Preparation method and application of high-activity statin B protein

Abstract

The invention relates to the technical field of biology, and provides a preparation method and application of high-activity statin B protein. The preparation method comprises the following steps: S1, mutating an alpha chain and a beta chain of statin B protein to obtain an alpha chain mutant and a beta chain mutant with amino acid sequences as shown in SEQ ID No.1 and SEQ ID No.2; and S2, inserting the alpha-chain mutant and the beta-chain mutant into an expression vector, transfecting to a CHO cell line to express recombinant protein, and purifying the recombinant protein to obtain the high-activity statin B protein. Compared with an INHB wild type, the expression yield of the INHB mutant co-expression sequence is improved in a breakthrough manner; and in the biological cell verification result, the activity is superior to that of a wild-type INHB standard substance, and the method provided by the invention accumulates a certain foundation for subsequent antibody development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing and applying a highly active inhibin B protein. Background Technology

[0002] Inhibin B (INHB) is a heterogeneous glycoprotein derived from germ cells, composed of a βB subunit linked to an α subunit. Its predicted molecular weight is approximately 28 kDa, and it belongs to the superfamily of transforming growth factor β (TGF-β). INHB is primarily secreted by Sertoli cells in the male testes and granulosa cells in the female ovaries. It regulates the pituitary gland and inhibits FSH secretion through negative feedback, and also affects the paracrine function of the gonads by regulating the amount of estradiol (E2).

[0003] INHB levels are closely related to male spermatogenesis, testicular volume, total sperm count, and female ovarian reserve and infertility caused by ovarian factors. It is an important biomarker for assessing testicular function and ovarian reserve, and is widely used in fertility assessment, optimization of assisted reproductive technology, and diagnosis and monitoring of reproductive system diseases (such as azoospermia, premature ovarian failure, and granulosa cell tumor).

[0004] Currently, the preparation of INHB faces significant technical bottlenecks. Prokaryotic expression systems (such as *E. coli*) lack glycosylation modification capabilities, resulting in bioactive expression products that easily form inclusion bodies. Natural extraction methods are limited in source, have low purity, and are costly, making it difficult to meet the needs of scientific research and clinical practice. While traditional eukaryotic expression systems (such as CHO and HEK293 cells) can achieve glycosylation modification, they suffer from low co-expression efficiency of α and β B subunits, difficulty in heterodimer assembly, low expression levels (typically at the ng / mL level), and complex purification processes. Therefore, how to construct an efficient and stable eukaryotic expression system to achieve synergistic expression and correct folding of the two INHB subunits, and obtain high-yield, highly bioactive recombinant INHB protein, has become a key technical challenge restricting its in-depth application in diagnostics, treatment, and basic research. Summary of the Invention

[0005] In view of this, the present invention proposes a method for preparing high-quality, high-yield, and highly active inhibin B protein and its application.

[0006] The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a method for preparing highly active inhibin B protein, comprising the following steps: S1, the α chain and β chain of inhibin B protein were mutated to obtain α chain mutants and β chain mutants with amino acid sequences as shown in SEQ ID No. 1 and 2; S2, α-chain mutants and β-chain mutants were inserted into the expression vector and transfected into CHO cell lines to express recombinant proteins. The recombinant proteins were then purified to obtain highly active inhibin B protein.

[0007] The carrier is the PTT5 carrier.

[0008] First, the α-chain mutant and the β-chain mutant are linked with a flexible linker peptide to obtain a co-expression sequence, which is then inserted into the expression vector.

[0009] The amino acid sequence of the flexible linker peptide is (GGGGS)3.

[0010] In a second aspect, the present invention provides a nucleic acid molecule that encodes an α-chain mutant and a β-chain mutant with amino acid sequences as shown in SEQ ID No. 1 and 2.

[0011] Thirdly, the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0012] Fourthly, the present invention provides a host cell comprising the above-mentioned recombinant expression vector.

[0013] Fifthly, the present invention provides a highly active inhibin B protein, which is prepared by the above method.

[0014] In a sixth aspect, the present invention provides the application of highly active inhibin B protein in the preparation of diagnostic reagents for male fertility assessment, female ovarian reserve function testing, or reproductive system tumor markers.

[0015] The preparation method and application of a highly active inhibin B protein of the present invention have the following advantages over the prior art: In the INHB expression stage, the expression of the co-expressed sequence of wild-type INHB failed, while the expression yield of the co-expressed sequence of INHB mutant showed a breakthrough improvement. Furthermore, in biological cell validation results, the activity of the INHB mutant was superior to that of the wild-type INHB standard. The method of this invention is highly efficient and convenient, significantly increasing the yield of INHB and laying a foundation for subsequent antibody development. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1The purity of purified INHB protein was determined by SDS-PAGE. (a) is the SDS-PAGE reducing gel of INHB mutant, (b) is the SDS-PAGE non-reducing gel of INHB mutant, and (c) is the SDS-PAGE reducing gel of INHB wild type.

[0018] Figure 2 A graph showing cell activity in rat pituitary cells as part of an experiment to inhibit FSH secretion. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] To obtain high-quality, high-yield inhibin B (INHB), this invention primarily achieves efficient expression of inhibin B protein through the following methods: The INHB α and β chains are point-mutated to obtain mutant sequences; two co-expression sequences are obtained by linking the wild-type α and β chains and the mutant α and β chains respectively using a flexible linker sequence (GGGGS)3; the PTT5 expression vector is selected, and the CHO cell line (CHO-K1) is used to optimize its glycosylation modification ability; after expression affinity purification, the activity of INHB is detected by biological assays.

[0021] The following examples will provide a detailed explanation.

[0022] Example 1: Obtaining the sequence and synthesizing the gene The α and β sequences related to the INHB wild type were obtained from NCBI, and mutation sites were predicted.

[0023] INHB α chain >233-366 (P05111): Glutamine (Q) at position 18 is mutated to glutamic acid (E); leucine (L) at position 65 is mutated to proline (P); threonine (T) at position 81 is mutated to alanine (A); and aspartic acid (D) at position 113 is mutated to N. The amino acid sequence of the α chain mutant is shown in SEQ ID No. 1.

[0024] α-chain wild-type sequence: >233-366 (P05111) STPLMSWPWSPSALRLL Q RPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCG L HIPPNLSLPVPGAPPT PAQPYSLLPGAQPCCAALPGTMRPLHVRTTS D GGYSFKYETVPNLLTQHCACI.

[0025] α-chain mutant sequence (SEQ ID No. 1): >233-366(P05111) STPLMSWPWSPSALRLL E RPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCG P HIPPNLSLPVPGAPP A PAQPYSLLPGAQPCCAALPGTMRPLHVRTTS N GGYSFKYETVPNLLTQHCACI.

[0026] INHB β chain >293-407 (P09529): The asparagine (N) at position 9 is mutated to threonine (T); the serine (S) at position 43 is mutated to threonine (T); and the arginine (R) at position 67 is mutated to lysine (K). The amino acid sequence of the β chain mutant is shown in SEQ ID No. 2.

[0027] Two co-expressed sequences were obtained by linking the α-chain and β-chain wild-type and the α-chain and β-chain mutants respectively using the flexible linker sequence (GGGGS)3.

[0028] β-chain wild-type sequence: >293-407 (P09529) GLECDGRT N LCCRQQFFIDFRLIGWNDWIIAPTGYYGNYCEG S CPAYLAGVPGSASSFHTAVVNQY R MRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA B-chain mutant sequence (SEQ ID No. 2): >293-407(P09529) GLECDGRT T LCCRQQFFIDFRLIGWNDWIIAPTGYYGNYCEG T CPAYLAGVPGSASSFHTAVVNQY K MRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA.

[0029] Co-expressed sequences of α-chain and β-chain wild-type: STPLMSWPWSPSALRLLQRPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCGLHIPPNLSLPVPGAPPTPAQPYSLLPGAQPCCAALPGTMRPLHVRTTSDGGYSFKYETVPNLLTQ HCACI(GGGGS)3GLECDGRTNLCCRQQFFIDFRLIGWNDWIIAPTGYYGNYCEGSCPAYLAGVPGSASSFHTAVVNQYRMRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA.

[0030] Co-expressed sequences of α-chain and β-chain mutants (SEQ ID No. 3): STPLMSWPWSPSALRLLERPPEEPAAHANCHRVALNISFQELGWERWIVYPPSFIFHYCHGGCGPHIPPNLSLPVPGAPPAPAQPYSLLPGAQPCCAALPGTMRPLHVRTTSNGGYSFKYETVPNLLTQ HCACI(GGGGS)3GLECDGRTTLCCRQQFFIDFRLIGWNDWIIAPTGYYGNYCEGTCPAYLAGVPGSASSFHTAVVNQYKMRGLNPGTVNSCCIPTKLSTMSMLYFDDEYNIVKRDVPNMIVEECGCA.

[0031] The above INHB wild-type (A) and mutant (B) sequences were sent to Qingke Biotechnology for gene synthesis according to the expression vector PTT5.

[0032] Example 2: Maternal Expression The synthesized plasmids (A and B) were transfected into CHO-K1 for transient expression, and the cells were collected 14 days later. The specific steps are as follows: 1. Cell resuscitation Preheat the water bath to 37°C and preheat the culture medium. Once the culture medium is preheated, add 30mL of preheated medium to a 125mL shake flask. After the water bath reaches 37°C, remove the cells to be thawed from the liquid nitrogen tank and quickly place them in the water bath. Shake rapidly to dissolve the cells, being careful not to let the cryovial tip touch the liquid surface. When the ice in the cryovial is almost completely melted, remove it, wipe it with alcohol, and place it in a clean bench. Gently transfer the cell suspension to a shake flask containing pre-added culture medium, avoiding repeated pipetting. Place the cells in a shaker with the following conditions: 37°C, 8% CO2 concentration, 110 rpm (50mm amplitude). Regularly monitor cell density. When the cell density reaches (1-3) × 10⁻⁶ cells / mL, the cell suspension is ready to be thawed. 6 When the cell count reaches 100 viable cells / mL (after approximately 2-3 days of growth), the cells are passaged at a density of 0.5 × 10⁻⁶ cells / mL. 6 live cells / mL.

[0033] 2. Cell passage When the cell density reaches 2x10 6 When the viable cell count is above 100%, passage the cells; prepare preheated fresh culture medium in advance, and add an appropriate amount of preheated culture medium to the shake flask; calculate the required cell suspension volume according to the passage density recommended in the table below.

[0034] Table 1. Subgeneration Density

[0035] Add the required volume of cell suspension to an appropriate amount of preheated fresh culture medium. Transfer the shake flask to a shaker. Culture conditions: 37℃, 8% CO2 concentration, rotation speed 110 (amplitude 50mm). Regularly monitor cell density until it reaches the passage density. Once the cell density reaches the passage density, if the cells are in good condition, they can be cryopreserved. Prepare the cryopreservation solution according to the formula. Prepare the cryopreservation solution fresh each time. Calculate the amount of cryopreservation solution to be prepared based on the number of cells to be cryopreserved, 1×10⁻⁶. 7 To cryopreserve one cell culture tube, first take 45% fresh culture medium, then add 10% DMSO, mix well, and pre-cool at 4°C. Prepare the cryopreservation tube, labeling it with the cell name, cell count, cryopreservation date, tube number code, and other special information. Transfer the cell suspension to be cryopreserved to a centrifuge tube, centrifuge at 800 rpm for 3 minutes, take 45% of the supernatant, add it to the pre-cryopreservation solution, mix well, and discard the remaining supernatant. Resuspend the cells in the cell pellet with an appropriate amount of cryopreservation solution to achieve a cell density of 1×10⁻⁶ cells / mL. 7 / mL; Take 1mL of cell suspension and add it to the cryovial, tighten the cap, place it in the cryopreservation box, and put the cryopreservation box in a -80℃ freezer; The next day, transfer the frozen cells to liquid nitrogen for storage; Fill in the cell storage form and proceed with cell storage; 3. Cell transfection The cell density reached 4 × 10⁻⁶ the day before transfection. 6 More than 10 live cells / mL; according to (2-3) × 10 6 Passage at a cell density of 1 live cells / mL; On the day of transfection, the cell density reached (5-6) × 10⁻⁶. 6 Transfection can be achieved with approximately one live cell / mL; Take two sterile centrifuge tubes and add A×5% (A = transfection system volume) of transfection dilution buffer (fresh culture medium) to each tube. Add the corresponding amount of plasmid to one tube and the corresponding amount of 25kDa linear PEI to the other tube. Let stand for 5 minutes, then add the PEI dilution buffer to the plasmid dilution buffer and mix well. Incubate at room temperature for 15-20 minutes, then add the mixture dropwise to the cells and place them in a shaker for culture.

[0036] Table 2 Transfection methods

[0037] Sugar levels were monitored daily after transfection. When the sugar level was below 3 g / L, sugar was supplemented to 6 / 8 g / L. Starting on the second day after transfection, add 5% PFF05 feed every other day; Approximately 7 days after transfection, 1 mL of cell suspension was taken as a preliminary identification sample. 7-14 days after transfection, when the cell state is below 50%, harvest the cell suspension (expression ends when the cell state is normal after 14 days), and then perform protein purification.

[0038] Example 3 Affinity Chromatography Equilibrate the packing material: Take out the Ni-NTA packing material in advance and wash it with equilibration solution (20mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol) to equilibrate it, so that the packing material is in the same buffer system as the protein, allowing the protein to bind more fully with the packing material.

[0039] Centrifugation incubation: Aliquot the supernatant from centrifugation into 100mL tubes, add 3mL of beads (Ni-NTA packing material, Borglon) and 10% glycerol to each tube. This method avoids protein aggregation or loss during subsequent purification. Incubate at 4℃-8℃ on a shaker at low speed for 1 hour.

[0040] Centrifugation: Collect the incubated supernatant and bead mixture (33 rpm × 5 min), discard the supernatant, and evenly distribute the centrifuged packing material into 4 empty 15 mL gravity columns.

[0041] Equilibration: Wash each gravity column with 5 column volumes of equilibration buffer (20 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol).

[0042] Washing: Wash the beads with washing buffer 1 (20 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol, 20 mM imidazole) for 5-10 column volumes, until no obvious blue color is detected by G250; wash the beads with washing buffer 2 (20 mM HEPES pH 7.4, 300 mM NaCl, 10% glycerol, 50 mM imidazole) for 5-10 column volumes, until no obvious blue color is detected by G250; do not collect the equilibrium products and washing products.

[0043] Elution 1: Add 5-10 mL of eluent (20 mM HEPES pH 7.4, 300 mM NaCl, 15% glycerol, 500 mM imidazole, 0.2%-0.5% SKL, 1 mM DTT), allow the eluent to fully bind with the beads at low temperature for 1 h, elute for 2 column volumes. Once no obvious blue color change is detected by G250 assay, collect the supernatant from 3 gravity columns, immediately add DTT to a final concentration of 2 Mm, store at low temperature, and perform SDS-page reduction and non-reduction verification to obtain the expression analysis of INHB A and B. Results are shown in [Figure 1]. Figure 1 And Table 3.

[0044] Table 3 Expression analysis of INHB mutant and wild type

[0045] Table 3 shows that in the INHB expression process, co-expression of wild-type and mutant sequences was performed separately. The expression of the wild-type INHB co-expressed sequence failed, while the expression yield of the INHB mutant co-expressed sequence was significantly improved.

[0046] Example 4: ELISA detection of protein activity I. Preparations before the experiment 1. Solution preparation Prepare complete culture medium: DMEM + 10% heat-inactivated FBS + 1% P / S. Preheat to 37°C.

[0047] Prepare pituitary cell digestion solution (0.25% trypsin + 0.1% collagenase in HBSS / PBS + 0.1% BSA), filter to sterilize. Preheat to 37°C.

[0048] Prepare the cell culture stop solution (0.05% trypsin + 0.02% EDTA in HBSS / PBS), and filter to sterilize. Preheat to 37°C.

[0049] Prepare HBSS / PBS (washing buffer) containing 0.1% BSA.

[0050] Prepare working solutions of test compounds: Dilute the stock solution to the desired concentration gradient (usually 10 times the final concentration) with complete culture medium.

[0051] Prepare the positive control working solution.

[0052] 2. Equipment preheating: Preheat biosafety cabinet, water bath, centrifuge (to room temperature), and incubator.

[0053] 3. Consumable sterilization: Ensure that all consumables that come into contact with cells are sterile.

[0054] 4. Pituitary gland acquisition: The experimental rats were euthanized in accordance with animal ethics (usually CO2 asphyxiation + cervical dislocation), and the pituitary gland was quickly and aseptically removed and placed in a pre-cooled culture dish containing HBSS / PBS + 0.1% BSA.

[0055] Note: The procedure should be performed quickly to avoid tissue drying and prolonged hypoxia.

[0056] II. Pituitary cell isolation and primary culture (operated in a biosafety cabinet) 1. Tissue Transfer and Washing: Transfer the pituitary gland to a new culture dish containing pre-cooled HBSS / PBS + 0.1% BSA. Carefully remove surrounding membrane tissue and blood, and wash the pituitary gland 2-3 times with HBSS / PBS + 0.1% BSA.

[0057] 2. Mechanical dispersion: The pituitary gland is cut into pieces of approximately 1 mm using sterile surgical scissors. 3 Small pieces.

[0058] 3. Enzyme digestion: Add an appropriate amount of preheated digestion solution (about 3-5 mL / pituitary gland), incubate in a 37°C water bath, and gently pipette every 5-10 minutes (using a sterile Pasteur tube or pipette).

[0059] Digestion typically takes 20-40 minutes, with close observation. Stop digestion when the tissue mass becomes noticeably loose and a large number of cells are free.

[0060] 4. Termination of Digestion and Cell Collection: Terminate digestion by adding an equal volume of preheated complete culture medium (containing FBS to inhibit trypsin activity). Filter the cell suspension through a 70μm or 100μm sterile cell strainer into a 50mL sterile centrifuge tube to remove undigested tissue fragments. Rinse the strainer and culture dish with a small amount of complete culture medium and transfer to the centrifuge tube.

[0061] 5. Centrifugation and washing: Centrifuge at 200-300 xg at room temperature for 5 minutes. Carefully discard the supernatant. Resuspend the cell pellet in 10 mL of preheated complete culture medium and gently pipette to mix. Repeat the centrifugation and washing once.

[0062] 6. Cell Counting and Resuspension: Resuspend the cell pellet in an appropriate amount of complete culture medium. Take a small amount of cell suspension and count viable cells using trypan blue staining or an automated cell counter. Adjust the cell concentration to the desired density (1.0 × 10⁻⁶). 5 -2.0×10 5 (cells / mL for 24-well plates).

[0063] 7. Seeding and Adhesion: Seed the cell suspension into multi-well plates (24-well plates, 1 mL per well) that have been pre-washed with complete culture medium. Gently agitate the plate to distribute the cells evenly. Place the culture plate in an incubator at 37°C, 5% CO2, and 95% humidity. Incubate for 24-48 hours to allow the cells to fully adhere and recover.

[0064] III. FSH Secretion Inhibition Test 1. Change the medium (after the cells recover): Carefully aspirate the old culture medium from the wells.

[0065] Gently wash the cells 1-2 times with pre-warmed HBSS / PBS containing 0.1% BSA (to remove residual serum and dead cells).

[0066] Key point: This step aims to remove the effect of serum on basal FSH secretion, putting cells in a "starved" state.

[0067] 2. Pre-incubation: Add basal medium containing 0.1% BSA (such as DMEM without phenol red) or low-serum medium (such as containing 1% FBS). Continue culturing for 2-4 hours or overnight. This allows the cells to adapt to low-serum / serum-free conditions and stabilize basal secretion.

[0068] 3. Add the test compound (inhibin B mutant protein): remove the pre-incubation medium.

[0069] Add fresh basal culture medium (containing 0.1% BSA, phenol red-free) containing different concentrations of the test compound (inhibin B mutant protein), positive control (inhibin B standard), or solvent. Typically, add 500 μL per well (4-well plate).

[0070] Key point: Duplicate wells (n=3-6) are required for each concentration and control. Setup: Blank wells: containing only basal culture medium (cell-free), used for background detection.

[0071] Basal secretion control group: basal culture medium + solvent (Vehicle Control).

[0072] Test compound set: basal culture medium + series of test compounds (inhibin B mutant protein).

[0073] Positive control group: basal culture medium + positive control of known effective concentration (inhibin B standard: Elabscience E-EL-H0313 standard).

[0074] Gently shake the board to mix.

[0075] 4. Incubation: Place the culture plate back into a 37°C, 5% CO2 incubator for the specified incubation time. The incubation time needs to be optimized, typically 4-24 hours (e.g., 6 hours, 18 hours). Excessive incubation time may lead to changes in cell state or FSH degradation.

[0076] Avoid disturbance: Try to avoid moving the culture plate during incubation.

[0077] IV. Sample Collection and Preservation 1. Collect the supernatant: After incubation, carefully remove the culture plate from the incubator. Gently aspirate the supernatant from each well using a pipette (avoiding adhering cells). Transfer the supernatant to a labeled sterile centrifuge tube or EP tube.

[0078] 2. Centrifugation: Centrifuge the collected supernatant at 4°C and 1000-2000 xg for 10 minutes to remove any detached cell debris. Carefully aspirate the clear supernatant and transfer it to a new, labeled centrifuge tube / EP tube.

[0079] 3. Storage: Immediately freeze the sample at -20°C or -80°C until FSH testing is performed. Avoid repeated freeze-thaw cycles.

[0080] V. Detection of FSH levels using ELISA The FSH level in rat pituitary cell culture supernatant was measured strictly according to the instructions of the selected rat FSH ELISA kit (manufacturer: Elabscience, catalog number: E-EL-R0391). The main steps included: 1. Add 100 μl of cell supernatant to a pre-coated ELISA plate and incubate at 37°C for 90 min.

[0081] 2. After discarding the liquid in the plate, immediately add 100 μl of biotinylated antibody working solution and incubate at 37°C for 60 min.

[0082] 3. Discard the liquid inside the plate and wash the plate 3 times.

[0083] 4. Add 100 μL of HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 min, discard the liquid in the plate, and wash the plate 5 times.

[0084] 5. Add 90 μL of substrate solution to each well and incubate at 37°C for about 15 min.

[0085] 6. Add 50 μL of stop solution to each well.

[0086] 7. Immediately take readings at a wavelength of 450 nm and process the data. See the results below. Figure 2 And Table 4.

[0087] Table 4. FSH detection data by ELISA method

[0088] Figure 2 Both Table 4 and Table 5 show that, in the results of biological cell verification, the activity of the INHB mutant of this application is superior to that of the wild-type INHB standard. This technology utilizes a highly efficient and convenient recombinant expression method, which increases the yield of INHB and lays a certain foundation for subsequent antibody development.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a highly active inhibin B protein, characterized in that: Includes the following steps: S1, the α chain and β chain of inhibin B protein were mutated to obtain α chain mutants and β chain mutants with amino acid sequences as shown in SEQ ID No. 1 and 2; S2, α-chain mutants and β-chain mutants were inserted into the expression vector and transfected into CHO cell lines to express recombinant proteins. The recombinant proteins were then purified to obtain highly active inhibin B protein.

2. The method for preparing a highly active inhibin B protein as described in claim 1, characterized in that: The carrier is the PTT5 carrier.

3. The method for preparing a highly active inhibin B protein as described in claim 1, characterized in that: First, the α-chain mutant and the β-chain mutant are linked with a flexible linker peptide to obtain a co-expression sequence, which is then inserted into the expression vector.

4. The method for preparing a highly active inhibin B protein as described in claim 3, characterized in that: The amino acid sequence of the flexible linker peptide is (GGGGS)3.

5. A nucleic acid molecule, characterized in that: The nucleic acid molecules encode amino acid sequences such as the α-chain mutant and β-chain mutant shown in SEQ ID No. 1 and 2.

6. A recombinant expression vector, characterized in that: It includes the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that: It includes the recombinant expression vector as described in claim 6.

8. A highly active inhibin B protein, characterized in that: It is prepared by the method described in any one of claims 1-4.

9. The use of the highly active inhibin B protein as described in claim 8 in the preparation of diagnostic reagents for male fertility assessment, female ovarian reserve function testing, or reproductive system tumor markers.

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