Application of Tex29 gene as specific biomarker in sperm acrosome reaction and male infertility disease

Abstract

The invention provides an application of a Tex29 gene as a specific biomarker in sperm acrosome reaction and male infertility diseases, and any one of the following applications of the biomarker and / or a substance for detecting the biomarker: an application in preparation of a product for researching a spermatogenesis process; application in preparation of a product for identifying a germ cell classification process; the invention also relates to an application in preparation of a product for screening sperm acrosome dysplasia. Application in preparation of a product for screening male infertility diseases; application in preparation of a product for diagnosing male sperm head malformation; application in preparation of products for diagnosing abnormal fertilization of sperms and eggs; application in preparation of a product for diagnosing male infertility; application in preparation of a product for diagnosing the acrosome reaction rate of sperms; wherein the biomarker is selected from a Tex29 gene, a TEX29 protein, mRNA (messenger ribonucleic acid) of the Tex29 gene and the like. The invention provides a novel biomarker and a therapeutic target for a kit for diagnosing male infertility diseases.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to the application of a Tex29 gene as a specific biomarker in sperm acrosome reaction and male infertility. Background Art

[0002] Male infertility is a major health problem. The latest data from the World Health Organization (WHO) show that about 15% of couples of childbearing age worldwide are unable to have children due to infertility, of which 50% are due to male factors, and the trend is increasing year by year. With the development of society and the process of modern industrialization, the incidence of male infertility has increased significantly worldwide, which has brought a heavy burden to individuals, families and society. The fertilization process of mammals involves a series of functional activities of sperm, such as the movement of sperm in the female reproductive tract, sperm superactivation and capacitation, acrosome reaction and sperm-egg fusion. Acrosome reaction (AR) is a necessary process in sperm fertilization. In IVF or natural fertilization in vivo, sperm AR is induced by zona pellucida glycoprotein receptors, and only zona pellucida induced AR has important physiological significance. Capacitated sperm are induced by factors to undergo AR, releasing enzymes in the acrosome to dissolve the corona radiata and zona pellucida (ZP) of the egg, thereby passing through the ZP and fusing with the oocyte to complete fertilization. The acrosome reaction has at least two functions, which is to enable sperm to penetrate the ZP and fuse with the egg membrane. Only sperm that complete the acrosome reaction can fuse with the oocyte. Recently, it has been found that human sperm acrosome reaction defects are also one of the causes of clinical "unexplained infertility". Therefore, detecting whether sperm undergoes acrosome reaction has become an important means to evaluate sperm function and study reproductive biology.

[0003] At present, the common method for judging male fertility is still based on the routine analysis of semen (including sperm morphology, density, quantity, motility and viability, etc.) according to the World Health Organization's Manual of Human Semen Examination and Processing (WHO 6th edition) combined with serum hormone level analysis, but the ability to predict male fertility is very limited. Routine semen analysis can only reflect the most basic sperm quality but not other characteristics of sperm, sperm functional defects, acrosome reaction and fertilization ability. Therefore, it is necessary to identify new biomolecular markers related to sperm abnormalities, fertilization abnormalities and male infertility to assist traditional routine semen analysis. Summary of the invention

[0004] The purpose of this application is to provide the application of Tex29 gene as a specific biomarker in sperm acrosome reaction and male infertility diseases. Tex29 gene and TEX29 protein can be used as biomarkers in the preparation of diagnostic kits for male sperm acrosome development abnormalities, sperm acrosome reaction abnormalities, fertilization abnormalities and male infertility, providing new targets for the diagnosis and treatment of male infertility-related diseases. The specific technical scheme is as follows:

[0005] The first aspect of the present application provides any of the following uses of a biomarker and / or a substance for detecting the biomarker:

[0006] A1) Use in the preparation of products for studying spermatogenesis;

[0007] A2) Use in the preparation of a product for identifying a germ cell sorting process;

[0008] A3) Use in the preparation of a product for screening sperm acrosome abnormalities;

[0009] A4) Use in the preparation of a product for screening male infertility diseases;

[0010] A5) Use in the preparation of a product for diagnosing male sperm head deformity;

[0011] A6) Use in the preparation of products for diagnosing abnormal sperm and egg fertilization;

[0012] A7) Use in the preparation of a product for diagnosing male infertility;

[0013] A8) Use in the preparation of a product for diagnosing sperm acrosome reaction rate;

[0014] Wherein, the biomarker is selected from Tex29 gene, TEX29 protein, mRNA of Tex29 gene, RNA of Tex29 gene or miRNA targeting TEX29 protein.

[0015] In some embodiments of the present application, the substance for detecting the biomarker is a reagent for detecting the presence of the Tex29 gene, TEX29 protein, mRNA of the Tex29 gene, RNA of the Tex29 gene, or miRNA targeting the TEX29 protein.

[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 TEX29 protein.

[0018] In some embodiments of the present application, the antibody that specifically binds to the TEX29 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.4 to SEQ ID NO.9.

[0019] In some embodiments of the present application, the nucleotide sequence of the Tex29 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex29 gene is shown as SEQ ID NO.2 or SEQ ID NO.20; or, the amino acid sequence of the TEX29 protein is shown as SEQ ID NO.3 or SEQ ID NO.21.

[0020] The second aspect of the present application provides a kit, wherein the kit includes a reagent for detecting the presence of a Tex29 gene, a TEX29 protein, an mRNA of a Tex29 gene, an RNA of a Tex29 gene, or an miRNA targeting a TEX29 protein, and 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 acrosome abnormalities;

[0024] A4) Screening for male infertility;

[0025] A5) Diagnosis of sperm head deformity in men;

[0026] A6) Diagnosis of abnormal sperm and egg fertilization;

[0027] A7) Diagnosis of male infertility;

[0028] A8) Diagnosis of sperm acrosome reaction rate In some embodiments of the present application, the kit includes an antibody that specifically binds to the TEX29 protein.

[0029] In some embodiments of the present application, the antibody that specifically binds to the TEX29 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.4 to SEQ ID NO.9.

[0030] In some embodiments of the present application, the kit includes at least one of a primer pair with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11, a primer pair with nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.13, and a primer pair with nucleotide sequences as shown in SEQ ID NO.14 and SEQ ID NO.15.

[0031] In some embodiments of the present application, the nucleotide sequence of the Tex29 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex29 gene is shown as SEQ ID NO.2 or SEQ ID NO.20; or, the amino acid sequence of the TEX29 protein is shown as SEQ ID NO.3 or SEQ ID NO.21.

[0032] 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.

[0033] In some embodiments of the present application, the method for judging the test results of the kit includes: if the test sample does not contain the Tex29 gene, TEX29 protein, Tex29 gene mRNA, Tex29 gene RNA or miRNA targeting the TEX29 protein, then the source of the test sample is suspected of suffering from male sperm head deformity, abnormal acrosome reaction, abnormal fertilization or male infertility.

[0034] 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:

[0035] B1) Promote sperm maturation and fertilization;

[0036] B2) Increase the sperm acrosome reaction rate;

[0037] B3) Improve sperm fertilization level;

[0038] B4) Improve male fertility;

[0039] B5) Repair abnormalities in the sperm head structure;

[0040] B6) Treatment of sperm dysfunction;

[0041] B7) Treatment of male sperm head deformity;

[0042] B8) Treatment of male infertility;

[0043] Wherein, the biomarker is selected from TEX29 protein or Tex29 gene.

[0044] Beneficial effects of this application:

[0045] The mRNA of the Tex29 gene described in the present application is specifically expressed in the testis and specifically localized on the acrosome of the elongated spermatids and epididymal sperm, indicating that the TEX29 protein is a new sperm acrosome protein. The Tex29 gene and TEX29 protein described in the present application can be used as biomarkers in the preparation of kits for diagnosing male sperm acrosome reaction rate, fertilization abnormalities, male infertility, etc., providing a new biomarker and a new target for treating male infertility for existing kits for diagnosing male infertility diseases.

[0046] 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

[0047] 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.

[0048] Figure 1A The amino acid sequence antigenicity analysis and antigen epitope prediction map of the mouse TEX29 protein in Example 1 of the present application;

[0049] Figure 1B The amino acid sequence antigenicity analysis and antigen epitope prediction map of the human TEX29 protein in Example 1 of the present application;

[0050] Figure 1C This is a diagram of the effectiveness test results of the TEX29 antibody prepared in Example 1 of the present application (immunohistochemistry of paraffin sections of mouse testis and epididymis);

[0051] Figure 2A This is a graph showing the expression results of the Tex29 gene in different tissues of wild-type mice in Example 2 of the present application;

[0052] Figure 2B This is a graph showing the expression results of the Tex29 gene in the testicular tissue of wild-type mice at different stages in Example 2 of the present application;

[0053] Figure 2C This is the localization map of the TEX29 protein in the testicular tissue cells of wild-type mice in Example 2 of the present application;

[0054] Figure 2D This is a result diagram showing the localization of TEX29 protein on the acrosome of wild-type mouse epididymal sperm and co-localization with acrosomal protein PNA in Example 2 of the present application;

[0055] Figure 3A This is a diagram for analyzing the Tex29 gene knockout strategy in Example 3 of the present application;

[0056] Figure 3B This is a diagram showing the Q-PCR identification results of wild-type mice and TEX29-KO mice in Example 3 of the present application;

[0057] Figure 3C This is a diagram showing the results of Western blotting (WB) of testicular tissue of wild-type mice and TEX29-KO mice in Example 3 of the present application;

[0058] Figure 3D The figure is the result of immunohistochemistry testis section of wild-type mice (Tex29+ / +) and TEX29-KO mice (Tex29- / -) in Example 3 of the present application;

[0059] Figure 3E This is a comparison of the sizes of the testicles and epididymis of wild-type mice (Tex29+ / +) and TEX29-KO mice (Tex29- / -) in Example 3 of the present application;

[0060] Figure 3F This is a statistical result diagram of the sizes of the testicles and epididymis of wild-type mice (Tex29+ / +) and TEX29-KO mice (Tex29- / -) in Example 3 of the present application;

[0061] Figure 3G This is a graph comparing the semen volume and motility of wild-type mice (Tex29+ / +) and TEX29-KO mice (Tex29- / -) in Example 3 of the present application;

[0062] Figure 4A This is a graph showing the results of TEX29 immunohistochemistry detection of sperm smear of wild-type mice (Tex29+ / +) in Example 4 of the present application;

[0063] Figure 4B This is a graph showing the results of TEX29 immunohistochemistry testing of male sperm smears in Example 4 of the present application;

[0064] Figure 5 This is a graph showing the changes in Tex29 expression in different male infertility patients in Example 5 of the present application. DETAILED DESCRIPTION

[0065] 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.

[0066] The present application provides any of the following applications of a biomarker and / or a substance for detecting the biomarker:

[0067] A1) Use in the preparation of products for studying spermatogenesis;

[0068] A2) Use in the preparation of a product for identifying a germ cell sorting process;

[0069] A3) Use in the preparation of a product for screening sperm acrosome abnormalities;

[0070] A4) Use in the preparation of a product for screening male infertility diseases;

[0071] A5) Use in the preparation of a product for diagnosing male sperm head deformity;

[0072] A6) Use in the preparation of products for diagnosing abnormal sperm and egg fertilization;

[0073] A7) Use in the preparation of a product for diagnosing male infertility;

[0074] A8) Use in the preparation of a product for diagnosing sperm acrosome reaction rate;

[0075] Wherein, the biomarker is selected from Tex29 gene, TEX29 protein, mRNA of Tex29 gene, RNA of Tex29 gene or miRNA targeting TEX29 protein.

[0076] The inventor of the present application found in the study that TEX29 protein is a newly discovered sperm head acrosome protein, which is specifically expressed in the testis. Tex29 gene and TEX29 protein can be used as molecular markers for the differentiation of round sperm into elongated sperm stages during spermatogenesis, and are used to study sperm differentiation, sperm acrosome reaction, evaluate fertilization ability and male reproductive dysfunction, etc. Tex29 gene and TEX29 protein described in the present application can be used as biomarkers for round sperm differentiation and biomarkers for sperm acrosome reaction and fertilization disorder diagnosis. In addition, the TEX29 human / mouse antibody designed and prepared can be used for the research of round sperm differentiation and maturation, distinguishing different germ cell types, detecting sperm acrosome reaction rate and diagnosis of male infertility.

[0077] In some embodiments of the present application, the substance for detecting the biomarker is a reagent for detecting the presence of the Tex29 gene, TEX29 protein, mRNA of the Tex29 gene, RNA of the Tex29 gene, or miRNA targeting the TEX29 protein.

[0078] 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.

[0079] In some embodiments of the present application, the reagent includes an antibody that specifically binds to the TEX29 protein.

[0080] In some embodiments of the present application, the antibody that specifically binds to the TEX29 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.4 to SEQ ID NO.9.

[0081] SEQ ID NO.4 (antigen of human Tex29 gene, 78-151aa): pev yihvfsaliv iiagafvitiiyrviqesrk ekaipvdval pqkssekael assssklglk paspgppsag p;

[0082] SEQ ID NO.5 (antigen of human Tex29 gene, 134-151aa): C-EDKDDVTGTITEAEETED

[0083] SEQ ID NO.6 (antigen of human Tex29 gene, 80-94aa): C-RVIQESRKEKAIPVD;

[0084] SEQ ID NO.7 (antigen of mouse Tex29 gene, 78-186aa): qvfftliwfvagafiiaviyrviqgtkkekkpl lpedsqveilksptpelipepipepipepi pepireppppvkktespieagcclwmkskpakdqpqketaapepps;

[0085] SEQ ID NO.8 (antigen of mouse Tex29 gene, 24-38aa, S replaces C): PLYDISDYNVTRERC;

[0086] SEQ ID NO.9 (antigen of mouse Tex29 gene, 78-91aa): IYRVIQGTKKEKKP-C.

[0087] In some embodiments of the present application, the nucleotide sequence of the Tex29 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex29 gene is shown as SEQ ID NO.2 or SEQ ID NO.20; or, the amino acid sequence of the TEX29 protein is shown as SEQ ID NO.3 or SEQ ID NO.21.

[0088]

[0089]

[0090] The second aspect of the present application provides a kit, wherein the kit includes a reagent for detecting whether a Tex29 gene, a TEX29 protein, an mRNA of a Tex29 gene, an RNA of a Tex29 gene, or a miRNA targeting a TEX29 protein exists, and the kit has at least one of the following uses:

[0091] A1) Study the process of spermatogenesis;

[0092] A2) Identification of the germ cell sorting process;

[0093] A3) Screening for sperm acrosome abnormalities;

[0094] A4) Screening for male infertility;

[0095] A5) Diagnosis of sperm head deformity in men;

[0096] A6) Diagnosis of abnormal sperm and egg fertilization;

[0097] A7) Diagnosis of male infertility;

[0098] A8) Diagnosis of sperm acrosome reaction rate.

[0099] The kit described in the present application can be used to evaluate the integrity of the sperm acrosome in vitro, initiate sperm capacitation, and induce sperm acrosome reaction to evaluate the ability of capacitated sperm to undergo acrosome reaction, thereby providing a theoretical basis for the analysis of the causes of infertility and the selection of assisted reproductive treatment options.

[0100] In some embodiments of the present application, the kit includes an antibody that specifically binds to the TEX29 protein.

[0101] In some embodiments of the present application, the antibody that specifically binds to the TEX29 protein is prepared using an antigen with an amino acid sequence as shown in any one of SEQ ID NO.4-SEQ ID NO.9. The TEX29 protein described in the present application is a newly discovered sperm acrosomal protein. According to the amino acid sequence of the TEX29 protein in humans / mouse, a human or mouse antigen as shown in any one of SEQ ID NO.4-SEQ ID NO.9 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.

[0102] In some embodiments of the present application, the kit includes at least one of a primer pair with nucleotide sequences as shown in SEQ ID NO.10 and SEQ ID NO.11, a primer pair with nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.13, and a primer pair with nucleotide sequences as shown in SEQ ID NO.14 and SEQ ID NO.15.

[0103] SEQ ID NO.10 (human Tex29 gene DNA upstream primer): AGCCAGCACCATCTATCCTCCAC;

[0104] SEQ ID NO.11 (human Tex29 gene DNA downstream primer): GCGTCTCAGTCAGTCCTCAGTTTC;

[0105] SEQ ID NO.12 (upstream primer of mRNA of human Tex29 gene): CGTGCTGGAAGTGAAGAACTCTCC;

[0106] SEQ ID NO.13 (downstream primer of mRNA of human Tex29 gene): GCCCTAACTTGCTGCTGGATGAG;

[0107] SEQ ID NO.14 (upstream primer of mRNA of human Tex29 gene): GAAAGAATCCTGGGCACCTCATCC;

[0108] SEQ ID NO.15 (downstream primer of mRNA of human Tex29 gene): TCCTCCTAGCATCACAGCAGTCTC.

[0109] In some embodiments of the present application, the nucleotide sequence of the Tex29 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex29 gene is shown as SEQ ID NO.2 or SEQ ID NO.20; or, the amino acid sequence of the TEX29 protein is shown as SEQ ID NO.3 or SEQ ID NO.21.

[0110] 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.

[0111] In some embodiments of the present application, the method for judging the test results of the kit includes: if the test sample does not contain the Tex29 gene, TEX29 protein, Tex29 gene mRNA, Tex29 gene RNA or miRNA targeting the TEX29 protein, then the source of the test sample is suspected of suffering from male sperm head deformity, abnormal acrosome reaction, abnormal fertilization or male infertility.

[0112] 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:

[0113] B1) Promote sperm maturation and fertilization;

[0114] B2) Increase the sperm acrosome reaction rate;

[0115] B3) Improve sperm fertilization level;

[0116] B4) Improve male fertility;

[0117] B5) Repair abnormalities in the sperm head structure;

[0118] B6) Treatment of sperm dysfunction;

[0119] B7) Treatment of male sperm head deformity;

[0120] B8) Treatment of male infertility;

[0121] Wherein, the biomarker is selected from TEX29 protein or Tex29 gene.

[0122] The fourth aspect of the present application provides the use of Tex29 gene, TEX29 protein, mRNA of Tex29 gene, RNA of Tex29 gene or miRNA targeting TEX29 protein as biomarkers in the process of spermatogenesis research, in the process of germ cell classification and identification, in the screening of abnormal sperm acrosome development, in the screening of male infertility diseases, in the diagnosis of abnormal sperm-egg fertilization, in the diagnosis of male infertility or in the diagnosis of sperm acrosome reaction rate.

[0123] The fifth aspect of the present application provides the use of TEX29 protein or Tex29 gene as a drug target in improving male fertility, treating sperm dysfunction, promoting sperm maturation and fertilization, improving sperm fertilization ability, improving sperm quality and improving sperm fertilization level and treating male infertility.

[0124] The TEX29 protein described in this application is a new sperm acrosome 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 TEX29 protein is produced, it can be used to detect the acrosome reaction rate of sperm. By detecting the cause of male fertilization abnormalities, detecting the sperm acrosome reaction rate can prevent and / or treat male infertility caused by male sperm fertilization abnormalities. The Tex29 gene or TEX29 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.

[0125] The present application also provides the use of the Tex29 gene or TEX29 protein. The Tex29 gene or TEX29 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.

[0126] Example

[0127] The following examples and comparative examples are given to more specifically describe the embodiments of the present application. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.

[0128] 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.

[0129] 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.

[0130] Example 1: Preparation of antibodies against TEX29 protein and detection of antibody effectiveness

[0131] 1. Preparation of TEX29 polyclonal antibody (rabbit anti-mouse)

[0132] The length of mouse TEX29 gene is 561bp, and TEX29 protein is composed of 186 amino acids. Antigenicity analysis of mouse TEX29 protein was performed, and the results are as follows Figure 1A As shown, it can be seen that the 78-186aa region of mouse TEX29 protein has 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 24-38aa region and the 78-91aa region were selected for peptide synthesis. The above two regions have good sequence specificity and antigenic epitopes and good amino acid hydrophilicity, and are used as mixed immunization.

[0133] SEQ ID NO.7 (antigen of mouse Tex29 gene, 78-186aa): qvfftliwfvagafiiaviyrviqgtkkekkpl lpedsqveilksptpelipepipepipepi pepireppppvkktespieagcclwmkskpakdqpqketaapepps;

[0134] SEQ ID NO.8 (antigen of mouse Tex29 gene, 24-38aa, S replaces C): PLYDISDYNVTRERC;

[0135] SEQ ID NO.9 (antigen of mouse Tex29 gene, 78-91aa): IYRVIQGTKKEKKP-C.

[0136] 2. Preparation of human TEX29 polyclonal antibody (rabbit anti-human)

[0137] Antigenicity analysis of human TEX29 protein was performed, and the results were as follows Figure 1BAs shown, it can be seen that the 78-151aa region of human TEX29 protein has 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 134-151aa region and the 80-94aa region were selected for peptide synthesis. The above two regions have good sequence specificity and antigenic epitope, and the amino acid hydrophilicity is also good, as mixed immunization.

[0138] SEQ ID NO.4 (antigen of human Tex29 gene, 78-151aa): pev yihvfsaliv iiagafvitiiyrviqesrk ekaipvdval pqkssekael assssklglk paspgppsag p;

[0139] SEQ ID NO.5 (antigen of human Tex29 gene, 134-151aa): C-EDKDDVTGTITEAEETED;

[0140] SEQ ID NO.6 (antigen of human Tex29 gene, 80-94aa): C-RVIQESRKEKAIPVD.

[0141] 3. Antibody Preparation

[0142] Based on the above sequence, the expression vector pGEX-4T-AB1 was constructed, and the antigen was expressed to prepare the antibody.

[0143] 1. Construction of prokaryotic protein expression vector

[0144] The analysis yielded an amino acid sequence with high antigenicity and antigen specificity, and then primers were designed for the sequence, followed by PCR amplification and cloning into a pGEX-4T-AB1 prokaryotic expression vector (Escherichia coli prokaryotic system) and identification.

[0145] 2. Expression and identification of recombinant proteins

[0146] 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:

[0147] 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;

[0148] 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;

[0149] 3) Collect 1 mL of bacterial precipitate, resuspend with sample buffer, heat and lyse, and centrifuge to collect the supernatant and precipitate;

[0150] 4) Prepare 10% SDS-PAGE gel, take 10-20 μL sample, and perform SDS-PAGE electrophoresis;

[0151] 5) After electrophoresis, the SDS-PAGE gel was stained with Coomassie Brilliant Blue R250 for 20 min;

[0152] 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;

[0153] 7) The positive plasmid with the highest expression level is preserved as the expression strain.

[0154] 3. Large-scale expression and extraction of recombinant proteins

[0155] The specific steps are as follows:

[0156] 1) Inoculate the identified expression plasmid into 15 mL LB liquid medium and shake at 37°C overnight;

[0157] 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;

[0158] 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);

[0159] 4) Collect the culture medium, centrifuge at 4°C, 5000×g for 10 min, discard the supernatant, and collect the bacterial precipitate;

[0160] 5) Wash the cells once with PBS, centrifuge at 5000 g for 10 min, discard the supernatant, and collect the cell pellet;

[0161] 6) Store at -20℃ for later use.

[0162] 4. Purification of recombinant protein

[0163] 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.

[0164] (1) Sample processing before loading: The specific steps are as follows:

[0165] 1) Resuspend the bacterial pellet of each 100 mL LB culture in 1 × 4 mL PBS;

[0166] 2) Add lysozyme to a final concentration of 1 mg / mL and digest on ice for 30 min;

[0167] 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 conditions are: ultrasound for 1 second, interval of 1 second, each ultrasound for 5 minutes, action 4 times, a total of 20 minutes;

[0168] 4) Centrifuge at 12,000 × g for 20 min at 4°C, and run 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;

[0169] 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.

[0170] (2) FPLC recombinant protein purification step, comprising the following steps in sequence:

[0171] 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.

[0172] 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.

[0173] 5. Animal immunization and identification of antiserum

[0174] (1) Animal immunization: Two New Zealand white rabbits (2.5-3.5 kg) were immunized with each purified antigen protein.

[0175] 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.

[0176] 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.

[0177] (2) Collection and storage of antiserum

[0178] Blood was collected from rabbits using the heart blood collection method, the steps are as follows:

[0179] 1) Place the rabbit on its back with its limbs tied to an animal holder or have an assistant fix the limbs;

[0180] 2) Use surgical scissors to cut the hair on the left chest of the rabbit, disinfect the skin with alcohol and wipe it dry;

[0181] 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;

[0182] 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;

[0183] 5) The drawn blood was immediately injected into a sterile 50 mL centrifuge tube and left at room temperature to coagulate;

[0184] 6) Place the retrieved blood in a 37°C incubator for 1 hour, then place it in a 4°C refrigerator overnight;

[0185] 7) After the blood coagulation and clot shrinkage, centrifuge at 3000×g for 15 min;

[0186] 8) Take the supernatant and divide it into portions and store it in a 4℃ refrigerator for later use.

[0187] (3) Antibody specificity identification:

[0188] Take the total bacterial protein, purified recombinant antigen protein, mouse testis and various tissue proteins.

[0189] 1) Perform Western blotting and use pre-immune serum as a negative control;

[0190] 2) Immunohistochemical detection and use of pre-immune serum as negative control;

[0191] 3) Immunocytochemistry detection and use of pre-immune serum as negative control.

[0192] The prepared antibodies were subjected to immunohistochemistry to detect their effectiveness and effectiveness test results. Figure 1C As shown in the figure, the newly prepared TEX29 antibody was used to perform immunohistochemistry in mouse testis and epididymis paraffin sections to detect the effectiveness and specificity of the antibody. From the results in the figure above, it can be seen that the antibody binding to the TEX29 protein is specifically localized in testicular round spermatids, elongated spermatids, epididymal sperm and sperm acrosomes, indicating that the prepared TEX29 antibody is effective and highly specific.

[0193] Example 2: Fluorescence quantitative PCR detection and immunohistochemistry detection

[0194] 1. 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 Tex29 was normalized to Gapdh using the 2-ΔCt method and reported as fold change. The expression level of Tex29 was used as an internal reference using GAPDH, and expression changes were detected. Table 1 shows the primers used.

[0195] Table 1 Primers

[0196] Primers application Sequence (5'-3') SEQ ID NO Tex29-FP Real-time quantitative PCR GAGTCTCGACTGCTGTITCTAC SEQ ID NO.24 Tex29-RP Real-time quantitative PCR GGAGTGGGAGACTTCAGTATTTC SEQ ID NO.25 GAPDH-FP Real-time quantitative PCR AGGTCGGTGTGAACGGATTTG SEQ ID NO.22 GAPDH-RP Real-time quantitative PCR TGTAGACCATGTAGTTGAGGTCA SEQ ID NO.23 Tex29-WT-FP Genotyping TTCAGAACGGGCCTATTTATTCT SEQ ID NO.18 Tex29-WT-RP Genotyping CATGGATGAGTCTCCACATCTC SEQ ID NO.19 Tex29-KO-FP Genotyping TGACCTCCAGCTACTTGTGAG SEQ ID NO.16 Tex29-KO-RP Genotyping CATCAGCTACTGCCTCCAAG SEQ ID NO.17

[0197] 2. Immunohistochemistry: Fresh testicles and epididymal tails of wild-type (WT) mice and male testicular biopsy tissue samples 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 15 minutes, 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 antibodies at room temperature for 1 hour. The sections were then washed, mounted and analyzed according to standard protocols. The antibodies used in this example include anti-human TEX29 antibodies (prepared in Example 1, 1:200) and anti-mouse TEX29 antibodies (prepared in Example 1, 1:200).

[0198] Figure 2A The Q-PCR expression diagram of Tex29 gene in different tissues of wild-type mice. Figure 2AThe Q-PCR results of different tissues of adult mice showed that Tex29 is a new testis-specific gene, which is specifically expressed only in adult mouse testis, but not in mouse heart, liver, kidney, lung, small intestine, spleen, muscle and other tissues. Figure 2B The following is a Q-PCR expression diagram of Tex29 gene in testicular tissue of wild-type mice at different stages. Figure 2B As shown, the Tex29 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 when round spermatozoa transform into elongated spermatozoa) and continued until adulthood. The results show that Tex29 can be used to study the process of spermatogenesis.

[0199] The expression of TEX29 protein in mouse testis tissue sections was detected by immunohistochemistry. Figure 2C As shown in the results, TEX29 protein is only localized in the cytoplasm of mouse round sperm, elongated spermatids and the acrosome of the head of epididymal sperm; the results show that TEX29 antibody can be used as a biomarker of sperm acrosome, and can be used in the study of sperm acrosome development and the identification of different types of germ cells. For example, mixed round spermatids and elongated spermatids (testicular supporting cells, interdig cells, spermatogonial stem cells, spermatocytes, round spermatids mixed with round spermatids and elongated spermatids). Further studies have found that: TEX29 protein is localized on the acrosome of mouse mature sperm and co-localized with sperm acrosome protein - PNA protein, such as Figure 2D As shown, it can be seen that immunofluorescence detection found that TEX29 protein is also located on the acrosome of mature sperm. The above results show that TEX29 is a new sperm acrosome protein that is specifically expressed in the testis. It is located in the cytoplasm of round sperm, elongated sperm and the acrosome of mature sperm, and can be used as a biomarker in the study of sperm acrosome development; this gene can not only be used to study the process of spermatogenesis and distinguish different types of germ cells, but also may be used to study the process of sperm acrosome development and the differentiation process of round sperm.

[0200] Example 3: Preparation and analysis of Tex29 knockout mice

[0201] 1. Using CRISPR / Cas9 technology, two sgRNA sequences targeting the Tex29 gene (Gene ID: 75528 in the NCBI database) were designed through the CRISPR online website (http: / / crispr.mit.edu), that is, sgRNAs (sgRNA-1: sgRNA-2) targeting exons 2-7 of Tex29 (ENSMUST00000033909.14) were constructed. After synthesis and annealing, they were connected to the pX458 vector expressing the Cas9 protein, and the Cas9 mRNA, sgRNA and donor plasmid were co-microinjected into C57BL / 6 mouse fertilized oocytes. Figure 3A This is a diagram of the Tex29 gene knockout strategy analysis. A bidirectional gRNA was designed near the exons of wild-type C57BL / 6N background mice to delete 14787bp of the Tex29 gene bases. Figure 3A The positions of the identification primers are marked in the middle.

[0202] 2. In advance, mate C57BL / 6N female mice that have been superovulated by hormone treatment with wild-type male mice to obtain a large number of blastocyst cells (at the same time, mate ligated male mice with C57BL / 6N female mice to produce pseudo-pregnant female mice), inject the gene-modified embryonic stem cells screened in the previous step into the blastocysts obtained in this step, and after a short period of in vitro culture, transplant the blastocysts back into the uterus of the pseudo-pregnant female mice to obtain F0 generation gene mutant mice.

[0203] 3. Use PCR method to identify the genotype of F0 mice, and the identification system is 25μL. The primer sequences are as follows:

[0204] TEX29 P1-F1: 5'-TGACCTCCAGCTACTTGTGAG-3' (SEQ ID NO. 16);

[0205] TEX29 P1-R1: 5'-CATCAGCTACTGCCTCCAAG-3' (SEQ ID NO. 17);

[0206] TEX29 P2-F1: 5'-TTCAGAACGGGCCTATTTATTCT-3' (SEQ ID NO. 18);

[0207] TEX29 P2-R1: 5'-CATGGATGAGTCTCCACATCTC-3' (SEQ ID NO. 19).

[0208] When TEX29 P-F1 / R1 (P1) was used as primer, no band was amplified for wild-type mice; when TEX29 PF2 / R2 (P2) was used as primer, a band of 904 bp was amplified; when P1 was used as primer for Tex29 knockout mice, a band of 444 bp was amplified when P2 was used as primer, and no band was found when P2 was used as primer, thus F1 mice carrying the Tex29 knockout allele could be identified.

[0209] 4. The heterozygous F1 mice with Tex29 gene knockout were mated with each other to obtain homozygous F2 mice with Tex29 gene knockout (hereinafter referred to as TEX29- / - mice) and wild-type mice (hereinafter referred to as TEX29+ / + mice). RNA from different tissues of wild-type male mice and testes 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 different tissues as templates. The expression level of Tex29 was measured using GAPDH as an internal reference, and expression changes were detected.

[0210] Testes and cauda epididymis were isolated from wild-type and Tex29-KO male mice and fixed in Bouin solution (Sigma-Aldrich, 158127-500G). Samples were embedded in paraffin and sliced ​​using a Leica slicer (Leica Biosystems, Germany). After standard dewaxing and hydration, the slices were blocked with 3% bovine serum albumin (BSA) at room temperature for 1 hour and then incubated with primary antibodies (prepared in Example 1) and anti-PNA (ThermoScrinicy, L21409) at 4°C overnight. After washing three times with PBST, the slices were incubated with secondary antibodies at room temperature for 1 hour. The slices were then cleaned, mounted, and analyzed separately.

[0211] 5. Identification and analysis of knockout mice. Figure 3B This is a Q-PCR identification result diagram of wild-type mice and homozygous knockout mice in Example 3 of the present application; it can be seen from the figure that the WT wild-type can amplify the Tex29 gene mRNA, while the TEX29 knockout homozygous mice do not amplify the signal. The above results indicate that the Tex29 gene is knocked out. Tex29- / - mice represent Tex29 gene knockout homozygous mice, and Tex29+ / + mice represent wild-type mice.

[0212] from Figure 3CIt can be seen that the WB test results of TEX29+ / + mice have TEX29 protein size bands; TEX29- / - mice do not have TEX29 protein size bands, indicating that the Tex29 gene has been knocked out and the protein is no longer expressed. Tex29- / - mice represent homozygous mice with Tex29 gene knockout, and Tex29+ / + mice represent wild-type mice.

[0213] 6. Immunohistochemical detection of testicular sections of wild-type mice and TEX29-KO mice. Figure 3D As shown, it can be seen that: TEX29-KO mice do not show coloration, indicating that the Tex29 gene has been knocked out. Tex29- / - mice represent homozygous mice for the Tex29 gene knockout, and Tex29+ / + mice represent wild-type mice.

[0214] 7. Analysis of the volume and weight of the testis and epididymis of Tex29- / - mice. Wild-type and Tex29 knockout mice grown to 8-12 weeks of age were killed by cervical dislocation. The mouse's abdominal hair was wiped with alcohol cotton balls, and the abdomen was cut open with dissecting scissors. The mouse's gonads were clamped with forceps, and the testicles, epididymis, and seminal vesicles were separated and measured and weighed.

[0215] Figure 3E and Figure 3F This is a comparison of the size of the testicles and epididymis of wild-type mice (TEX29+ / + mice) and homozygous TEX29 knockout mice (TEX29- / - mice) in Example 3 of the present application, and the results show that there is no significant difference. The above results show that there is no obvious difference in the gonad volume between adult male TEX29- / - mice and TEX29+ / + mice. There is no significant difference in the testicular size and weight of TEX29- / - mice and wild-type mice, indicating that TEX29 has little effect on the number of sperm. Because the amino acid sequence of mouse TEX29 is only 58% similar to that of human TEX29. The results caused by the human TEX29 mutation may differ from the clinical phenotype of mice.

[0216] 8. CASA experiment to detect sperm motility and quantity analysis of TEX29- / - mice. Methods: The epididymis of three adult male TEX29- / - mice was placed in CASA buffer, the epididymis was cut open, and the sperm was kept at 37°C and 5% CO. 2Incubate in an incubator for 5 minutes, 60 minutes, 90 minutes, and 120 minutes, then collect sperm and analyze sperm motility. Sperm counting: Place the mouse epididymis in CASA buffer, cut the epididymis (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 drop 10μL 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 photographic field of view, and the sperm dilution multiple and total volume, calculate the corresponding number of sperm. Among them, the formula of CASA buffer: 120mM NaCl, 4.8mMKCl, 1.2mM MgSO 4 , 1 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 21 mM sodium DL-lactate (Na-dl-lactate), 5 mM glucose, 25 mM NaHCO 3 , 0.25 mM sodium pyruvate (Napyruvate), 0.4 μg / mL phenol red (Phenol red), 3 mg / mL bovine serum albumin (BSA V).

[0217] Figure 3G The results of the comparison of semen volume and motility of wild-type mice (TEX29+ / + mice) and homozygous Tex29 knockout mice (TEX29- / - mice) in Example 3 of the present application are shown in Figure 3. The results show that there is no significant difference in semen volume and sperm motility between adult male TEX29- / - mice and TEX29+ / + mice. This shows that mouse TEX29 has little effect on semen volume and sperm motility.

[0218] 9. Take 3 adult male TEX29- / - mice and mate with several female wild-type mice for 3 months. During mating, observe whether there are sperm plugs in the reproductive tract of female wild-type mice. The results show that sperm plugs exist in the reproductive tract of female wild-type mice, but the plug rate has a downward trend. The above results show that Tex29 gene knockout may be related to the fertilization process of mice. Because the amino acid sequence of mouse TEX29 is only 58% similar to that of human TEX29, the inventors believe that the consequences of human TEX29 mutations may be more serious.

[0219] Example 4: Tex29 acrosome reaction-related detection

[0220] By evaluating the sperm acrosome function, it is helpful to guide the clinical selection of infertility treatment options and help improve the success rate of assisted reproductive technology. The study of this embodiment was approved by the Ethics Committee of Peking University Third Hospital and the informed consent of the patients was obtained. Ten discarded semen samples of male patients with abnormal fertilization were collected 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.

[0221] Methods: Fresh or liquid nitrogen frozen semen samples were taken and then subjected to density gradient centrifugation to obtain highly active spermatozoa and remove contaminants such as leukocytes, germ cells and dead cells. The sperm suspension was incubated at 37°C and 5% CO 2 Incubate in the incubator for 3 hours (loosen the tube cap to allow gas exchange) to induce capacitation. Add Ca 2+ The acrosome reaction was induced by adding Hepes buffer to the control tube and incubated at 37°C for 15 minutes. A small amount of suspension was taken from each tube to evaluate sperm motility; 10 μL of sperm suspension was taken from each tube and evenly spread on a glass slide and dried naturally in the air. Then, the specific TEX29 antibody prepared in Example 1 was used for immunohistochemistry experiments, and the sperm acrosome reaction was observed under a fluorescence microscope and the acrosome reaction rate was calculated. The judgment criteria were as follows according to the recommendations of the World Health Organization:

[0222] 1) Ca 2+ The acrosome reaction rate induced by the vector (%) = the acrosome reaction rate of the test tube - the acrosome reaction rate of the control tube;

[0223] 2) Ca 2+ The normal value of the acrosome reaction rate induced by the vector is about 15%. If it is less than 15%, it is abnormal. If it is between 10-15%, it indicates that the sperm function may be abnormal.

[0224] 3) If the acrosome reaction rate of the control tube is greater than 15%, it indicates that spontaneous acrosome reaction occurred prematurely.

[0225] When AR>15%, IUI treatment can be considered; when AR<15%, it is recommended to abandon IUI and choose IVF / ICSI treatment; when AR<3% or AR<15% of the optimal sperm, the fertilization rate is reduced and half-ICSI is recommended.

[0226] Figure 4A This is a sperm smear of wild-type mice (Tex29+ / +) in Example 4 of the present application, and the result of TEX29 immunohistochemistry detection. Figure 4AThe results show that most sperm are stained (yellow arrows), and a small number of sperm are not stained (red arrows). The results show that TEX29 antibody can be used as a biomarker to detect the acrosome reaction of mouse sperm. By counting the number of stained or unstained sperm, the acrosome reaction rate of sperm can be counted and the function of sperm acrosome can be evaluated.

[0227] Figure 4B This is the result of TEX29 immunohistochemistry test on the male sperm smear in Example 4 of this application. Figure 4B The results show that most sperm are stained (yellow arrows), sperm with deformed heads and a small number of sperm are not stained (red arrows). The results show that TEX29 antibody can be used as a biomarker to detect the acrosome reaction of human sperm. By counting the number of stained or unstained sperm, the acrosome reaction rate of sperm can be counted and the function of the human sperm acrosome can be evaluated.

[0228] Example 5: Screening of male infertile patients with Tex29 mutation gene and semen analysis

[0229] The study of this example was approved by the Ethics Committee of Peking University Third Hospital and the patients' informed consent was obtained. 150 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.

[0230] Methods: TIANamp Micro DNA Kit was used to extract DNA from the patient's blood. According to the instructions, the patient's blood genomic DNA was used as a template, and the exon sequence of the Tex29 gene was amplified through PCR reaction and sent for sequencing. After the sequencing results were returned, sequence alignment was performed to screen patients with Tex29 mutant genes. The primer sequences are shown in SEQ ID NO.10 and SEQ ID NO.11. At the same time, with the patient's consent, discarded testicular biopsy samples from male infertility patients caused by non-genetic factors who were to undergo IVF-ET treatment were collected, RNA was extracted, reverse transcribed and amplified to detect changes in Tex29 expression. Analysis of results: For male infertility patients caused by non-genetic factors who were to undergo IVF-ET treatment, PCR amplification (such as Figure 5 ) and sequencing, the primer sequences are shown in SEQ ID NO.12 and SEQ ID NO.13 to detect the changes in Tex29 expression.

[0231] Figure 5The following is a graph showing the expression changes of Tex29 in different male infertility patients. The RT-PCR results in the figure show that the expression of Tex29 in different patients (21Y03670, 21Y03671, 21Y03672, 21Y03675 and 21Y05112) is different. The expression of Tex29 gene is almost undetectable in patients with azoospermia (21Y05111) and Sertoli cell syndrome (21Y05112, 21Y05113). The above results show that TEX29 can be used as a biomarker to detect the spermatogenesis of the patient's testicles and to determine whether the patient has azoospermia or male infertility according to the sperm production.

[0232] Through transcriptome sequencing and whole genome sequencing screening, a patient with a Tex29 mutant gene was obtained (as shown in Table 2), and it was found that the Tex29 gene mutation occurred in its 5th exon, that is, the 356th base C in the Tex29 gene sequence mutated to T (c.356C>T), and the nucleotide sequence of its mRNA is shown in SEQ ID NO.2 and SEQ ID NO.20; the 119th amino acid of the TEX29 polypeptide mutated to V (p.A1 19V), and the amino acid sequence of the TEX29 mutant protein is shown in SEQ ID NO.3 and SEQID NO.21. The above results indicate that the TEX29 (c.356C>T) mutation site can be used as a molecular marker for the diagnosis of male infertility-related diseases.

[0233] The sperm of the patients with the above-mentioned Tex29 mutation defective gene was analyzed; semen was collected after 5 days of abstinence, liquefied at 37℃ for 30 minutes, and CASA (computer-assisted sperm analysis) was used to detect semen volume, concentration, sperm morphology, motility and viability. The results are as follows: It was found that the sperm concentration, percentage of motile sperm, percentage of forward-moving sperm and percentage of normal morphological sperm in the patients with TEX29 mutation gene were all decreased (the patient had done it twice and the average value was taken).

[0234] Clinical data analysis of the patient revealed that the patient had been suffering from infertility for 8 years since his marriage. The patient had undergone four artificial inseminations (IUI) and four IVFs, all of which failed. After examination, his semen parameters were abnormal and he was diagnosed with male primary infertility. Semen examination showed that his sperm density was normal, but the motility was low. Sperm morphology examination examined a total of 202 sperm, and 202 of them were found to have head deformities, accounting for 100% of the head deformities. Patients with sperm head deformities are prone to low fertilization rates (abnormal fertilization) or fertilization failure, which in turn causes male infertility. After long-term treatment, the patient's semen quality did not improve. Whole exome sequencing revealed a mutation in the TEX29 (c.356C>T) gene. Because the amino acid sequence of mouse TEX29 is only 58% similar to that of human TEX29. The inventor believes that the consequences of human TEX29 mutations may be more serious, including decreased sperm motility, abnormal head morphology, and decreased fertilization rate. The TEX29 (c.356C>T) mutation site can be used as a molecular marker for the diagnosis of male sperm head deformity and male infertility. The above results show that after entering the ovulation induction cycle, it is very important to test the male's sperm fertilization function, including testing the acrosome reaction rate and sperm head deformity to reduce the risk of conventional IVF fertilization failure, and provide a basis for the differential diagnosis of the etiology of clinical male infertility.

[0235] Table 2 Mutation information of a patient with Tex29 mutation

[0236] Patient digital number ACMG Evidence Mutation type Mutations protein Clinical phenotype 21Y09313 VUS pp3 Likely benign c.356C>T p.A119V Sperm head deformity

[0237] 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 screening sperm acrosome abnormalities; A4) Use in the preparation of a product for screening male infertility diseases; A5) Use in the preparation of a product for diagnosing male sperm head deformity; A6) Use in the preparation of products for diagnosing abnormal sperm and egg fertilization; A7) Use in the preparation of a product for diagnosing male infertility; A8) Use in the preparation of a product for diagnosing sperm acrosome reaction rate; in, The biomarker is selected from the group consisting of Tex29 gene, TEX29 protein, mRNA of Tex29 gene, RNA of Tex29 gene or miRNA targeting TEX29 protein.

2. The use according to claim 1, wherein: The substance is a reagent for detecting the presence of Tex29 gene, TEX29 protein, mRNA of Tex29 gene, RNA of Tex29 gene or miRNA targeting TEX29 protein; 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 TEX29 protein; preferably, the antibody that specifically binds to the TEX29 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.4 to SEQ ID NO.

9.

4. The use according to claim 2, wherein: The nucleotide sequence of the Tex29 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex29 gene is shown as SEQ ID NO.2 or SEQ ID NO.20; or, the amino acid sequence of the TEX29 protein is shown as SEQ ID NO.3 or SEQ ID NO.

21.

5. A kit, wherein: The kit includes a reagent for detecting whether a Tex29 gene, a TEX29 protein, an mRNA of a Tex29 gene, an RNA of a Tex29 gene, or a miRNA targeting a TEX29 protein exists, and 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 acrosome abnormalities; A4) Screening for male infertility; A5) Diagnosis of sperm head deformity in men; A6) Diagnosis of abnormal sperm and egg fertilization; A7) Diagnosis of male infertility; A8) Diagnosis of sperm acrosome reaction rate.

6. The kit according to claim 5, wherein The kit includes an antibody that specifically binds to the TEX29 protein; preferably, the antibody that specifically binds to the TEX29 protein is prepared using an antigen having an amino acid sequence as shown in any one of SEQ ID NO.4 to SEQ ID NO.

9.

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.10 and SEQ ID NO.11, a primer pair having nucleotide sequences as shown in SEQ ID NO.12 and SEQ ID NO.13, and a primer pair having nucleotide sequences as shown in SEQ ID NO.14 and SEQ ID NO.

15.

8. The kit according to claim 5, wherein The nucleotide sequence of the Tex29 gene is shown as SEQ ID NO.1; or, the nucleotide sequence of the mRNA of the Tex29 gene is shown as SEQ ID NO.2 or SEQ ID NO.20; or, the amino acid sequence of the TEX29 protein is shown as SEQ ID NO.3 or SEQ ID NO.

21.

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 test result of the kit includes: if the test sample does not contain Tex29 gene, TEX29 protein, Tex29 gene mRNA, Tex29 gene RNA or miRNA targeting TEX29 protein, the source of the test sample is suspected of suffering from male sperm head deformity, abnormal acrosome reaction, abnormal fertilization 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) Increase the sperm acrosome reaction rate; B3) Improve sperm fertilization level; B4) Improve male fertility; B5) Repair abnormalities in the sperm head structure; B6) Treatment of sperm dysfunction; B7) Treatment of male sperm head deformity; B8) Treatment of male infertility; in, The biomarker is selected from TEX29 protein or Tex29 gene.

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