Application of NUCB1 in reproductive capacity diagnosis and assisted reproductive evaluation

Abstract

The invention provides an application of NUCB1 in reproductive capacity diagnosis and assisted reproductive evaluation, and particularly provides an application of an NUCB1 gene, mRNA, cDNA, protein or a detection reagent thereof. The invention finds for the first time that (1) the reproductive capacity of a mouse with NUCB1 gene deletion is lost or obviously reduced no matter whether the mouse is male or female; (2) during assisted reproduction, the superovulation capacity of the female rats is obviously reduced; therefore, the NUCB1 gene or the protein thereof can be (i) used as a marker for detecting and diagnosing reproductive capacity and (ii) used as a marker for judging female superovulation potential during assisted reproduction.

Description

Application of NUCB1 in fertility diagnosis and assisted reproduction evaluation Technical Field

[0001] The present invention relates to the fields of reproduction and diagnosis. More specifically, the present invention relates to the application of NUCB1 in reproductive capacity diagnosis and assisted reproduction assessment. Background Art

[0002] Reproductive health is a core component of population health. The World Health Organization lists infertility as one of the three major diseases of the 21st century (after cancer and cardiovascular and cerebrovascular diseases). Factors such as environmental impacts, increased work pressure, and delayed childbearing age in women have led to an increasing incidence of infertility. Globally, the infertility rate is over 10%. According to the "China Infertility Status Survey Report" jointly released by the China Population Association and the National Health and Family Planning Commission in August 2016, the infertility prevalence in my country is between 12.5% ​​and 15%. Based on this, the number of infertility patients in my country is estimated to be around 50 million. It is estimated that by 2025, the infertility prevalence in my country will increase to 18%, and the number of patients will reach approximately 60 million.

[0003] Therefore, being able to predict or diagnose fertility is particularly meaningful. Currently, there are a variety of tests to examine reproductive capacity or health, such as endocrine and gynecological examinations for women, and semen and endocrine examinations for men. Although these tests can provide some initial assessment, in most cases, their clinical value is controversial, and new methods are needed to ensure more accurate assessment.

[0004] The three main treatment options for infertility are conventional medication, surgery, and assisted reproductive technology (ART), with ART being the treatment of last resort. Approximately 20% of these women, or approximately 10 million women, require ART to resolve their fertility issues. Furthermore, ART boasts an overall pregnancy rate of 40%-60%, significantly higher than the other two treatment options. Therefore, ART has become the primary treatment for infertility. Driven by the two-child policy, ART has become an important option for older mothers. According to statistics, demand for ART services in my country grew at a compound annual growth rate of 13.6% from 2015 to 2020.

[0005] With the surge in the number of infertile women, assisted reproductive technology (ART) has become an important way for these women to achieve conception. Controlled ovarian stimulation (COT) is crucial for obtaining high-quality and sufficient oocytes, achieving ideal pregnancy and live birth rates. Poor ovarian response (POR), primarily manifested by a poor response to gonadotropins and the inability to obtain oocytes of sufficient quality and quantity, has become a major challenge in assisted reproduction. Therefore, biomarkers that can predict or indicate ovarian responsiveness to gonadal hormones are crucial.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a diagnostic indicator, a kit and a method for detecting and diagnosing reproductive ability and evaluating assisted reproduction.

[0008] The present invention provides the use of the secretory protein NUCB1 in reproductive capacity diagnosis and assisted reproduction assessment. Specifically, the present invention provides a use of a NUCB1 gene, mRNA, cDNA, protein, or a detection reagent thereof. The present invention first discovered that mice lacking the NUCB1 gene (1) lose or significantly reduce reproductive capacity, regardless of whether they are male or female; and (2) the superovulation ability of female mice is significantly reduced during assisted reproduction. Therefore, the NUCB1 gene or its protein can be used (i) as a marker for detecting and diagnosing reproductive capacity, and (ii) as a marker for determining the superovulation potential of female mice during assisted reproduction.

[0009] The first aspect of the present invention provides a use of a NUCB1 gene, mRNA, cDNA, protein or a detection reagent thereof, (i) as a marker for detecting and diagnosing reproductive ability; (ii) as a marker for determining the superovulation ability of females during assisted reproduction; and / or (iii) for preparing a diagnostic reagent or kit for detecting and diagnosing reproductive ability; and / or (iv) for preparing a diagnostic reagent or kit for determining the superovulation ability of females during assisted reproduction.

[0010] In another preferred embodiment, the diagnostic reagents include: antibodies, primers, probes, sequencing libraries, nucleic acid chips or protein chips.

[0011] In another preferred embodiment, the NUCB1 protein includes a full-length protein or a protein fragment.

[0012] In another preferred embodiment, the NUCB1 gene, mRNA, cDNA, or protein is derived from mammals, preferably from rodents (such as mice, rats), primates and humans, and more preferably from patients with low fertility or poor response to superovulation.

[0013] In another preferred embodiment, the accession number of the NUCB1 gene is Gene ID: 4924.

[0014] In another preferred embodiment, the accession number of the NUCB1 mRNA is NM_006184.6.

[0015] In another preferred example, the accession number of the NUCB1 protein is NP_006175.2.

[0016] In another preferred embodiment, the subjects for determining reproductive capacity include mammals and non-mammals.

[0017] In another preferred embodiment, the mammals include humans and non-human mammals.

[0018] In another preferred embodiment, the detection is tissue sample detection, blood sample detection and / or serum sample detection, preferably, blood sample detection.

[0019] In another preferred embodiment, the detection includes immunohistochemistry, immunoblotting, fluorescence quantitative PCR detection, enzyme-linked immunosorbent assay (ELISA) quantitative detection method, solid-phase enzyme-linked immunosorbent spot technology (ELISPOT), micro-sample multi-index flow cytometry protein quantification technology (CBA), and Luminex-liquid suspension chip system.

[0020] In another preferred embodiment, the detection reagent is used to perform in vitro immunohistochemistry, immunoblotting, fluorescence quantitative PCR detection, enzyme-linked immunosorbent assay (ELISA) quantitative detection method, solid-phase enzyme-linked immunosorbent spot technology (ELISPOT), micro-sample multi-index flow cytometry protein quantification technology (CBA), Luminex-liquid suspension chip system method to detect the content of NUCB1 in blood or serum or cell or tissue samples, and the content of NUCB1 is significantly downregulated compared with the content of NUCB1 in blood or serum or cell or tissue samples in the general population (normal population).

[0021] In another preferred embodiment, the significant downregulation means that the NUCB1 content is decreased by ≥60%, preferably ≥80%, more preferably ≥90% or 95%, such as 100%, compared with the NUCB1 content in blood, serum, cell or tissue samples in the general population (normal population).

[0022] In another preferred embodiment, the detection reagents include immunohistochemistry, immunoblotting, fluorescence quantitative PCR detection, enzyme-linked immunosorbent assay (ELISA) quantitative detection method, solid-phase enzyme-linked immunosorbent spot technology (ELISPOT), micro-sample multi-index flow cytometry protein quantification technology (CBA), and Luminex-liquid suspension chip system method conventional supporting reagents.

[0023] In another preferred embodiment, the detection reagent includes a specific antibody for NUCB1, a specific binding molecule for NUCB1, a specific amplification primer, a probe or a chip.

[0024] In another preferred embodiment, the NUCB1 protein or its specific antibody or specific binding molecule is coupled with or carries a detectable label.

[0025] In another preferred embodiment, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0026] In another preferred embodiment, the NUCB1-specific antibody is a monoclonal antibody or a polyclonal antibody.

[0027] A second aspect of the present invention provides a kit for (i) detecting and diagnosing reproductive ability; and / or (ii) judging the superovulation ability of females during assisted reproduction, the kit comprising a container containing a detection reagent for detecting the NUCB1 gene, mRNA, cDNA, or protein; and a label or instructions indicating that the kit is used for (i) detecting and diagnosing reproductive ability; and / or (ii) judging the superovulation ability of females during assisted reproduction; and optional additional components.

[0028] In another preferred embodiment, the diagnosis of reproductive capacity refers to determining the ability, situation or difficulty of fertility.

[0029] In another preferred embodiment, the determination of the superovulation ability of a female during assisted reproduction refers to determining the potential or quantity of oocytes that a female can obtain before superovulation drugs or treatment when retrieving eggs during assisted reproduction.

[0030] In another preferred example, the judgment includes prejudgment or advance prediction.

[0031] In another preferred embodiment, the additional components are other markers for diagnosing reproductive capacity or judging the superovulation capacity of assisted reproduction.

[0032] In another preferred embodiment, the other markers for diagnosing reproductive ability or judging the ability of assisted reproductive superovulation are selected from the following group: anti-Mullerian hormone AMH (anti mullerian hormone), inhibin B (inhibin B), follicle-stimulating hormone FSH (Follicle-stimulating hormone), or a combination thereof.

[0033] In another preferred embodiment, the detection reagent for detecting NUCB1 gene, mRNA, cDNA, or protein includes:

[0034] (1) anti-NUCB1 protein specific antibodies; and / or

[0035] (2) Specific primers for specifically amplifying the mRNA or cDNA of the NUCB1 gene.

[0036] In another preferred embodiment, the label or instructions indicate that the kit is used for:

[0037] (a) When the expression level B of NUCB1 in the blood, serum, cells or tissues of the test subject is lower than the control value A, it indicates that the fertility of the test subject is lower than that of the general population (normal control group);

[0038] (b) When the expression level B of NUCB1 in the blood, serum, cells or tissues of the test subject is lower than the control value A, it indicates that the number of oocytes obtained by the test subject during in vitro superovulation for assisted reproduction is lower than that of the general group (normal control group).

[0039] In another preferred embodiment, the control value A refers to the average tissue expression level or blood content of the general population (normal population).

[0040] In another preferred embodiment, the mammals include humans and non-human mammals.

[0041] In another preferred embodiment, the B is lower than A, which means B / A<1 / 2, more preferably, B / A<1 / 3, and even more preferably, B / A<1 / 4.

[0042] A third aspect of the present invention provides a method for diagnosing reproductive capacity and determining the ability of females to undergo superovulation during assisted reproduction, the method comprising:

[0043] (a) providing a test sample from a subject;

[0044] (b) detecting the expression level B of the NUCB1 gene or protein in the test sample;

[0045] (c) comparing the expression level B of the NUCB1 gene or protein determined in step (b) with that of the control;

[0046] Among them, compared with the control, the expression level B of the NUCB1 gene or its protein in the test sample is lower than the control value A, indicating that the fertility of the subject is lower than that of the general group (normal control group); and / or the number of oocytes obtained by female subjects during assisted reproductive in vitro superovulation is lower than that of the general group (normal control group).

[0047] In another preferred embodiment, the control value A refers to the average tissue expression level or blood content of the general population (normal population).

[0048] In another preferred embodiment, the B is lower than A, which means B / A<1 / 2, preferably, B / A<1 / 3, and more preferably, B / A<1 / 4.

[0049] In another preferred embodiment, the subject is a human or non-human mammal.

[0050] In another preferred embodiment, the test sample is blood, serum, cells or tissue of a patient with low fertility or poor response to superovulation.

[0051] In another preferred embodiment, the detection step (b) comprises detecting the amount of NUCB1 mRNA, or the amount of secreted NUCB1 in blood or serum; and / or detecting the amount of NUCB1 protein in tissues.

[0052] In another preferred embodiment, the expression level of NUCB1 protein in the sample is detected by fluorescent quantitative PCR or immunohistochemistry.

[0053] In another preferred embodiment, the expression level of the NUCB1 gene in the sample is detected by fluorescent quantitative PCR or quantitative chip method.

[0054] In another preferred embodiment, the concentration of NUCB1 protein in the sample is detected by enzyme-linked immunosorbent assay.

[0055] In another preferred embodiment, the method is non-diagnostic and non-therapeutic. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows that the reproductive capacity of NUCB1 knockout mice is significantly reduced. A. Wild-type mice give birth to mice on average about 20 days after mating. Compared with wild-type mice (WT), the reproductive capacity of NUCB1 knockout mice is significantly reduced, the time is doubled, and some are infertile (infertility is counted as 50 days); B. There is no difference in the number of mice born per litter between wild-type mice and NUCB1 knockout mice; C, D. The reproductive capacity and number of mice born per litter of NUCB1 heterozygous male mice and NUCB1 heterozygous female mice are no different from those of wild-type mice.

[0057] Figure 2 shows that the reproductive capacity of NUCB2 gene knockout mice is not affected. A. NUCB2 heterozygous male mice mated with NUCB2 heterozygous female mice, NUCB2 knockout male mice mated with wild-type female mice, and NUCB2 knockout female mice mated with wild-type male mice. In all three mating (pairing) situations, the babies were born at normal times and were comparable to the wild type. B. NUCB2 heterozygous male mice were paired with NUCB2 heterozygous female mice, NUCB2 knockout male mice were paired with wild-type female mice, and NUCB2 knockout female mice were paired with wild-type male mice. There was no difference in the number of mice born per litter in the three pairing situations, and it was comparable to the wild type.

[0058] Figure 3 shows the superovulation of NUCB1 and NUCB2 knockout female mice in response to hormone treatment. A. Flowchart of superovulation treatment; B. The number of superovulations in NUCB1 knockout female mice was significantly reduced; C. The superovulation ability of NUCB2 knockout female mice was not affected. DETAILED DESCRIPTION

[0059] After extensive and intensive research, the inventors have discovered and experimentally demonstrated for the first time that the nucleobindin-1 (NUCB1) gene or protein can be used (i) as a marker for detecting and diagnosing reproductive capacity and (ii) as a marker for determining the ability of females to undergo superovulation during assisted reproduction. Based on this, the inventors have completed the present invention.

[0060] In the present invention, "nucleobindin-1" and "NUCB1 protein" are used interchangeably and have the same meaning. "nucleobindin-2" and "NUCB2 protein" are used interchangeably and have the same meaning.

[0061] sample

[0062] As used herein, the terms "sample" or "specimen" refer to a material that is specifically associated with a subject, from which specific information about the subject can be determined, calculated, or inferred. A sample can consist entirely or in part of biological material from the subject. A sample can also be a material that has been in contact with the subject in a manner that allows testing of the sample to provide information about the subject. A sample can also be a material that has been in contact with other materials that were not from the subject, but that enable the first material to be subsequently tested to determine information about the subject, for example, a sample can be a cleaning solution from a probe or scalpel. A sample can be a source of biological material other than that in contact with the subject, as long as a person skilled in the art can still determine information about the subject from the sample.

[0063] Express

[0064] As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or a gene or gene portion, and also includes the appearance of a detection substance associated with expression. For example, cDNA, the binding of a binding ligand (such as an antibody) to a gene or other oligonucleotide, protein or protein fragment, and the chromogenic portion of the binding ligand are all included within the scope of the term "expression." Thus, an increase in half-dot density on an immunoblot such as a western blot also falls within the scope of the term "expression" based on biological molecules.

[0065] Control value

[0066] As used herein, the term "control value" refers to a value that is statistically correlated with a particular outcome when compared to an assay result. In a preferred embodiment, a reference or control value is determined based on a statistical analysis of studies comparing the expression of the NUCB1 gene or protein with known clinical outcomes. Some such studies are shown in the Examples section herein. However, studies from the literature and user experience with the methods disclosed herein can also be used to generate or adjust reference or control values. Reference or control values ​​can also be determined by taking into account circumstances and outcomes specifically related to the patient's medical history, genetics, age, and other factors.

[0067] In the present invention, the gene knockout mouse data showed that the fertility and ovulation response of mice with complete NUCB1 knockout were significantly reduced; the fertility and ovulation response of heterozygous mice (with half the NUCB1 content) did not change significantly.

[0068] Therefore, when the expression level B of NUCB1 in the blood of the test subject of the present invention is lower than the control value A, that is, B / A<1 / 2, better, B / A<1 / 3, and even better, B / A<1 / 4, it indicates that the fertility of the test subject is lower than that of the general group (normal control group), the infertility or pregnancy time is significantly longer than that of the general group (normal control group), or the number of oocytes obtained by the test subject during in vitro superovulation for assisted reproduction is lower than that of the general group (normal control group).

[0069] NUCB1 protein and polynucleotide

[0070] In the present invention, the terms "protein of the present invention," "NUCB1 protein," and "NUCB1 polypeptide" are used interchangeably to refer to proteins or polypeptides having a NUCB1 amino acid sequence. These include NUCB1 proteins with or without an initiator methionine. Furthermore, the term also encompasses full-length NUCB1 and fragments thereof. The NUCB1 protein referred to herein includes its complete amino acid sequence, secreted proteins, mutants, and functionally active fragments thereof.

[0071] NUCB1 is a member of a family of secreted calcium-binding proteins. The NUCBs (Nucleobindins) family includes NUCB1 and NUCB2. The biological functions and molecular mechanisms of NUCB1 remain largely unknown.

[0072] The human NUCB1 protein is 461 amino acids long (Accession No. NP_006175.2), and the mouse NUCB1 protein is NP_001157134.1.

[0073] In the present invention, the terms "NUCB1 gene" and "NUCB1 polynucleotide" are used interchangeably and refer to nucleic acid sequences having NUCB1 nucleotide sequences.

[0074] The full-length genome of the human NUCB1 gene is 23060 bp (NCBI GenBank accession number is NC_000019.10), and the full-length mRNA sequence of its transcription product is 2406 bp (NCBI GenBank accession number is NM_006184.6).

[0075] The full-length genome of the mouse NUCB1 gene is 17513 bp (NCBI GenBank accession number NC_000073.7), and the full-length mRNA sequence of its transcription product is 2418 bp (NCBI GenBank accession number NM_001163662.2).

[0076] The protein sequence similarity between human and mouse NUCB1 is 89%.

[0077] It should be understood that when encoding the same amino acid, the substitution of nucleotides in the codon is acceptable. In addition, it should be understood that when the nucleotide substitution produces a conservative amino acid substitution, the change of nucleotides is also acceptable.

[0078] When an amino acid fragment of NUCB1 is obtained, a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the NUCB1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified in each time are spliced ​​together in the correct order.

[0079] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0080] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0081] Currently, DNA sequences encoding the protein of the present invention (or its fragments and derivatives) can be obtained entirely through chemical synthesis, and the DNA sequences can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

[0082] The polynucleotide sequences of the present invention can be used to express or produce recombinant NUCB1 polypeptides using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0083] (1) Transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the human NUCB1 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0084] (2) Host cells cultured in a suitable culture medium;

[0085] (3) Isolate and purify proteins from culture medium or cells.

[0086] In the present invention, the NUCB1 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0087] Methods well known to those skilled in the art can be used to construct expression vectors containing a NUCB1-encoding DNA sequence and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0088] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0089] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0090] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces bacteria cells; fungal cells, such as yeast; plant cells; insect cells; animal cells, etc.

[0091] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0092] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0093] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0094] Specific antibodies

[0095] In the present invention, the terms "antibody of the present invention" and "anti-NUCB1 specific antibody" are used interchangeably.

[0096] The present invention also includes polyclonal antibodies and monoclonal antibodies, particularly monoclonal antibodies, that are specific for human NUCB1 polypeptides. Here, "specificity" refers to the ability of the antibody to bind to human NUCB1 gene products or fragments. Preferably, it refers to antibodies that can bind to human NUCB1 gene products or fragments but do not recognize and bind to other non-related antigenic molecules. The antibodies of the present invention include those that can bind to and inhibit human NUCB1 protein, as well as those that do not affect the function of human NUCB1 protein. The present invention also includes antibodies that can bind to modified or unmodified forms of human NUCB1 gene products.

[0097] The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a mouse antibody but still retain an antibody portion from a human.

[0098] The antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human NUCB1 gene products or antigenic fragments thereof can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human NUCB1 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. The antibodies of the present invention can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256;495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). The antibodies of the present invention include antibodies that can block the function of human NUCB1 protein as well as antibodies that do not affect the function of human NUCB1 protein. The various antibodies of the present invention can be obtained using fragments or functional regions of human NUCB1 gene products using conventional immunological techniques. These fragments or functional regions can be prepared using recombinant methods or synthesized using a peptide synthesizer. Antibodies that bind to unmodified forms of the human NUCB1 gene product can be produced by immunizing animals with the gene product produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (e.g., glycosylated or phosphorylated proteins or polypeptides) can be obtained by immunizing animals with the gene product produced in eukaryotic cells (e.g., yeast or insect cells).

[0099] Antibodies against human NUCB1 can be used in immunohistochemistry to detect NUCB1 in specimens (especially tissue or serum samples). Because NUCB1 has an extracellular domain, when this extracellular domain sheds and enters the blood, the soluble NUCB1 extracellular domain can become a target for serum detection.

[0100] Detection method

[0101] Taking advantage of the fact that NUCB1 is closely related to reproductive capacity and / or the ability of female superovulation during assisted reproduction, the present invention also provides a method for detecting reproductive capacity and / or the ability of female superovulation during assisted reproduction.

[0102] In a preferred embodiment of the present invention, the present invention provides a high-throughput second-generation sequencing method for detecting NUCB1 as well as Sanger sequencing, fluorescence quantitative PCR (qPCR), in situ immunofluorescence (FISH) and immunohistochemistry.

[0103] Detection kit

[0104] Based on the fact that NUCB1 is closely related to reproductive ability and / or female superovulation ability during assisted reproduction, NUCB1 can be used as a diagnostic marker for reproductive ability and / or female superovulation ability during assisted reproduction.

[0105] The present invention also provides a kit for (i) detecting and diagnosing reproductive ability; and / or (ii) judging the superovulation ability of females during assisted reproduction, which contains a detection reagent for detecting the NUCB1 gene, mRNA, cDNA, or protein; and a label or instructions, wherein the label or instructions indicate that the kit is used for (i) detecting and diagnosing reproductive ability; and / or (ii) judging the superovulation ability of females during assisted reproduction.

[0106] Wherein, the label or instructions indicate the following:

[0107] (a) When the expression level B of NUCB1 in the blood, serum, cells or tissues of the test subject is lower than the control value A, it indicates that the fertility of the subject is lower than that of the general population (normal control group);

[0108] (b) When the expression level B of NUCB1 in the blood, serum, cells or tissues of the test subject is lower than the control value A, it indicates that the number of oocytes obtained by the subject during in vitro superovulation for assisted reproduction is lower than that of the general group (normal control group).

[0109] In another preferred embodiment, the control value A refers to the average tissue expression level or blood content of the general population (normal population).

[0110] Detection methods and kits

[0111] The present invention relates to diagnostic assays for quantitative and localized detection of human NUCB1 protein or mRNA levels. These assays are well known in the art. The human NUCB1 protein levels detected in these assays can be used to (i) diagnose reproductive capacity and / or (ii) determine the ability of females to undergo superovulation for assisted reproduction.

[0112] One method for detecting whether NUCB1 protein is present in a sample is to use a specific antibody against NUCB1 protein for detection, which comprises: contacting the sample with the specific antibody against NUCB1 protein; and observing whether an antibody complex is formed. The formation of the antibody complex indicates the presence of NUCB1 protein in the sample.

[0113] NUCB1 protein or its polynucleotides can be used for the diagnosis and treatment of diseases associated with NUCB1 protein. Part or all of the polynucleotides of the present invention can be immobilized as probes on microarrays or DNA chips for analysis of differential gene expression in tissues and genetic diagnosis. Anti-NUCB1 antibodies can be immobilized on protein chips for detection of NUCB1 protein in samples.

[0114] The main advantages of the present invention include:

[0115] (1) The present invention first discovered and experimentally demonstrated that the nucleobindin-1 (NUCB1) gene or its protein can be used (i) as a marker for detecting and diagnosing reproductive ability and (ii) as a marker for determining the superovulation ability of females during assisted reproduction.

[0116] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

[0117] General Methods

[0118] 1. Mouse reproductive capacity test

[0119] Wild-type C57BL / 6 mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) and mice with NUCB1 and NUCB2 genotypes (obtained from Professor Li Lin's laboratory at the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences) were housed in an SPF-grade animal facility on a normal diet (13.5% kcal) with a 12-hour lights-off (19:30-07:30) and a 12-hour lights-on (07:30-19:30). The specific mouse mating cage treatment protocol was as follows: mating cages were set up in a 1:1 ratio, with one male and one female per cage. Two weeks after the mating cages were set up, the males were removed and absorbent paper was placed in the cages for the females to nest. Around 20 days after birth, the number of births was counted. For NUCB1 knockout mice, the above protocol failed to produce offspring. A new protocol was implemented: four weeks after the mating cages were set up, the males were removed and absorbent paper was placed in the cages for the females to nest. The number of births was counted, and births were counted. In this case, if the mice were still infertile, the births were counted as 50 days.

[0120] 2. Superovulation of female mice

[0121] On the first day, pregnant mare serum gonadotropin (PMSG) 7.5 IU / female mouse was injected; on the third day (48 hours), human chorionic gonadotropin (hCG) 7.5 IU / female mouse was injected, and the number of oocytes was counted on the fourth day (64 hours).

[0122] 3. Data Statistical Analysis

[0123] Data are presented as mean ± standard error (mean ± sem). P values ​​were determined by t-test (student's test) or one-way ANOVA, and significance is indicated as *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0124] Example 1: Fertility of mice lacking the NUCB1 gene is significantly reduced

[0125] As shown in Figure 1A, three mouse pairing (mating) groups were set up: (1) wild-type male mice mated with wild-type female mice. This group produced mice on average about 20 days; (2) NUCB1 gene knockout male mice mated with wild-type female mice; (3) NUCB1 gene knockout female mice mated with wild-type male mice. Compared with the mice in group (1), the NUCB1 gene deletion significantly reduced the reproductive capacity of the mice, doubled the reproductive time, and some were infertile (the reproductive time of infertility was recorded as 50 days). As shown in Figure 1B, there was no difference in the number of offspring mice born in each mating cage in the three pairing situations in 1A. As shown in Figures 1C and 1D, NUCB1 heterozygous male mice were paired with NUCB1 heterozygous female mice. In this case, the reproductive capacity and number of mice born per litter were no different from those of the wild type.

[0126] Example 2 Fertility of mice lacking the NUCB2 gene is not affected

[0127] As shown in Figure 2A, three mouse pairings (matings) were set up: (1) NUCB2 heterozygous male mice mated with NUCB2 heterozygous female mice, (2) NUCB2 knockout male mice mated with wild-type female mice, and (3) NUCB2 knockout female mice mated with wild-type male mice. All three conditions produced offspring on schedule and were identical to wild-type mice. As shown in Figure 2B, there was no difference in the number of offspring born in each mating cage under the three pairing conditions in 2A.

[0128] Example 3 Female mice lacking the NUCB1 gene responded poorly to superovulation, whereas lacking the NUCB2 gene had no significant effect

[0129] Figure 3A shows the most commonly used experimental design for mouse superovulation. Specifically, progesterone is injected on day 1, human chorionic gonadotropin is injected on day 3 (48 hours), and the number of oocytes is counted on day 4 (64 hours).

[0130] Figures 3B and 3C show that female mice lacking the NUCB1 gene (6 weeks old) responded poorly to superovulation, with the number of ovulations significantly lower than that of wild-type female mice, while female mice lacking the NUCB2 gene (8 weeks old) had no significant effect.

[0131] The gene knockout mouse data of the present invention show that the fertility and ovulation response of mice with complete NUCB1 knockout are significantly reduced; the fertility and ovulation response of heterozygous mice (with half the NUCB1 content) do not change significantly.

[0132] The present invention is the first to discover that nucleobindin-1 (NUCB1) can be used (i) as a marker for detecting and diagnosing reproductive capacity and (ii) as a marker for determining female superovulation capacity during assisted reproduction. However, the NUCB2 gene from the same family does not have this function.

[0133] All documents mentioned in this application are incorporated herein by reference, just as if each document were individually incorporated by reference. It should also be understood that after reading the above disclosure, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of NUCB1 gene, mRNA, cDNA, protein or a detection reagent thereof, characterized in that: (i) used as a marker for detecting and diagnosing reproductive capacity; (ii) used as a marker for determining the superovulation capacity of females during assisted reproduction; and / or (iii) used for preparing a diagnostic reagent or kit for detecting and diagnosing reproductive capacity; and / or (iv) used for preparing a diagnostic reagent or kit for determining the superovulation capacity of females during assisted reproduction.

2. The use according to claim 1, characterized in that The detection is a tissue sample detection, a blood sample detection and / or a serum sample detection, preferably, a blood sample detection.

3. The use according to claim 1, characterized in that The detection reagent is used to perform in vitro immunohistochemistry, immunoblotting, fluorescence quantitative PCR detection, enzyme-linked immunosorbent assay (ELISA) quantitative detection method, solid phase enzyme-linked immunosorbent spot technology (ELISPOT), trace sample multi-index flow protein quantification technology (CBA), Luminex-liquid suspension chip system method to detect the content of NUCB1 in blood or serum or cell or tissue samples, and the content of NUCB1 is significantly downregulated compared with the content of NUCB1 in blood or serum or cell or tissue samples in the general population (normal population).

4. A kit for (i) detecting and diagnosing reproductive capacity; and / or (ii) determining the superovulation capacity of females during assisted reproduction, characterized in that: The kit contains a container containing a detection reagent for detecting NUCB1 gene, mRNA, cDNA, or protein; and a label or instructions, wherein the label or instructions indicate that the kit is used for (i) detecting and diagnosing reproductive ability; and / or (ii) judging the superovulation ability of females during assisted reproduction; and optional additional components.

5. The kit according to claim 4, characterized in that The diagnosis of reproductive capacity refers to determining the ability, condition or difficulty of fertility.

6. The kit according to claim 4, characterized in that The determination of the superovulation ability of a female during assisted reproduction refers to determining the potential or quantity of oocytes that a female can obtain when taking eggs during assisted reproduction, before using superovulation drugs or treatments.

7. The kit according to claim 4, characterized in that The additional components are other markers used to diagnose reproductive capacity or determine the superovulation capacity of assisted reproduction.

8. The kit according to claim 7, characterized in that The other markers for diagnosing reproductive ability or judging the superovulation ability of assisted reproduction are selected from the following group: anti-Mullerian hormone AMH (anti mullerian hormone), inhibin INH B (inhibin B), follicle-stimulating hormone FSH (Follicle-stimulating hormone), or a combination thereof.

9. A method for diagnosing reproductive capacity and determining the superovulation capacity of females during assisted reproduction, characterized in that: The method comprises: (a) providing a test sample from a subject; (b) detecting the expression level B of the NUCB1 gene or protein in the test sample; (c) comparing the expression level B of the NUCB1 gene or protein determined in step (b) with that of the control; Among them, compared with the control, the expression level B of the NUCB1 gene or its protein in the test sample is lower than the control value A, which indicates that the fertility of the subject is lower than that of the general group (normal control group); and / or the number of oocytes obtained by female subjects during in vitro superovulation during assisted reproduction is lower than that of the general group (normal control group).

10. The method according to claim 9, characterized in that The B being lower than A means B / A<1 / 2, preferably, B / A<1 / 3, more preferably, B / A<1 / 4.

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