPCR and Western blot experiments.

[0037] qPCR: (1) Total RNA extraction: Collected tissues were ground in liquid nitrogen and 1 ml of TRIzol (Invitrogen) was added. Total RMA was extracted according to the standard procedure. (2) cDNA was reverse transcribed using the PrimeScript RT Reagent Kit and then... qPCR SYBR Green Master Mix (No Rox) was used for qPCR amplification, and the primers were: forward primer GGCAAACTTGAAGAGCAGAAACC (SEQ ID NO. 1) and reverse primer TCACGGTAGTCCAGGAGAGCAA (SEQ ID NO. 2).

[0038] WB: (1) Gel preparation: take equal volume of lower gel solution and lower gel buffer, 2 mL each, mix well; add 40 μL of modified coagulant, mix well, then pour into the glass plate, and after the lower gel is solidified (about 15 min), pour off the upper water or alcohol; after solidification, take equal volume of upper gel solution and colored upper gel buffer, 0.5 mL each, mix well, add 10 μL of modified coagulant, mix well, pour into the glass plate, and insert the comb; after the upper gel is solidified, pull out the comb. (2) Electrophoresis: mix the protein sample to be tested with the loading buffer, then add to the sample well in the gel. At the same time, add the molecular weight marker to the sample well; (3) Membrane transfer: soak the PVDF membrane in anhydrous methanol in advance; prepare the membrane transfer device. Then put the membrane transfer tank with the membrane transfer device into an ice box, and use a constant current of 300 mA for membrane transfer for 100 min. (4) Blocking: block with 5% BSA / TBST for 1-2 h; (5) primary antibody incubation: dilute the primary antibody (TCTP protein, Cell Signaling Technology, #8441) with 5% BSA / TBST to 1:1000 dilution, and put the cut PVDF membrane into the primary antibody, incubate on a 4°C room shaker overnight; then take out the membrane, equilibrate at room temperature for 30 min; wash the membrane with 1xTBST for 3 times, 10 min each time; (6) secondary antibody incubation: dilute the secondary antibody (proteintech, SA00001-2) with 1xTBST, incubate for 1-2 h; wash the membrane with 1xTBST for 3 times, 10 min each time; (7) prepare the chemiluminescence solution for development and exposure.

[0039] It was confirmed by Realtime qPCR and Western blot experiments that the expression of mRNA and encoded TCTP protein of TCTP gene in mouse fetal lung tissue gradually increased with the progress of pregnancy, and reached a peak at delivery (see FIG. 1 and FIG. 2). Figure 8 ).

[0040] Example 2

[0041] To further confirm the correlation between TCTP gene and the initiation of delivery and premature birth, a mifepristone (RU486) induced mouse preterm labor model was constructed. The construction method was as follows: after 8-week female mice were pregnant for 8 days, mifepristone (RU486; 50 μg / 100 μL propylene glycol) or propylene glycol control was injected subcutaneously on the nape, and the fetal lung was taken by cesarean section at 15.5 days of pregnancy, qPCR experiment and WB experiment were performed, and the method was the same as that in Example 1.

[0042] The experimental results found that the mRNA and protein expression levels of TCTP in the fetal lung of the preterm mouse (RU-PTL) were significantly higher than those in the fetal lung of the control group mouse (injected with DMSO, P15.5d cesarean section) at the same gestational age (see Figure 2). Figure 9 The above results suggest that the expression level of TCTP in the fetal lung is closely related to the initiation of delivery.

[0043] Example 3

[0044] 0.5 ml of peripheral blood of 126 pregnant women was collected, including 68 cases of premature pregnant women who delivered less than 37 weeks and 58 cases of full-term non-labor control pregnant women, and the TCTP protein content was detected (amino acid sequence: miiyrdlishdemfsdiykireiadglclevegkmvsrtegniddsliggnasaegpegegtestvitgvdivmnhhlqetsftkeaykkyikdymksikgkleeqrpervkpfmtgaaeqikhilanfknyqffigenmnpdgmvalldyredgvtpymiffkdglkmekc, SEQ ID NO. 3). The specific detection method is as follows:

[0045] (1) The whole blood was placed in a test tube without anticoagulant, and was naturally agglomerated at room temperature for 30-60 min until the blood was coagulated. Centrifugation was performed at a speed of 2000-3000 rpm for 5-10 min, and 250 ul of supernatant was taken as serum.

[0046] (2) The TCTP protein content in the serum was detected by ELISA kit (CUSABIO, item number: CSB-EL024134HU), and the specific method included making standard curve and detecting sample to be tested.

[0047] ①Making standard curve: 1 ml of sample diluent was added to the TCTP recombinant protein standard, 250 ul of sample diluent was added to 7 centrifuge tubes, and then 100 uL was taken for detection after dilution according to the instruction.

[0048] ② Take 100 μL of the serum sample for testing. Add the standard and the sample to be tested to a 96-well plate pre-coated with TCTP antigen, fix the plate on, and incubate at 37 degrees Celsius for 2 hours;

[0049] ③ Discard the liquid, shake dry, add 100u1 of BioHome labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour;

[0050] ④ Discard the liquid inside the hole, wash the plate 3 times, soaking for 2 minutes each time, and then spin dry;

[0051] ⑤ Add 100 μL of horseradish peroxide-labeled avidin working solution to each well, attach the plate, incubate at 37°C for 1 hour, discard the wave medium inside the well, spin dry, wash the plate 5 times, soaking for 2 minutes each time, and spin dry.

[0052] ⑥ Add 90 μL of substrate solution sequentially, and incubate at 37°C in the dark for 30 minutes for color development;

[0053] ⑦ Add 50 μL of stop solution to terminate the reaction. Then, use a microplate reader to measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm.

[0054] ⑧ Use Curve Expert 1.4.zip software (https: / / www.cusabio.cn / statics / 200822010756694.rar) to plot the standard curve (see appendix). Figure 10 The TCTP protein content in the sample to be tested was calculated.

[0055] The results showed that TCTP protein was significantly increased in the preterm birth group (see appendix). Figure 11 The first and third quartiles of TCTP protein levels in the full-term group were 41 pg / ml and 63 pg / ml, respectively; while in the preterm group, the first and third quartiles were 77 pg / ml and 94 pg / ml, respectively. Therefore, we recommend a serum TCTP level greater than 70 pg / ml as the standard for detecting high risk of preterm birth. In 126 samples, the detection rate of preterm birth using this method was 91%, with a false positive rate of 8.6% (5 cases) in 58 full-term deliveries.

[0056] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0057] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a product for detecting TCTP protein in the preparation of a reagent for the diagnosis of non-invasive prematurity, characterized in that, The amino acid sequence of the TCTP protein is shown as SEQ ID NO.

3.

2. Use according to claim 1, characterized in that, The product is a kit.

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