GNRH recombinant protein vaccine as well as preparation method and application thereof

Abstract

The invention discloses a GNRH recombinant protein vaccine as well as a preparation method and application thereof. The GNRH recombinant protein is a recombinant protein containing a GnRH peptide fragment and comprises human CD5 signal peptide and five GnRH repetitive peptide fragments which are connected in series, the sequence of the human CD5 signal peptide connected in series is shown as a sequence 3, and the sequence of the five GnRH repetitive peptide fragments is shown as a sequence 1 in a sequence table. A vaccine prepared from the recombinant protein containing the GnRH peptide fragment can generate a high-level anti-GnRH antibody and effectively reduce the hormone level of male animals, so that testis atrophy and sexual characteristics are inhibited. The vaccine is stable in preparation process, low in cost, suitable for large-scale production and beneficial to industrial application.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and specifically to a GNRH recombinant protein vaccine and a preparation method thereof. Background Art

[0002] With the development of the pet economy, more and more people are keeping dogs and cats as pets. While pet dogs and cats bring convenience and enjoyment to humans, they can also cause unnecessary trouble. When in estrus, adult cats howl loudly, become restless, become extra clingy, or even become aggressive. Male dogs in estrus may urinate randomly, mount, become abnormally excited, lose their appetite, or become aggressive. Some pets emit an unpleasant odor during estrus, affecting indoor air quality. For these reasons, many pet owners choose to have their pets sterilized. However, surgical sterilization is not only expensive but also carries risks of anesthesia, bleeding, infection, and even death. For some valuable pets, sterilization can render them infertile. Therefore, researchers are constantly searching for alternatives. In recent years, immunocastration, as a new, animal-friendly, and painless alternative, has been successfully studied both domestically and internationally.

[0003] The core antigen of immune castration is gonadotropin-releasing hormone (GnRH). In 1971, Polish-American biologist Viktor Schally first isolated GnRH, also known as luteinizing hormone-releasing hormone (LHRH), from the porcine hypothalamus, for which he was awarded the 1977 Nobel Prize in Medicine. GnRH, located at the upper end of the hypothalamic-pituitary-gonadal axis (HPG axis), is the core initiating and controlling hormone for the physiological functions of the entire reproductive axis and the optimal target hormone for reproductive immune regulation. Studies have shown that antibodies produced by active immunization with GnRH can neutralize endogenous GnRH, thereby inhibiting the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), inducing gonadal atrophy, terminating gametogenesis, suppressing reproductive behavior, and leading to infertility in both male and female animals.

[0004] GNRH monomers cannot be used directly due to their small molecular weight and short half-life, failing to maintain sufficient immunity after immunization in animals. However, conjugating GNRH peptides to proteins or directly linking them to recombinant proteins can significantly enhance immune efficacy. Furthermore, recombinant protein production is mature and cost-effective, providing more reliable vaccine quality and facilitating large-scale production. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a recombinant protein containing a GnRH peptide segment, which is expressed in HEK293 mammalian cells and has antigenicity and immunogenicity similar to that of the natural protein, and has the characteristic of strong immunogenicity.

[0006] A second object of the present invention is to provide nucleic acid molecules encoding the above-mentioned recombinant proteins, as well as recombinant eukaryotic vectors and recombinant cells into which these nucleic acid molecules are inserted for recombinant expression of the above-mentioned recombinant proteins.

[0007] The third object of the present invention is to provide a method for preparing and using the above-mentioned recombinant protein containing the GnRH peptide segment.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a recombinant protein containing a GnRH peptide segment, which comprises a tandem human CD5 signal peptide and five GnRH repeat peptide segments, wherein the tandem human CD5 signal peptide sequence is shown in Sequence 3, and the sequence of the five GnRH repeat peptide segments is shown in Sequence 1 in the sequence table.

[0010] Preferably, the recombinant protein is composed of a human CD5 signal peptide sequence, a sequence encoding five GnRH repeat peptide segments, and a purification tag 6×His sequence, which are fused in series.

[0011] Preferably, the gene sequence encoding five GnRH repeat peptide segments has the amino acid sequence shown in Sequence 1; the human CD5 signal peptide has the amino acid sequence shown in Sequence 3.

[0012] The present invention also provides a nucleic acid molecule encoding the aforementioned recombinant protein, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a human CD5 signal peptide and a nucleotide sequence encoding five GnRH repeat peptide segments.

[0013] Specifically, the nucleic acid molecule sequence encoding the human CD5 signal peptide is shown in Sequence 4, and the nucleic acid molecule sequence encoding five GnRH repeat peptide segments is shown in Sequence 2.

[0014] The present invention also provides a recombinant vector, which comprises the aforementioned nucleic acid molecule and, in addition, an expression control sequence operatively linked to the nucleic acid molecule sequence.

[0015] In the present invention, "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in host cells. For example, vectors include plasmids, phages, cosmids, artificial chromosomes, phages, and animal viruses. Types of animal viruses used as vectors include lentiviruses, adenoviruses, herpes viruses, poxviruses, baculoviruses, and papillomaviruses. A vector may contain multiple expression control elements, including promoter sequences, reverse transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0016] In a specific embodiment of the present invention, the recombinant vector is constructed by inserting the nucleic acid molecule shown above into the pCDNA3.1 vector (Thermo Fisher Scientific).

[0017] The present invention also provides a method for constructing the aforementioned recombinant vector, which comprises inserting the aforementioned nucleic acid molecule into a pCDNA3.1 vector.

[0018] Specifically, the construction method of the recombinant vector of the present invention is as follows:

[0019] The human CD5 signal peptide (coding sequence such as sequence 4), 5 GnRH repeat peptide segments and 6×His sequence were merged respectively, and the termination codon TAA was added to the C-terminus of the gene sequence to artificially synthesize the complete gene sequence expressing the recombinant protein containing the GnRH peptide segment. The gene sequence was then cloned into the expression vector pCDNA3.1 using molecular cloning methods to construct a recombinant eukaryotic expression plasmid.

[0020] Specifically, the recombinant expression vector is constructed by inserting the nucleotide sequence shown in sequence 5 in the sequence table between the Nhe I and Not I enzyme recognition sites of the expression vector pCDNA3.1 to obtain a recombinant eukaryotic expression plasmid pCDNA3.1-GnRH that expresses a recombinant protein containing a GnRH peptide segment.

[0021] The present invention also provides a recombinant cell, into which the aforementioned nucleic acid molecule is introduced or the aforementioned recombinant vector is transfected.

[0022] The term "recombinant cell" as used herein refers to a cell into which a nucleic acid molecule or vector has been introduced, and includes many cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells and Sf9, or fibroblasts, CHO cells, COS cells, BHK cells, HEK293 cells, etc.

[0023] In a specific embodiment of the present invention, the recombinant cell is a HEK293 cell transformed with the above-mentioned recombinant expression vector.

[0024] The present invention also provides a method for producing a recombinant protein containing a GnRH peptide segment using the recombinant cells described above, the method comprising culturing the recombinant cells described above under suitable conditions and recovering the recombinant protein, the method comprising the following steps: transfecting a host cell or the recombinant cell described above with a recombinant expression vector, harvesting the culture supernatant of the transfected cells multiple times, and / or culturing the recombinant cells in a target culture medium, harvesting the culture supernatant for purification, and obtaining the recombinant protein containing the GnRH peptide segment described above.

[0025] Use of the above-mentioned recombinant protein containing GnRH peptide segment or recombinant eukaryotic expression vector or the recombinant cell according to claim 5 in the preparation of a vaccine for inhibiting animal reproductive behavior.

[0026] Preferably, the animal is a dog or a cat.

[0027] This vaccine, prepared from a recombinant protein containing a GnRH peptide, can produce high levels of anti-GnRH antibodies and effectively reduce hormone levels in male animals, causing testicular atrophy and suppressing sexual characteristics. The vaccine preparation process is stable, low-cost, and can be produced on a large scale, facilitating industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a map of the construction of a recombinant eukaryotic expression plasmid containing a GnRH peptide segment.

[0029] Figure 2 The figure shows the SDS-PAGE detection results of the recombinant protein containing GnRH peptide after expression and purification. Lane 1 is the purified GnRH eukaryotic expression protein (marked by a black arrow), and lane 2 is PageRuler pre-stained protein Marker (ThermoFisher Scientific Company).

[0030] Figure 3 These are the antibody test results of male cats at different times after immunization, including GNRH recombinant protein vaccine, GNRH synthetic peptide vaccine, commercial control drug, and blank control.

[0031] Figure 4The results of antibody testing in male dogs at different times after immunization are shown. They are GNRH recombinant protein vaccine, GNRH synthetic peptide vaccine, commercial control drug, and blank control.

[0032] Figure 5 These are the testosterone test results of male cats at different times after immunization, including GNRH recombinant protein vaccine, GNRH synthetic peptide vaccine, commercial control drug, and blank control.

[0033] Figure 6 These are the testosterone test results of male dogs at different times after immunization, including GNRH recombinant protein vaccine, GNRH synthetic peptide vaccine, commercial control drug, and blank control.

[0034] Figure 7 The changes in organ volume of male cats at different times after immunization are shown, including GNRH recombinant protein vaccine, GNRH synthetic peptide vaccine, commercial control drug, and blank control.

[0035] Figure 8 The changes in organ volume of male dogs at different times after immunization are shown, including GNRH recombinant protein vaccine, GNRH synthetic peptide vaccine, commercial control drug, and blank control. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are further described below with reference to specific examples. It should be understood by those skilled in the art that modifications and variations may be made within the spirit and scope of the claims, but all fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources.

[0038] The plasmid pCDNA3.1 used in the following examples was purchased from Thermo Fisher Scientific; the transfection reagent PEI was purchased from Alfa Aesar; HEK293 cells were purchased from the American Type Culture Collection (ATCC); and the HisTrap HP 5 ml nickel prepacked column and HiLoad Superdex 16 / 600 200 pg gel filtration column were purchased from GE.

[0039] Example 1: Expression and purification of recombinant protein containing GnRH peptide

[0040] When expressing GnRH protein using an E. coli expression system, the expression yield was found to be low; when expressing GnRH protein using an insect cell-baculovirus expression system, the protein precipitated. In comparison, in the present invention, the codon-optimized expression gene is secreted and expressed in a 293 mammalian cell expression system to obtain the target protein GnRH. This results in low levels of impurities in the supernatant, and subsequent scale-up of subunit vaccine production requires only a single step of tangential flow ultrafiltration and concentration to meet the purity requirement of over 80%. The industrialized process is essentially endotoxin-free, while maintaining similar advantages to natural GnRH, resulting in excellent safety and immunogenicity after administration to target animals.

[0041] 1. Vector construction and synthesis

[0042] First, the human CD5 signal peptide (having the amino acid sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4), five GnRH repeat peptide segments (the amino acid sequence is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2) and the 6×His sequence were combined, and the termination codon TAA was added to the C-terminus of the gene sequence to artificially synthesize the complete gene sequence expressing the GnRH repeat peptide segment (as shown in SEQ ID NO: 5); then, the fragment was cloned into the Nhe I and Not I enzyme recognition sites of the expression vector pCDNA3.1 using molecular cloning methods to construct the recombinant eukaryotic expression plasmid pCDNA3.1-GnRH ( Figure 1 ), this step was directly handed over to Suzhou GeneWeizhi Gene Synthesis Co., Ltd.

[0043] 2. Cultivation of recombinant bacterial solution

[0044] The artificially synthesized positive bacterial liquid was punctured and inoculated into 5 ml of LB medium containing ampicillin antibiotics, and cultured overnight at 37°C and 220 rpm with shaking.

[0045] 3. Extraction of recombinant eukaryotic expression plasmid

[0046] Refer to the TIANGEN Plasmid Extraction Kit (Cat. No. DP103) and add anhydrous ethanol to the rinse solution PW according to the instructions before use.

[0047] (1) Column equilibration step: Add 500 μl of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0048] (2) Take 5 ml of overnight culture solution and add it to a centrifuge tube. Centrifuge at 12,000 rpm for 1 min and remove the supernatant as much as possible.

[0049] (3) Add 250 μl of solution P1 to the centrifuge tube containing the bacterial pellet and thoroughly suspend the bacterial pellet using a pipette or vortex oscillator.

[0050] (4) Add 250 μl of solution P2 to the centrifuge tube and gently invert it 6 to 8 times to fully lyse the bacteria.

[0051] (5) Add 350 μl of solution P3 to the centrifuge tube and immediately mix thoroughly by gently inverting the tube 6-8 times. A white flocculent precipitate will form. Centrifuge at 12,000 rpm for 10 min.

[0052] (6) Use a pipette to transfer the supernatant collected in the previous step to the adsorption column CP3 (place the adsorption column in the collection tube), taking care not to aspirate the precipitate. Centrifuge at 12,000 rpm for 30-60 seconds. Discard the waste liquid in the collection tube and place the adsorption column CP3 in the collection tube.

[0053] (7) Add 600 μl of rinse solution PW to the adsorption column CP3, centrifuge at 12,000 rpm for 30 to 60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.

[0054] (8) Repeat step (7).

[0055] (9) Place the adsorption column CP3 in a collection tube and centrifuge at 12,000 rpm for 2 min to remove the remaining rinse solution in the adsorption column.

[0056] (10) Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μl of elution buffer EB to the middle part of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge it at 12,000 rpm for 2 minutes to collect the plasmid solution into the centrifuge tube.

[0057] (11) Take 2 μl of recombinant plasmid DNA and measure its concentration using Nanodrop2000 A260.

[0058] 4. Transfection of recombinant eukaryotic expression plasmid and expression of recombinant protein containing GnRH repeat peptide

[0059] (1) Transfection of vector DNA: HEK293 cells were seeded in a 15 cm diameter cell culture dish. When the cell density reached 1.2×10 7 ~1.5×10 7 The recombinant expression plasmid pCDNA3.1-GnRH was transfected with PEI (Alfa Aesar) at a concentration of 1 mg / ml.

[0060] The specific operation is as follows: add 1ml OPTI-MEM medium to a 4ml Eppendorf tube, then add 30μg of recombinant expression plasmid pCDNA3.1-GnRH in turn and mix gently; add 850μl OPTI-MEM medium to another 1.5ml Eppendorf tube, then add 150μl PEI transfection reagent and mix; then add the OPTI-MEM medium containing the transfection reagent to the Eppendorf tube containing the recombinant plasmid and mix gently. After standing at room temperature for 15 minutes, add it to the culture dish with HEK293 cells, culture at 37°C and 5% CO2 for 5 hours, and replace the medium with serum-free DMEM medium.

[0061] (2) Expression of recombinant protein: After culturing the HEK293 cells transfected with the recombinant DNA plasmid for 72 h, the culture supernatant was harvested and stored at 4°C until use; serum-free DMEM medium was added to the cell culture dish again, and the supernatant was harvested after further culturing for 72 h.

[0062] 5. Purification of expressed GnRH recombinant protein

[0063] (1) 1000 ml of culture supernatant was filtered through a 0.22 μm filter membrane and loaded onto a HisTrap HP 5 ml nickel pre-packed column using a peristaltic pump at a flow rate of 1 ml / min. After loading, the column was washed with a buffer containing 20 mM imidazole (20 mM NaCl, 50 mM Tris-HCl, pH 8.0) on an AKTA purifier to remove impurities and unbound proteins. The target protein was then washed with a buffer containing 100 mM imidazole and then with a buffer containing 300 mM imidazole. 20 μl of protein sample was collected at each stage, 5 μl of 5× loading buffer was added, and the column was placed on ice after boiling in a water bath for 10 minutes.

[0064] (2) Take 10 μl of protein sample for SDS-PAGE electrophoresis, and collect the sample containing the target protein based on the SDS-PAGE electrophoresis results for the next gel filtration chromatography.

[0065] (3) The sample obtained in the previous step was purified by AKTA purification instrument through GE HiLoad Superdex 16 / 600 200pg column to obtain the target protein. 10μl protein sample was taken for SDS-PAGE electrophoresis identification. There was a clear target band around 15KDa ( Figure 2 ).

[0066] (4) The target protein was concentrated using a 10kD ultrafiltration tube. The protein concentration was determined to be 2 mg / ml using Nanodrop2000. The volume was 40 ml, and the expression level of GnRH protein was calculated to be as high as 100 mg / L.

[0067] 6. Chemical synthesis of five GnRH repeat peptides

[0068] Five GnRH repeating peptide segments were synthesized, purified, and freeze-dried using the Fmoc chemical method to prepare a synthetic peptide antigen with a purity of approximately 80%.

[0069] Example 2: Preparation of GnRH vaccine

[0070] 1. Preparation of antigen aqueous phase

[0071] 1.1 Preparation of GNRH recombinant protein vaccine GNRH recombinant protein antigen was sterile filtered, and after concentration determination, the protein antigen was diluted to 100 μg / ml with sterile water for injection.

[0072] 1.2 Preparation of GNRH synthetic peptide vaccine: The freeze-dried GNRH synthetic peptide was dissolved in distilled water, diluted to 100 μg / ml, and sterile filtered.

[0073] 2. Adjuvant: Prepare sterile SEPPIC MONTANIDE ISA 201VG adjuvant.

[0074] 3. Vaccine Preparation: Under sterile conditions, heat the antigen aqueous phase and 201 adjuvant to 32°C. Activate magnetic stirring and set the hot plate to 32°C. Slowly add the aqueous phase to the adjuvant in a 1:1 mass ratio. Maintain thorough stirring at >31°C for 5 minutes. After stirring, cool in a 20°C cold bath for 1 hour. Prepare the GNRH protein vaccine and synthetic peptide vaccine, respectively, at a concentration of 50 μg / ml for both the protein and synthetic peptide antigens.

[0075] Example 3: Evaluation of the immune effect of GnRH vaccine in dogs and cats

[0076] 1. Experimental Animals

[0077] 8 adult male dogs and 8 male cats.

[0078] 2. Test methods and results

[0079] 2.1 Eight adult male dogs and eight male cats were divided equally into four groups, with two animals in each group. Groups 1 and 2 were injected subcutaneously in the neck with either GNRH protein vaccine or GNRH synthetic peptide vaccine, 1.0 ml per animal. Two weeks after immunization, the animals received a booster immunization with the same vaccine and the same dose. They were housed normally and had free access to food and water. Group 3 was injected with Virbac's fast-acting aphrodisiac implant as a control. Group 4 was not immunized and served as a blank control. Blood was collected one day before immunization and every two weeks after immunization, and serum was separated for antibody and hormone testing. Testicular volume was measured on the day of the second immunization and four weeks after the second immunization in male dogs and male cats.

[0080] 2.2 Antibody determination The GNRH protein was coated on the enzyme-labeled plate at 1ug / ml and coated overnight at 2-8°C. Blocked with 1.5% BSA blocking solution, the serum to be tested was diluted in multiples with PBS, added to the enzyme-labeled plate, and incubated at 37°C for 1 hour. The diluted rabbit anti-cat secondary antibody and goat anti-dog secondary antibody were added respectively, and incubated at 37°C for 1 hour. The substrate solution TMB was added for color development, and the stop solution was added to terminate the reaction. The OD value at a wavelength of 450nm was read on the enzyme-labeled instrument. It can be seen that the antibodies in the dog and cat GNRH protein vaccine groups began to rise from the second week, reached a peak in the 4th to 6th week, and continued to be stable until the 12th week. The GNRH synthetic peptide vaccine also produced higher antibodies after immunization, but it slowly declined after 6-8 weeks. Higher antibodies were also detected in the market drug control group from the 4th week, and remained at a high level until 12 weeks. The antibody levels of the GNRH protein vaccine and the market drug were similar, and the overall antibody level was higher than that of the synthetic peptide vaccine, and the duration was longer. The blank control group always maintained a low antibody level ( Figure 3 、 Figure 4 ).

[0081] 2.3 Hormone determination was performed using the iodine-125 testosterone radioimmunoassay. It can be seen that testosterone levels in both the cat GNRH protein vaccine and synthetic peptide vaccine groups decreased significantly from the 2nd to 4th week, while the canine GNRH protein vaccine group maintained a relatively stable decrease until 12 weeks, while the synthetic peptide vaccine group showed a rebound in hormone levels from 6th to 8th week. The marketed drug control group also began to decrease from the 2nd to 4th week and remained stable until 12 weeks. The blank control group fluctuated at a high level throughout the whole period ( Figure 5 、 Figure 6 ).

[0082] 2.4 Organ Monitoring The testicular volume of male dogs and cats in each experimental group was measured on day 0 and 4 weeks after the second vaccination. Vernier calipers were used for measurement, volume = length × height × width, and the average value was taken after measuring the volume of both testicles. It can be seen that the testicular volume of male dogs and cats in the GNRH protein vaccine group decreased by 16.9% and 15.1% respectively 4 weeks after the second vaccination compared with the day of the second vaccination, the testicular volume of male dogs and cats in the synthetic peptide vaccine group decreased by 10.6% and 11.9% respectively 4 weeks after the second vaccination compared with the day of the second vaccination, and the testicular volume of male dogs and cats in the market drug control group decreased by 16.4% and 14.8%. The testicular volume of dogs and cats in the blank control group increased by 1.2% and decreased by 5.3% respectively ( Figure 7 、 Figure 8 ).

[0083] From the experimental results, it can be seen that the GNRH protein vaccine can more effectively reduce the hormone levels of male dogs and cats than the GNRH repeat peptide group, and cause organ atrophy to a certain extent.

Claims

1. A recombinant protein containing a GnRH peptide segment, comprising a tandem human CD5 signal peptide and five GnRH repeat peptide segments, wherein: The sequence of the tandem human CD5 signal peptide is shown in Sequence 3, and the sequence of the five GnRH repeat peptide segments is shown in Sequence 1 in the sequence listing.

2. The recombinant protein according to claim 1, characterized in that The recombinant protein is composed of a human CD5 signal peptide sequence, a sequence encoding five GnRH repeating peptide segments and a purification tag 6×His sequence, which are sequentially fused in series.

3. A recombinant eukaryotic expression vector for expressing a recombinant protein containing a GnRH peptide segment according to claim 1 or 2, characterized in that: The recombinant protein containing the GnRH peptide segment and the coding gene of the human CD5 signal peptide as described in claim 2 are cloned into the starting vector pCDNA3.1 to obtain a recombinant eukaryotic expression vector expressing the GnRH peptide segment gene; wherein, the amino acid sequence of the human CD5 signal peptide is shown in Sequence 3, and the nucleotide sequence of the human CD5 signal peptide is shown in Sequence 4.

4. The recombinant eukaryotic expression vector according to claim 3, characterized in that The recombinant eukaryotic expression vector is a recombinant eukaryotic expression vector expressing the GnRH peptide gene obtained by cloning the nucleotide fragment shown in sequence 5 in the sequence table between the Nhe I and Not I enzyme recognition sites of pCDNA3.1 and screening.

5. A recombinant cell line containing the recombinant eukaryotic expression vector according to claim 3 or 4.

6. The method for preparing a recombinant protein containing a GnRH peptide segment according to claim 1 or 2, characterized in that: The recombinant protein eukaryotic expression vector according to claim 3 or 4 is transfected into HEK293 mammalian cells, and the recombinant protein containing the GnRH peptide segment is obtained through culture and purification.

7. Use of the recombinant protein containing the GnRH peptide segment according to claim 1 or 2, the recombinant eukaryotic expression vector according to claim 3 or 4, or the recombinant cell line according to claim 5 in the preparation of a vaccine for inhibiting animal reproductive behavior.

8. The use according to claim 7, characterized in that The animal is a dog or a cat.

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