WO2025051101A1 - Anti-meiob monoclonal antibody, mutation detection primer set, aso molecule, small molecule drug against meiob proteins, and use thereof - Google Patents

Abstract

Provided are an anti-MEIOB monoclonal antibody, a mutation detection primer set, an ASO molecule, a small molecule drug against MEIOB proteins, and the use thereof. The monoclonal antibody can specifically recognize MEIOB proteins, and has good specificity and a high titer. Compared with a polyclonal antibody, the monoclonal antibody has higher recognition specificity. The mutation detection primer set can detect the discovered hMEIOB gene mutation sites and potential mutation sites in the hMEIOB gene. The ASO molecule can target and bind to the transcription product of the hMEIOB gene so as to inhibit the expression thereof. The full thio-modification of the molecule improves the stability and half-life of the molecule, and the molecule itself is non-toxic and degradable. The small molecule targeting MEIOB proteins can be used for blocking MEIOB functions and spermiogenesis so as to achieve a contraceptive effect, and can be used for reducing the expression level of MEIOB proteins and cell proliferation capacity in tumor cells so as to realize tumor-targeted therapy.

Description

Anti-MEIOB monoclonal antibodies, mutation detection primer sets, ASO molecules, and small molecule drugs targeting MEIOB protein and their applications Technical Field

The present invention belongs to the field of biotechnology, and relates to an anti-MEIOB monoclonal antibody, a mutation detection primer set, an ASO molecule, and a small molecule drug targeting a MEIOB protein and applications thereof.

Background Art

Genetic factors (gene mutations) account for a large proportion of gamete (sperm or egg) disorders. By exploring the genetic mechanism, it was found that the precise completion of meiosis is crucial for the stable transmission of genetic information. During the homologous chromosome synapsis stage, a large number of meiosis-specific genes should be expressed in time and space and perform specific functions. As one of the many genes, as early as 2013, through the meiotic proteomics study of the model animal C57/6J mouse, the inventor (Luo Mengcheng) was the first to discover a single-stranded DNA binding protein MEIOB (meiosis specific with OB domain) involved in the synapsis of homologous chromosomes in the early stage of meiosis I. Through the study of its function, it was found that abnormal mouse MEIOB can lead to the arrest of male sperm development at the prophase of meiosis I and the early apoptosis of female follicles; with the progress of the study of human meiosis-related proteins, it was discovered in 2014 that MEIOB was nominated as a new cancer antigen due to its high expression characteristics in cancer and may be used as a target for immunotherapy; in 2016, transcriptome analysis of testicular tissues of 19 testicular cancer patients confirmed that MEIOB belongs to extremely highly expressed testicular cancer-related genes (extremely highly expressed CT genes, EECTGs); in 2017, a family member with azoospermia was analyzed and the results showed that MEIOB was a new type of cancer antigen and a potential target for immunotherapy. Whole genome sequencing was performed, and MEIOB mutants were found for the first time as genetic pathogenic factors; in 2019, some scholars found that familial primary ovarian insufficiency was associated with human MEIOB abnormalities; in 2020, NGS (next-generation sequencing) analysis of a cohort of 25 people with idiopathic oligospermia or non-obstructive azoospermia found that most people had abnormal hMEIOB gene sequences; in another study in the same year, exome sequencing of meiosis-related genes was performed on a spermatogenesis block cohort of 147 people, and it was found that some patients carried hMEIOB pathogenic gene variants. At present, a variety of hMEIOB mutations have been analyzed, but the uncertainty of gene mutations is high, and new mutation patterns will appear in the future. In addition, the role of MEIOB in spermatogenesis has not been clearly explored, and corresponding research strategies are also being gradually implemented.

In summary, detecting the MEIOB gene sequence and protein expression in infertile patients is of great practical significance for clarifying the etiology and targeted treatment of idiopathic infertility. It is urgent to develop a monoclonal antibody against MEIOB protein with good titer and high specificity, and by targeted inhibition of MEIOB gene expression or protein function, the spermatogenesis process can be blocked to achieve a contraceptive effect.

Summary of the invention

In order to solve the technical problem, the present invention provides a monoclonal antibody against MEIOB, a mutation detection primer set, an ASO molecule, and a small molecule drug targeting MEIOB protein and its application. The monoclonal antibody can specifically recognize MEIOB protein, has good antibody specificity and high titer, and has stronger recognition specificity than polyclonal antibodies; the mutation detection primer set can detect the discovered hMEIOB gene mutation sites and potential mutation sites in the hMEIOB gene; the antisense oligonucleotide molecule (ASO molecule) targeting the mRNA of the transcription product of the MEIOB gene can target and bind to the transcription product of the hMEIOB gene to inhibit its expression, and its full thiolation modification improves the stability and half-life of the molecule, and the molecule itself is non-toxic and degradable; the small molecule targeting the MEIOB protein can be used to block the MEIOB function and sperm formation to achieve a contraceptive effect, and can also be used to reduce the expression level of the MEIOB protein in tumor cells and the cell proliferation ability to achieve targeted therapy.

The present invention provides an anti-MEIOB monoclonal antibody, which can recognize human MEIOB protein and comprises a complete heavy chain and a complete light chain:

The amino acid sequence of the light chain is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2.

Furthermore, the monoclonal antibody further comprises:

An antibody with the same function obtained by replacing, deleting and/or adding one or more amino acids to the amino acid sequence of the monoclonal antibody;

or comprising a heavy chain variable region having an amino acid sequence having at least 80% homology to the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology to the light chain variable region;

Or an antibody obtained by connecting a tag to the N-terminus and/or C-terminus of the monoclonal antibody.

Further, the _VH and/or _VL amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequences. Antibodies having _VH and _VL regions that are highly (i.e., 80% or more) homologous to the _above sequences can be obtained by _mutagenesis , and then the encoded altered antibodies are tested for retained function using the functional assays described herein.

The monoclonal antibodies include human antibodies, humanized antibodies or chimeric antibodies.

Furthermore, the variable region genes can be converted into scFv genes. Once the DNA fragments encoding the _VH and _VL fragments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA technology, for example, the variable region genes can be converted into full-length antibody chain genes, Fab fragment genes or scFv genes.

In these operations, the DNA fragment encoding _VL or _VH is operably linked to another DNA fragment encoding another protein such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" means that two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

Furthermore, the present invention provides a nucleic acid molecule encoding the monoclonal antibody, wherein the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

Furthermore, the present invention provides an expression vector comprising the nucleic acid, and the expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell.

The vector may be a conventional vector; specifically, it may be a plasmid vector, a phage vector, or a viral vector;

Furthermore, the present invention provides an engineered bacterium or eukaryotic host cell comprising the expression vector.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in preparing a MEIOB protein reagent or kit.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in preparing the quality control antibody of the MEIOB protein colloidal gold detection kit.

Furthermore, the present invention provides a colloidal gold rapid detection test strip for MEIOB protein, comprising:

Bottom plate,

A sample absorption pad, a conjugate pad, a chromatography matrix and a water absorption pad are bonded to the surface of the bottom plate and overlapped in sequence; wherein,

The surface of the conjugate pad is coated with a colloidal gold complex coated with a monoclonal antibody of the MEIOB protein; a quality control line C is provided on the side of the chromatography matrix close to the conjugate pad, and a detection line T is provided on the side of the chromatography matrix close to the absorbent pad; the quality control line C is coated with an anti-mouse IgG secondary antibody; and the detection line T is coated with a monoclonal antibody of the MEIOB protein.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in identifying point mutants formed by mutation of the MEIOB gene.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in detecting truncations formed by MEIOB gene mutations, and specifically, the use of the monoclonal antibody of the MEIOB protein in preparing recognition antibodies for a MEIOB truncated protein detection kit.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in purifying the MEIOB protein in tissues and cells, and specifically, the use of the monoclonal antibody of the MEIOB protein in preparing affinity antibodies for a MEIOB protein purification kit.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in the enrichment pretreatment of the MEIOB protein in the sample before mass spectrometry detection, and specifically, the use of the monoclonal antibody of the MEIOB protein in the preparation of affinity antibodies for a MEIOB protein spectrum detection kit.

Furthermore, the present invention provides the use of the monoclonal antibody of the MEIOB protein in the enrichment of nucleic acids in the early stage of gene high-throughput sequencing, and specifically, the use of the monoclonal antibody of the MEIOB protein in the preparation of affinity antibodies for a MEIOB protein targeted gene high-throughput sequencing detection kit.

Furthermore, the present invention provides a primer set for detecting MEIOB gene mutations, which can detect the discovered hMEIOB (human MEIOB gene) gene mutation sites and potential mutation sites in the hMEIOB gene, including the following primer pairs:

The nucleotide sequences of the primer set for exon 1 are shown in SEQ ID NO.3 and SEQ ID NO.4;

The nucleotide sequences of the primer set for exon 2 are shown in SEQ ID NO.5 and SEQ ID NO.6;

The nucleotide sequences of the primer set for exon 3 are shown in SEQ ID NO.7 and SEQ ID NO.8;

The nucleotide sequences of the primer set for exon 4 are shown in SEQ ID NO.9 and SEQ ID NO.10;

The nucleotide sequences of the primer set for exon 5 are shown in SEQ ID NO.11 and SEQ ID NO.12;

The nucleotide sequences of the primer set for exon 6 are shown in SEQ ID NO.13 and SEQ ID NO.14;

The nucleotide sequences of the primer set for exon 7 are shown in SEQ ID NO.15 and SEQ ID NO.16;

The nucleotide sequences of the primer sets for exons 8 and 9 are shown in SEQ ID NO. 17 and SEQ ID NO. 18;

The nucleotide sequences of the primer set for exon 10 are shown in SEQ ID NO.19 and SEQ ID NO.20;

The nucleotide sequences of the primer set for exon 11 are shown in SEQ ID NO.21 and SEQ ID NO.22;

The nucleotide sequences of the primer set for exon 12 are shown in SEQ ID NO.23 and SEQ ID NO.24;

The nucleotide sequences of the primer set for exon 13 are shown in SEQ ID NO.25 and SEQ ID NO.26.

Preferably, the primer set can cover the entire exons of the human MEIOB gene and the introns at the ends of the exons.

The primer set can be used for PCR reaction to amplify different exon sequences of hMEIOB, and the different sequences can cover the entire exon of the hMEIOB gene and the intron at the end of each exon.

Furthermore, the present invention provides a kit for detecting human MEIOB gene mutations containing the primer set.

Specifically, a kit for detecting non-obstructive azoospermia or premature ovarian insufficiency containing the primer set is provided.

Furthermore, the present invention provides an antisense oligonucleotide molecule targeting the mRNA of the transcription product of the MEIOB gene, which is a single-stranded DNA with a nucleotide sequence of ATTTATCGTGCAGCCCAAAGT, a sequence length of 21 bp, 3 locked nucleic acids at each end, and a fully thiolated modification.

The antisense oligonucleotide molecules recognize mRNA through base complementarity and inhibit the expression of MEIOB protein.

Furthermore, the present invention provides an application of the antisense oligonucleotide molecule targeting the mRNA of the transcription product of the MEIOB gene, which is used to reduce the mRNA content of the MEIOB gene in cells by co-incubating the antisense oligonucleotide molecule with a culture medium, or to reduce the mRNA content of the MEIOB gene in the testis by injecting the antisense oligonucleotide molecule into the testis.

Specifically, an application of the antisense oligonucleotide molecule targeting the mRNA of the transcription product of the MEIOB gene is provided, and the mRNA content of the hMEIOB gene in the cell is reduced by co-incubating the antisense oligonucleotide molecule with the culture medium.

Alternatively, another application of the antisense oligonucleotide molecule targeting the mRNA of the MEIOB gene transcription product is provided, and the mRNA content of the mMEIOB gene (mouse MEIOB gene) in the mouse testis is reduced by testicular injection of the antisense oligonucleotide molecule, the administration method is local scrotal injection, the administration dose is 0.33nmol/g, and the observation time is 72 hours.

Specifically, it is used to prepare a product for inhibiting the expression of mRNA, a transcription product of the MEIOB gene, or inhibiting the expression of the MEIOB protein.

Furthermore, the present invention provides a small molecule targeting MEIOB protein and its analogs, at least one of which has a molecular structure as shown below:

The small molecule targeting the MEIOB protein can inhibit the binding of MEIOB to SPATA22 by binding to the MEIOB protein.

The small molecules and analogs thereof targeting the MEIOB protein are reversibly bound to the MEIOB protein and can be rapidly dissociated from the surface of the MEIOB protein as the concentration decreases.

Furthermore, the present invention provides an application of the small molecule and its analogs targeting MEIOB protein, which can inhibit the production of sperm in the testicles by oral administration or local subcutaneous injection of the small molecule and its analogs targeting MEIOB protein.

Furthermore, the present invention provides an application of the small molecule targeting MEIOB protein and its analogs for preparing a product that inhibits the binding of MEIOB and SPATA22 in cells or inhibits the production of sperm in testicles.

Furthermore, the present invention provides an application of the small molecule targeting MEIOB protein and its analogs for preparing contraceptive drugs.

Furthermore, the present invention provides an application of the small molecule targeting MEIOB protein and its analogs for preparing drugs against tumors with high MEIOB expression.

The present invention has the following advantages and beneficial effects:

The anti-MEIOB monoclonal antibody of the present invention can specifically recognize the MEIOB protein, and has good antibody specificity and high titer.

The anti-MEIOB monoclonal antibody provided by the present invention has good recognition specificity for both in vitro purified human MEIOB protein and intracellular exogenously expressed MEIOB protein; compared with polyclonal antibodies, the monoclonal antibody has stronger recognition specificity.

The anti-MEIOB monoclonal antibody of the present invention can specifically recognize complete MEIOB protein, point mutant protein, and truncated protein, and based on this, it can achieve the enrichment of MEIOB protein or MEIOB-bound gene nucleic acid sequence, and then be used for protein enrichment mass spectrometry detection, genome sequencing, protein function analysis after purification and crystal structure analysis. The antibody has good specificity and high titer.

The primer set provided by the present invention can amplify intron sequences containing 100 bp bases on both sides of the exon, so it can be used to detect the production of abnormal spliceosomes caused by intron mutations at the ends of exons. In addition to identifying all existing mutation types, it can also be used to detect new unknown mutations.

The (antisense oligonucleotide molecule targeting the mRNA of the transcription product of the MEIOB gene) ASO molecule provided by the present invention can target and bind to the transcription product of the MEIOB gene to inhibit its expression, and its full thiolation modification improves the stability and half-life of the molecule, and the molecule itself is non-toxic and degradable.

The MEIOB protein-targeted small molecule provided by the present invention has the ability to inhibit the binding of MEIOB to SPATA22, can prevent sperm production, and achieve a contraceptive effect.

The MEIOB protein targeted small molecule provided by the present invention has the ability to inhibit the function of MEIOB in tumor cells, can inhibit the growth of tumor cells with high expression of MEIOB, and achieve the purpose of tumor targeted therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

FIG1 is a diagram showing the purification of human MEIOB protein used to immunize mice in Example 1;

Figure 2 shows the results of SDS-PAGE detection of the purification of monoclonal antibodies against human MEIOB protein; the amount of antibody loaded increases from left to right;

Figure 3 is an ELISA experiment to detect the titer of anti-human MEIOB protein monoclonal antibody; the abscissa is the antibody dilution ratio, and the ordinate is the A450 absorbance;

Figure 4 is a Western Blot experiment to identify the specific recognition between the purified anti-human MEIOB protein monoclonal antibody and the MEIOB protein. The sample is the purified MEIOB fusion protein with a His tag. The left figure shows the recognition of the sample by the Anti-human-MEIOB antibody and the right figure shows the recognition of the sample by the Anti-His tag antibody.

Figure 5 is a Western Blot experiment to identify the specific recognition between anti-human MEIOB protein polyclonal antibody, monoclonal antibody and MEIOB protein, respectively. The sample is a purified MEIOB fusion protein with a His tag; the left picture shows the recognition of the sample by the anti-human MEIOB protein polyclonal antibody and the right picture shows the recognition of the sample by the anti-human MEIOB protein monoclonal antibody;

FIG6 shows a human MEIOB overexpressing cell line expressing mCherry fusion in a human U2OS cell line, the left picture shows the purchased Anti-mCherry antibody, and the right picture shows the detection results of Anti-MEIOB polyclonal and monoclonal antibodies;

FIG7 is the antibody coding sequence identification result;

FIG8 is a schematic diagram showing the expression and degradation levels of point mutants and truncations of the MEIOB protein detected by antibodies;

FIG9 is a schematic diagram showing antibody binding to MEIOB protein to promote the latter's dispersion suitable for structural analysis;

FIG10 is a schematic diagram showing the application value of antibodies in proteomics and genomics by binding to MEIOB;

FIG11 is the result of RT-QPCR test to identify that ASO reduces the level of hMEIOB gene mRNA in cells;

FIG12 is the result of RT-QPCR test to identify that ASO reduces the mMEIOB gene mRNA level in mouse testis;

FIG13 is a live cell fluorescence imaging showing the results of small molecules targeting hMEIOB affecting the binding of MEIOB and SPATA22;

FIG. 14 is the result of HE staining of testicular tissue showing that small molecules targeting mMEIOB affect spermatogenesis in seminiferous tubules.

Figure 15 shows the results of sperm counting in the cauda epididymis;

Figure 16 shows the results of Western Blot detection of MEIOB protein levels in HELA, SiHA, C33A, A2780, MD231, and THP-1 cell lines;

Figure 17 is the result of Western Blot identification of small molecules targeting hMEIOB to reduce the level of MEIOB protein in C33A cells;

Figure 18 is the gray value result of Western Blot identification of small molecules targeting hMEIOB to reduce the level of MEIOB protein in C33A cells;

FIG19 is the result of CCK8 identifying small molecules targeting hMEIOB to reduce the proliferation ability of C33A cells;

FIG20 is the result of CCK8 identifying the effect of different concentrations of small molecules targeting hMEIOB on reducing the proliferation ability of C33A cells after 72 hours of addition;

FIG21 is the result of CCK8 identifying small molecules targeting hMEIOB to reduce the proliferation ability of A2780 cells;

FIG. 22 shows the results of CCK8 identification of the ability of small molecules targeting hMEIOB at different concentrations to reduce the proliferation of A2780 cells after 72 hours of addition.

DETAILED DESCRIPTION

The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

The monoclonal antibodies of the present application and their preparation methods and application effects are described in detail below in conjunction with the examples and experimental data. The specific experimental conditions and methods not specified in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd edition), Science Press, 1992; D.L. Spector et al., Cell Experiment Guide, Science Press, 2001, or the conditions recommended by the manufacturer.

Example 1

Monoclonal antibody and preparation method thereof

1. Cloning construction and prokaryotic protein expression

(1) Clone the MEIOB CDS sequence into the Pet42b vector to obtain the recombinant vector Pet42b-MEIOB.

The primer pair sequences used were: forward primer (5’-ATGGCGAACAGCTTCGCAGCACGTATTTTT-3’; SEQ ID NO: 3); reverse primer (5’-CACATGTTTCTGACCACTCAGGTTACGACTCGC-3’; SEQ ID NO: 4), cloned into the NdeI-XhoⅠ restriction site of Pet42b vector.

(2) Protein expression and purification:

①Plasmid transformation:

The constructed MEIOB prokaryotic expression plasmid was transformed into BL21 competent cells, spread on LB plates containing kanamycin resistance, and after a single clone grew out, the single clone was picked and shaken.

② Shake bacteria & express:

Shake overnight bacteria, transfer to large amount the next morning, dilute 1:100-1:50, shake bacteria at 37℃ until _OD600 = 0.4-0.8, induce with IPTG (0.1-0.3mM), and induce expression at 16-25℃ for 24 hours.

③Collecting and splitting bacteria:

The induced bacterial solution was collected by centrifuge at 4℃, washed with Milli Q water, and an appropriate amount of Lysis buffer, as well as proteasome inhibitors, lysozyme, and PMSF (1:100) were added, placed on ice at 30℃, and ultrasonically lysed. After lysing, high-speed centrifuge was used at 4℃ for 2 times, each time for 30 minutes, and the supernatant was retained.

④ Combined with Ni NTA beads:

Wash Ni NTA beads three times with lysis buffer, centrifuging at 2500 rpm for 3 min each time. After washing, add to the supernatant and combine at 4°C for 4-6 hours.

⑤Wash beads & elute protein:

Centrifuge and remove the supernatant. Wash the Ni NTA beads with lysis buffer for 30 minutes, and then wash twice with Wash buffer, 30 minutes each time. After washing, aspirate the liquid and elute the protein with Elution buffer for 4-6 hours.

⑥Concentrated & dialyzed protein

If the protein concentration is low, concentrate it using a concentrator tube and replace the PBS buffer while concentrating; if the protein concentration is high, dialyze directly with PBS.

⑦Protein storage

The dialyzed protein was quantified by Coomassie Brilliant Blue staining, then 10-20% glycerol was added, quick-frozen in liquid nitrogen, and stored at -80°C.

(3) Protein purification related reagent formula

Lysis buffer: Tris-HCL: 100 mM, NaCl: 300 mM, NP-40: 0.05%, pH = 8.0;

Washing buffer: Tris-HCL: 100mM, NaCl: 300mM, Imidazole: 20-50mM, NP-40: 0.05%, pH = 8.0;

Washing buffer: Tris-HCL: 100mM, NaCl: 300mM, Imidazole: 250mM, NP-40: 0.05%, pH = 8.0;

The protein purification diagram is shown in Figure 1.

2. Animal immunization

Purified human MEIOB protein was used as an antigen to immunize mice. Three 6-8 week old Balb/C mice were selected. Freund's complete adjuvant was used for the first main injection, and Freund's incomplete adjuvant was used for subsequent booster injections. Both were fully mixed with an equal volume of antigen before injection. The immunization method used multiple abdominal injections. The immunization dose was 100 μg antigen/experimental mouse for the main injection and 50 μg antigen/experimental mouse for the booster injection.

The immunization cycle is shown in Table 1 ;

Table 1

3. Antiserum detection

(1) Take a small amount of blood from the tail vein of mice and prepare antiserum.

(2) ELISA method was used to detect the antiserum titer.

4. Cell fusion and subcloning

(1) Myeloma cell preparation

One week before fusion, SP2/0 cells were revived and cultured normally to the logarithmic phase.

(2) Preparation of spleen cells

The mice to be fused were selected and killed by cervical dislocation on the day of fusion. The spleens were removed and spleen cells were collected and counted according to standard procedures.

(3) Cell fusion

Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10, and cell fusion was performed according to the standard procedure. Then, HAT DMEM complete medium was used for culture. Hybridoma cells were visible 3 days after fusion. 1/2 HAT complete medium was replaced on the 7th day, and 1/2 HT medium was replaced on the 8th day. Screening tests were started about 10 days after fusion.

Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope, where multiple growing hybridoma cells were seen, proving that the fusion operation was successful.

(4) Fusion screening

Pipette 100 μL/well of cell supernatant for indirect ELISA detection. Determine the positive wells based on the ELISA results. Use a single-channel pipette to pick out the positive wells detected on the entire plate and conduct a second re-test to further confirm the positive wells.

(5) Subcloning

The positive wells in the rescreening were subjected to two rounds of subcloning. Because the positive well cell lines obtained in the first subcloning were not stable yet and might contain multiple hybridoma cells, it was generally believed that the hybridoma cells after the second subcloning were a single cell line and were confirmed to be positive.

For the first subclone, the cells in the limiting dilution positive wells were transferred to multiple wells and cultured with HT DMEM medium. The wells were observed under a microscope for about 7 days and the wells with cloned growth were detected by indirect ELISA. The wells with high OD values were taken as positive wells. The cells in the positive wells were picked for the second subclone, and the stable positive hybridoma cell lines were detected as the cells for the final preparation of monoclonal antibodies. The culture was expanded to obtain hybridoma cell lines.

5. Ascites preparation and antibody purification

(1) Ascites preparation

The above positive cells are expanded and cultured and injected into the abdominal cavity of Balb/C mice (sensitized with Freund's incomplete adjuvant). Generally, bulging of the abdomen of the mice can be seen in 7-10 days, indicating the formation of ascites. When the mice have obvious ascites, the ascites is extracted in time.

(2) Ascites purification

The ascites of the above cells was purified, and the purity of the antibody after purification was greater than 90%.

(3) Purification by ammonium octanoate sulfate + DEAE ion column method

(4) The ascites was centrifuged, the light yellow liquid was aspirated and the volume was calculated. It was diluted 1:3 with 4 volumes of 60 mM acetate buffer (pH 4.0), and caprylic acid was added dropwise (final concentration was 25 μL/mL diluted ascites). The mixture was stirred at room temperature for 30 min, and then allowed to stand at 4 °C for more than 2 h to allow it to fully precipitate.

(5) 10000 r/min, 4°C, 20 min, collect the supernatant, add 1/10 volume of 10× PBS (0.1 M, pH 7.4), add 0.277 g of solid ammonium sulfate (45% saturated ammonium sulfate is 0.291 g/mL at 0°C) per ml of the above mixture, and continue to stand for at least 60 min.

(6) 10000 r/min, 4°C, 20 min, discard the supernatant, dissolve the precipitate in a small amount of PBS, and dialyze against PBS at 4°C overnight.

(7) Detection of antibody concentration and purity. The antibody concentration was measured to be 1.5 mg/mL. The purity of the purified antibody was detected by Coomassie Brilliant Blue staining test. The results in Figure 2 showed heavy chain and light chain bands, with no other mixed bands, indicating that the antibody purity was high.

6. Antibody light chain and heavy chain sequencing

(1) Cultivation of hybridoma cells

After the hybridoma cell line was revived, it was cultured and when the cell number expanded to about 1×10 ⁷ , the cells were collected by centrifugation at 1000 rpm for 5 min.

(2) Extraction of cellular RNA

Under the ultra-clean bench environment, add 1mL Trizol reagent to the centrifuged cells, let stand for 5 minutes, add 200L chloroform, shake vigorously for 15 seconds, let stand at room temperature for 5 minutes, centrifuge at 12000rpm for 15 minutes, draw the upper water layer into a new EP tube, add 0.5mL isopropanol, let stand for 10 minutes at -20℃. Centrifuge at 12000rpm for 10 minutes. Discard the supernatant, add 1mL 75% ethanol, centrifuge at 7500rpm for 5 minutes, dry the precipitate, and add 50μL RNase-free double distilled water. Identify the purity and quantify by agarose electrophoresis, and store at -80℃ for later use.

(3) Reverse transcription to prepare cDNA

1 μL of total cell RNA, 6 μL of RNase Free ddH ₂ O, 0.5 μL of oligo dT Primer, 0.5 μL of PRIME Script RT Enzyme Mix I, 2 μL of 5× Prime Script Buffer, mix well, incubate at 37°C for 15 min, and 85°C for 5 s.

(4) Amplification of cDNA

Use mouse IgG _VH and _VL primer libraries to amplify the above cDNAs. 2×PCR mix 20μL, cDNA 2μL, upstream primer 2μL, downstream primer 2μL, add water to 40μL. Perform PCR reaction according to the following reaction conditions: incubate at 98℃ for 5min, denature at 98℃ for 30s, anneal at 63℃ for 20s, extend at 72℃ for 25s, and extend at 72℃ for 5min after 40 cycles.

(5) Agarose gel electrophoresis and gel recovery

The above PCR products were subjected to agarose gel electrophoresis, the electrophoresis results were observed, and the amplified products with molecular weights of 700-800 bp and 1400-1600 bp were sent for sequencing.

(6) The antibody light chain sequencing results are shown in SEQ ID NO: 1.

(7) The antibody heavy chain sequencing results are shown in SEQ ID NO: 2.

Table 2

Example 2

Antibody titer testing

The titer of the antibody was determined by ELISA experiment. 50 ng of antigen (prokaryotic purified human MEIOB protein) was coated on each well of the plate, and the purified monoclonal antibody was diluted according to the proportion in Figure 3. The A450 absorbance of each well was measured. The results are shown in Table 3 and Figure 3;

Table 3

As shown in Table 3 and Figure 3, the antibody titer can reach 1:3000.

Example 3

Antibody specificity testing

1. Use in vitro purified human MEIOB protein to detect antibody specificity.

The His-tagged MEIOB protein was purified in vitro by prokaryotes, and the recognition specificity of the purified antibody was detected by Western Blot experiment, as shown in Figure 4. The left and right figures are Western Blot experiments to detect the specificity of anti-human-MEIOB antibody and anti-histag antibody, respectively. The results showed that the anti-human-MEIOB monoclonal antibody has good recognition specificity for MEIOB.

2. Testing antibody specificity at the cellular level

A cell line overexpressing the human MEIOB gene was constructed in the human U2OS cell line, and the specificity of the monoclonal antibody was identified by immunoblotting, as shown in FIG5 . The method for constructing the cell line overexpressing the human MEIOB gene in the embodiment of the present invention is as follows: the MEIOB gene sequence is cloned into the PB511B-1 vector (with restriction sites of EcoR I and BamH I), and the cells are transfected together with the PB210PA-1 plasmid encoding the transposase, and the cell line stably expressing the MEIOB gene is obtained by puromycin screening. As shown in FIG5 , the monoclonal antibody (Polyclonal hMEIOB Antibody) can more specifically detect the expression of the human MEIOB protein than the polyclonal antibody (Monoclonl hMEIOB Antibody).

3. Comparison of specificity between anti-human MEIOB monoclonal antibody and commercially available antibodies.

There is no MEIOB monoclonal antibody on the market yet. We used the tag protein as the detection target, purchased an antibody that recognizes the tag protein (mCherry), and compared the antigen recognition specificity of three antibodies (anti-human MEIOB polyclonal, monoclonal antibody and anti-mCherry antibody). The results are shown in Figure 6. Although both can recognize the MEIOB protein, the monoclonal antibody we purified has a single band, indicating better specificity.

Example 4

Antibody coding sequence detection

1. Sequence the antibody coding sequence after library construction. Use a plasmid extraction kit (Tiangen, #DP106-02) to extract the plasmid loaded with the antibody sequence, then use DNA gel electrophoresis to identify the presence of the plasmid and use a gel recovery kit to remove interfering sequences, and use an ultraviolet spectrophotometer to measure the purity and concentration of the plasmid DNA; select a suitable sequencing primer (pCMV-F) and perform DNA amplification according to Sanger's dideoxy chain termination method, send the reaction product to a capillary electrophoresis instrument for analysis, and determine the composition of the DNA sequence by detecting different fluorescent colors; use bioinformatics software such as Snapgene to edit and align the sequencing results to obtain accurate antibody coding sequence information, as shown in Figure 7.

Example 5

Antibodies detect MEIOB point mutants and truncations

1. Analysis of existing point mutants and truncations of MEIOB protein.

It has been reported that patients with non-obstructive azoospermia or primary ovarian insufficiency may have single residue mutations in the MEIOB protein, such as P.N64I, P.S106R, P.S106A, P.A326T, or partial fragment deletions, such as P.V87fs, P.V177fs, P.V228fs, P.R272fs, P.M358fs, P.S366fs, and P.T406fs; and the above changes will lead to limited or missing protein function. Through analysis, it was found that single amino acid residue mutations can lead to protein conformational instability and increased degradation levels, and the number of MEIOB protein hydrolysis fragments in the sample increased; in addition, the remaining protein cannot function efficiently due to unstable conformation, and the content of MEIOB protein expressed by normal tissues and cells is reduced. For truncated proteins, due to the loss of the functional region of the large fragment, the residual MEIOB fragment cannot perform normal functions, and the molecular weight is different from normal; at the same time, the fragment loss often occurs near the translation termination region (carboxyl terminus) while the translation start fragment (amino terminus) is relatively intact. Based on this, through protein immunoblotting (WB), monoclonal antibodies that recognize a single site can evaluate the expression and degradation levels of MEIOB point mutants and truncations, as shown in Figure 8.

Example 6

Antibody enrichment of MEIOB for protein function analysis

1. Currently, the structure of MEIOB protein has not been resolved. Through early expression, it was found that MEIOB is very easy to aggregate to form inclusion bodies and precipitate. This poor dispersion ability is not conducive to protein crystallization and subsequent analysis. Monoclonal antibodies have a large molecular weight (150kD), so they produce a large steric hindrance after mixing with MEIOB, which can play a role in dispersing MEIOB protein; at the same time, monoclonal antibodies will present a single binding mode with MEIOB, which increases the order of the protein-antibody complex in the crystal lattice, as shown in Figure 9, promoting the superposition of the diffraction values of the complex, and finally obtaining a diffraction intensity sufficient for measurement, which is conducive to protein structure analysis.

2. Monoclonal antibodies are used to explore the functional mechanism of MEIOB. The protein has a large molecular weight (~50kD) and may have potential modification sites on the surface; especially in the protein degradation stage after the function is fulfilled, whether it is degraded through ubiquitination modification and which residue mediates the modification has not been reported. MEIOB protein is a protein specifically expressed by reproductive tissues. Because there is no suitable germ cell line to express this protein, and heterologous expression cannot simulate the microenvironment of meiosis and the protein expressed in time and space, the analysis of its biological function can only be explored by isolating animal tissues; in addition, the method of constructing a model animal for expressing tagged proteins is costly and has a long cycle. In summary, monoclonal antibodies can quickly enrich MEIOB proteins without tags in reproductive tissues. While enriching MEIOB proteins through antibodies, functional complexes formed with the participation of MEIOB can be obtained at the same time, and the monoclonal antibody-dependent MEIOB enrichment provides convenience for the following experiments.

3. Monoclonal antibodies are used for proteomics analysis. Proteins enriched by monoclonal antibodies can be further used for mass spectrometry detection. The analysis can identify proteins that directly or indirectly interact with MEIOB; the antibodies can then be used in pull-down assays to analyze potential interaction sites. In addition, MEIOB proteins enriched by antibodies can be used for epigenetic modification analysis to identify potential modification sites on the surface of MEIOB and the corresponding modified proteins, thereby clarifying the effects of modification on protein function and biological processes, as shown in Figure 10.

4. Monoclonal antibodies are used for genomic analysis. MEIOB participates in the meiotic process, specifically by promoting the synapsis of homologous chromosomes in collaboration with interacting proteins. When high-affinity antibodies are enriched for MEIOB, gene fragments with affinity to the MEIOB protein complex can be obtained at the same time; by aligning the gene sequence to the genome, information on the preference of chromosome recombination sites can be obtained, as shown in Figure 10.

Example 7

Identification of gene point mutations:

1. Primer design. The genomic sequence of the human MEIOB gene was analyzed to locate each exon and the 100 bp intron sequences on both sides, and the exon containing the aforementioned introns on both sides was selected as the target sequence; the upstream and downstream primers were generated using Snapgene software to ensure that the annealing temperature of all primers was around 63°C, see Table 4.

Table 4 Primer sequence information

2. Peripheral blood genome extraction. Draw 0.5 mL of blood from the subject's vein, add 0.8 mL of normal saline and mix well, centrifuge at 10,000 rpm for 1 min at room temperature; remove the supernatant, keep 0.1 mL of precipitate, add 0.4 mL of TE buffer to resuspend the cells, add 5 μL of 10 mg/mL proteinase K and 50 μL of 10% SDS to lyse the cells, and digest overnight in a 37°C water bath; add 1/3 volume of saturated sodium chloride solution, mix thoroughly, let stand for 10 min, and centrifuge at 10,000 rpm for 10 min; take the supernatant to a new tube, add an equal volume of chloroform and mix thoroughly, and centrifuge at 1 0000rpm for 10 min; aspirate the upper aqueous phase to a new tube, add 1/10 volume of 3M NaAc (pH 5.2) and mix well, add 0.7 times volume of isopropanol and mix well, centrifuge at 10000rpm for 5 min; remove the supernatant, add 0.5mL of 70% ethanol to wash the isopropanol, invert and mix, centrifuge at 10000rpm for 5 min, remove the supernatant and repeat once; open the lid to allow the ethanol to evaporate completely, add 50μL of sterilized double distilled water, and fully dissolve at 55°C; use Nanodrop instrument to measure the DNA concentration, and then store at -20°C.

3. PCR amplification of target sequence. The reaction system is 40 μL, add 20 μL of 2×Taq polymerase reaction premix, 100 ng of DNA template, 2 μL of 10 μM upstream and downstream primers and appropriate amount of double distilled water (ddH ₂ O), mix well, and use the following parameters for PCR amplification reaction: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, cycle 30 times, continue extension for 5 min after the cycle, and keep warm at 12°C.

4. Target sequence gene sequencing. Obtain the target sequence through the gel recovery kit, and determine the purity and concentration of the plasmid DNA using a UV spectrophotometer; select the respective sequencing primers (the default is F) and perform DNA amplification according to Sanger's dideoxy chain termination method, send the reaction product to a capillary electrophoresis instrument for analysis, and determine the composition of the DNA sequence by detecting different fluorescent colors; use bioinformatics software such as Snapgene to edit and align the sequencing results to obtain accurate coding sequence information.

5. DNA sequence comparison. Comparing the sequencing results with the standard nucleic acid information and the existing mutation information (Table 5) can reveal whether the subject's MEIOB gene is normal, whether it conforms to the existing mutation, and whether there is a new type of gene mutation.

Table 5 Known mutation information

Example 8

ASO molecules reduce the level of hMEIOB gene mRNA in cells:

1. Construction of hMEIOB stable expression cell line. The hMEIOB mRNA was obtained from the testis, reverse transcribed into cDNA, and the full-length hMEIOB gene sequence was amplified and constructed into the pcDNA3.1 plasmid; to facilitate the observation of gene expression, the green fluorescent protein (GFP) coding sequence was fused to the end of hMEIOB; the sequenced recombinant plasmid was transfected into the U2OS cell line, and the cell line stably expressing hMEIOB was screened using 500 μg/mL of G418.

2. ASO molecule synthesis. Based on the mRNA sequence of hMEIOB, a complementary ASO sequence with a length of 21 nucleotides was designed; an automated oligonucleotide synthesizer was used to gradually build the ASO chain by adding single nucleotides; the ASO was treated with a thiolation reagent and then purified to remove unreacted reagents and other impurities; it was purified by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry to ensure that its quality and purity met the experimental requirements.

3. Co-culture of ASO molecules with cells and identification of hMEIOB gene expression by RT-QPCR. The synthesized ASO molecules were co-cultured with the hMEIOB gene stably transfected cell line constructed above at a final concentration of 4 μM for 72 hours, and the cells were recovered to extract mRNA and reverse transcribed to obtain a PCR reaction template; the relative mRNA level of hMEIOB in the cells after the addition of ASO was detected by RT-QPCR reaction and the difference was statistically analyzed; the mRNA level of hMEIOB in U2OS cells decreased by 72% after the addition of ASO, indicating that ASO can effectively interfere with the expression of hMEIOB (Figure 11).

Embodiment 9

ASO molecules reduce the mRNA level of the mMeiob gene in mouse testis

1. Mouse preparation: Select 6-8 week old C57BL/6J male mice, weigh them, calculate the drug dosage (0.33 nmol/g), and anesthetize the mice by intraperitoneal injection.

2. Testicular injection of ASO molecules. Squeeze the abdomen of the mouse to make the testicles return to the scrotum; use a microsyringe to draw up the ASO solution and inject the drug directly into the testicles through the scrotal skin; put the mouse back into the cage, wait for it to wake up, and then transfer the cage to the animal room to continue feeding for 72 hours.

3. RT-QPCR identification of mMeiob gene expression. mRNA in testicular tissue was extracted and reverse transcribed into cDNA; the relative mRNA level of mMeiob in mouse testis after the addition of ASO was detected by RT-QPCR reaction and the difference was statistically analyzed; the results showed that the relative mRNA level of mMeiob in mouse testis decreased after ASO (0.044±0.005 vs. 0.037±0.002) (Figure 12).

Example 10

Small molecules targeting MEIOB protein inhibit the interaction between MEIOB and SPATA22 in cells

1. Construction of cell lines stably expressing MEIOB and SPATA22. pcDNA3.1 vectors expressing MEIOB protein fused with green fluorescent protein (GFP) and SPATA22 fused with red fluorescent protein (mCherry) were constructed respectively, and co-transfected into U2OS cell lines. Cell lines stably expressing the two fluorescent fusion proteins were screened by G418.

2. Screening of small molecules targeting MEIOB protein. Through virtual screening, candidate small molecules were obtained, and small molecule compounds ZJH-I-OMe (CID_10847865), ZJH-I-OGer, ZJH-I-OEt, ZJH-I-OiPr, ZJH-I-OCD3, ZJH-I-OH, ZJH-I-HHG, ZJH-I-103-B, and ZJH-I-103-C were further synthesized.

Table 6 shows the names and affinity constants of the nine compounds.

The affinity constants (KD) of these 9 small molecule compounds were detected by biomembrane interference technology. The results showed that the KD values of ZJH-I-OMe (CID_10847865) and ZJH-I-HHG were lower, indicating that they had higher affinity with MEIOB protein (Table 6). The following experiments selected ZJH-I-OMe (CID_10847865) for verification.

3. Co-culture of small molecules with cells. CID_10847865 small molecules at a concentration of 40 μM were added to the culture medium and co-cultured with U2OS cells in a 37°C CO ₂ incubator for 48 hours.

4. Fluorescence microscopy was used to detect cell interactions. The distribution of GFP protein and mCherry protein was observed under 488nm and 596nm excitation light, respectively. The fluorescence results showed that both MEIOB and SPATA22 could be localized in the nucleus in U2OS cells in the culture medium without small molecules. In the culture medium with small molecules, MEIOB was distributed in the cytoplasm, while SPATA22 was localized in the nucleus alone, indicating that small molecules affect the binding of the two proteins (Figure 13).

Embodiment 11

Small molecules targeting MEIOB protein inhibit spermatogenesis in mouse testis

1. Mouse preparation: Select 6-8 week old C57BL/6J male mice, weigh them, and calculate the dosage (0.3 mg/20 g) and frequency of administration (once every 2 days, 7 times in total).

2. Small molecule scrotal injection. Use a microsyringe to draw up the small molecule premix (the drug is dissolved in 10 μL DMSO), pull up the mouse scrotal skin, and ensure that the solution is injected subcutaneously; put the mouse back into the cage and continue to feed it.

3. Preparation of paraffin sections and HE staining of testicular tissue. Mice were killed by cervical dislocation, the testicles were separated and fixed, paraffin tissue sections were prepared, the tissues were stained by HE staining, and the structural changes of seminiferous tubules were observed under a microscope after sealing. After drug administration, cavitated seminiferous tubules appeared in the testicles of mice (Figure 14).

4. Count sperm in the tail of epididymis. Separate the epididymis of mice and soak it in 1 mL PBS solution. Cut the tissue into pieces to fully release sperm. Pipette 10 μL of solution and add 90 μL PBS to dilute it. Pipette 10 μL from the dilution solution into the cell counting plate. Cover it with a coverslip and heat it at 50°C for 5 minutes to stop sperm movement. Count sperm under a microscope. The number of sperm in the epididymis of mice after medication decreased (Figure 15).

Example 12

Small molecules targeting MEIOB protein reduce the proliferation ability of tumor cells with high MEIOB expression

1. Screening of tumor cell lines with high expression of MEIOB. Whole cell proteins of cervical cancer cell lines (HELA, SiHA, C33A), breast cancer cell lines (A2780, MD231) and leukemia cell lines (THP-1) were collected, and the expression level of MEIOB protein was detected by Western Blot. The results showed that MEIOB was highly expressed in cervical cancer cell line C33A and breast cancer cell lines A2780 and MD231 (Figure 16).

2. Western Blot was used to detect the effect of small molecules on the expression level of MEIOB in cells. Small molecules of different concentrations were added to the culture medium and incubated with C33A cells in a 37°C CO ₂ incubator for 72 hours. The protein expression level of MEIOB in cells was detected by Western Blot. Western Blot results showed that the protein level of MEIOB in cells decreased with the increase of small molecule concentration, indicating that small molecules targeting MEIOB protein inhibited the protein expression level of MEIOB in C33A cell lines (Figures 17 and 18).

3. CCK8 detection of cell proliferation. C33A and A2780 cells were inoculated in 96-well plates and incubated in a 37°C CO2 incubator. After the cells adhered to the wall, small molecules of different concentrations were added to the culture medium and incubated with C33A and A2780 cells respectively. The cells were removed at 0, 24, 48, 72, and 96 hours, and 10 μL CCK8 detection reagent was added to each well. After continuing to culture for 2 hours, the 450nm light absorption value was read with an enzyme reader. The CCK8 results showed that the cell viability of C33A and A2780 cells decreased after the addition of small molecules targeting the MEIOB protein (Figures 19-22).

The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims (20)

A monoclonal antibody against MEIOB, characterized in that: the monoclonal antibody can recognize human MEIOB protein, the monoclonal antibody comprises a complete heavy chain and a complete light chain; the amino acid sequence of the light chain is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2. A monoclonal antibody against MEIOB, characterized in that: the monoclonal antibody is an antibody obtained by connecting a label to the N-terminus and/or C-terminus of the monoclonal antibody according to claim 1. A nucleic acid molecule encoding the anti-MEIOB monoclonal antibody according to claim 1 or 2, characterized in that the nucleic acid molecule comprises a nucleic acid molecule encoding the complete heavy chain and a nucleic acid molecule encoding the complete light chain. An expression vector comprising the nucleic acid molecule of claim 3, characterized in that the expression vector is capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell. An engineered bacterium or eukaryotic host cell comprising the expression vector according to claim 4. An application of the monoclonal antibody against the MEIOB protein according to claim 1 or 2, characterized in that it is used for preparing a MEIOB protein detection reagent or kit. An application of the monoclonal antibody of the MEIOB protein according to claim 1 or 2, characterized in that it is used to prepare the quality control antibody of the MEIOB protein colloidal gold detection kit. A colloidal gold rapid detection test strip for MEIOB protein, characterized in that it comprises: Bottom plate, A sample absorption pad, a conjugate pad, a chromatography matrix and a water absorption pad are adhered to the surface of the bottom plate and overlapped in sequence; The surface of the conjugate pad is coated with a colloidal gold complex coated with a monoclonal antibody to the MEIOB protein according to claim 1 or 2; a quality control line C is provided on the side of the chromatography matrix close to the conjugate pad, and a detection line T is provided on the side of the chromatography matrix close to the absorbent pad; the quality control line C is coated with an anti-mouse IgG secondary antibody; and the detection line T is coated with a monoclonal antibody to the MEIOB protein according to claim 1 or 2. An application of a monoclonal antibody against the MEIOB protein according to claim 1 or 2, characterized in that it is used to identify point mutants formed by MEIOB gene mutations, or to detect truncations formed by MEIOB gene mutations, or to purify MEIOB protein in tissues and cells, or to enrich and pretreat MEIOB protein in samples in the early stages of mass spectrometry detection, or to enrich nucleic acids in the early stages of high-throughput gene sequencing. A primer set for detecting MEIOB gene mutation, characterized in that it can detect the discovered hMEIOB gene mutation site and the potential mutation site in the hMEIOB gene, and includes the following primer pairs: The nucleotide sequences of the primer set for exon 1 are shown in SEQ ID NO.3 and SEQ ID NO.4; The nucleotide sequences of the primer set for exon 2 are shown in SEQ ID NO.5 and SEQ ID NO.6; The nucleotide sequences of the primer set for exon 3 are shown in SEQ ID NO.7 and SEQ ID NO.8; The nucleotide sequences of the primer set for exon 4 are shown in SEQ ID NO.9 and SEQ ID NO.10; The nucleotide sequences of the primer set for exon 5 are shown in SEQ ID NO.11 and SEQ ID NO.12; The nucleotide sequences of the primer set for exon 6 are shown in SEQ ID NO.13 and SEQ ID NO.14; The nucleotide sequences of the primer set for exon 7 are shown in SEQ ID NO.15 and SEQ ID NO.16; The nucleotide sequences of the primer sets for exons 8 and 9 are shown in SEQ ID NO.17 and SEQ ID NO.18; The nucleotide sequences of the primer set for exon 10 are shown in SEQ ID NO.19 and SEQ ID NO.20; The nucleotide sequences of the primer set for exon 11 are shown in SEQ ID NO.21 and SEQ ID NO.22; The nucleotide sequences of the primer set for exon 12 are shown in SEQ ID NO.23 and SEQ ID NO.24; The nucleotide sequences of the primer set for exon 13 are shown in SEQ ID NO.25 and SEQ ID NO.26. An application of a primer set for detecting MEIOB gene mutation, characterized in that it is applied to prepare a product for diagnosing non-obstructive azoospermia or premature ovarian insufficiency. An antisense oligonucleotide molecule targeting the mRNA of the transcription product of the MEIOB gene, characterized in that it is a single-stranded DNA with a nucleotide sequence of ATTTATCGTGCAGCCCAAAGT, a sequence length of 21 bp, 3 locked nucleic acids at each end, and a modification method of full thiolation modification. The antisense oligonucleotide molecule targeting the mRNA of the MEIOB gene transcription product according to claim 12 is characterized in that the antisense oligonucleotide molecule recognizes mRNA through base complementarity, inhibits the expression of MEIOB protein or inhibits the expression of mRNA of the MEIOB gene transcription product. An application of an antisense oligonucleotide molecule targeting the mRNA of the MEIOB gene transcription product according to claim 12 or 13, characterized in that it is used to prepare a product that inhibits the expression of the mRNA of the MEIOB gene transcription product or inhibits the expression of the MEIOB protein. An application of the antisense oligonucleotide molecule targeting the mRNA of the MEIOB gene transcription product according to claim 12 or 13, characterized in that it is used for preparing contraceptive drugs. A small molecule targeting MEIOB protein and its analogs, characterized in that its molecular structure is at least one of the following:
The small molecules and analogs thereof targeting MEIOB protein according to claim 16 are characterized in that the small molecules and analogs thereof targeting MEIOB protein can inhibit the binding of MEIOB and SPATA22 by binding to MEIOB protein, and the binding of the small molecules and analogs thereof targeting MEIOB protein to MEIOB protein is reversible and can be rapidly dissociated from the surface of MEIOB protein as the concentration decreases. An application of a small molecule targeting MEIOB protein and its analogs according to claim 16 or 17, characterized in that it is used to prepare a product that inhibits the binding of MEIOB and SPATA22 in cells or inhibits the production of sperm in testicles. An application of the small molecule targeting MEIOB protein and its analogs according to claim 16 or 17, characterized in that they are used for preparing contraceptive drugs. An application of the small molecule targeting MEIOB protein and its analogs according to claim 16 or 17, characterized in that they are used to prepare drugs against tumors with high MEIOB expression.