KR20120102216A - Analytical method for diagnosing recurrent pregnancy loss and kit therefor - Google Patents

Abstract

PURPOSE: A method for providing information for diagnosing recurrent pregnancy loss is provided to simply and early diagnose recurrent pregnancy loss using blood. CONSTITUTION: An analysis for providing information needed for diagnosing recurrent pregnancy loss comprises a step of measuring expression of proteins of sequence number 1 in blood. A gene encoding the protein of sequence number 1 has a base of sequence number 2. The protein expression is measured by western blotting. A kit for diagnosing recurrent pregnancy loss contains a molecule which is able to measure the expression of the protein of sequence number 1, antibody which specifically binds to the protein, a substrate, ligand, or a cofactor. The molecule is labeled with a detectable probe.

Description

Analytical method for diagnosing recurrent pregnancy loss and kit therefor}

The present invention relates to an analytical method for providing information necessary for diagnosing recurrent pregnancy loss and a kit for diagnosing habitual miscarriage.

Recurrent pregnancy loss (RPL) is a reproductive disease that affects about 2-5% of women (CB Coulam, et al., Am. J. Reprod. Immunol. , 1997, 38, 57-74). It is called addictive miscarriage when at least three miscarriages occur in succession within 20-28 weeks of gestation (PG Crosignani and BL Rubin, Hum. Reprod., 1991, 6, 609-610; S. Harlap and PH Shiono, Lancet, 1980, 2 , 173-176; MK Pandey, R. Rani and S. Agrawal, Arch.Gynecol. Obstet., 2005, 272, 95-108).

Despite many studies of habitual miscarriage, the exact causes and mechanisms are unknown, due to the difficulty of sampling and various pregnancy conditions. Many recent studies have reported that habitual miscarriage is associated with a variety of causes, including immunity, dissection, endocrine, heredity, infection, and chromosomal abnormalities. Moreover, the complex signaling processes involved in the implantation and placental formation process are thought to be associated with habitual miscarriage. Natural killer cells of the uterus are located adjacent to the embryo and secrete various cytokines such as IL-1β, IL-6, IL-11, and TGF-β, which are involved in the immune response and promote implantation. In contrast, apoptosis-related genes, such as p53, which prevent successful implantation, are also secreted. Moreover, many other genes are expressed in patients with habitual miscarriage, which are known to be associated with maternal immune response, angiogenesis, apoptosis and thrombosis (HK Choi, BC Choi, SH Lee, JW Kim). , KY Cha and KH Baek, Mol.Reprod. Dev. , 2003, 66, 24-31; MJ Jasper, KP Tremellen and SA Robertson, Am. J. Reprod. Immunol. , 2007, 73, 74-84.).

Recently, proteomics studies have been used to identify post-translational modifications in various diseases including reproductive and infertility. This is useful for identifying altered protein expression as well as proteins associated with the onset of the disease. This study has also been applied to habitual miscarriage, and it has been reported that immune- and thrombus-related proteins are associated with habitual miscarriage (YS Kim, MS Kim, SH Lee, BC Choi, JM Lim, KY Cha and KH Baek, Proteomics, 2006, 6, 3445-3454; KH Baek, EJ Lee and YS Kim, Trends Mol.Med., 2007, 13, 310-317).

In the previous study, we carried out a proteomics study with follicular samples from patients with habitual miscarriage and completed the component component C3c chain E, fibrinogen γ, antithrombin, angio Tensinogen (angiotensinogen), hemopexin precursor (hemopexin precursor) was confirmed to be inappropriately expressed (YS Kim, MS Kim, SH Lee, BC Choi, JM Lim, KY Cha and KH Baek, Proteomics, 2006, 6, 3445-3454; S. Angelucci, D. Ciavardelli, F. Giuseppe Di, E. Eleuterio, M. Sulpizio, GM Tiboni, F. Giampietro, P. Palumbo and C. Di Ilio, Biochim. Biophys. Acta, 2006, 1764 , 1775-1785). Amniotic fluid, like follicular fluid, is a reproductive fluid. However, these reproductive fluids are difficult to collect from patients and use for therapeutic purposes. Therefore, identifying marker proteins expressing specific expression patterns in patients with habitual miscarriage from blood, one of the body fluids that play a pivotal role in the system of delivery, is an easy way to diagnose patients with habitual miscarriage early, It will be useful for the development of diagnostic kits that can be diagnosed.

The present inventors searched for marker proteins capable of diagnosing habitual miscarriage at a molecular biological level through proteomics analysis in order to develop a method for diagnosing the habitual miscarriage in various ways. The present inventors performed 2-dimensional electrophoresis, MALDLI TOF-MS, and protein sequencing on blood obtained from normal and habitual abortion patients. Found.

Accordingly, an object of the present invention is to provide an analysis method using a protein specifically expressed only in a habitual abortion patient in order to provide information necessary for the diagnosis of the habitual abortion.

In addition, an object of the present invention is to provide a diagnostic kit for habitual miscarriage comprising a molecule capable of measuring the expression of the protein.

According to one aspect of the present invention, in order to provide information necessary for the diagnosis of habitual miscarriage, an analysis method is provided that includes measuring the presence or absence of the expression of the protein of SEQ ID NO: 1 in blood isolated from a female.

In the analysis method of the present invention, the determination of the expression of the protein can be carried out by measuring the expression of the gene encoding the protein of SEQ ID NO: 1, preferably the gene having a nucleotide sequence of SEQ ID NO: 2. In addition, the measurement of the expression level of the protein may be performed by Western blotting.

According to another aspect of the present invention, there is provided a diagnostic kit for a habitual miscarriage comprising a molecule capable of measuring the expression of the protein of SEQ ID NO: 1, wherein the molecule specifically binds to the protein, an antibody, a substrate, a ligand, or an auxiliary A kit for diagnosing a habitual miscarriage, which is a cofactor, is provided.

The molecule may be labeled with a detectable label, and the kit may be in the form of a protein chip in which an antibody against a protein of SEQ ID NO: 1 is immobilized on a substrate.

In accordance with the present invention, it has been found that a particular protein, ie, the protein of SEQ ID NO: 1, is specifically expressed in blood isolated (ie, collected) from a woman, including pregnant women. Therefore, the analytical method and diagnostic kit according to the present invention can be conveniently used for early diagnosis of habitual abortion patients using blood obtained from a woman including a pregnant woman.

Figure 1 shows the results of two-dimensional electrophoresis of proteins extracted from blood extracted from patients with habitual miscarriage.
2 shows 15 spots with increased expression in patients with habitual miscarriage.
3 shows six spots with reduced expression in habitual miscarriage patients.
4 is a Western blot analysis of α-2-macroglobulin (163 kDa), α-1-antichymotrypsin (AACT) (68 kDa), and IGFBP1 (36 kDa) in patients with habitual miscarriage.
5 is a Western blot analysis of ITI-H4 in the habitual miscarriage patients and the control group.
Figure 6 shows the results of LC-MS / MS analysis to identify the cleavage site of the ITI-H4 fragment. FIG. 6A shows the matching sequence of ITI-H4 (120 kDa), and FIG. 6B shows the matching sequence of ITI-H4 (36 kDa).

The inventors performed an analysis on blood from normal and habitual abortion patients to find proteins specifically expressed in habitual abortion patients. We are addictive in comparison with normal people at polypeptide level via 2-dimensional electrophoresis (2-DE), MALDI-TOF-MS, and nano-LC MS / MS of blood obtained from patients with habitual miscarriage. Factors showing differences in expression in abortion patients were identified and digested from the specific protein, ITI-H4 (inter-α trypsin inhibitor-heavy chain 4), by biochemical analysis such as Western blotting. It was confirmed that the protein fragment of kDa (SEQ ID NO: 1) is specifically expressed in patients with habitual miscarriage.

Accordingly, the present invention provides an analysis method comprising the step of measuring the presence or absence of the expression of the protein of SEQ ID NO: 1 in blood isolated from a woman in order to provide information necessary for the diagnosis of habitual miscarriage.

In the analysis method of the present invention, the blood separated from the woman, preferably the blood of the pregnant woman in the early stage of pregnancy refers to the blood separated by blood collection by a conventional method. In general, pregnant women undergo regular or irregular examinations at a gynecology hospital from the time they are informed or notified that they are pregnant, and clinicians take samples of blood and the like for various tests. The analytical method of the present invention can be used to determine the habitual miscarriage of the collected and separated blood.

In the analysis method of the present invention, the protein of SEQ ID NO: 1 is a 36 kDa protein cleaved from 120 kDa of ITI-H4, and has the amino acid sequence of SEQ ID NO: 1. In addition, the gene encoding the amino acid sequence of SEQ ID NO: 1 may have a nucleotide sequence of SEQ ID NO: 2.

Determination of the expression of the protein can be carried out by measuring the expression of the gene encoding the protein of SEQ ID NO: 1, preferably the gene having a nucleotide sequence of SEQ ID NO: 2. Determination of the expression of the gene can be carried out by measuring in accordance with methods commonly used in the field of biotechnology. In addition, the measurement of the expression of the protein may be performed by a method such as western blotting.

The present invention also provides a diagnostic kit for a habitual miscarriage comprising a molecule capable of measuring the expression of the protein of SEQ ID NO: 1, wherein the molecule specifically binds to the protein, an antibody, a substrate, a ligand, or a cofactor (cofactor) The present invention provides a kit for diagnosing a habitual miscarriage.

A molecule capable of determining the expression of the protein (ie, the protein of SEQ ID NO: 1) disclosed in accordance with the present invention may be an antibody, a substrate, a ligand, or a cofactor that specifically binds to the protein. have. The protein of SEQ ID NO: 1 may be used to prepare a polyclonal antibody or monoclonal antibody according to methods commonly used in the field of biotechnology, and a diagnostic kit comprising the antibody may be prepared. In addition, the kit of the present invention may be prepared to include a substrate, a ligand, or a cofactor for the protein of SEQ ID NO: 1.

In the diagnostic kit of the present invention, the molecule capable of measuring the expression of the protein of SEQ ID NO: 1 may be labeled with a detectable label (eg, chromophore, etc.). In addition, the diagnostic kit of the present invention may be, for example, in the form of a protein chip in which an antibody to a protein of SEQ ID NO: 1 is immobilized on a substrate.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by these examples.

Example

1. Test Method

(1) sample

IEF (isoelectric focusing) method using proteomics analysis was used. Blood was collected from 29 addictive abortion patients who experienced two or more miscarriages during the first trimester. Blood samples were collected from five healthy healthy subjects who had no experience with miscarriage or other obstetric diseases. The normal person had no experience of miscarriage and had at least one birth. Patient information including abortion experience for each habitual abortion patient is shown in Table 1 below. Intravenous blood samples were stored at -80 ° C until use. All information is written from the participants, and the study was approved by the institutional Ethics Committee (Institutional Review Board, IRB) of the CHA.

No. age Continuous miscarriage experience 36 kDa (ITI-H4) Expression One 30 3 O 2 32 5 O 3 35 4 O 4 31 3 X 5 34 2 O 6 39 3 O 7 30 5 O 8 32 5 X 9 37 5 O 10 31 3 O 11 33 4 X 12 27 3 O 13 28 2 X 14 37 4 O 15 37 3 O 16 34 4 O 17 46 3 X 18 29 4 O 19 35 3 X 20 27 3 X 21 33 2 O 22 27 2 O 23 32 3 X 24 30 2 O 25 35 2 X 26 36 2 X 27 30 3 O 28 29 3 O 29 30 2 O

(2) 2-DE and Image Analysis

Using Multiple Affinity Removal Column (MARC) (Agilent, Wilmington, DE, USA) to remove abundant proteins (albumin, transferrin, IgG, IgA, haptoglobin, antitrypsin, etc.) that are most commonly found in serum It was. A 4.6 mm x 50 mm column was used with binding to 20 μl serum. Chromatographic separation of abundant proteins by MARC was performed using a mobile phase reagent kit according to manufacturer's instructions. That is, the crude serum sample was diluted to one fifth with buffer A containing a protease inhibitor (Complete ^™ , Roche, Mannheim, Germany), and then 16,000 g at room temperature for 1-2 minutes using a 0.22 μm spin filter. Purification by centrifugation. Samples were collected in fractions and stored at -20 ° C until use. This concentrated serum was precipitated for 1 hour at -20 ℃ using a cooled 10% TCA solution to separate from 2-DE gel. After washing with cold acetone the pellet was again dissolved in sample buffer for 2-DE.

IEF was performed using a multiphor II system (Amersham Biosciences, Uppsala, Sweden). Samples containing 1 mg of serum protein were mixed with a sufficient amount of rehydration buffer (7 M urea, 2 M thiourea, 4.5% CHAPS, 100 mM DTE, 40 mM Tris, pH 8.8) to make a total of 350 μl. . Samples were stored in 18 cm Immobiline Drystrips, pH 3-10 nonlinear (Amersham Biosciences, Uppsala, Sweden) and subjected to IEF at 80000 Vh. For two-dimensional separation, a 9-16% linear gradient polyacrylamide gel was separated for 5 hours at 40 mA. After fixing the protein for 1 hour with a solution containing 40% methanol and 5% phosphoric acid, the gel was stained with Coomassie brilliant blue G-250 (CBB G-250) for 12 hours. This stained gel was scanned with a GS-710 imaging densitometer (Bio-Rad, Hercules, Calif., USA) and analyzed by Image Master ^™ 2D Platinum software (Amersham Bioscience, Uppsala, Sweden).

(3) Gel Digestion and Mass Spectrum Analysis

Spots were cut from the gel and then digested with trypsin (Promega, Madison, Wis., USA). Forus R2 and oligo R3 resins (Applied Biosystems, Foster City, Calif., USA) were used for peptide desalination and concentration. MALDI-TOF MS and MS / MS were prepared using the 4800 MALDI-TOF / TOF ^™ Analyzer (Applied Biosystems, Foster City) using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. , CA, USA). The pressure of the TOF analyzer was 7.6 x 10 ^-7 Torr. Mass spectral values were calculated with a voltage of 20 kV, 500 laser pulses, and trypsin auto-digested peaks (m / z 842.5090 and 2211.1046). Data was obtained through Data Explorer 4.4 (PerSeptive Biosystems), and monoisotopic masses were obtained from the NCBInr protein database (Ver. 20090707) of the human (5043617 sequences; 1746788941 residues) and the MASCOT search engine (http: // www). .matrixscience.com).

(4) LC-MS / MS

To identify cleavage sites of ITI-H4, Nano LC-MS / MS analysis was performed with an Agilent 1100 Series nano-LC and LTQ-mass spectrometer (Thermo Electron, Waltham, Mass., USA). Capillary column (150 mm x 0.075 mm) purchased from Proxeon (Odense C, Denmark) for LC-MS / MS analysis and Magic C18 stationary phase (Michrom Bioresources, Auburn, CA, USA). Mobile phase A for LC separation used an aqueous 0.1% formic acid solution and mobile phase B used formic acid in 0.1% acetonitrile. Gradients were set at 5% -35% B 50 minutes, 40% -60% B 20 minutes, 60% -80% B 5 minutes. Flow rate was maintained at 300 nL / min after injection. The MS / MS scan range was 400-1800 m / z, with each MS / MS averaged 1 microscan on LTQ. The temperature of the ion transfer tube was maintained at 200 ° C., and the spray was 1.5.0-2.0 kV. Normalized collision energy for MS / MS was set to 35%. The mass tolerance for precursors and fragment ions was 1.5 Da and 0.8 Da, respectively. For protein identification, a MASCOT ion score of> 30 was used. To increase the reliability, we used δCn≥0.1 at 1+, Rsp≤4, Xcorr≥1.5 at 2+, Xcorr≥2.0 at 3+, Xcorr≥2.5 at 3+, and peptide probability> 0.1. Peptides were oxidized to methionine and carboxyamidomethylated and carboxymethylated with cysteine.

(5) Western blot analysis

SDS-PAGE was performed on serum diluted 1/2 with phosphate buffered saline (PBS). 30 μg of protein was separated on 10% SDS-PAGE and blotted onto nitrocellulose membrane. Membrane was α-2-diluted with anti-IGFBP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-AACT (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1 to 200 It was reacted with a blocking solution containing macroglobulin (Abcam, Cambridge, UK) antibody. In addition, to confirm the expression of ITI-H4, blocking including anti-ITI-H4 (sc-21987 and sc-34471, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies in which the membranes were diluted 1: 200 each Reacted with the solution. All membranes were then reacted with secondary antibodies to donkey anti-goat IgG-HRP or goat anti-mouse IgG-HRP diluted to 10,000. Finally, the protein was detected using the ECL system (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands obtained by western blot were scanned and compared using Fluor-S ^™ MultiImager (Bio-Rad, Hercules, CA, USA).

2. Results

(1) 2-DE analysis using blood samples from patients with habitual miscarriage

A comparative proteomics study was performed using blood samples from habitual abortion patients and normal subjects. Blood samples were used to address the difficulty of sampling tissue or follicular fluid from habitual abortion patients. An immunoaffinity column (MARC) was used to remove abundant proteins present in the blood. The proteomic analysis confirmed the protein expressed differently in the blood samples of normal and habitual abortion patients, as shown in FIG.

(2) Identifying proteins with differences in expression in patients with habitual miscarriage

To compare protein expression levels, blood samples of three habitual abortion patients were prepared, and one sample was prepared by mixing three normal bloods as a control. As a result of 2-DE analysis, 514 spots in the control group and 549 spots, 563 spots and 553 spots in each habitual abortion patient sample were identified. The expression level was confirmed by comparing the volume ratio of the relative spots of the protein separated from the gel, and the comparative quantitative analysis of the spots more than two times low-expression is shown in Table 2 below, and the comparison of the spots more than two times low-expression The quantitative analysis results are shown in Table 3 below.

In the samples of patients with habitual miscarriage, 15 spots more than doubled and 7 spots more than doubled (see FIGS. 2 and 3). The increased 15 spots in the samples of patients with habitual miscarriage were 305 of a, 402, 428, 453, 475, c 590, 616, 653, d 778, e 669, 684, 704, 714, 956, And spot 530 of f (FIG. 2). The seven spots decreased relative to the normal subject's sample are 207 of a, 215, 238, 320, 351 of c, and 702, 709 of c (FIG. 3).

The spots showing the difference in expression level were identified by MALDI-TOF / MS. Which proteins are the 22 spots are shown in Table 4 (protein with increased expression) and Table 5 (protein with reduced expression).

* Score: -10 X log (P) [P is the probability that the observed match is a random event. It is based on the NCBInr protein database using the MASCOT search engine as MS / MS data]

* Score: -10 X log (P) [P is the probability that the observed match is a random event. It is based on the NCBInr protein database using the MASCOT search engine as MS / MS data]

ITI-H4 (spots 475, 669) and AACT (spots 402) are involved in immune responses, IGFBP-1 (spots 305) is involved in metabolism, and macroglobulin α2 (spots 714) is associated with thrombosis do. Of these, ITI-H4 was identified identically at spots 475 and 669 with different molecular weights. This indicates that the two spots of ITI-H4 will be truncated proteins of the same but different size, and the fragmentation will be associated with habitual miscarriage.

(3) Western blot analysis

Western blot analysis was performed on proteins with increased expression levels (36 kDa of ITI-H4, AACT, IGFBP-1, α2-macroglobulin) and reduced proteins (120 kDa of ITI-H4) in 2-DE assay. The results were verified. Figure 4 shows that α2-macroglobulin, AACT (α1-antichymotrypsin), and IGFBP 1 (insulin-like growth factor-binding protein 1) are not significant with habitual miscarriage. Interestingly, ITI-H4, which showed increasing expression, showed very weak expression compared to normal samples (FIG. 5). To confirm the reason, Western blot analysis was performed on the 36 kDa and 85 kDa cleaved forms of ITI-H4. As shown in FIG. 5, the expression of cleaved 36 kDa of ITI-H4 was strongly expressed as compared to the normal sample in the habitual abortion patient. Therefore, spot 475 was confirmed to be 85 kDa of ITI-H4 cleaved rather than 120 kDa of ITI-H4. We conducted a MALDI-TOF / MS analysis to determine the presence of 120 kDa of ITI-H4 in gels analyzed using blood samples from normal individuals. As a result, it was confirmed that the reduced spot number 207 in the sample of the habitual miscarriage patient was ITI-H4 of 120 kDa (Fig. 3 and Table 5). In addition, more habitual patients and normal blood samples were used to confirm the significance of the results (FIG. 5). Of the total 29 habitual abortion patients, 19 showed expression of 36 kDa ITI-H4, which is shown in Table 1 above. Although expression of 120 kDa ITI-H4 was observed in 7 patients with habitual abortion, the expression of 36 kDa ITI-H4 was 65.51% in the patients with habitual abortion. These results show that ITI-H4 can be used as a biomarker for patients with habitual miscarriage.

(4) Confirmation of cleavage site using LC-MS / MS

LC-MS / MS analysis was performed to identify the 120 kDa and 36 kDa ITI-H4 fragmentation sites identified in Western blot analysis (FIGS. 5 and 6). Reportedly, ITI-H4 is fragmented from 621G to 650Q (GSTFFKYYLQGAKIPKPEASFSPRRGWNRQ) (J. Song, M. Patel, CN Rosenzweig, Y. Chan-Li, LJ Sokoll, ET Fung, NH Choi-Miura, M). Goggins, DW Chan and Z. Zhang, Clin. Chem. (Washington, DC) , 2006, 52, 1045-1063). In the LC-MS / MS analysis performed by the inventors, the protein sequence of 36 kDa ITI-H4 was confirmed only at the fragmentation site (FIG. 6a), and the protein sequence of 120 kDa ITI-H4 was confirmed throughout. (FIG. 6B).

3. Consideration

Pregnancy is a complex process regulated by various intracellular regulatory processes such as maternal immunity and angiogenesis. Therefore, habitual miscarriage is caused by abnormalities of various intracellular control processes that occur during pregnancy. 50-60% of habitual miscarriages have been reported to be associated with chromosomal abnormalities, anatomical abnormalities, and infections in fetuses, but 40-50% have no known cause. In this regard, we recently discovered and reported for the first time a specific protein specifically expressed in the follicular sample of a habitual abortion patient using 2-DE and MALDI-TOF / MS analysis (YS Kim, MS). Kim, SH Lee, BC Choi, JM Lim, KY Cha and KH Baek, Proteomics , 2006, 6, 3445-3454).

In this study, 22 proteins with different expression patterns were identified using proteomics technology. Among them, five proteins were selected and confirmed by Western blot analysis in the samples of habitual abortion patients and normal patients. As a result, it was confirmed that the expression patterns of ITI-H4 among the five proteins are significantly different in the habitual miscarriage patients. Interestingly, 120 kDa of ITI-H4 was sectioned into 85 kDa and 36 kDa in a sample of a habitual miscarriage. Sectioning of the habitual abortion patients decreased ITI-H4 at 120 kDa but increased the two divided bands. We also identified using LC-MS / MS techniques to analyze where fragmentation takes place at ITI-H4 (FIG. 6). As a result, the amino acid sequence of 36 kDa ITI-H4 was identified only at the back of the predicted fragment site (FIG. 6B), and the amino acid sequence of 120 kDa ITI-H4 was identified at the front and back of the predicted fragment. (FIG. 6A).

Therefore, fragmentation occurring in ITI-H4 is believed to be involved in habitual miscarriage. In other words, 36 kDa of ITI-H4 was identified in 65.51% of addictive abortion patients, and ITI-H4 fragmentation may be closely related to one of the causes of habitual abortion. ITI-H4 expression in the blood samples of patients can be used as an effective biomarker for pregnancy immunity.In particular, the diagnosis using blood samples can reduce the time it takes to diagnose habitual abortions. Will be useful.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Analytical method for diagnosing recurrent pregnancy loss and kit          therefor <130> PN0473 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 310 <212> PRT <213> Homo sapiens <400> 1 Gly Ser Thr Phe Phe Lys Tyr Tyr Leu Gln Gly Ala Lys Ile Pro Lys   1 5 10 15 Pro Glu Ala Ser Phe Ser Pro Arg Arg Gly Trp Asn Arg Gln Ala Gly              20 25 30 Ala Ala Gly Ser Arg Met Asn Phe Arg Pro Gly Val Leu Ser Ser Arg          35 40 45 Gln Leu Gly Leu Pro Gly Pro Pro Asp Val Pro Asp His Ala Ala Tyr      50 55 60 His Pro Phe Arg Arg Leu Ala Ile Leu Pro Ala Ser Ala Pro Pro Ala  65 70 75 80 Thr Ser Asn Pro Asp Pro Ala Val Ser Arg Val Met Asn Met Lys Ile                  85 90 95 Glu Glu Thr Thr Met Thr Thr Gln Thr Pro Ala Pro Ile Gln Ala Pro             100 105 110 Ser Ala Ile Leu Pro Leu Pro Gly Gln Ser Val Glu Arg Leu Cys Val         115 120 125 Asp Pro Arg His Arg Gln Gly Pro Val Asn Leu Leu Ser Asp Pro Glu     130 135 140 Gln Gly Val Glu Val Thr Gly Gln Tyr Glu Arg Glu Lys Ala Gly Phe 145 150 155 160 Ser Trp Ile Glu Val Thr Phe Lys Asn Pro Leu Val Trp Val His Ala                 165 170 175 Ser Pro Glu His Val Val Val Thr Arg Asn Arg Arg Ser Ser Ala Tyr             180 185 190 Lys Trp Lys Glu Thr Leu Phe Ser Val Met Pro Gly Leu Lys Met Thr         195 200 205 Met Asp Lys Thr Gly Leu Leu Leu Leu Ser Asp Pro Asp Lys Val Thr     210 215 220 Ile Gly Leu Leu Phe Trp Asp Gly Arg Gly Glu Gly Leu Arg Leu Leu 225 230 235 240 Leu Arg Asp Thr Asp Arg Phe Ser Ser His Val Gly Gly Thr Leu Gly                 245 250 255 Gln Phe Tyr Gln Glu Val Leu Trp Gly Ser Pro Ala Ala Ser Asp Asp             260 265 270 Gly Arg Arg Thr Leu Arg Val Gln Gly Asn Asp His Ser Ala Thr Arg         275 280 285 Glu Arg Arg Leu Asp Tyr Gln Glu Gly Pro Pro Gly Val Glu Ile Ser     290 295 300 Cys Trp Ser Val Glu Leu 305 310 <210> 2 <211> 930 <212> DNA <213> Homo sapiens <400> 2 ggttccactt tcttcaaata ttatctccag ggagcaaaaa taccaaaacc agaggcttcc 60 ttttctccaa gaagaggatg gaatagacaa gctggagctg ctggctcccg gatgaatttc 120 agacctgggg ttctcagctc caggcaactt ggactcccag gacctcctga tgttcctgac 180 catgctgctt accacccctt ccgccgtctg gccatcttgc ctgcttcagc accaccagcc 240 acctcaaatc ctgatccagc tgtgtctcgt gtcatgaata tgaaaatcga agaaacaacc 300 atgacaaccc aaaccccagc ccccatacag gctccctctg ccatcctgcc actgcctggg 360 cagagtgtgg agcggctctg tgtggacccc agacaccgcc aggggccagt gaacctgctc 420 tcagaccctg agcaaggggt tgaggtgact ggccagtatg agagggagaa ggctgggttc 480 tcatggatcg aagtgacctt caagaacccc ctggtatggg ttcacgcatc ccctgaacac 540 gtggtggtga ctcggaaccg aagaagctct gcgtacaagt ggaaggagac gctattctca 600 gtgatgcccg gcctgaagat gaccatggac aagacgggtc tcctgctgct cagtgaccca 660 gacaaagtga ccatcggcct gttgttctgg gatggccgtg gggaggggct ccggctcctt 720 ctgcgtgaca ctgaccgctt ctccagccac gttggaggga cccttggcca gttttaccag 780 gaggtgctct ggggatctcc agcagcatca gatgacggca gacgcacgct gagggttcag 840 ggcaatgacc actctgccac cagagagcgc aggctggatt accaggaggg gcccccggga 900 gtggagattt cctgctggtc tgtggagctg 930

Claims (7)

In order to provide information necessary for the diagnosis of habitual miscarriage, the analysis method comprising the step of measuring the presence or absence of the expression of the protein of SEQ ID NO: 1 in blood isolated from the female. The analysis method according to claim 1, wherein the measurement of the expression of the protein is performed by measuring the expression of the gene encoding the protein of SEQ ID NO: 1. The analysis method according to claim 2, wherein the gene encoding the protein of SEQ ID NO: 1 has a nucleotide sequence of SEQ ID NO: 2. The method of claim 1, wherein the measurement of the expression of the protein is performed by western blotting. A diagnostic kit for a habitual miscarriage comprising a molecule capable of measuring the expression of a protein of SEQ ID NO: 1, wherein the molecule is an antibody, substrate, ligand, or cofactor that specifically binds to the protein. Diagnostic kit of miscarriage. The diagnostic kit of habitual miscarriage according to claim 5, wherein the molecule is labeled with a detectable label. The diagnostic kit for habitual miscarriage according to claim 5, wherein the antibody against the protein of SEQ ID NO: 1 is in the form of a protein chip immobilized on a substrate.

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