PAPPA2-171aa protein and application thereof

Abstract

The invention discloses a PAPPA2-171aa protein and an application of the PAPPA2-171aa protein. The amino acid sequence of the PAPPA2-171aa protein is as shown in SEQ ID NO: 1. The PAPPA2-171aa protein which can be used for inhibiting invasion and migration of trophoblast cells is obtained through screening, through overexpression of the PAPPA2-171aa, it is found that the invasion and migration capacities of the trophoblast cells are obviously inhibited, it is indicated that the PAPPA2-171aa protein can participate in regulation and control of occurrence and development of preeclampsia, the PAPPA2-171aa protein can be used for screening drugs for treating preeclampsia, and the PAPPA2-171aa protein can be used for preparing drugs for treating preeclampsia. And a new research direction is provided for developing a medicine for treating preeclampsia.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a PAPPA2-171aa protein and its applications. Background Technology

[0002] Preeclampsia (PE) is a hypertensive disorder that occurs during pregnancy, affecting approximately 5-8% of pregnant women. PE / eclampsia is associated with 10-15% of maternal mortality. PE is linked to maternal eclampsia and HELLP syndrome, as well as fetal preterm birth, intrauterine growth restriction, and perinatal mortality. PE has adverse effects on the health and quality of life of both the mother and offspring. Women with a history of PE have a 2-fold increased risk of cardiovascular complications and a 5-12-fold increased risk of end-stage renal disease later in life. Over the past 30 years, various factors related to the pathogenesis of PE have been reported, including hypoxia and oxidative stress, uterine spiral artery remodeling, endothelial dysfunction, maternal vascular destruction, superficial trophoblast invasion, and inflammation. However, the molecular mechanisms regulating the development and progression of PE remain unclear. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a PAPPA2-171aa protein that can be effectively used to inhibit the invasion and migration function of trophoblast cells.

[0004] The present invention also proposes a B having the above-mentioned A.

[0005] According to one aspect of the invention, a protein is provided, said protein being any one of the following (1)-(3):

[0006] (1) A protein with an amino acid sequence as shown in SEQ ID NO: 1;

[0007] (2) A protein having the same function as the protein described in (1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.

[0008] (3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence in (1) or (2).

[0009] In some embodiments of the present invention, the protein is PAPPA2-171aa protein.

[0010] In a second aspect of the invention, a nucleic acid molecule encoding the aforementioned protein is proposed.

[0011] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the PAPPA2-171aa protein is shown in SEQ ID NO:6.

[0012] In a third aspect of the invention, a biomaterial related to the above-mentioned nucleic acid molecule is provided, which is any one of 1) to 7) below:

[0013] 1) Expression cassettes containing the aforementioned nucleic acid molecules;

[0014] 2) Recombinant vectors containing the above-mentioned nucleic acid molecules;

[0015] 3) A recombinant vector containing the expression cassette described in 1);

[0016] 4) Recombinant microorganisms containing the above-mentioned nucleic acid molecules;

[0017] 5) Recombinant microorganisms containing the expression cassette described in 1);

[0018] 6) Recombinant microorganisms containing the recombinant vector described in 2);

[0019] 7) Recombinant microorganisms containing the recombinant vector described in 3).

[0020] In a fourth aspect of the invention, the application of the above-described protein, the nucleic acid molecule encoding the above-described protein, and the biomaterial is proposed, wherein the application is in the preparation of a product that inhibits the invasion and migration of trophoblast cells.

[0021] In some embodiments of the present invention, the trophoblast cells include HTR8-SVneo and JEG-3.

[0022] In some embodiments of the invention, the application is in the preparation of products for the prevention and / or treatment of preeclampsia.

[0023] In some embodiments of the present invention, the product includes pharmaceuticals.

[0024] In some embodiments of the present invention, the pharmaceutical product further includes scientifically acceptable excipients.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from one or more of carriers, diluents, binders, lubricants, and wetting agents.

[0026] In some embodiments of the present invention, the form of the medicine is selected from one or more of the following: solution, injection, spray, nasal drops, aerosol, powder spray, tablet, capsule, and granule.

[0027] In some embodiments of the invention, the application is in the preparation of drugs for screening the prevention and / or treatment of preeclampsia.

[0028] In some embodiments of the invention, the application is in the preparation of products for detecting preeclampsia.

[0029] According to a fourth aspect of the present invention, an antibody for detecting the above-mentioned protein is provided, the sequence of which is shown in SEQ ID NO:2.

[0030] According to a fifth aspect of the invention, the use of an agent overexpressing PAPPA2-171aa in the preparation of products that inhibit trophoblast cell invasion and migration, inhibit MMP-2 protein expression and / or MMP-9 protein expression, or prevent and / or treat preeclampsia is proposed.

[0031] In some embodiments of the present invention, the product includes pharmaceuticals.

[0032] In some embodiments of the present invention, the reagent for overexpressing PAPPA2-171aa includes the PAPPA2-171aa overexpression plasmid.

[0033] In some embodiments of the present invention, the PAPPA2-171aa overexpression plasmid is a plasmid vector containing a nucleic acid molecule encoding the PAPPA2-171aa protein.

[0034] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the PAPPA2-171aa protein is shown in SEQ ID NO:6.

[0035] According to a sixth aspect of the invention, a medicament for the prevention and / or treatment of preeclampsia is provided, said medicament comprising a reagent for increasing the content of the aforementioned protein.

[0036] In some embodiments of the present invention, the reagent includes the PAPPA2-171aa overexpression plasmid.

[0037] In some embodiments of the present invention, the PAPPA2-171aa overexpression plasmid is a plasmid vector containing a nucleic acid molecule encoding the PAPPA2-171aa protein.

[0038] In some embodiments of the present invention, the sequence of the nucleic acid molecule encoding the PAPPA2-171aa protein is shown in SEQ ID NO:6.

[0039] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The present invention obtains PAPPA2-171aa protein that can be used to inhibit the invasion and migration of trophoblast cells through screening. By overexpressing PAPPA2-171aa, it was found that the invasion and migration ability of trophoblast cells were significantly inhibited, indicating that PAPPA2-171aa protein can participate in the regulation of the occurrence and development of preeclampsia. It can be used to screen drugs for the treatment of preeclampsia, providing a new research direction for the development of drugs for the treatment of preeclampsia. Attached Figure Description

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0041] Figure 1 This is a schematic diagram of a plasmid in an embodiment of the present invention;

[0042] Figure 2 The images shown are qPCR and WB detection results in the embodiments of the present invention, where A is the qPCR detection result image, B is the WB detection result image, and "*" indicates P < 0.05.

[0043] Figure 3 This is a mass spectrometry detection result diagram of Flag-171aa in an embodiment of the present invention;

[0044] Figure 4 This is a diagram showing the scratch test results in an embodiment of the present invention;

[0045] Figure 5 The images shown are Transwell and WB detection results in this embodiment of the invention, where A is the Transwell detection result image and B is the WB detection result image.

[0046] Figure 6 Figure 1 shows the expression of PAPPA2-171aa in preeclampsia placental tissue and its clinical correlation with the present invention. Figure 2 shows the results of immunohistochemical detection; Figure 3 shows the results of immunohistochemical detection; Figure 4 shows the results of PAPPA2-171aa expression and systolic blood pressure; Figure 5 shows the results of PAPPA2-171aa expression and diastolic blood pressure; Figure 6 shows the results of PAPPA2-171aa expression and 24-hour urinary protein level. "****" indicates P < 0.0001. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] Example 1

[0049] This embodiment provides a polypeptide protein PAPPA2-171aa, with the following sequence:

[0050] MKCAITCQRGFALQASSGQYIRPMQKEILLTCSSGHWDQNVSCLPVDCGVPDPSL

[0051] VNYANFSCSEGTKFLKRCSISCVPPAKLQGLSPWLTCLEDGLWSLPEVYCKLECDAPPI

[0052] ILNANLLLPHCLQDNHDVGTICKYECKPGYYVAESAEGKVRKCVSIDQERPEQFLFF

[0053] (SEQ ID NO:1).

[0054] The PAPPA2-171aa protein was artificially synthesized by Jiangsu Saisofi Company.

[0055] Example 2

[0056] This embodiment provides a specific antibody (Anti-PAPPA2-171aa) for detecting the polypeptide protein PAPPA2-171aa. The expression of PAPPA2-171aa (approximately 19 kDa) can be detected by Western blotting using the Anti-PAPPA2-171aa antibody.

[0057] The sequence of the antibody Anti-PAPPA2-171aa is shown below: VAESAEGKVRKCVSIDQERPEQFLFF (SEQ ID NO:2).

[0058] Example 3

[0059] This embodiment provides the application of the polypeptide protein PAPPA2-171aa in inhibiting the migration and invasion of trophoblast cells.

[0060] The experiment used hsa_circ_0111277 (a protein previously studied by the inventor's laboratory that inhibits trophoblast cell migration and invasion, and has been disclosed in Circular RNA circ_0111277attenuates human trophoblast cell invasion and migration by regulating miR-494 / HTRA1 / Notch-1 signal pathway in pre-eclampsia) as a positive control. The specific verification steps are as follows:

[0061] 1. Construct the wild-type plasmid hsa_circ_0111277, the ATG mutant plasmid hsa_circ_0111277-mut, and the PAPPA2-171aa plasmid.

[0062] The schematic diagrams of the wild-type plasmid hsa_circ_0111277, the ATG mutant plasmid hsa_circ_0111277-mut, and the PAPPA2-171aag plasmid are shown below. Figure 1 As shown, all of them were synthesized by Jiangsu Saisofi Company.

[0063] The hsa_circ_0111277 sequence is as follows:

[0064] ACTGCCTCCAGGACAACCACGACGTGGGCACCATCTGCAAATATGAATGCAA

[0065] ACCAGGGTACTATGTGGCAGAAAGTGCAGAGGGTAAAGTCAGGAAGTGTGTTTCA

[0066] ATAGACCAGGAGAGGCCAGAGCAATTTTTATTTTTTTGACAACTGATGGCCTAGTT

[0067] CCCGGAGAGCATCAGCAGCCGACAGTGACTCTCTACCTGACCGATGTCCGTGGAA

[0068] GCAACCACTCTCTTGGAACCTATGGACTGTCATGCCAGCATAATCCACTGATTATCA

[0069] ATGTGACCCATCACCAGAATGTCCTTTTCCACCATACCACCTCAGTGCTGCTGAATT

[0070] TCTCATCCCCACGGGTCGGCATCTCAGCTGTGGCTCTAAGGACATCCTCCCGCATT

[0071] GGTCTTTCGGCTCCCAGTAACTGCATCTCAGAGGACGAGGGGCAGAATCATCAGG

[0072] GACAGAGCTGTATCCATCGGCCCTGTGGGAAGCAGGACAGCTGTCCGTCATTGCT

[0073] GCTTGATCATGCTGATGTGGTGAACTGTACCTCTATAGGCCCAGGTCTCATGAAGT

[0074] GTGCTATCACTTGTCAAAGGGGATTTGCCCTTCAGGCCAGCAGTGGGCAGTACATC

[0075] AGGCCCATGCAGAAGGAAATTCTGCTCACATGTTCTTCTGGGCACTGGGACCAGA

[0076] ATGTGAGCTGCCTTCCCGTGGACTGCGGTGTTCCCGACCCGTTCTTTGGTGAACTAT

[0077] GCAAACTTCTCCTGCTCAGAGGGAACCAAATTTCTGAAACGCTGCTCAATCTCTTG

[0078] TGTCCCACCAGCCAAGCTGCAAGGACTGAGCCCATGGCTGACATGTCTTGAAGAT

[0079] GGTCTCTGGTCTCTCCCTGAAGTCTACTGCAAGTTGGAGTGTGATGCTCCCCCCTATT

[0080] ATTCTGAATGCCAACTTGCTCCTGCCTC (SEQ ID NO: 3).

[0081] The basic plasmid vector and insert sequence used by Jiangsu Saisofe Biotechnology Co., Ltd. to synthesize the corresponding plasmid vector are shown below:

[0082] (1) Constructing the hsa_circ_0111277 plasmid

[0083] Vector: pCDH-CMV-MCS-EF1-copGFP-T2A-Puro;

[0084] Insert sequence:

[0085]

[0086] (2) Construct the hsa_circ_0111277-mut plasmid

[0087] Vector: pCDH-CMV-MCS-EF1-copGFP-T2A-Puro;

[0088] Insert sequence:

[0089]

[0090] (2) Construction of PAPPA2-171aa plasmid

[0091] Vector: pCDH-CMV-MCS-EF1-copGFP-T2A-Puro;

[0092] Insert sequence:

[0093] ATGAAGTGTGCTATCACTTGTCAAAGGGGATTTGCCCTTCAGGCCAGCAGTGGGCAGTACATCAGGCCCATGCAGAAGGAAATTCTGCTCACATGTTCTTCTGGGCACTGGGACCAGAATGTGAGCTGCC TTCCCGTGGACTGCGGTGTTCCCGACCCGTCTTGGTGAACTATGCAAACTTCTCCTGCTCAGAGGGAACCAAATTTCTGAAACGCTGCTCAATCTCTTGTGTCCCACCAGCCAAGCTGCAAGGACTGAG CCCATGGCTGACATGTCTTGAAGATGGTCTCTGGTCTCTCCCTGAAGTCTACTGCAAGTTGGAGTGTGATGCTCCCCCTATTCTGAATGCCAACTTGCTCCTGCCTCACTGCCTCCAGGACAACCAC GACGTGGGCACCATCTGCAAATATGAATGCAAACCAGGGTACTATGTGGCAGAAAGTGCAGAGGGTAAAGTCAGGAAGTGTGTTTCAATAGACCAGGAGAGGCCAGAGCAATTTTTATTTTTTTGA(SEQ IDNO:6).

[0094] 2. To investigate the cell migration, invasion, and secretion functions of HTR8-SVneo and JEG-3 cell lines stably transfected with PAPPA2-171aag plasmid, and to clarify the molecular function of PAPPA2-171aa in trophoblast cells.

[0095] The hsa_circ_0111277 plasmid, hsa_circ_0111277-mut plasmid, and PAPPA2-171aag plasmid, along with the blank control plasmid, were used to transfect pCDH-CMV-NC (specifically, the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro plasmid, purchased from Jiangsu Saisuofei Company) into the feeder cell lines HTR8-SVneo and JEG-3 according to the following groupings:

[0096] HTR-8 / SVneo control group: HTR-8 / SVneo cell line transfected with pCDH-CMV-NC blank plasmid;

[0097] HTR-8 / SVneo wild-type group: HTR-8 / SVneo cell line transfected with hsa_circ_0111277 plasmid;

[0098] HTR-8 / SVneo mutant group: HTR-8 / SVneo cell lines were transfected with the hsa_circ_0111277-mut plasmid;

[0099] HTR-8 / SVneo protein expression group: PAPPA2-171aag plasmid transfected into HTR-8 / SVneo cell line;

[0100] JEG-3 control group: JEG-3 cell line transfected with pCDH-CMV-NC blank plasmid;

[0101] JEG-3 wild-type group: JEG-3 cell line transfected with hsa_circ_0111277 plasmid;

[0102] JEG-3 mutant group: hsa_circ_0111277-mut plasmid in JEG-3 transfected cell lines;

[0103] JEG-3 protein expression group: PAPPA2-171aa plasmid transfected into JEG-3 cell line.

[0104] The specific methods for cell transfection are as follows:

[0105] ① Cell plating:

[0106] Adherent cells: One day before transfection, seed 0.5-2 × 10⁶ cells with 500 μL of antibiotic-free medium. 5 The trophoblast cell lines (HTR8-SVneo or JEG-3) were used to achieve 70-90% confluence by the next day.

[0107] Suspension cells: Before preparing the DNA-Lip2000 complex, inoculate 4-8 × 10⁴ cells with 500 μL of antibiotic-free medium. 5 Cells are sufficient.

[0108] ② For each transfected sample, perform the following operations:

[0109] Add 50 μl of serum-free culture medium (such as OPTI-MEMⅠ medium) and 0.8 μg of DNA (the corresponding plasmid for each group) to centrifuge tubes, mix gently, and prepare DNA dilution solutions.

[0110] Add 50 μL of serum-free culture medium (such as OPTI-MEMⅠ medium) and 2.0 μL of Lip2000 (make sure to mix well before use) to another centrifuge tube, mix gently to prepare Lip2000 dilution, and let stand at room temperature for 5 minutes.

[0111] Mix the DNA dilution buffer and Lip2000 dilution buffer gently, and let stand at room temperature for 20 minutes to form a DNA-Lip2000 complex. The DNA-Lip2000 complex is stable at room temperature for 6 hours.

[0112] ③ Add the DNA-Lip2000 complex to the inoculated cells and gently shake the culture plate back and forth to disperse the complex evenly.

[0113] ④ After culturing in a 37℃ CO2 incubator for 4-6 hours, replace the culture medium and continue culturing for 18-48 hours.

[0114] ⑤ To screen for stable cell lines, seed the cells into fresh culture medium at a ratio of 1:10 or higher 24 hours after transfection, and add selective culture medium the next day for screening.

[0115] 48 h after transfection, nucleic acid molecules were extracted from each group of cells and cDNA was obtained by reverse transcription. The cDNA was used as a template to detect the expression of hsa_circ_0111277 in the cells by qPCR. The primer sequences used for qPCR detection are shown in Table 1, and the reaction system is shown in Table 2. The cycling parameters for the fluorescent PCR reaction were: 95℃ for 2 min; then the cycling phase was: 95℃ for 10 sec, 60℃ for 30 sec (collecting fluorescence), for 40 cycles.

[0116] Table 1

[0117]

[0118] Table 2

[0119]

[0120] Meanwhile, proteins were extracted from the protein expression group cells, and the expression of PAPPA2-171aa (approximately 19KD) was detected by Western blotting using the antibody prepared in Example 2 (Anti-PAPPA2-171aa (purchased from Anmart, catalog number PK47184) and GenScript antibody were used for detection). The steps of the Western blotting detection are as follows: (1) The extracted protein was mixed with 5× loading buffer at a ratio of 4:1 and denatured in a 95℃ metal bath for 5 min; (2) Electrophoresis and electrotransfer: SDS-PAGE gel electrophoresis was performed, and 30 μg of total protein was accurately added to each well. The stacking gel voltage was constant at 200V, and the gel running time was 45 minutes; the constant voltage was set at 100V for 60 mins, and the current was observed: the Tank transfer current start < 250mA, stop < 350mA was transferred into the polyvinylidene fluoride (PVDF) membrane; (3) Blocking: The PVDF membrane was placed in the blocking solution (containing 5% deionization solution). (3) Blocking in TBST of milk fat for 2 hours; (4) Primary antibody (purchased from Anmart, catalog number PK47184) incubation: TBST containing 3% BSA as diluent (pH 7.4), dilute the primary antibody at a ratio of 1:1000, and place the PVDF membrane in the primary antibody overnight at 4°C; wash with TBST for 7 minutes × 2 times; (5) Secondary antibody (purchased from Anmart, catalog number M21008) incubation: select the appropriate secondary antibody according to the source of the primary antibody, the secondary antibody diluent is the same as the blocking buffer, and the blocking buffer used can be directly prepared. Jackson's mouse and rabbit secondary antibodies are diluted 1:5000 (stock solution stored at -20°C), incubated at room temperature for 1 hour, and washed with TBST for 7 minutes × 3 times; (6) Development: PVDF membrane is immersed in ECL reagent for 2 minutes and then photographed with a fully automated chemiluminescence detector; protein analysis is performed using Imagelab software.

[0121] The results are as follows Figure 2 As shown in the figure, qPCR results indicate successful overexpression of wild-type hsa_circ_0111277 and ATG mutant hsa_circ_0111277; WB results indicate successful overexpression of PAPPA2-171aa. Sequencing of the corresponding positive recombinant cells also confirmed successful expression of the corresponding proteins.

[0122] Simultaneously, PAPPA2-171aa was inserted into a plasmid containing 6 flag tags to construct a 171aa-6Flag fusion plasmid containing 6×flags (synthesized by Jiangsu Saisofe Company). The control group was a blank plasmid pCDH-CMV-MCS-EF1-copGFP-T2A-Puro. After transfecting 293T cells with the fusion plasmid and the blank plasmid, the expression of Flag-171aa (molecular weight of about 28 kDa) was detected by Western blotting. The specific amino acid sequence of the PAPPA2-171aa peptide across the cleavage site was identified by liquid chromatography-tandem mass spectrometry, and the presence of PAPPA2-171aa protein was verified by mass spectrometry.

[0123] The results are as follows Figure 3 As shown, the PAPPA2-171aa protein was verified.

[0124] 3. Detection of the application of peptide protein PAPPA2-171aa in inhibiting trophoblast cell migration and invasion

[0125] The cell migration, invasion, and secretion functions of recombinant HTR8-SVneo and JEG-3 cell lines stably transfected with different plasmid groups were examined to clarify the molecular function of PAPPA2-171aa in trophoblast cells. Specifically, the experiments were conducted as follows:

[0126] (1) Transfected at 0, 6h, 12h and 24h, the cell migration ability of each group was observed by scratch assay;

[0127] (2) 48 h after transfection, Transwell was used to observe the invasive ability of cells in each group;

[0128] (3) 48 h after transfection, the expression levels of MMP-2 and MMP-9 were detected in each group of cells by WB.

[0129] The scratch test procedure is as follows:

[0130] 1) After trypsin digestion, experimental cells in the logarithmic growth phase of each group were seeded into 6-well plates, 1 × 10⁶ cells per well. 6 Each cell.

[0131] 2) After the cells have completely adhered to the culture medium, change the medium and add mitomycin C at a final concentration of 1 μg / ml for 1 hour to inhibit cell division.

[0132] 3) Place a 200μL syringe tip against the edge of the cap and make a vertical scratch on the bottom of the plate.

[0133] 4) Wash three times with PBS to remove the scraped cells, and add basal culture medium.

[0134] 5) Place in an incubator for further culture, and take pictures of 8 scratch fields of view at 0 and 24 hours respectively.

[0135] 6) Use Image-J software to measure the distance and calculate the migration rate. Migration rate = (0h - other time points) / 0h.

[0136] The Transwell experiment is as follows:

[0137] 1) Dissolve Matrigel overnight at 4°C. Dilute it with pre-cooled basal medium at a ratio of Matrigel (BDBiocoat 354234): medium = 1:3. Take 40 μl and add it to a pre-cooled Transwell chamber. Do this slowly to avoid generating bubbles.

[0138] 2) Incubate at 37°C for 2 hours to allow Matrigel to solidify.

[0139] 3) Add 100 μl and 600 μl of basal culture medium to the upper and lower chambers respectively, and hydrate overnight at 37°C. Remove the culture medium the next day.

[0140] 4) Take experimental cells from each group in the logarithmic growth phase, wash them once with PBS, digest them into single-cell suspensions with 0.25% trypsin, take an appropriate amount of cell suspension, and centrifuge at 800 rpm for 5 minutes.

[0141] 5) Remove the supernatant, resuspend the cells in basal culture medium, count the cells, and adjust the cell concentration to 1×10⁻⁶ cells / mL using basal culture medium. 6 / ml, take 100μl and add it to the upper chamber of the Transwell chamber, and add 600μl of complete culture medium to the lower chamber.

[0142] 6) After incubating in an incubator for 72 hours, wash three times with PBS. Wipe away cells from the upper chamber surface with a cotton swab, add 2 ml of fixative to the well plate, place the Transwell plate in the well, and fix for 15 minutes. Note: The Transwell plate must be completely immersed in the liquid.

[0143] 7) Wash with PBS 3 times, 5 minutes each time.

[0144] 8) Add 1 ml of staining solution to the well plate, immerse Transwell in the solution, and stain for 10 minutes.

[0145] 9) Discard the staining solution, add 1 ml of color-mixing solution A, add one drop of color-mixing solution B, mix quickly, and observe under a microscope.

[0146] Note: Do not apply stain solution B directly to the stained cells. Adjust the amount of stain solution B according to the desired staining color of your cells. If the color is too dark, add another drop of stain solution B until you achieve a satisfactory color. Once you achieve a satisfactory result, discard all liquid and wash once with PBS.

[0147] 10) Observation under an upright microscope: Remove the membrane with a blade, place it on a glass slide, add 1-2 drops of mounting solution, cover with a coverslip, and observe under a microscope. Observation under an inverted microscope: Directly photograph the Tr under a 100x or 200x microscope according to the cell size, and count the number of cells that pass through it.

[0148] The results are as follows Figure 4-5 As shown, from Figure 4 As can be seen from the scratch and Transwell assays, overexpression of wild-type hsa_circ_0111277 inhibited trophoblast cell migration and invasion, while ATG mutant hsa_circ_0111277 lost its inhibitory ability, and overexpression of PAPPA2-171aa inhibited cell migration and invasion.

[0149] from Figure 5 As can be seen from the results, Western blot analysis showed that overexpression of wild-type hsa_circ_0111277 inhibited the expression of MMP-2 and MM-9, and overexpression of PAPPA2-171aa inhibited the expression of MMP-2 and MM-9, while ATG mutant hsa_circ_0111277 lost its inhibitory ability.

[0150] The results showed that overexpression of the PAPPA2-171aa polypeptide sequence significantly inhibited the invasion and migration of trophoblast cells, consistent with the results of the positive control.

[0151] Example 4: Expression of PAPPA2-171aa in placental tissue during preeclampsia and its clinical relevance.

[0152] Using an antibody synthesized by Genscript (an antibody designed for the sequence CVSIDQERPEQFLFF), immunohistochemical staining was performed on the expression of PAPPA2-171aa in placental tissues from 32 patients with preeclampsia and 28 normal late-pregnancy patients, and its correlation with blood pressure / urinary protein was analyzed. The clinical characteristics of the patients are shown in Table 3.

[0153] Table 3 Clinical characteristics of patients

[0154]

[0155] Test results as follows Figure 6 As shown, from Figure 6 As shown in the immunohistochemical results of Figures A and B, PAPPA2-171aa is a protective molecule, which is significantly highly expressed in PE samples compared to normal late-pregnancy placental tissue; it can also be used as a target for the detection of preeclampsia samples for accurate detection of preeclampsia samples.

[0156] from Figure 6As can be seen from Figures C and D, the expression of PAPPA2-171aa is correlated with both systolic and diastolic blood pressure, but the correlation between the expression of PAPPA2-171aa and diastolic blood pressure is not significant.

[0157] from Figure 6 As can be seen from Figure E, the expression of PPAPPA2-171aa is negatively correlated with the level of 24-hour urinary protein.

[0158] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A protein, characterized in that, The protein is any one of the following (1)-(3): (1) A protein with an amino acid sequence as shown in SEQ ID NO: 1; (2) A protein having the same function as the protein described in (1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

1. (3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence in (1) or (2).

2. A nucleic acid molecule encoding the protein as described in claim 1.

3. A biomaterial related to the nucleic acid molecule of claim 2, characterized in that, It is any one of the following 1) to 7): 1) Expression cassettes containing the aforementioned nucleic acid molecules; 2) Recombinant vectors containing the above-mentioned nucleic acid molecules; 3) A recombinant vector containing the expression cassette described in 1); 4) Recombinant microorganisms containing the above-mentioned nucleic acid molecules; 5) Recombinant microorganisms containing the expression cassette described in 1); 6) Recombinant microorganisms containing the recombinant vector described in 2); 7) Recombinant microorganisms containing the recombinant vector described in 3).

4. The use of the protein of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of products that inhibit trophoblast cell invasion and migration, MMP-2 protein expression, and / or MMP-9 protein expression.

5. The use of the protein of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of products for the prevention and / or treatment of preeclampsia.

6. The use of the protein of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of drugs for screening the prevention and / or treatment of preeclampsia.

7. The use of the protein of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of a product for detecting preeclampsia.

8. An antibody for detecting the protein as described in claim 1, characterized in that, The sequence of the antibody is shown in SEQ ID NO:

2.

9. The use of the reagent for overexpressing the protein as described in claim 1 in the preparation of products that inhibit trophoblast cell invasion and migration, inhibit MMP-2 protein expression and / or MMP-9 protein expression, or prevent and / or treat preeclampsia.

10. A drug for the prevention and / or treatment of preeclampsia, characterized in that, The drug includes a reagent for increasing the protein content as described in claim 1.

Images