Tex26 gene as specific biomarker for diagnosing abnormal sperm maturation and male infertility disease and application of Tex26 gene

Abstract

The invention provides a Tex26 gene as a specific biomarker for diagnosing abnormal sperm maturation and male infertility diseases and application of the Tex26 gene. The biomarker and / or the substance for detecting the biomarker are / is applied to the preparation of a product for researching the spermatogenesis process, and any one of the following applications of the biomarker and / or the substance for detecting the biomarker; application in preparation of a product for identifying a germ cell classification process; application in preparation of a product for diagnosing abnormal sperm maturation; application in preparation of a product for diagnosing abnormal fertilization; application in preparation of products for screening and diagnosing male infertility diseases; application in the preparation of male contraceptives and / or male contraceptive vaccine products; wherein the biomarker is selected from a Tex26 gene, a TEX26 protein, an mRNA (messenger Ribonucleic Acid) of the Tex26 gene, an RNA (Ribonucleic Acid) of the Tex26 gene or a miRNA (Micro Ribonucleic Acid) targeting the TEX26 protein. The invention provides a novel biomarker and a therapeutic target for a kit for diagnosing sperm maturation and male infertility diseases.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a Tex26 gene as a specific biomarker for diagnosing sperm maturation abnormalities and male infertility and its application. Background Art

[0002] Clinical infertility refers to a couple who have been unable to conceive after 12 months of trying. It is estimated that 30%-50% of infertility is caused by male factors. The causes of male infertility are complex, including testicular dysfunction, endocrine diseases, lifestyle factors (such as smoking and obesity), congenital anatomical factors, gonadal toxin exposure and aging, which can all lead to infertility or decreased fertility. Sperm dysfunction is considered the main cause of male infertility and one of the main indications for assisted reproductive technology (ART).

[0003] Mature sperm with normal morphology and motility are vital to male reproduction. The latest evidence shows that sperm dysfunction is the result of testicular and / or epididymal dysfunction. The epididymis plays an important role in the normal maturation and fertilization of sperm. Although the primitive sperm produced in the testicles have the external morphology of sperm, they do not have the ability to move, recognize sperm and egg, and fertilize. After the sperm enters the epididymis, it moves along the head, body, and tail of the epididymis, and undergoes a series of drastic changes in morphology, biochemical metabolism, and physiological functions before it can acquire the ability to move, recognize sperm and egg, and fertilize. This process is the maturation process of sperm in the epididymis. This process is of great significance to the occurrence of fertilization.

[0004] Sperm maturation is regulated by the epididymis. The epididymis plays an important role in maintaining intraluminal ion concentration, supporting sperm motility, and storing and protecting sperm. During epididymal maturation, the sperm proteome undergoes significant changes. Epididymal dysfunction can lead to abnormal protein secretion and interfere with sperm maturation, semen parameters, and sperm DNA integrity, resulting in excessive white blood cell concentration, excessive immature germ cells and fragments, abnormal sperm maturation, and decreased sperm motility, which in turn causes idiopathic infertility. At present, the data on key biomarkers of sperm maturation are still very limited. Screening and determining these markers can help diagnose the potential causes of sperm dysfunction, help evaluate sperm dysfunction, fertilization ability, and improve the diagnosis and treatment of male infertility diseases. It is also of great significance for the early detection, early diagnosis, and pathogenesis research of male infertility.

[0005] The study also found that some proteins are taken up during sperm maturation and become sperm membrane antigens. These antigens can become suitable candidates for contraceptive vaccines, bringing new ideas to the research of male contraception. By detecting these biomarkers, we can improve the male reproductive capacity and contraceptive evaluation system and provide a certain theoretical basis for the development of male contraceptive vaccines. Summary of the invention

[0006] The purpose of this application is to provide a Tex26 gene as a specific biomarker for diagnosing abnormal sperm maturation and male infertility and its application. The Tex26 gene and TEX26 protein can be used as biomarkers in the preparation of kits for diagnosing abnormal sperm maturation, abnormal fertilization, male infertility and male contraception, providing a new target for the diagnosis and treatment of male infertility-related diseases and the development of male contraceptives. The specific technical solution is as follows:

[0007] The first aspect of the present application provides any of the following uses of a biomarker and / or a substance for detecting the biomarker:

[0008] A1) Use in the preparation of products for studying spermatogenesis;

[0009] A2) Use in the preparation of a product for identifying a germ cell sorting process;

[0010] A3) Use in the preparation of a product for diagnosing sperm maturation abnormalities;

[0011] A4) Use in the preparation of a product for diagnosing fertilization abnormalities;

[0012] A5) Use in the preparation of products for screening and diagnosing male infertility diseases;

[0013] A6) Application in the preparation of male contraceptive drugs and / or male contraceptive vaccine products;

[0014] Wherein, the biomarker is selected from Tex26 gene, TEX26 protein, mRNA of Tex26 gene, RNA of Tex26 gene or miRNA targeting TEX26 protein.

[0015] In some embodiments of the present application, the substance for detecting the biomarker is a reagent for detecting whether the biomarker exists in the Tex26 gene, TEX26 protein, mRNA of the Tex26 gene, RNA of the Tex26 gene, or miRNA targeting the TEX26 protein, or the substance is a reagent for detecting whether the expression level of the TEX26 protein, mRNA of the Tex26 gene, RNA of the Tex26 gene, or miRNA targeting the TEX26 protein has changed, or the substance is a reagent for detecting whether there is a sequence mutation in the Tex26 gene, TEX26 protein, or mRNA of the Tex26 gene.

[0016] In some embodiments of the present application, the test sample detected when detecting the biomarker substance is selected from at least one of blood, oral tissue, testicular tissue and sperm.

[0017] In some embodiments of the present application, the reagent includes an antibody that specifically binds to the TEX26 protein.

[0018] In some embodiments of the present application, the antibody that specifically binds to the TEX26 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.12-SEQ ID NO.13.

[0019] In some embodiments of the present application, the nucleotide sequence of the Tex26 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex26 gene is shown as SEQ ID NO.2 or SEQ ID NO.3; or, the amino acid sequence of the TEX26 protein is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0020] The second aspect of the present application provides a kit, wherein the kit includes a reagent for detecting whether a Tex26 gene, a TEX26 protein, an mRNA of a Tex26 gene, an RNA of a Tex26 gene, or a miRNA targeting a TEX26 protein exists, or the substance is a reagent for detecting whether the expression amount of a TEX26 protein, an mRNA of a Tex26 gene, an RNA of a Tex26 gene, or a miRNA targeting a TEX26 protein changes, or the substance is a reagent for detecting whether a sequence mutation exists in a Tex26 gene, a TEX26 protein, or an mRNA of a Tex26 gene; the kit has at least one of the following uses:

[0021] A1) Study the process of spermatogenesis;

[0022] A2) Identification of the germ cell sorting process;

[0023] A3) Screening for sperm maturation abnormalities;

[0024] A4) Diagnosis of fertilization abnormalities;

[0025] A5) Screening and diagnosis of male infertility;

[0026] A6) Preparation of male contraceptive pills and / or male contraceptive vaccines.

[0027] In some embodiments of the present application, the kit includes an antibody that specifically binds to the TEX26 protein.

[0028] In some embodiments of the present application, the antibody that specifically binds to the TEX26 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.12-SEQ ID NO.13.

[0029] In some embodiments of the present application, the kit includes at least one of a primer pair having nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7, a primer pair having nucleotide sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, and a primer pair having nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11.

[0030] In some embodiments of the present application, the nucleotide sequence of the Tex26 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex26 gene is shown as SEQ ID NO.2 or SEQ ID NO.3; or, the amino acid sequence of the TEX26 protein is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0031] In some embodiments of the present application, the test sample of the kit is selected from at least one of blood, oral tissue, testicular tissue and sperm.

[0032] In some embodiments of the present application, the method for judging the test results of the kit includes: if the Tex26 gene, TEX26 protein, Tex26 gene mRNA, Tex26 gene RNA or miRNA targeting TEX26 protein is not present in the test sample, or the expression level of TEX26 protein, Tex26 gene mRNA, Tex26 gene RNA or miRNA targeting TEX26 protein in the test sample changes, or the sequence of Tex26 gene, TEX26 protein or Tex26 gene mRNA in the test sample mutates, then the source of the test sample is suspected of suffering from male sperm maturation abnormality, fertilization abnormality or male infertility.

[0033] The third aspect of the present application provides the use of a biomarker as a drug target in the preparation of a drug having any of the following uses:

[0034] B1) Promote sperm maturation and fertilization;

[0035] B2) Improve sperm maturation rate;

[0036] B3) Improve sperm fertilization level;

[0037] B4) Improve male fertility;

[0038] B5) Treatment of sperm dysfunction;

[0039] B6) Treatment of male infertility;

[0040] B7) preparing male contraceptive pills and / or male contraceptive vaccines;

[0041] Wherein, the biomarker is selected from TEX26 protein or Tex26 gene.

[0042] Beneficial effects of the present application: The mRNA of the Tex26 gene described in the present application is specifically expressed in the testis and epididymis, and is specifically localized in the tail of the elongated spermatid cell and the tail of the epididymal sperm, indicating that the TEX26 protein is a new protein localized in the elongated sperm tail and the epididymal sperm tail. The Tex26 gene and TEX26 protein described in the present application can be used as biomarkers in the preparation of kits for diagnosing male sperm maturation abnormalities, fertilization abnormalities, male infertility, male contraceptives and / or male contraceptive vaccines, etc., providing a new biomarker and a new target for diagnosing and treating male infertility and preparing male contraceptives for existing kits for diagnosing male infertility diseases.

[0043] Of course, implementing any product or method of the present application does not necessarily require achieving all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0045] Figure 1A This is a diagram of the tertiary spatial structure analysis of the mouse TEX26 protein in Example 1 of the present application;

[0046] Figure 1B The amino acid sequence antigenicity analysis and antigen epitope prediction map of the mouse TEX26 protein in Example 1 of the present application;

[0047] Figure 1C This is a graph showing the effectiveness test results of the TEX26 antibody prepared in Example 1 of the present application (immunohistochemistry of mouse testis sections);

[0048] Figure 1D This is a graph showing the effectiveness test results of the TEX26 antibody prepared in Example 1 of the present application (fluorescent immunohistochemistry of mouse epididymal sperm);

[0049] Figure 2A This is a graph showing the expression results of the Tex26 gene in different tissues of wild-type mice in Example 2 of the present application;

[0050] Figure 2B This is a graph showing the expression results of the Tex26 gene in the testicular tissue of wild-type mice at different stages in Example 2 of the present application;

[0051] Figure 2C This is the localization map of the TEX26 protein in the testicular tissue cells of wild-type mice in Example 2 of the present application;

[0052] Figure 2D This is a diagram showing the localization results of TEX26 protein in wild-type mouse epididymal sperm in Example 2 of the present application.

[0053] Figure 2E This is a diagram showing the RT-PCR identification results of Tex26 gene KO mice in Example 2 of the present application;

[0054] Figure 3A This is a comparison diagram of the testicular transcriptome sequencing results of TEX26- / - mice and wild-type mice in Example 3 of the present application;

[0055] Figure 3B This is a graph showing the GO analysis results in Example 3 of the present application;

[0056] Figure 3C This is a diagram of the Reactome analysis results in Example 3 of the present application;

[0057] Figure 3D This is the KEGG analysis result diagram in Example 3 of the present application;

[0058] Figure 4A This is a diagram showing the localization results of the Tex26 gene in the caput epididymides of wild-type mice in Example 4 of the present application;

[0059] Figure 4B This is a diagram showing the localization results of the Tex26 gene in the epididymis of wild-type mice in Example 4 of the present application;

[0060] Figure 4C This is a diagram showing the localization results of the Tex26 gene in the cauda epididymis of wild-type mice in Example 4 of the present application;

[0061] Figure 5 This is a graph showing the expression changes of the Tex26 gene in different male infertility patients in Example 5 of the present application. DETAILED DESCRIPTION

[0062] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the present application belong to the scope of protection of the present application.

[0063] The present application provides any of the following applications of a biomarker and / or a substance for detecting the biomarker:

[0064] A1) Use in the preparation of products for studying spermatogenesis;

[0065] A2) Use in the preparation of a product for identifying a germ cell sorting process;

[0066] A3) Use in the preparation of a product for diagnosing sperm maturation abnormalities;

[0067] A4) Use in the preparation of a product for diagnosing fertilization abnormalities;

[0068] A5) Use in the preparation of products for screening and diagnosing male infertility diseases;

[0069] A6) Application in the preparation of male contraceptive drugs and / or male contraceptive vaccine products;

[0070] Wherein, the biomarker is selected from Tex26 gene, TEX26 protein, mRNA of Tex26 gene, RNA of Tex26 gene or miRNA targeting TEX26 protein.

[0071] The inventors of the present application have found in their research that TEX26 protein is a newly discovered sperm tail localization protein, which is specifically expressed in the testis and epididymis. Tex26 gene and TEX26 protein etc. can be used as molecular markers for the differentiation of round sperm into elongated sperm stages during spermatogenesis, and can also be used as molecular markers for the maturation of epididymal tail sperm, to study the differentiation of round sperm, sperm maturation, assessment of fertilization ability and male reproductive dysfunction, etc. Tex26 gene and TEX26 protein described in the present application can be used as biomarkers for the differentiation of round sperm and biomarkers for the diagnosis of sperm maturation and fertilization disorders. In addition, the TEX26 human / mouse antibody designed and prepared can be used for the research of the differentiation and maturation of round sperm, identification of different germ cell types, detection of abnormal sperm maturation, abnormal fertilization and diagnosis of male infertility diseases.

[0072] In some embodiments of the present application, the substance for detecting the biomarker is a reagent for detecting whether the Tex26 gene, TEX26 protein, mRNA of the Tex26 gene, RNA of the Tex26 gene, or miRNA targeting the TEX26 protein exists, or the substance is a reagent for detecting whether the expression amount of the TEX26 protein, mRNA of the Tex26 gene, RNA of the Tex26 gene, or miRNA targeting the TEX26 protein changes, or the substance is a reagent for detecting whether the Tex26 gene, TEX26 protein, or mRNA of the Tex26 gene has a sequence mutation. In the present application, the sequence mutation of the Tex26 gene and the mRNA of the Tex26 gene refers to a mutation of the nucleotide sequence, which can be a mutation of one nucleotide site or a mutation of multiple nucleotide sites; the sequence mutation of the TEX26 protein refers to a mutation of the amino acid sequence, which can be a mutation of one amino acid site or a mutation of multiple amino acid sites.

[0073] In some embodiments of the present application, the test sample detected when detecting the biomarker substance is selected from at least one of blood, oral tissue, testicular tissue and sperm.

[0074] In some embodiments of the present application, the reagent includes an antibody that specifically binds to the TEX26 protein.

[0075] In some embodiments of the present application, the antibody that specifically binds to the TEX26 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.12 to SEQ ID NO.13.

[0076] SEQ ID NO.12 (antigen of mouse Tex26 gene, 90-193aa): DTEFRYNYQIPAQIPELKDFSFKYGCYASLPVASQGLVPSVLSSYIRNEERTKKQTTYECDYGKACLDFLTILDSFTPSQVHDYLQSVSYKDRQILERFIHSHC;

[0077] SEQ ID NO.13 (antigen of mouse Tex26 gene, 102-115aa, S replaces C): QIPELKDFSFKYGC.

[0078] In some embodiments of the present application, in the 5'-3' direction, the Tex26 gene is composed of the nucleotide sequence shown in SEQ ID NO.1, the nucleotide sequence shown in SEQ ID NO.20 and the nucleotide sequence shown in SEQ ID NO.21 connected in sequence (it should be noted that, in order to facilitate the preparation of the sequence table, the Tex26 gene is divided into the upper section SEQ ID NO.1, the middle section SEQ ID NO.20 and the lower section SEQ ID NO.21) (>NC_000013.11:30932656-30975500TEX26[organism=Homo sapiens][GeneID=122046][chromosome=13]); or, the nucleotide sequence of the mRNA of the Tex26 gene is as shown in SEQ ID NO.2 or SEQ ID NO.3; or, the amino acid sequence of the TEX26 protein is as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0079]

[0080]

[0081] SEQ ID NO.4 (human TEX26 protein): MEQPGPRAPDPSLLCHHNLQPTDDPNWDSYATTMR TAFTPKTGAVPALIRQNGIRRLGYTYSLSDPILNQTQYSDEYTWKSHSKEDLIKTETSRGIKSHKSHLNEDIFLWTLPHCQQTGTLKNCLPWKIPASMKEVNKALSNQFISLTKRDFVDRSKAQKIKK SSHLSLEWKKLLPQPPDTEFRRNYQIPAKIPELQDFSFKYGCYSSLPVASQGLVPSVLHSYLRNQEHTKKQTTYQSDYDKTYPDFLMLLNSFTSSQVKEYLQSLSYKDRQIIDRFIRTHCDTNKKKK.

[0082] SEQ ID NO. 5 (human TEX26 protein): MKEVNKALSNQFISLTKRDFVDRSKAQKIKKSSH LSLEWKKLLPQPPDTEFRRNYQIPAKIPELQDFSFKYGCYSSLPVASQGLVPSVLHSYLRN QEHTKKQTTYQSDYDKTYPDFLMLLNSFTSSQVKEYLQSLSYKDRQIIDRFIRTHCDTNK KKK.

[0083] The second aspect of the present application provides a kit, wherein the kit includes a reagent for detecting whether a Tex26 gene, a TEX26 protein, an mRNA of a Tex26 gene, an RNA of a Tex26 gene, or a miRNA targeting a TEX26 protein exists, or the substance is a reagent for detecting whether the expression amount of a TEX26 protein, an mRNA of a Tex26 gene, an RNA of a Tex26 gene, or a miRNA targeting a TEX26 protein changes, or the substance is a reagent for detecting whether a sequence mutation exists in a Tex26 gene, a TEX26 protein, or an mRNA of a Tex26 gene; the kit has at least one of the following uses:

[0084] A1) Study the process of spermatogenesis;

[0085] A2) Identification of the germ cell sorting process;

[0086] A3) Screening for sperm maturation abnormalities;

[0087] A4) Diagnosis of fertilization abnormalities;

[0088] A5) Screening and diagnosis of male infertility;

[0089] A6) Preparation of male contraceptive pills and / or male contraceptive vaccines.

[0090] The kit described in the present application can be used to evaluate the integrity of sperm maturation in vitro, and to initiate sperm capacitation and induce sperm maturation reaction to evaluate the ability of capacitated sperm to undergo maturation reaction, thereby providing a theoretical basis for the analysis of the causes of infertility and the selection of assisted reproductive treatment options.

[0091] In some embodiments of the present application, the kit includes an antibody that specifically binds to the TEX26 protein.

[0092] In some embodiments of the present application, the antibody that specifically binds to the TEX26 protein is prepared using an antigen whose amino acid sequence is shown in any one of SEQ ID NO.12-SEQ ID NO.13. The TEX26 protein described in the present application is a newly discovered sperm maturation protein. According to the amino acid sequence of the TEX26 protein in humans / mouse, a human or mouse antigen as shown in any one of SEQ ID NO.12-SEQ ID NO.13 is designed, and then an antibody is prepared. The function of the antibody is not limited to WB, IHC / ICC / IF, IP / Co-IP experiments.

[0093] In some embodiments of the present application, the kit includes at least one of a primer pair having nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7, a primer pair having nucleotide sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, and a primer pair having nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11.

[0094] SEQ ID NO.6 (upstream primer of mRNA of human Tex26 gene): CCCAACTGGGATTCCTATGCT;

[0095] SEQ ID NO.7 (downstream primer of mRNA of human Tex26 gene): CGTTTTGGCGAATTAAGGCAG;

[0096] SEQ ID NO.8 (upstream primer of mRNA of human Tex26 gene): TAATTCGCCAAAACGGTATCAGA;

[0097] SEQ ID NO.9 (downstream primer of mRNA of human Tex26 gene): TGACAGTGAGGTAGTGTCCAC;

[0098] SEQ ID NO.10 (upstream primer of mRNA of human Tex26 gene): GTTGCTTCTCAGGGTCTAGTGC;

[0099] SEQ ID NO.11 (downstream primer of mRNA of human Tex26 gene): AGGTTTTGTCGTAGTCACTTTGG.

[0100] In some embodiments of the present application, the nucleotide sequence of the Tex26 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex26 gene is shown as SEQ ID NO.2 or SEQ ID NO.3; or, the amino acid sequence of the TEX26 protein is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0101] In some embodiments of the present application, the test sample of the kit is selected from at least one of blood, oral tissue, testicular tissue and sperm.

[0102] In some embodiments of the present application, the method for judging the test results of the kit includes: if the Tex26 gene, TEX26 protein, Tex26 gene mRNA, Tex26 gene RNA or miRNA targeting TEX26 protein is not present in the test sample, or the expression level of TEX26 protein, Tex26 gene mRNA, Tex26 gene RNA or miRNA targeting TEX26 protein in the test sample changes, or the sequence of Tex26 gene, TEX26 protein or Tex26 gene mRNA in the test sample mutates, then the source of the test sample is suspected of suffering from male sperm maturation abnormality, fertilization abnormality or male infertility.

[0103] The third aspect of the present application provides a use of a biomarker as a drug target in the preparation of a drug having any of the following uses:

[0104] B1) Promote sperm maturation and fertilization;

[0105] B2) Improve sperm maturation rate;

[0106] B3) Improve sperm fertilization level;

[0107] B4) Improve male fertility;

[0108] B5) Treatment of sperm dysfunction;

[0109] B6) Treatment of male infertility;

[0110] B7) preparing male contraceptive pills and / or male contraceptive vaccines;

[0111] Wherein, the biomarker is selected from TEX26 protein or Tex26 gene.

[0112] The fourth aspect of the present application provides the use of Tex26 gene, TEX26 protein, mRNA of Tex26 gene, RNA of Tex26 gene or miRNA targeting TEX26 protein as biomarkers in spermatogenesis research, in germ cell classification and identification, in screening for abnormal sperm maturation and development, in screening and diagnosis of male infertility diseases, in diagnosing abnormal sperm-egg fertilization, or in the preparation of male contraceptives and / or male contraceptive vaccines.

[0113] The fifth aspect of the present application provides the use of TEX26 protein or Tex26 gene as a drug target in improving male fertility, treating sperm dysfunction, promoting sperm maturation and fertilization, improving sperm fertilization ability, improving sperm quality, improving sperm fertilization level and treating male infertility.

[0114] The TEX26 protein described in this application is a new sperm maturation-related protein, which can be used as a biomarker for the classification and identification of germ cells and for the study of spermatogenesis. By detecting whether the TEX26 protein is produced, it can be used to detect the maturity of sperm. By detecting the cause of male fertilization abnormalities, detecting the maturity of sperm can prevent and / or treat male infertility caused by male sperm fertilization abnormalities. The Tex26 gene or TEX26 protein can be used as a biomarker in the preparation of a kit for diagnosing male infertility, providing a new molecular marker and therapeutic target for existing kits for diagnosing male infertility.

[0115] The present application also provides the use of Tex26 gene or TEX26 protein. Tex26 gene or TEX26 protein can be used as a molecular marker to prepare a kit for diagnosing male infertility, and can also be used as a drug target to prepare a product that can prevent and diagnose male infertility and / or perform fertilization assessment; promote sperm maturation and fertilization; improve sperm fertilization ability; improve sperm quality; and improve one or more functions of male fertility.

[0116] Example

[0117] Hereinafter, embodiments of the present invention are described in more detail. Various tests and evaluations are performed according to the following methods. In addition, the percentage contents mentioned herein, unless otherwise specified, refer to mass percentages for solid-liquid mixing and solid-solid mixing, and refer to volume percentages for liquid-liquid mixing.

[0118] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0119] The experimental animals used in the following examples were all raised and bred at the Animal Experiment Center of Peking University Health Science Center, and had free access to water and food during the experiments.

[0120] In the following examples, WT indicates wild-type mice, and TEX26- / - indicates Tex26 gene knockout homozygous mice.

[0121] Example 1: Preparation of antibodies against TEX26 protein and detection of antibody effectiveness

[0122] 1. Preparation of TEX26 polyclonal antibody (rabbit anti-mouse)

[0123] Mouse TEX26 protein is composed of 211 amino acids, and its tertiary structure analysis is shown in the figure below: Figure 1A The results of antigenicity analysis of the amino acid sequence of mouse TEX26 protein are shown in Figure 1B As shown. It can be seen that the 90-193aa and 102-115aa regions of the mouse TEX 26 protein have good specificity and antigenic epitope prediction, and a certain secondary structure is conducive to protein expression. The expression vector pGEX-4T-AB1 was constructed, and the antigen was expressed by the Escherichia coli prokaryotic system, injected into rabbits, and rabbit polyclonal antibodies were prepared; and the 90-193aa region and the 102-115aa region were selected for peptide synthesis. The above two region sequences are human / mouse homologous sequences, with good specificity and antigenic epitopes and good amino acid hydrophilicity, as mixed immunization.

[0124] SEQ ID NO.12 (antigen of mouse Tex26 gene, 90-193aa): DTEFRYNYQIPAQIPELKDFSFKYGCYASLPVASQGLVPSVLSSYIRNEERTKKQTTYECDYGKACLDFLTILDSFTPSQVHDYLQSVSYKDRQILERFIHSHC;

[0125] SEQ ID NO. 13 (antigen of mouse Tex26 gene, 102-115aa, S replaces C): QIPELKDFSFKY GC.

[0126] 2. Antibody Preparation

[0127] Based on the above sequence, the expression vector pGEX-4T-AB1 was constructed, and the antigen was expressed to prepare the antibody.

[0128] 1. Construction of prokaryotic protein expression vector

[0129] The analysis obtained an amino acid sequence with high antigenicity and antigen specificity, and then primers were designed for the sequence, followed by PCR amplification and cloning in a pGEX-4T-AB1 prokaryotic expression vector (Escherichia coli prokaryotic system) and identification.

[0130] 2. Expression and identification of recombinant proteins

[0131] The identified expression plasmid is transformed into the expression plasmid. After the identification is correct, part of the bacteria is preserved and part of the bacteria is shaken to express the recombinant protein. The specific steps are as follows:

[0132] 1) Pick 4-6 single colonies and inoculate them into a 5 mL LB culture tube and culture until OD 600 Reach 0.4-0.6;

[0133] 2) Add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.2, 0.4, 0.6, 0.8 and 1 mmol / L to each tube, and induce with shaking at 37°C (set different temperatures of 18°C, 20°C, 25°C, 30°C, 32°C, and 37°C to detect the effect of different temperatures on the induction expression of recombinant proteins) for 3-4 hours, and use the uninduced bacterial solution as a negative control;

[0134] 3) Collect 1 mL of bacterial precipitate, resuspend with sample buffer, heat and lyse, and centrifuge to collect the supernatant and precipitate;

[0135] 4) Prepare 10% SDS-PAGE gel, take 10-20 μL sample, and perform SDS-PAGE electrophoresis;

[0136] 5) After electrophoresis, the SDS-PAGE gel was stained with Coomassie Brilliant Blue R250 for 20 min;

[0137] 6) Decolorize with decolorizing solution and analyze the protein expression results to determine whether the size of the induced recombinant protein is consistent with the expected one, and which temperature and concentration of IPTG have the best induction effect;

[0138] 7) The positive plasmid with the highest expression level is preserved as the expression strain.

[0139] 3. Large-scale expression and extraction of recombinant proteins

[0140] The specific steps are as follows:

[0141] 1) Inoculate the identified expression plasmid into 15 mL LB liquid medium and shake at 37°C overnight;

[0142] 2) On the second day, transfer the bacterial solution to 250 mL LB medium at a ratio of 1:100 (volume ratio) and culture for 1.5-3 hours until the OD 600 The value reaches 0.4-0.6;

[0143] 3) Add IPTG to induce expression for 3-4 hours (the concentration of IPTG and induction temperature are based on the optimal parameters of small-amount shaking induction);

[0144] 4) Collect the culture medium, centrifuge at 4°C, 5000×g for 10 min, discard the supernatant, and collect the bacterial precipitate;

[0145] 5) Wash the cells once with PBS, centrifuge at 5000 g for 10 min, discard the supernatant, and collect the cell pellet;

[0146] 6) Store at -20℃ for later use.

[0147] 4. Purification of recombinant protein

[0148] The identified recombinant protein was purified using Amersham's GST Trap Chelating HP (5 mL) purification column and high performance liquid chromatography (HPLC instrument), and the concentration was determined.

[0149] (1) Sample processing before loading: The specific steps are as follows:

[0150] 1) Resuspend the bacterial pellet of each 100 mL LB culture in 1 × 4 mL PBS;

[0151] 2) Add lysozyme to a final concentration of 1 mg / mL and digest on ice for 30 min;

[0152] 3) Add PMSF (phenylmethylsulfonyl fluoride) at a ratio of 1:100 (volume ratio), place the beaker on ice, and disrupt the cells with ultrasound; the ultrasound conditions are: ultrasound for 1 second, interval of 1 second, ultrasound for 5 minutes each time, action for 4 times, a total of 20 minutes;

[0153] 4) Centrifuge at 12,000 × g for 20 min at 4°C, and perform electrophoresis on the supernatant and precipitate to determine whether the target protein is expressed in a soluble form or in the form of inclusion bodies;

[0154] 5) If the expression is in soluble form, take the supernatant and filter it with a 0.22μm filter membrane; the filtered liquid is the sample for column loading. If the expression is in inclusion body form, the inclusion body is renatured and then filtered with a 0.22μm filter membrane; the filtered liquid is the sample for column loading. Inclusion body renaturation steps: collect bacterial precipitate and add 10mL TE1 (10mM Tris-HCl (pH7.0), 1mM EDTA), blow evenly, and shake until fully uniform; then use ultrasonic disruption for 30min (5s disruption / 9s interval), 8000×g, 4℃, centrifuge for 30min, collect precipitate; then add 10mL TE2 (10mM Tris-HCl (pH7.0), 1mM EDTA, 1% TritonX-100 (V / V)), blow evenly, shake for 20min, 7000×g, 4℃, centrifuge for 20min, collect precipitate. Then add 10mL TE3 (2M urea, 10mM Tris-HCl (pH7.0), 1mM EDTA), blow evenly, shake for 20min, centrifuge at 7000×g, 4℃ for 20min, and collect the precipitate. Finally, add 3mL denaturing solution or Buffer B (8M urea, 10mM Tris-HCl (pH7.0), 10mM DTT), blow evenly, shake for 20min, transfer to a 37℃ shaker and shake at 200×g for about 2h until the precipitate is fully dissolved. Centrifuge at 8000×g, 4℃ for 20min, collect the supernatant and filter with a 0.22μm filter membrane.

[0155] (2) FPLC recombinant protein purification step, comprising the following steps in sequence:

[0156] Double distilled water, 1×PBS, Elution buffer (10mM glutathione, pH=8 Tris-HCl) and 20% ethanol aqueous solution, pass through a 0.22μm filter and ultrasonically degas for 20min; wash the pump (3mL / min double distilled water); connect the GSTrap FF 5mL column to the FPLC (5CV, 1-3mL / min double distilled water); 1×PBS until the baseline is washed (5CV 1-3mL / min); add the recombinant protein sample (1mL / min); elute to remove impurities (40CV, 1×PBS until the baseline is washed); collect the eluted peak protein (100% 1mL / min Elution buffer); 1×PBS until the baseline is washed (5CV 1-3mL / min); pass the 20% ethanol aqueous solution through the column; disassemble the GSTrap FF 5mL column; wash the pump; wash the tube with 20% ethanol aqueous solution, and the purification is completed.

[0157] The purified recombinant antigen protein and total bacterial protein were subjected to Western blotting electrophoresis (10% SDS-PAGE gel); after electrophoresis, Coomassie brilliant blue staining was performed for 20 minutes, and after decolorization, the purification effect of the recombinant protein and the presence of mixed bands were observed. The recombinant antigen protein was quantitatively measured by the Brandford method and the protein concentration was measured and stored at -80°C for future use.

[0158] 5. Animal immunization and identification of antiserum

[0159] (1) Animal immunization: Two New Zealand white rabbits (2.5-3.5 kg) were immunized with each purified antigen protein.

[0160] First immunization: Use a 2mL syringe to absorb 200μg / rabbit recombinant antigen protein and an equal volume of complete Freund's adjuvant to fully emulsify and inject. Use the multi-point injection method to immunize animals. That is, select 8-10 points on both sides of the rabbit spine for subcutaneous injection, and inject 1 point on both sides of the shoulder (or arm), and inject about 0.2mL at each point.

[0161] Second immunization: Two weeks later, take 200μg / rabbit of recombinant antigen protein and emulsify it with an equal volume of incomplete Freund's adjuvant. The animal is immunized by the same multi-point injection method, with 0.2mL injected subcutaneously at each point. After that, the injection is given once every week, and after 2-3 consecutive booster injections, blood is collected to determine the titer. During the measurement, 0.5-1.0mL of blood is collected from the ear vein, the serum is separated, and the antibody titer of the immune serum is determined by Western blot or ELISA test. If the titer does not meet the requirements, after the second immunization, the antigen emulsified with Freund's incomplete adjuvant (FIA) is used for another 1-2 times. The injection site, dose and interval are the same as the second time, and blood is tested again to measure the antibody titer. If the titer meets the requirements, blood can be drawn to collect serum.

[0162] (2) Collection and storage of antiserum

[0163] Blood was collected from rabbits using the heart blood collection method, the steps are as follows:

[0164] 1) Place the rabbit on its back with its limbs tied to an animal holder or have an assistant fix the limbs;

[0165] 2) Use surgical scissors to cut the hair on the left chest of the rabbit, disinfect the skin with alcohol and wipe it dry;

[0166] 3) Touch the part of the heart where the heart beats the strongest with your left thumb and keep the heart fixed on the left side of the chest;

[0167] 4) Use a 50mL syringe (connected to a 16-gauge needle), tilt the needle at 45 degrees, and pierce the heart at the strongest point of the heartbeat to draw blood;

[0168] 5) The drawn blood was immediately injected into a sterile 50 mL centrifuge tube and left at room temperature to coagulate;

[0169] 6) Place the retrieved blood in a 37°C incubator for 1 hour, then place it in a 4°C refrigerator overnight;

[0170] 7) After the blood coagulation and clot shrinkage, centrifuge at 3000×g for 15 min;

[0171] 8) Take the supernatant and divide it into portions and store it in a 4℃ refrigerator for later use.

[0172] (3) Antibody specificity identification

[0173] Take the total bacterial protein, purified recombinant antigen protein, mouse testis and various tissue proteins.

[0174] 1) Perform Western blotting and use pre-immune serum as a negative control;

[0175] 2) Immunohistochemical detection and use of pre-immune serum as negative control;

[0176] 3) Immunocytochemistry detection and use of pre-immune serum as negative control.

[0177] The prepared antibodies were subjected to immunohistochemistry to detect their effectiveness. The effectiveness test results were as follows: Figure 1C As shown. The newly prepared TEX26 antibody was used to perform immunohistochemistry in mouse testis paraffin sections to detect the effectiveness and specificity of the antibody. As can be seen from the results in the above figure, the antibody binding to the TEX26 protein is specifically localized in the cytoplasm of testicular round spermatids and elongated spermatids, indicating that the prepared TEX26 is a round spermatid and elongated sperm localization protein with high specificity and effectiveness. This antibody can be used to study spermatogenesis and round sperm differentiation; it can also be used as a specific marker to identify and distinguish different germ cells (such as spermatogonia, spermatocytes and round spermatids).

[0178] The newly prepared TEX26 antibody was used to perform immunofluorescence staining on mouse epididymal sperm to test the effectiveness and specificity of the antibody. The results of the TEX26 antibody effectiveness test (mouse epididymal sperm fluorescent immunohistochemistry) are shown in the figure. Figure 1D As shown in the above figure, it can be seen that the antibody binding to the TEX26 protein is specifically localized on the sperm tail (sperm tail, red), indicating that TEX26 is a sperm tail localized protein. The above results show that the prepared TEX26 antibody is effective and highly specific.

[0179] Example 2: Fluorescence quantitative PCR detection and immunohistochemistry detection

[0180] Fluorescence quantitative PCR detection: Trizol reagent (Thermo Fisher) was used to extract total RNA from mouse testes in different tissues and at different times. 500 ng of total RNA was reverse transcribed using Prime-Script RT reagent kit (Takara RR047A). Real-time fluorescence quantitative PCR detection was performed using cDNA products from different samples as templates. The expression level of the Tex26 gene was normalized to Gapdh using the 2-ΔCt method and reported as fold change. The expression level of the Tex26 gene was normalized to Gapdh using GAPDH as an internal reference, and expression changes were detected. Table 1 shows the primers used.

[0181] Table 1 Primers

[0182] Primers application Sequence (5'-3') SEQ ID NO Tex26-FP Real-time quantitative PCR GAAATCACTCCTTCCCCGGC SEQ ID NO.14 Tex26-RP Real-time quantitative PCR GTGGTCTGCTTCTTTGTGCG SEQ ID NO.15 GAPDH-FP Real-time quantitative PCR AGGTCGGTGTGAACGGATTTG SEQ ID NO.16 GAPDH-RP Real-time quantitative PCR TGTAGACCATGTAGTTGAGGTCA SEQ ID NO.17

[0183] Immunohistochemistry: Fresh testicles, epididymis and testicular biopsy tissue samples of wild-type (WT) mice and male infertility patients were fixed in 4% paraformaldehyde (Servicebio, G1101-500ML) and immunostained for 24 hours. Paraffin sections were boiled in 10mM sodium citrate buffer (pH6.0) for 15min, gradually cooled to room temperature, and washed 3 times with PBS containing 0.1% Triton X-100. The sections were blocked with 3w / v% bovine serum albumin (BSA) at room temperature for 1 hour and then incubated with the primary antibody at 4°C overnight. Next, the sections were incubated with the corresponding secondary antibody at room temperature for 1 hour. The sections were then operated and analyzed according to standard protocols. The antibodies used in this example include anti-human / mouse TEX26 antibodies (primary antibodies, prepared in Example 1, the volume ratio of the primary antibody dilution is 1:200).

[0184] Figure 2A The figure is the real-time fluorescence quantitative PCR (Q-PCR) expression diagram of Tex26 gene in different mouse tissues. Figure 2A The Q-PCR results of different tissues of adult mice showed that Tex26 is a new testis- and epididymis-specific expression gene. It is specifically expressed only in the testis and epididymis of adult mice, but not in the heart, liver, kidney, lung, small intestine, spleen, muscle and other tissues of mice. Figure 2B The real-time fluorescence quantitative PCR (Q-PCR) expression diagram of Tex26 gene expression in mouse testis tissue at different stages. Figure 2BAs shown, the Tex26 gene initially began to be weakly expressed and transcribed at 18 days after birth, and then transcription increased from 24 days (this stage is exactly the time period for round spermatids to transform into elongated sperm) and continued until adulthood. The results show that the Tex26 gene can be used as a specific marker to study the process of spermatogenesis and round sperm differentiation.

[0185] The expression and localization of TEX26 protein in mouse testis tissue sections were detected by immunohistochemistry. Figure 2C As shown in the results, TEX26 protein is only located in the cytoplasm of mouse round spermatids and elongated spermatids; the results show that TEX26 antibody can be used as a biomarker for the production of round spermatids and can be applied to the study of spermatogenesis and the identification of different types of germ cells. For example, flow cytometry can be used to sort round spermatids or elongated spermatids and exclude other mixed cells (such as testicular supporting cells, interstitial cells, spermatogonial stem cells, spermatocytes, etc.) or to identify the purity of different sorted germ cells. The study further found that: TEX26 protein is located on the tail of mouse epididymal sperm, such as Figure 2D As shown in the figure. As can be seen from the figure, immunofluorescence detected that the TEX26 protein is located on the tail of mature sperm in the cauda epididymis and is involved in the entire process of epididymal sperm maturation. The above results show that TEX26 is a new sperm maturation-related protein that is specifically expressed in the testis and epididymis. It is specifically located in the cytoplasm of round sperm, elongated sperm and the tail of mature epididymal sperm, and is involved in the entire process of epididymal sperm maturation; the Tex26 gene can not only be used to study the process of spermatogenesis, the differentiation process of round sperm and distinguish different types of germ cells, but also can be used as a biomarker for sperm maturation research to study the process of sperm maturation and diagnose sperm maturity.

[0186] According to the Tex26 genome structure, the Tex26 gene has multiple transcription products. According to the Tex26 gene knockout sequence, primers were designed. The PCR method was used to identify whether the Tex26 gene in the testis of the Tex26 gene KO mouse was expressed, and the identification system was 25 μL. The primer sequences are shown in Table 2.

[0187] Table 2 Primers

[0188] Primers application Sequence (5'-3') SEQ ID NO GAPDH-FP Real-time quantitative PCR AGGTCGGTGTGAACGGATTTG SEQ ID NO.16 GAPDH-RP Real-time quantitative PCR TGTAGACCATGTAGTTGAGGTCA SEQ ID NO.17 Tex26-WT-FP Genotyping CACACTTTCTGACATCATCTTTAGC SEQ ID NO.18 Tex26-WT-RP Genotyping CATACACTGGATTAGCCACATACA SEQ ID NO.19

[0189] Testicular RNA from TEX26- / -KO mice and wild-type male mice at 8 weeks postpartum was extracted using Trizol reagent (Invitrogen 15596018) and quantified using a spectrophotometer. 500 ng of total RNA was added for reverse transcription using Prime-Script RT reagent kit (Takara RR047A). Real-time fluorescence quantitative PCR was performed using cDNA products from testicular tissues of different WT mice as templates. The expression level of the Tex26 gene was measured using GAPDH as an internal reference, and expression changes were detected. The results are shown in Figure 2E As shown in the figure, it can be seen that the amplified band of the Tex26 gene can be detected in the wild-type mice W1 and the wild-type mice W2, while the amplified band of the Tex26 gene cannot be detected in the TEX26- / -KO1 mice and TEX26- / -KO2. In summary, the Tex26 gene has been knocked out and the Tex26 gene knockout mice have been successfully prepared.

[0190] Example 3: Tex26 gene knockout affects the expression of 6652 sperm proteins and changes in multiple signaling pathways

[0191] Total RNA was extracted from the testicular tissue of Tex26 knockout mice as follows: After the testicular tissue was washed with PBS, it was placed in an RNase free EP tube. An appropriate amount of Triozol (50-100 mg sample plus 1 mL Trizol) (Sigma) was added to each sample to break and mix the sample at room temperature. Centrifuge at 4°C, 12000rpm for 10 min, transfer the supernatant to a new RNase free EP tube, and discard the undissolved tissue. Incubate at room temperature for 5 min to ensure that the nucleoprotein complex is fully dissociated. Add an appropriate amount of chloroform (1 mL Trizol plus 0.2 mL chloroform) according to the initial amount of Trizol added, shake vigorously by hand for 15 s, and let stand at room temperature for 2-3 min. Centrifuge at 4°C, 12000rpm for 15 min, and separate the samples. Carefully aspirate the upper aqueous phase into a new EP tube (approximately aspirate 80% of the upper aqueous phase), and avoid aspirating the middle phase and the lower red organic phase. Add an appropriate amount of 100% isopropanol according to the initial amount of Trizol (1 mL Trizol plus 0.5 mL isopropanol), and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 12000 rpm for 10 minutes. Remove the supernatant and keep the white RNA precipitate at the bottom of the tube. Add 75 vol% ethanol solution (DEPC-H 2 O preparation, add 1mL 75vol% ethanol solution to every 1mL Trizol), shake several times, centrifuge at 7500rpm for 5min at 4℃, and discard the liquid. Repeat this step once. Open the lid and let it stand at room temperature to dry the RNA precipitate. Add appropriate amount of RNase-free water, pipette several times, and redissolve the RNA. Send the RNA sample to BGI for transcriptome sequencing.

[0192] The results are as follows: Figure 3A This is a comparison chart of the testicular transcriptome sequencing results of TEX26- / - mice and wild-type mice in Example 3 of the present application; it can be seen that GO and KEGG analysis showed that 6652 genes / proteins were differentially expressed (P < 0.05), and 24852 genes were not significantly expressed; among them, 4233 genes were upregulated and 2419 genes were downregulated.

[0193] Figure 3B It is the GO analysis result diagram in Example 3 of this application; Tex26 gene is specifically expressed in the testis and may be involved in spermatogenesis. It was found in the up-regulated differentially expressed genes that 182 genes closely related to spermatogenesis were up-regulated after Tex26 gene knockout; because Tex26 gene is located in the cytoplasm of round spermatids and elongated sperm in the testis, it may be involved in the differentiation process of round spermatids. It was found in the up-regulated differentially expressed genes that 73 genes related to spermatocyte (round spermatid) differentiation were up-regulated; the above results indicate that Tex26 gene may be involved in spermatogenesis and the differentiation process of round spermatids. The study found that 54 genes related to the sperm acrosome had changes in expression. The above results indicate that Tex26 gene may be involved in regulating the formation process of sperm head acrosome, and its absence will affect the expression changes of genes related to sperm acrosome, and sperm acrosome is closely related to sperm-egg fertilization.

[0194] Immunohistochemistry showed that the Tex26 gene was located in the sperm tail and epididymal sperm tail, and may be involved in sperm movement and epididymal sperm maturation. Analysis of differentially expressed genes found that 88 genes related to sperm flagellar movement were upregulated or downregulated; among them, 56 genes related to the "9+2" structure of the sperm flagellar axis were upregulated or downregulated; the above results showed that the loss of the Tex26 gene would affect the expression of genes related to the structure and movement of the sperm tail, and the sperm tail structure, especially the flagellar axis, was closely related to the movement of sperm; further immunohistochemistry showed that the Tex26 gene was also located in the middle of the sperm tail, which was highly coincident with the location of sperm mitochondria, indicating that the Tex26 gene may be involved in the metabolic regulation of sperm energy. Analysis of differentially expressed genes found that 78 genes related to oxidative phosphorylation were upregulated or downregulated; the above results showed that the loss of the Tex26 gene may affect the changes in the expression of genes related to ATP energy metabolism.

[0195] Figure 3CThis is a further analysis result diagram of Reactome in Example 3 of the present application; it can be seen that the gene signaling pathways related to oxidative phosphorylation and ATP synthesis have changed, affecting the mitochondrial electron transport chain and energy metabolism, and further affecting the synthesis of sperm ATP energy. Sperm mitochondrial energy metabolism is closely related to sperm motility and movement.

[0196] Figure 3D This is the KEGG analysis result diagram in Example 3 of the present application; it can be seen that the genes related to sperm tail dynein have undergone significant expression changes, and sperm tail dynein is closely related to sperm movement.

[0197] The above results show that Tex26 gene knockout will directly affect the expression changes of 6652 sperm proteins, especially proteins related to sperm differentiation, sperm acrosome, sperm motor proteins, sperm tail structure assembly proteins, sperm tail mitochondrial electron transport chain and ATP metabolism-related pathway proteins; this shows that the decreased expression of the Tex26 gene has an important effect on sperm motility, fertilization ability, and sperm maturation.

[0198] Example 4: Detection of Tex26 epididymal sperm maturation function

[0199] Because the sperm produced in the testicles do not have the ability to move, recognize the sperm and egg, and fertilize. After entering the epididymis, the sperm runs along the epididymal head, body, and tail, and undergoes a series of drastic changes in morphological structure, biochemical metabolism, and physiological function before it can acquire the ability to move, recognize the sperm and egg, and fertilize. By evaluating the mature function of sperm (fertilization ability and motility), it helps to guide the selection of infertility treatment options in the clinic and help improve the success rate of assisted reproductive technology.

[0200] The mouse epididymis sperm parameters (sperm motility, density and number, etc.) were tested by CASA (computer-assisted sperm analysis) experiment. The details were as follows: the mouse epididymis was placed in CASA buffer, the epididymis was cut open, and the sperm was kept in a humidified environment at 37°C and 5% CO. 2Incubate in an incubator for 5 minutes, 60 minutes, 90 minutes, and 120 minutes, and then collect sperm to analyze sperm motility. Sperm counting: Place the mouse epididymis in CASA buffer, cut the epididymis (cut the epididymal head, epididymal body, and epididymal tail separately, record the number of cuts and ensure that the number and size of each cut are uniform), maintain 37°C, wait for the sperm to swim out for 30 minutes, and then pass through a 40μm filter. After diluting 10 times, mix well, and take 10μL and drop it onto the Biorad cell counting plate. Take pictures with a microscope, and use ImageJ to analyze the number of cells in each field of view. According to the thickness of the counting plate, the conversion formula for the size of the photographed field of view, and the sperm dilution multiple and total volume, the corresponding number of sperm is calculated. Among them, the formula of CASA buffer: 120mM NaCl, 4.8mM KCl, 1.2mM MgSO 4 , 1 mM CaCl 2 , 1.2mM KH 2 PO 4 , 21mM sodium DL-lactate (Na-dl-lactate), 5mM glucose, 25mM NaHCO 3 , 0.25 mM sodium pyruvate (Napyruvate), 0.4 μg / mL phenol red (Phenol red), and 3 mg / mL bovine serum albumin (BSA V).

[0201] Take the epididymal tissue samples of wild-type (WT) mice, cut the epididymal head, epididymal body and epididymal tail respectively, fix them in 4% paraformaldehyde (Servicebio, G1101-500ML), and immunostain for 24 hours. Paraffin sections were boiled in 10mM sodium citrate buffer (pH6.0) for 15min, gradually cooled to room temperature, and washed 3 times with PBS containing 0.1% Triton X-100. The sections were blocked with 3w / v% bovine serum albumin (BSA) at room temperature for 1 hour, and then incubated with the primary antibody at 4°C overnight. Next, the sections were incubated with the corresponding secondary antibody at room temperature for 1 hour. The sections were then washed, mounted and analyzed according to standard protocols. The antibody used in this example is anti-human / mouse TEX26 antibody (primary antibody, prepared in Example 1, the volume ratio of the primary antibody dilution is 1:200).

[0202] Figure 4A This is the immunohistochemistry result of the epididymal caput in Example 4 of the present application. It can be seen from the figure that there are few sperm in the epididymal caput and it is in the early stage of epididymal sperm maturation with limited fertilization ability. At this time, the TEX26 protein is not localized.

[0203] Figure 4BThis is the immunohistochemistry result of the epididymis in Example 4 of the present application. It can be seen that the number of sperm in the epididymis begins to increase and is in the middle stage of epididymal sperm maturation. The fertilization ability increases, and at this time, the TEX26 protein begins to be localized.

[0204] Figure 4C This is the immunohistochemistry result of the epididymal tail in Example 4 of the present application. It can be seen that the epididymal tail has the largest number of sperm and is in the late stage of epididymal sperm maturation and has the ability to fertilize. At this time, the TEX26 protein is localized in the tail of the mature epididymal sperm and the expression level of TEX26 protein is also the highest.

[0205] The above results indicate that the expression pattern of TEX26 protein in the epididymis is consistent with the maturation pattern of epididymal sperm (possessing fertilization ability and motility), indicating that Tex26 gene / TEX26 protein can be used as a biomarker for epididymal sperm maturation and for the assessment of sperm maturity (motility, fertilization ability and sperm maturation).

[0206] Example 5: Detection of Tex26 gene expression in male infertile patients and semen analysis

[0207] The study of this example was approved by the Ethics Committee of Peking University Third Hospital and the patients' informed consent was obtained. 120 discarded semen and testicular biopsy samples were collected from patients with male infertility-related diseases caused by non-genetic factors who were to undergo IVF-ET treatment at Peking University Third Hospital, and patients with organic lesions of the reproductive system, untreated endocrine disorders, and patients with semen abnormalities caused by known causes such as drug or alcohol abuse, chromosomes, AZF abnormalities, cryptorchidism or mumps within two years were excluded.

[0208] Methods: TIANamp Micro DNA Kit was used to extract the patient's blood DNA. According to the instructions, the patient's blood genomic DNA was used as a template to amplify the exon sequence of the Tex26 gene through PCR reaction and send it for sequencing. After the sequencing results were returned, the sequences were compared to screen patients with Tex26 mutant genes. At the same time, with the consent of the patients, discarded testicular biopsy samples of male infertility patients due to non-genetic factors who were to undergo IVF-ET treatment were collected, RNA was extracted, and RT-PCR amplification was performed to detect changes in Tex26 gene expression. The primer sequences used are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11.

[0209] Figure 5The following is a graph showing the changes in Tex26 gene expression in different male infertility patients. The RT-PCR results in the figure show that there are differences in the expression of Tex26 gene in different male infertility patients (21Y07610, 21Y07611, 21Y07711, 21Y07712 and 21Y07616). The expression of Tex26 gene could not be detected by immunohistochemistry in patients with azoospermia and Sertoli cell syndrome because they had no sperm. No bands were amplified in infertile patients 21Y05111 and 21Y05113, indicating that the Tex26 gene was mutated or missing. The results showed that the Tex26 gene can be used as a biomarker to detect the spermatogenesis of the patient's testicles and male infertility-related diseases. The above results indicate that it is very important to perform sperm function testing on the male after entering the ovulation induction cycle, including testing semen parameters and sperm maturity to reduce the risk of IVF fertilization failure, and provide a basis for the etiology identification and diagnosis of clinical male infertility. At the same time, when selecting assisted reproductive technology programs, the best infertility treatment program can also be selected for patients (for example, patients with deficiency give priority to intracytoplasmic sperm injection to improve fertilization rate), so as to achieve personalized medicine, improve the efficiency of medical resources, and reduce the economic loss and psychological burden of patients. This application provides new biomarkers and therapeutic targets for the diagnosis and treatment of male infertility-related diseases.

[0210] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. Any of the following uses of a biomarker and / or a substance for detecting the biomarker: A1) Use in the preparation of products for studying spermatogenesis; A2) Use in the preparation of a product for identifying a germ cell sorting process; A3) Use in the preparation of a product for diagnosing sperm maturation abnormalities; A4) Use in the preparation of a product for diagnosing fertilization abnormalities; A5) Application in the preparation of products for screening and diagnosing male infertility diseases: A6) Application in the preparation of male contraceptive drugs and / or male contraceptive vaccine products; in, The biomarker is selected from the group consisting of Tex26 gene, TEX26 protein, mRNA of Tex26 gene, RNA of Tex26 gene or miRNA targeting TEX26 protein.

2. The use according to claim 1, wherein: The substance is a reagent for detecting whether the substance exists in the Tex26 gene, TEX26 protein, mRNA of the Tex26 gene, RNA of the Tex26 gene or miRNA targeting the TEX26 protein, or the substance is a reagent for detecting whether the expression amount of the TEX26 protein, mRNA of the Tex26 gene, RNA of the Tex26 gene or miRNA targeting the TEX26 protein has changed, or the substance is a reagent for detecting whether there is a sequence mutation in the Tex26 gene, TEX26 protein or mRNA of the Tex26 gene; preferably, the test sample for the test is selected from at least one of blood, oral tissue, testicular tissue and sperm.

3. The use according to claim 2, wherein: The reagent includes an antibody that specifically binds to the TEX26 protein; preferably, the antibody that specifically binds to the TEX26 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.12 to SEQ ID NO.

13.

4. The use according to claim 2, wherein: The nucleotide sequence of the Tex26 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex26 gene is shown as SEQ ID NO.2 or SEQ ID NO.3; or, the amino acid sequence of the TEX26 protein is shown as SEQ ID NO.4 or SEQ ID NO.

5.

5. A kit, wherein: The kit includes a reagent for detecting whether a Tex26 gene, a TEX26 protein, an mRNA of a Tex26 gene, an RNA of a Tex26 gene, or a miRNA targeting a TEX26 protein exists, or the substance is a reagent for detecting whether the expression amount of a TEX26 protein, an mRNA of a Tex26 gene, an RNA of a Tex26 gene, or a miRNA targeting a TEX26 protein changes, or the substance is a reagent for detecting whether a sequence mutation exists in a Tex26 gene, a TEX26 protein, or an mRNA of a Tex26 gene; the kit has at least one of the following uses: A1) Study the process of spermatogenesis; A2) Identification of the germ cell sorting process; A3) Screening for sperm maturation abnormalities; A4) Diagnosis of fertilization abnormalities; A5) Screening and diagnosis of male infertility; A6) Preparation of male contraceptive pills and / or male contraceptive vaccines.

6. The kit according to claim 5, wherein The kit includes an antibody that specifically binds to the TEX26 protein; preferably, the antibody that specifically binds to the TEX26 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.12 to SEQ ID NO.

13.

7. The kit according to claim 5, wherein The kit comprises at least one of a primer pair having nucleotide sequences as shown in SEQ ID NO.6 and SEQ ID NO.7, a primer pair having nucleotide sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, and a primer pair having nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.

11.

8. The kit according to claim 5, wherein The nucleotide sequence of the Tex26 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex26 gene is shown as SEQ ID NO.2 or SEQ ID NO.3; or, the amino acid sequence of the TEX26 protein is shown as SEQ ID NO.4 or SEQ ID NO.

5.

9. The kit according to claim 5, wherein The test sample of the kit is selected from at least one of blood, oral tissue, testicular tissue and sperm; Preferably, the method for judging the detection result of the kit includes: if the Tex26 gene, TEX26 protein, Tex26 gene mRNA, Tex26 gene RNA or miRNA targeting TEX26 protein is not present in the test sample, or the expression level of TEX26 protein, Tex26 gene mRNA, Tex26 gene RNA or miRNA targeting TEX26 protein in the test sample changes, or the sequence of Tex26 gene, TEX26 protein or Tex26 gene mRNA in the test sample mutates, then the source of the test sample is suspected of suffering from male sperm maturation abnormality, fertilization abnormality or male infertility.

10. Use of biomarkers as drug targets in the preparation of drugs for any of the following purposes: B1) Promote sperm maturation and fertilization; B2) Improve sperm maturation rate; B3) Improve sperm fertilization level; B4) Improve male fertility; B5) Treatment of sperm dysfunction; B6) Treatment of male infertility; B7) preparing male contraceptive pills and / or male contraceptive vaccines; in, The biomarker is selected from TEX26 protein or Tex26 gene.

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