A TEX101 recombinant protein-specific antibody, encoding gene, and use thereof

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a specific antibody against TEX101 recombinant protein, an encoding gene, and uses thereof. The specific antibody is produced using hybridoma cell technology, wherein the antigen used is the TEX101 recombinant protein, whose amino acid sequence is shown in SEQ ID No. 1. The specific antibody is a monoclonal antibody, whose heavy chain variable region has an amino acid sequence shown in SEQ ID No. 2, and whose light chain variable region has an amino acid sequence shown in SEQ ID No. 3. The monoclonal antibody obtained by the present invention has high specificity, effectively improves the ability to detect sperm quality, and can be widely used in clinical testing.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a monoclonal antibody targeting TEX101 recombinant protein and uses thereof. Background Art

[0002] TEX101, or testis-expressed protein 101, is a cell-specific glycoprotein. The TEX101 gene is located at position 19q13.31 on the long arm of human chromosome 19. The protein consists of a 25-amino acid signal peptide region and a 225-amino acid mature protein region, with a molecular weight of approximately 24,093. TEX101 is present on the surface of stem cells as a glycosylphosphatidylinositol-anchored protein. During male gonadal development, it first appears on the membranes of early pregnancy cells in the immature spermatic cord of the fetal testis. Although TEX101 is not expressed on spermatogonia, it reappears on meiotic cells and testicular spermatozoa after puberty. TEX101 is subsequently released from the surface of epididymal spermatozoa during transport through the capus epididymis.

[0003] TEX101 is a biomarker for male infertility. In male infertility patients, particularly those with azoospermia, TEX101 levels in seminal plasma are significantly reduced. Human fertility has been declining over time due to factors such as environmental pollution, unhealthy lifestyles, and the spread of sexually transmitted diseases. Currently, there are relatively few clinical methods for using TEX101 antibodies to assess sperm quality. The challenge of selecting TEX101 peptides as recombinant proteins for immunoprecipitation and preparation of monoclonal antibodies that can specifically and efficiently recognize TEX101 proteins in biological samples is a pressing issue. Therefore, the development of highly specific monoclonal antibodies and detection kits is crucial for the clinical diagnosis of TEX101. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a recombinant TEX101 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] SEQ ID NO.1:

[0006] VTSYSNYCEDSFCNDKDSLSQFWEFSETTAST.

[0007] Preferably, the recombinant TEX101 protein of the present invention is obtained by truncating the disclosed UniProtKB-Q9BY14 (TX101_HUMAN).

[0008] Another aspect of the present invention is to provide a monoclonal antibody prepared by hybridoma cell technology using the recombinant TEX101 protein shown in SEQ ID NO.1 as an immunogen, wherein the monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.3.

[0009] Specifically, the preparation method of the monoclonal antibody or its antigen-binding fragment of the present invention is to immunize 6-week-old female BALB / c mice with TEX101 recombinant protein as an antigen, collect immune spleen cells, fuse them with NS-1 myeloma cells to construct hybridoma cells, and separate and purify the monoclonal antibody produced by the hybridoma cells.

[0010] Specifically, the following steps are included:

[0011] (1) Animal immunization

[0012] Six 6-week-old female BALB / c mice were immunized with TEX101 recombinant protein as an antigen. The immunization procedure was as follows: an equal amount of recombinant antigen emulsified with Freund's complete adjuvant was injected subcutaneously at two points on the back, with a total immunization dose of 30 μg / mouse; two weeks later, an equal amount of recombinant antigen emulsified with Freund's incomplete adjuvant was injected subcutaneously at two points, with the dose remaining unchanged. A third routine immunization was performed three weeks later, and blood was collected from the tail three weeks after each immunization, and the antibody titer produced by the mice was detected by indirect ELISA; after an interval of at least one month, the recombinant antigen was injected into the tail vein for the last time on the third day before fusion, with a dose of 15 ug / mouse, for a booster immunization.

[0013] (2) Preparation of NS-1 myeloma cells

[0014] One week before fusion, NS-1 myeloma cells stored in liquid nitrogen in the laboratory were revived and cultured in 24-well cell culture plates. 15% fetal bovine serum DMEM culture medium was used for subculture for one week, and the cell concentration was adjusted to 10 6 During fusion, select myeloma cells in the logarithmic growth phase, discard the culture medium in the original bottle, add an appropriate amount of serum-free DMEM culture medium, gently blow off the cells, transfer them to a 50mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, wash the cells twice, discard the supernatant, resuspend the cell pellet in serum-free culture medium, count and set aside.

[0015] (3) Preparation of immune spleen cells

[0016] An immunized BALB / c mouse was sacrificed by orbital bleeding and then disinfected by soaking in 75% alcohol for 5 minutes.

[0017] In a clean bench, fix the sterilized mouse on a dissecting board with the forelimbs fixed and the hind limbs crossed (left hind limb on top). Use forceps to grasp the skin of the lower abdomen, make a small cut, tear the skin open to expose the peritoneum, change a set of forceps and scissors, cut the peritoneum open, expose the spleen, change another set of instruments, grasp the spleen with forceps, and use scissors to remove the fat tissue and connective tissue adhering to the cells.

[0018] After rinsing the spleen with serum-free culture medium, transfer it to a sterile glass culture dish containing 5 mL of serum-free culture medium and place it on a 100-mesh copper grid. Use sterile scissors to cut the spleen into 3-5 pieces, gently grind it with the inner core of a syringe, and gently rinse the copper grid with serum-free culture medium to collect the spleen cell suspension.

[0019] The cells were centrifuged at 1000 rpm for 5 min, washed twice, the supernatant was discarded, and the cell pellet was suspended in serum-free culture medium and then counted.

[0020] (4) Preparation of feeder cells

[0021] Take an unimmunized BALB / c mouse, bleed its orbital area, and soak it in 75% alcohol for 5 minutes;

[0022] Move the mouse to a clean clean bench and fix it on the dissecting table in a supine position. Cut the abdominal skin to expose the abdomen. Make a small cut in the peritoneum (in the center of the abdomen). Then use a pipette to aspirate 3 mL of serum-free culture medium into the mouse's abdominal cavity. Pipet several times, then aspirate the liquid and place it in a 50 mL centrifuge tube. Repeat this process. This is the peritoneal macrophage.

[0023] The cells were centrifuged at 1000 rpm for 10 min and the supernatant was removed. The cells were resuspended and counted. The cells were suspended in HAT medium and placed in a 37°C, 5% CO2 incubator for later use.

[0024] (5) Cell fusion

[0025] Mix NS-1 myeloma cells and immune spleen cells at a ratio of 1:10 in a 50mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Use a pipette to remove any remaining liquid and gently tap the bottom of the tube with your index finger to loosen the cell pellet. Hold the tube containing the cell mixture by the bottom. Then, slowly add 0.5mL of 50% PEG solution (pre-warmed to 37°C) over 1 minute, shaking the tube.

[0026] Use both hands to work for 90 seconds, then slowly add 10 mL of 37°C pre-warmed DMEM medium. The specific method is: add 1 mL dropwise in the first minute, 1 mL in the second minute, 3 mL in the third to fourth minute, and the remaining 5 mL after 5 minutes. Each addition should be done slowly, and the tube should be gently shaken continuously. After completion, centrifuge at 600 rpm / min for 5 minutes and discard the supernatant.

[0027] The fused cell pellet was gently suspended in 2.5 ml of complete culture medium, added to 22.5 ml of semi-solid culture medium, mixed well, and poured into a 3.5 cm diameter dish, about 2 ml per dish, and cultured at 37°C, 5% CO2;

[0028] After one week, white spots can be seen on the surface of the culture medium in the dish. Under the microscope, these are cell clones. Under sterile conditions, the dispersed, single cell clusters in the dish are aspirated and placed into the culture medium of a 96-well plate and cultured.

[0029] (6) Screening of hybridoma cells

[0030] The antigen concentration was adjusted to 1 ng / ul, the coating volume per well was 100ul, and the coating was carried out overnight at 4°C.

[0031] The next day after blocking, shake off the coating liquid in the wells, add 200ul of 3% BSA blocking solution to each well, and block at 37℃ for 1.5h;

[0032] Add primary antibody and wash the plate 5 times, shake dry vigorously. Use anti-fish parvalbumin fusion cell culture supernatant as primary antibody and incubate at 37°C for 1 hour;

[0033] Add secondary antibody and wash the plate 5 times, shake dry vigorously, use HRP-goat anti-mouse IgG as secondary antibody, dilute at 1:4000, add 100 μl to each well, and incubate at 37°C for 1 h;

[0034] Add 50 μl of TMB colorimetric solution A / B to each well and incubate at 37°C for 15 min;

[0035] Termination was accomplished by adding 50 μl of 2 mM sulfuric acid to each well;

[0036] Read the plate at A450nm and record the data. A positive result is considered if the ratio of the reading of the assay well to the negative control value is greater than 2.1.

[0037] (7) Cloning of hybridoma cells

[0038] Wells that test positive twice consecutively by indirect ELISA should be subcloned by limiting dilution. A mouse feeder cell layer should be prepared the day before cloning. Gently remove the hybridoma cells to be cloned from the culture wells and count the number of viable cells using a hemocytometer. Dilute the cells with complete medium to 5, 10, and 50 cells per ml. Add 100 μL / well of each of these three cell suspensions to a 96-well culture plate containing pre-prepared feeder cells, so that each well contains 0.5, 1, and 5 cells, respectively. Change the medium halfway on day 4 of culture. Carefully observe and record the cell growth in each well on days 5-6. On days 10-14 after cloning, when the cells have filled approximately 1 / 4-1 / 2 of the bottom of the well, change the medium completely and perform indirect ELISA the following day. If specific anti-TEX101 antibodies are detected in the wells, select the wells with high antibody titers, single clones, and good morphology for further cloning using the same method. Typically, two cloning cycles are performed. After two times, the hybridoma cells in the positive wells can be moved to a 24-well culture plate. When the hybridoma cells in the 24-well plate grow well, the hybridoma cells can be frozen.

[0039] (8) Production of monoclonal antibodies

[0040] The established hybridoma cells were expanded and cultured, and at the same time, ascites was induced in BALB / c mice to produce monoclonal antibodies and purify them.

[0041] Another aspect of the present invention is to provide a kit for detecting sperm quality, wherein the kit comprises the monoclonal antibody of the present invention.

[0042] Another aspect of the present invention is to provide an application of the monoclonal antibody of the present invention, wherein the application is used to prepare a detection kit for detecting sperm quality.

[0043] Beneficial technical effects of the present invention:

[0044] Based on existing research, the present invention truncates the human TEX101 protein and uses the shorter recombinant protein as an antigen to prepare monoclonal antibodies. This antigen has a relatively simple structure, low synthesis cost, and a well-defined epitope. This helps induce the body to produce antibodies against specific epitopes, thereby reducing cross-reactivity with non-target antigens. The resulting monoclonal antibodies have high specificity and can quickly and efficiently detect TEX101 proteins, making them widely applicable for in vitro testing of sperm quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0046] Figure 1 :WB image of specific identification of monoclonal antibody 2E5;

[0047] Figure 2 : ELISA standard curve of monoclonal antibody 2E5. DETAILED DESCRIPTION

[0048] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0049] Example 1: Preparation of monoclonal antibodies

[0050] (1) Animal immunization

[0051] TEX101 recombinant protein (VTSYSNYCEDSFCNDKDSLSQFWEFSETTAST

[0052] , SEQ ID No. 1) was used as an antigen to immunize six 6-week-old female BALB / c mice. The immunization procedure was as follows: an equal amount of recombinant antigen emulsified in Freund's complete adjuvant was injected subcutaneously at two points on the back, with a total immunization dose of 30 μg / mouse; two weeks later, an equal amount of recombinant antigen emulsified in Freund's incomplete adjuvant was injected subcutaneously at two points, with the same dose. Three weeks later, a third routine immunization was performed. Three weeks after each immunization, blood was collected from the tail, and the antibody titer produced by the mice was detected by indirect ELISA; after an interval of at least one month, the recombinant antigen was injected into the tail vein for the last time on the third day before fusion, with a dose of 15 μg / mouse, for a booster immunization.

[0053] (2) Preparation of NS-1 myeloma cells

[0054] One week before fusion, NS-1 myeloma cells stored in liquid nitrogen in the laboratory were revived and cultured in 24-well cell culture plates. 15% fetal bovine serum DMEM culture medium was used for subculture for one week, and the cell concentration was adjusted to 10 6During fusion, select myeloma cells in the logarithmic growth phase, discard the culture medium in the original bottle, add an appropriate amount of serum-free DMEM culture medium, gently blow off the cells, transfer them to a 50mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, wash the cells twice, discard the supernatant, resuspend the cell pellet in serum-free culture medium, count and set aside.

[0055] (3) Preparation of immune spleen cells

[0056] An immunized BALB / c mouse was sacrificed by orbital bleeding and then disinfected by soaking in 75% alcohol for 5 minutes.

[0057] In a clean bench, fix the sterilized mouse on a dissecting board with the forelimbs fixed and the hind limbs crossed (left hind limb on top). Use forceps to grasp the skin of the lower abdomen, make a small cut, tear the skin open to expose the peritoneum, change a set of forceps and scissors, cut the peritoneum open, expose the spleen, change another set of instruments, grasp the spleen with forceps, and use scissors to remove the fat tissue and connective tissue adhering to the cells.

[0058] After rinsing the spleen with serum-free culture medium, transfer it to a sterile glass culture dish containing 5 mL of serum-free culture medium and place it on a 100-mesh copper grid. Use sterile scissors to cut the spleen into 3-5 pieces, gently grind it with the inner core of a syringe, and gently rinse the copper grid with serum-free culture medium to collect the spleen cell suspension.

[0059] The cells were centrifuged at 1000 rpm for 5 min, washed twice, the supernatant was discarded, and the cell pellet was suspended in serum-free culture medium and then counted.

[0060] (4) Preparation of feeder cells

[0061] Take an unimmunized BALB / c mouse, bleed its orbital area, and soak it in 75% alcohol for 5 minutes;

[0062] Move the mouse to a clean clean bench and fix it on the dissecting table in a supine position. Cut the abdominal skin to expose the abdomen. Make a small cut in the peritoneum (in the center of the abdomen). Then use a pipette to aspirate 3 mL of serum-free culture medium into the mouse's abdominal cavity. Pipet several times, then aspirate the liquid and place it in a 50 mL centrifuge tube. Repeat this process. This is the peritoneal macrophage.

[0063] The cells were centrifuged at 1000 rpm for 10 min and the supernatant was removed. The cells were resuspended and counted. The cells were suspended in HAT medium and placed in a 37°C, 5% CO2 incubator for later use.

[0064] (5) Cell fusion

[0065] Mix NS-1 myeloma cells and immune spleen cells at a ratio of 1:10 in a 50mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Use a pipette to remove any remaining liquid and gently tap the bottom of the tube with your index finger to loosen the cell pellet. Hold the tube containing the cell mixture by the bottom. Then, slowly add 0.5mL of 50% PEG solution (pre-warmed to 37°C) over 1 minute, shaking the tube.

[0066] Use both hands to work for 90 seconds, then slowly add 10 mL of 37°C pre-warmed DMEM medium. The specific method is: add 1 mL dropwise in the first minute, 1 mL in the second minute, 3 mL in the third to fourth minute, and the remaining 5 mL after 5 minutes. Each addition should be done slowly, and the tube should be gently shaken continuously. After completion, centrifuge at 600 rpm / min for 5 minutes and discard the supernatant.

[0067] The fused cell pellet was gently suspended in 2.5 ml of complete culture medium, added to 22.5 ml of semi-solid culture medium, mixed well, and poured into a 3.5 cm diameter dish, about 2 ml per dish, and cultured at 37°C, 5% CO2;

[0068] After one week, white spots can be seen on the surface of the culture medium in the dish. Under the microscope, these are cell clones. Under sterile conditions, the dispersed, single cell clusters in the dish are aspirated and placed into the culture medium of a 96-well plate and cultured.

[0069] (6) Screening of hybridoma cells

[0070] The antigen concentration was adjusted to 1 ng / ul, the coating volume per well was 100ul, and the coating was carried out overnight at 4°C.

[0071] The next day after blocking, shake off the coating liquid in the wells, add 200ul of 3% BSA blocking solution to each well, and block at 37℃ for 1.5h;

[0072] Add primary antibody and wash the plate 5 times, shake dry vigorously. Use anti-fish parvalbumin fusion cell culture supernatant as primary antibody and incubate at 37°C for 1 hour;

[0073] Add secondary antibody and wash the plate 5 times, shake dry vigorously, use HRP-goat anti-mouse IgG as secondary antibody, dilute at 1:4000, add 100 μl to each well, and incubate at 37°C for 1 h;

[0074] Add 50 μl of TMB colorimetric solution A / B to each well and incubate at 37°C for 15 min;

[0075] Termination was accomplished by adding 50 μl of 2 mM sulfuric acid to each well;

[0076] Read the plate at A450nm and record the data. A positive result is considered if the ratio of the reading of the assay well to the negative control value is greater than 2.1.

[0077] (7) Cloning of hybridoma cells

[0078] Wells that test positive twice consecutively by indirect ELISA should be subcloned by limiting dilution. A mouse feeder cell layer should be prepared the day before cloning. Gently remove the hybridoma cells to be cloned from the culture wells and count the number of viable cells using a hemocytometer. Dilute the cells with complete medium to 5, 10, and 50 cells per ml. Add 100 μL / well of each of these three cell suspensions to a 96-well culture plate containing pre-prepared feeder cells, so that each well contains 0.5, 1, and 5 cells, respectively. Change the medium halfway on day 4 of culture. Carefully observe and record the cell growth in each well on days 5-6. On days 10-14 after cloning, when the cells have filled approximately 1 / 4-1 / 2 of the bottom of the well, change the medium completely and perform indirect ELISA the following day. If specific anti-TEX101 antibodies are detected in the wells, select the wells with high antibody titers, single clones, and good morphology for further cloning using the same method. Typically, two cloning cycles are performed. After two times, the hybridoma cells in the positive wells can be moved to a 24-well culture plate. When the hybridoma cells in the 24-well plate grow well, the hybridoma cells can be frozen.

[0079] (8) Production of monoclonal antibodies

[0080] The established hybridoma cells were expanded and cultured, and at the same time, ascites was induced in BALB / c mice to produce monoclonal antibodies and purify them.

[0081] Finally, a positive monoclonal antibody with high titer, 2E5, was selected, and sequencing and structural analysis were performed on monoclonal antibody 2E5. Sequencing analysis identified monoclonal antibody 2E5 as an IgG antibody. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.2, the amino acid sequence of its light chain variable region is shown in SEQ ID NO.3, the amino acid sequence of its heavy chain constant region is shown in SEQ ID NO.4, and the amino acid sequence of its light chain constant region is shown in SEQ ID NO.5.

[0082] SEQ ID NO.2:

[0083] QVQLVQSGAEVKKPGASVKVSCKASQLLVHWVRQAPGQGLEWMGDLSVTGSARFTISRDNSKNTLYLQMNSLRAEDTAVYYCFVSYLYNEGVSVFTYEFWGQGTLVTVSS.

[0084] SEQ ID NO.3:

[0085] DIQMTQSPSSLSASVGDRVTITCPSLWVTCWYQQKPGKAPKLLIYSLAVLRSFGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVTKLPNILQRASKAPFGQGTKVEIK.

[0086] SEQ ID NO.4:

[0087] 。

[0088] SEQ ID NO.5:

[0089] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0090] Example 2: Specificity identification of monoclonal antibodies

[0091] (1) Collect human semen and extract seminal plasma proteins as sample proteins;

[0092] (2) Add the protein sample to SDS gel for electrophoresis separation;

[0093] (3) Transfer the proteins in the gel to the PVDF membrane;

[0094] (4) Block with 5% skim milk powder at room temperature for 1 h;

[0095] (5) Add the monoclonal antibody 2E5 from Example 1, a positive antibody (commercially available anti-TEX101 antibody, ab69522), and a negative antibody (commercially available anti-β-Actin antibody, ab8226). Dilute the antibodies according to their dilution ratio and incubate at 4°C overnight.

[0096] (6) Washing: Wash the membrane 3-5 times with TBST, 10 minutes each time;

[0097] (7) Secondary antibody incubation: add secondary antibody matching the primary antibody and incubate at room temperature for 1 h;

[0098] (8) Washing: Wash the membrane again with TBST;

[0099] (9) Color development: adding a colorimetric substrate for color development reaction and then imaging.

[0100] The results showed that monoclonal antibody 2E5 could specifically bind to TEX protein 101 ( Figure 1 ).

[0101] Example 3: Establishment of a standard curve for monoclonal antibody 2E5

[0102] (1) Select 2E5 as the standard and dilute it in the following order from high to low concentration. The dilution gradient is 1000 ng / mL → 500 ng / mL → 250 ng / mL → 125 ng / mL → 62.5 ng / mL → 31.25 ng / mL → 15.625ng / mL → 7.8125 ng / mL → 3.90625 ng / mL → 1.953125 ng / mL.

[0103] (2) Coat the TEX101 protein into the wells of an ELISA plate, 100 μl per well, with an antigen concentration of 10 μg / ml. Incubate the ELISA plate at 37°C for 2 h. Wash the plate with PBST and repeat the wash five times.

[0104] (3) Add 5% skim milk powder for blocking, incubate at 37°C for 2 h, wash the ELISA plate with PBST, and repeat the washing process 5 times.

[0105] (4) Add the diluted standard to the wells of the ELISA plate, 100 μl per well, incubate at 37°C for 2 h, wash the ELISA plate with PBST, and repeat the wash 5 times.

[0106] (5) Add enzyme-labeled secondary antibody to each well, 100 μl per well, incubate at 37°C for 1 h, wash the ELISA plate with PBST, and repeat the wash 5 times.

[0107] (6) Add TMB for color development, 100 μl per well, incubate at 37°C for 30 min, add stop solution, 50 μl per well, to terminate the color development reaction.

[0108] (7) Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 450 nm and record the result.

[0109] The results showed that the 2E5 monoclonal antibody had a good fit and the minimum detection limit was 57.68452 ng / ml ( Figure 2 ).

[0110] Example 4: In vitro detection of sperm quality using monoclonal antibody 2E5

[0111] Ten semen samples from healthy men and ten semen samples from azoospermia patients were collected and numbered in random order (numbered 1-20 respectively). The monoclonal antibody 2E5 was used to detect the above 20 semen samples by ELISA method.

[0112] The test results are as follows:

[0113] Table 1

[0114]

[0115] The results showed that 10 TEX101-positive samples and 10 TEX101-negative samples were detected, which was consistent with the actual situation, proving that the 2E5 monoclonal antibody of the present invention can detect TEX101 protein and can be used to prepare a detection kit for in vitro detection of sperm quality.

[0116] The above descriptions are merely optional embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present description and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A recombinant TEX101 protein, characterized in that: The amino acid sequence of the recombinant TEX101 protein is shown in SEQ ID NO.

1.

2. A monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The monoclonal antibody or its antigen-binding fragment specifically binds to the TEX101 protein, and comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

3.

3. A coding gene, characterized in that The gene encodes the heavy chain variable region and light chain variable region of the monoclonal antibody or antigen-binding fragment thereof according to claim 2.

4. An expression vector, characterized in that The expression vector comprises the coding gene according to claim 3.

5. A host cell, characterized in that The host cell contains the expression vector according to claim 4.

6. A method for producing the monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The method comprises culturing the host cell of claim 5 in a culture medium under conditions sufficient to produce the monoclonal antibody or antigen-binding fragment thereof, and then isolating and purifying the produced monoclonal antibody or antigen-binding fragment thereof.

7. A use of the monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The application is to prepare a test kit for detecting sperm quality.

8. A kit for detecting sperm quality in vitro, characterized in that: The kit comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 2.

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