Protein, nucleic acid, expression vector, castration vaccine, method and application

Abstract

The invention relates to the field of animal immune castration vaccines, in particular to a protein, a nucleic acid, an expression vector, a castration vaccine, a method and application. The protein is as shown in SEQ ID NO: 1. The nucleic acid is as shown in SEQ ID NO: 2. The expression vector comprises the nucleic acid. The castration vaccine contains the protein, the nucleic acid or the expression vector as an active ingredient. The method comprises the step of preparing the nucleic acid and / or the expression vector. The application comprises preparation of castration vaccines. The protein, the nucleic acid, the expression vector and / or the castration vaccine can generate immune response in a mouse body, reduce testosterone and estrogen levels in the mouse body and generate an obvious trend effect.

Description

Technical Field

[0001] The present application relates to the field of animal immune castration vaccines, and specifically to proteins, nucleic acids, expression vectors, castration vaccines, methods and applications. Background Art

[0002] Immunocastration of animals is a method that has many technical advantages over physical and chemical castration. The definition of immunocastration in the Veterinary Dictionary is: a technique that uses immunization to inhibit the development of sexual organs in order to achieve the purpose of castration. It mainly achieves the purpose of fertility control by inducing sufficiently high levels of antibodies against antigens that play an important role in the reproductive process, blocking the reproductive hormone regulation pathway through antigen-antibody reactions. Immunocastration has the advantages of being safe and convenient, having a mild effect, low animal stress, and being reversible. It has great advantages over surgical and chemical castration. However, the main problem is how to select suitable antigens with sufficient immunogenicity to obtain contraceptive products.

[0003] The core part of immunocastration is to select one or a combination of multiple reproductive-related hormones or receptors as targets, express exogenous castration genes in animals to produce antibodies and then neutralize endogenous hormones to obtain significant immune effects. At present, the main antigens for immunization are the zona pellucida (ZP) around the oocyte, which plays an important role in fertilization, gonadotropin-releasing hormone (GnRH), prolactin receptor (LH), follicle-stimulating hormone (FSH), etc. The above antigens can induce higher antibody levels through appropriate coupling and adjuvants, thus achieving a castration effect. However, the zona pellucida antigen (ZP) can only be used for female animals, and its castration effect on cats is uncertain. The immune duration of prolactin receptor antigen (LH) and follicle-stimulating hormone antigen (FSH) is relatively short. Summary of the invention

[0004] The present application example constructs a protein, which is used as the active ingredient of castration vaccine, and can obtain a protein vaccine with high activity, good immunogenicity and high biosafety. The protein can produce an immune response in mice, reduce the levels of testosterone and estrogen in mice, and produce a significant trend effect.

[0005] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0006] In a first aspect, the embodiment discloses a protein having an amino acid sequence as shown in SEQ ID NO:1.

[0007] In a second aspect, the embodiment discloses a nucleic acid having a nucleotide sequence as shown in SEQ ID NO:2.

[0008] In a third aspect, the embodiments disclose a castration vaccine, which has a protein as shown in SEQ ID NO: 1 as an active ingredient.

[0009] In a fourth aspect, the embodiments disclose a castration vaccine, which has the nucleic acid shown in SEQ ID NO: 2 as an active ingredient.

[0010] In a fifth aspect, the embodiment discloses an expression vector, which is a plasmid having a nucleotide sequence as shown in SEQ ID NO:2.

[0011] In a sixth aspect, an embodiment discloses a castration vaccine, which uses the expression vector described in the fifth aspect as an active ingredient.

[0012] In the seventh aspect, an embodiment discloses a method for preparing an expression vector, comprising: synthesizing a nucleic acid as shown in SEQ ID NO:4; performing PCR amplification on the nucleic acid as shown in SEQ ID NO:4 using a primer pair; obtaining a linearized fragment of a first expression vector; connecting the PCR amplification product of the nucleic acid as shown in SEQ ID NO:4 with the linearized fragment of the first expression vector; transforming the obtained connection product into Escherichia coli; screening positive clones from transformants; and extracting a second expression vector from the positive clones or their culture.

[0013] In an eighth aspect, an embodiment discloses a method for preparing a protein as shown in SEQ ID NO: 1, comprising: transferring the expression vector described in the fifth aspect into Pichia pastoris; screening positive clones from the transformants to obtain an expression strain; inoculating the expression strain into a culture medium for inducing expression, and collecting the protein as shown in SEQ ID NO: 1 from the culture.

[0014] In the ninth aspect, the embodiments disclose the use of the protein described in the first aspect, the nucleic acid described in the second aspect, or the expression vector described in the fifth aspect in the preparation of a castration vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The Western-Blot test results of the supernatant containing the target protein at different fermentation times provided in the examples. Figure 1 A is the detection graph, M is the protein maker, lanes 1-6 are the supernatant samples of the expression strain at 24h, 48h, 72h, 96h, 120h and 144h of fermentation, lane 7 is the supernatant sample of the control strain at 48h of fermentation, and lane 8 is the supernatant sample of the empty plasmid strain at 48h of fermentation. Figure 1 B is a statistical graph of gray values ​​of lanes 1 to 6.

[0016] Figure 2The Western-Blot test results of the supernatant containing the target protein obtained under different methanol induction conditions provided in the examples. Figure 2 A is the detection graph, M is the protein maker, lanes 1-6 are the supernatant samples obtained by fermenting the expression strains in the presence of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0% methanol, respectively, lane 7 is the supernatant sample obtained by fermenting the control strain in the presence of 1.5% methanol for 48 h, and lane 8 is the supernatant sample obtained by fermenting the empty plasmid strain in the presence of 1.5% methanol for 48 h. Figure 2 B is a statistical graph of gray values ​​of lanes 1 to 6.

[0017] Figure 3 The Western-Blot test results of the supernatant containing the target protein obtained at different fermentation temperatures provided in the examples. Figure 2 A is the detection graph, M is the protein maker, lanes 1-6 are the supernatant samples obtained by the expression strains under fermentation conditions of 26°C, 27°C, 28°C, 29°C, 30°C and 31°C, respectively, lane 7 is the supernatant sample obtained by the control strain under fermentation conditions of 30°C, and lane 8 is the supernatant sample obtained by the empty plasmid strain under fermentation conditions of 30°C. Figure 2 B is a statistical graph of gray values ​​of lanes 1 to 6.

[0018] Figure 4 The Western-Blot test results of the supernatant containing the target protein obtained at different fermentation pH values ​​provided in the examples. Figure 2 A is the detection diagram, M is the protein maker, lanes 1-7 are the supernatant samples obtained by the expression strain under the fermentation conditions of pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, and pH 7.0, respectively, lane 8 is the supernatant sample obtained by the control strain under the fermentation condition of pH 7.0, and lane 9 is the supernatant sample obtained by the empty plasmid under the fermentation condition of pH 7.0. Figure 4 B is a statistical graph of gray values ​​of lanes 1 to 7.

[0019] Figure 5 This is the protein profile obtained by strong anion exchange chromatography of the fermentation supernatant of the expression strain provided in the example. Figure 5 A is the protein absorption peak spectrum, and the number of tubes to be tested is collected from the beginning of the peak; Figure 5 B is the protein SDS-PAGE map, Figure 5 C is the protein WB result, M is the protein marker, and lanes 1-13 are the proteins eluted from the even-numbered tubes in tubes 8-32.

[0020] Figure 6The statistical results of testosterone (T) and estradiol (E2) in the blood were collected every 2 weeks after the initial immunization provided in the example.

[0021] Figure 7 The results of mouse testis and epididymis tissues were collected every 2 weeks after the initial immunization provided in the examples. Figure 7 A. Testis images of mice in the control group (C) and test group (T) at 2W, 4W and 6W. Figure 7 B is Figure 7 A. Statistical results of mouse testis weight. Figure 7 C is Figure 7 A. Statistical results of mouse testicular organ index obtained. Figure 7 D Pictures of mouse epididymal tissues of the control group (C) and the test group (T) at 2W, 4W and 6W. Figure 7 E is Figure 7 A: Statistical results of mouse epididymal tissue weight obtained. Figure 7 F is Figure 7 A. Statistical results of mouse epididymal tissue organ index.

[0022] Figure 8 HE staining of testicular tissue sections taken every two weeks after the initial immunization provided in the examples. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0024] The term "castration" as used herein, also known as castration, refers to the removal of an animal's gonads or elimination of the gonadal function of an animal by physical, chemical or immunological means. The purpose of castration is to eliminate the sexual desire and reproductive ability of livestock or poultry, making the temperament of livestock or poultry docile and hardy, and easier to manage; it can also improve the meat quality and increase the yield of meat animals; it can also control the mating behavior in the herd, which is conducive to the reproduction and breeding of good breeds.

[0025] On the one hand, the embodiment discloses a protein having an amino acid sequence as shown in SEQ ID NO: 1. The protein can also exist in the form of a dimer or a polymer. When the protein is used to immunize an animal, it can effectively stimulate the animal's immune response, improve the immune effect, and enhance the level of neutralizing antibodies. It is also safer and has a shorter safety evaluation cycle, and has broad application prospects in production.

[0026] In one aspect, the embodiments disclose a nucleic acid having a nucleotide sequence as shown in SEQ ID NO: 2. The nucleic acid can be transcribed and translated in a host cell to obtain a protein as shown in SEQ ID NO: 1. The nucleic acid can be a DNA molecule, an RNA molecule, or a chimera of a DNA and RNA molecule, as long as it can be transcribed and translated in a host cell to obtain a protein as shown in SEQ ID NO: 1. For example, the nucleic acid can have a nucleotide sequence encoding a protein as shown in SEQ ID NO: 1.

[0027] The term "coding sequence" or "nucleotide sequence encoding a specific protein" is a DNA sequence that is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' end (amino terminus) and a translation stop codon at the 3' end (carboxyl terminus). Coding sequences can include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (such as mammalian) DNA, and even synthetic DNA sequences. The transcription termination sequence is usually located at the 3' end of the coding sequence.

[0028] In another aspect, the embodiment further discloses an expression vector, which is a plasmid having a nucleotide sequence as shown in SEQ ID NO: 2. The nucleic acid as shown in SEQ ID NO: 2 is operably linked to a basic plasmid. The basic plasmid can be a plasmid capable of replicating, transcribing, translating and expressing proteins in animals or microorganisms, such as pPICZαA, pCAG series plasmids or pCIG plasmids.

[0029] The condition that a coding sequence is "operably" linked to the base plasmid is that RNA polymerase will transcribe the two coding sequences into mRNA, and then translate them into the chimeric polypeptide encoded by the two coding sequences. These coding sequences need not be contiguous to each other, as long as the transcribed sequences are ultimately processed to produce the desired chimeric protein. A control sequence is "operably linked" to a coding sequence when it controls the transcription of the coding sequence.

[0030] A control sequence "directs the transcription of a coding sequence" in a cell when RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, and then into the polypeptide encoded by the coding sequence.

[0031] A "host cell" is a cell that has been or is capable of being transformed or is capable of being transformed by an exogenous DNA sequence.

[0032] When such exogenous DNA is introduced into the cell membrane, when the exogenous DNA "transforms" the cell, the exogenous DNA may or may not be integrated (covalently linked) into the chromosomal DNA to form the genome of the cell. In prokaryotes and yeast, for example, the exogenous DNA can remain as a free element, such as a plasmid. As for eukaryotic cells, stably transformed cells are cells in which the exogenous DNA has been integrated into the chromosome so that it can be inherited to daughter cells through chromosome replication. The ability of eukaryotic cells to establish cell lines or clones composed of a population of daughter cells containing the exogenous DNA demonstrates this stability.

[0033] Based on this, the embodiments also disclose a castration vaccine. In some embodiments, the castration vaccine has the protein shown in SEQ ID NO: 1 as an active ingredient. In some embodiments, the castration vaccine has the nucleic acid shown in SEQ ID NO: 2 or an expression vector carrying the nucleotide shown in SEQ ID NO: 2 as an active ingredient.

[0034] Among them, "active ingredient" is an ingredient that can produce an immune response in an animal.

[0035] An "immune response" to an antigen or vaccine is the development of a cellular and / or antibody-mediated immune response in a host to the desired composition or vaccine. Typically, such a response includes, but is not limited to, one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or γδ T cells specifically against the antigen or antigens in the desired composition or vaccine.

[0036] In some embodiments, the castration vaccine can be used for animals, such as mammals, rodents, avian animals, etc. According to some embodiments, the castration vaccine can be used for mice, rats, cats, dogs, cattle, sheep, goats, pigs, rabbits, horses, chickens, ducks, etc.

[0037] In some embodiments, the castration vaccine further comprises at least one immune adjuvant.

[0038] The implementation methods of the present application will be described in more detail below, but this does not constitute a limitation on the implementation methods of the present application.

[0039] 1. Synthesis of the first expression vector

[0040] The first expression vector is a plasmid carrying the nucleotide sequence shown in SEQ ID NO: 3. Specifically, the plasmid is pPICZαA (Cat. No. HG-VJI0296, Aonogen).

[0041] 2. Synthesis of the second expression vector

[0042] The second expression vector is a plasmid carrying the nucleotide sequence shown in SEQ ID NO: 2. The step of synthesizing the second expression vector includes: inserting the nucleotide sequence shown in SEQ ID NO: 4 into the 5' end of the nucleotide sequence shown in SEQ ID NO: 3 of the first expression vector by homologous recombination to obtain the second expression vector.

[0043] In some embodiments, the step of synthesizing the second expression vector specifically includes:

[0044] 1) synthesizing the nucleic acid shown in SEQ ID NO:4;

[0045] 2) performing PCR amplification on the nucleic acid shown in SEQ ID NO: 4 using F1 (SEQ ID NO: 5) and R1 (SEQ ID NO: 6) as a primer pair;

[0046] 3) obtaining a linearized fragment of the first expression vector;

[0047] 4) connecting the PCR amplification product of the nucleic acid shown in SEQ ID NO: 4 to the linearized fragment of the first expression vector;

[0048] 5) The ligation product obtained in step 4) is transformed into Escherichia coli;

[0049] 6) Screening positive clones from transformants;

[0050] 7) Extracting and obtaining the second expression vector from the positive clone or its culture.

[0051] In some steps 2), the PCR amplification system contains 1 μL of plasmid vector, 10 μL of 2×Rapid Taq MasterMix, 11 μL of F1, 11 μL of R1, and the balance of ddH2O in 20 μL. The PCR amplification steps include: 98°C pre-denaturation for 5 min, 98°C denaturation for 10 s, 51°C annealing for 15 s, 72°C extension for 40 s, 35 cycles, and 72°C extension for 10 min.

[0052] In some steps 3), the first expression vector is PCR amplified using primer pair F2 (SEQ ID NO: 7) and R2 (SEQ ID NO: 8) to obtain a linearized vector of the first expression vector. The PCR amplification system contains 20 μL of the plasmid vector 1 μL, 2×Rapid Taq Master Mix 10 μL, F2 1 μL, R2 1 μL, and the balance ddH2O. The PCR amplification steps include: 98°C pre-denaturation for 5 min, 98°C denaturation for 10 s, 54°C annealing for 15 s, 72°C extension for 40 s, 35 cycles, and 72°C extension for 10 min.

[0053] In some steps 4), the ligation reaction system comprises 20 μL of the linearized vector of the first expression vector, 1 μL of the PCR amplification product of the nucleic acid shown in SEQ ID NO: 4, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and the balance of ddH2O. The ligation reaction conditions include: 37°C for 30 min and then immediately cooled on ice.

[0054] In some steps 5), the ligation product is transformed into E. coli Trelief 5α competent cells.

[0055] In some steps 6), positive clones are screened from transformants by bacterial solution PCR verification. Wherein, the colony PCR reaction system contains 20 μL of bacterial solution: 1 μL of bacterial solution, 10 μL of 2×Rapid Taq Master Mix, 1 μL of F3 (SEQ ID NO: 9), 1 μL of R3 (SEQ ID NO: 10) and the remainder of ddH2O. The bacterial solution PCR reaction steps include: pre-denaturation at 98°C for 5 min, denaturation at 98°C for 10 s, annealing at 51°C for 15 s, extension at 72°C for 40 s, 35 cycles, and extension at 72°C for 10 min. The bacterial solution PCR verification step also includes: electrophoresis detection of the bacterial solution PCR reaction product. If the target band at about 2182 bp is detected, it is confirmed to be a positive clone.

[0056] Referring to the above steps, a pPICZαA plasmid containing only SEQ ID NO: 4 was also prepared.

[0057] 3. Construction of expression strain

[0058] In some steps, the second expression vector is transferred into competent Pichia pastoris X33 cells, and positive clones are screened from the transformants, which are expression strains. Specifically, the second expression vector is digested to obtain linearized fragments; electroporated into competent Pichia pastoris X33 cells; sterile and pre-cooled 1 mol / L sorbitol solution is immediately added to the electroporated Pichia pastoris X33 cells, and incubated in a 30°C constant temperature incubator for 1 hour; in a clean bench, the incubated bacterial solution is spread on a YPDS plate added with 100 μg / mL Zeocin, and the plate is cultured at 30°C for 2-10 days until colonies are formed, and the yeast genome is extracted after the grown colonies are cultured, and colony PCR detection is performed using F4 (SEQ ID NO: 11) and R4 (SEQ ID NO: 12) primers, and the detection conditions are the same as the above steps.

[0059] Referring to the above steps, the pPICZαA plasmid and the pPICZαA plasmid containing only SEQ ID NO: 4 were respectively transformed into Pichia pastoris X33 to obtain a control strain and an empty plasmid strain, respectively.

[0060] 4. Protein Expression

[0061] In some steps, the above expression strain is inoculated into a culture medium for inducing expression, and the protein shown in SEQ ID NO: 1 is collected from the culture.

[0062] In some steps, the protein expression step includes:

[0063] 1) Pick single colonies of the expression strain, control strain and empty plasmid strain respectively into 5 ml YPDS liquid medium, add 20 μL Zeocin (100 mg / mL), and culture at 30°C and 250 rpm / min for 16-18 h until the OD value reaches 2-4;

[0064] 2) Pipette 2 ml of bacterial solution into 100 mL of BMGY medium, add 100 μL of bleomycin (100 mg / mL), and culture at 30°C, 250 rpm / min for 16-18 h until the OD value reaches 2-4;

[0065] 3) Centrifuge at 1500 g for 8 min at room temperature and discard the supernatant.

[0066] 4) Resuspend the cells in 100 mL BMMY medium and start induction at 30°C and 250 rpm / min;

[0067] 5) Every 12 hours, 1.5 mL of bacterial solution was aspirated and 1.5 mL of 100% sterile methanol was added to a final methanol concentration of 1.5%. The bacterial solution was stored at -20°C for a total of 144 hours of induction; empty plasmid yeast (negative control) and MBP yeast (positive control) were induced at the same time;

[0068] 6) Centrifuge 1.5 mL of the remaining bacterial solution at 4°C, 12,000 rpm for 10 min to collect the protein supernatant;

[0069] 7) 80 μL of protein was mixed with 20 μL of 5×SDS loading buffer, boiled at 100° C. for 10 min, and centrifuged at 12,000 rpm for 4 min. The supernatant was the solution containing the protein shown in SEQ ID NO: 1.

[0070] In some test examples, Western-Blot detection was performed on the supernatant obtained above. Figure 1As shown, it is shown that the protein shown in SEQ ID NO: 1 fluctuates with the increase of induction time, and the size of the target protein expressed by the expression strain is predicted to be 61.2 kDa, the size of the target protein expressed by the control strain is 43.3 kDa, and no target protein is expressed by the empty plasmid. Figure 1 As shown, the expression level of the target protein reached the maximum at 48h of induction.

[0071] In some embodiments, the methanol induction conditions of the expression strain are also optimized. For example, the final concentration of methanol in the above step 5) is prepared as 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%, and methanol of the corresponding concentration is added every 12 hours, induced for 48 hours, centrifuged at 4°C and 15000g for 15 minutes, 80 μL of protein supernatant is drawn and mixed with 20 μL of 5×SDS loading buffer, boiled at 100°C for 10 minutes, 12000rpm, centrifuged for 4 minutes, and the supernatant is drawn for Western-blot test.

[0072] Figure 2 The protein expression levels at 48 h of induction under different methanol concentrations are shown. Figure 2 As shown, the expression level of the target protein reached the maximum when the methanol concentration in the BMMY fermentation medium was 1.5%.

[0073] In some embodiments, the fermentation temperature of the expression strain is also optimized. For example, the expression strain is fermented at 26°C, 27°C, 28°C, 29°C, 30°C or 31°C, 1.5% methanol is added every 12 hours, induced for 48 hours, centrifuged at 4°C, 15000g for 15 minutes, 80 μL of protein supernatant is drawn and mixed with 20 μL of 5×SDS loading buffer, boiled at 100°C for 10 minutes, 12000rpm, centrifuged for 4 minutes, and the supernatant is drawn for Western-blot test. The conditions are the same as those in the above embodiment.

[0074] Figure 3 The protein expression levels after 48 h of induction at different temperatures are shown. Figure 3 As shown, with the increase of induction temperature, the expression level of the target protein gradually increased and reached the maximum at 30°C.

[0075] In some embodiments, the fermentation pH of the expression strain is also optimized. For example, the expression strain is subjected to protein fermentation test in BMMY medium at pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5 or pH 7.0, respectively, the temperature is controlled at 30°C, 1.5% methanol is added every 12 hours, induced for 48 hours, centrifuged at 4°C and 15000g for 15 minutes, 80 μL of protein supernatant is drawn and mixed with 20 μL of 5×SDS loading buffer, boiled at 100°C for 10 minutes, centrifuged at 12000rpm for 4 minutes, and the supernatant is drawn for Western-blot test.

[0076] Figure 4 The protein expression levels at different fermentation medium pHs after 48 h of induction are shown. Figure 4 As shown, the expression level of the target protein reached the maximum when the pH of the culture medium was 7.0.

[0077] It can be seen that the optimal induction expression conditions for expressing the protein shown in SEQ ID NO: 1 are: induction for 48 hours, methanol concentration of 1.5%, temperature of 30° C., and medium pH 7.0.

[0078] 5. Protein purification

[0079] In order to separate the target protein from the protein supernatant, the induced protein supernatant was subjected to ion exchange chromatography using a strong anion exchange resin. ① Preparation: Filter the induced protein supernatant with a 0.45 micron filter membrane; and replace the sample with 3 times the equilibrium solution to reduce the conductivity. ② Cleaning: Rinse the storage solution in the chromatography column with 3-5 column volumes of deionized water. ③ Balance: Use 5 column volumes of equilibrium solution to balance the chromatography column so that the filler is in the same buffer system as the protein of the day, which plays a role in protecting the protein. ④ Loading: Add the sample to the balanced chromatography column at a flow rate of 1.5mL / min to ensure that the target protein is fully in contact with the filler and improve the recovery rate of the target protein. ⑤ Washing: Wash with 5-10 column volumes of equilibrium solution until the ultraviolet absorption reaches a stable baseline. Remove non-specifically adsorbed impurities. ⑥ Elution: Use elution buffer to wash with a linear gradient, set 20 column volumes to separate proteins with different binding strengths, and collect samples from the protein absorption peak. ⑦ Cleaning and storage: Use 6 column volumes of 0.2M NaOH and 6 column volumes of deionized water to balance the filler, and finally store it at 4 degrees to prevent the filler from being contaminated by bacteria. ⑧ Detection: The samples obtained during the purification process were detected by SDS-PAGE and WB to determine the purification effect.

[0080] Figure 5 The protein profile obtained by strong anion exchange chromatography is shown in FIG. Figure 5As shown, tubes 22-26 contain the target protein with correct bands, and there are fewer bands of other impurity proteins.

[0081] 6. Immune effect test

[0082] (1) Animal husbandry and management

[0083] 60 3-week-old SPF male Kunming mice were purchased from the Experimental Animal Center of Huazhong Agricultural University. After pre-feeding for 1 week, they were randomly divided into groups and entered the experimental period. They were kept in a special animal room in this laboratory, and the breeding temperature was controlled at about 25°C. The animal room maintained a 12h / 12h light and dark cycle. The light time was from 8:00 am to 8:00 pm, and the lights were turned off from 8:00 pm to 8:00 am the next day. All experimental mice were fed and drank freely. The animal room was equipped with air conditioning and ventilation equipment to strictly control temperature and humidity. Cages were implemented, with 5 mice per cage. Sanitary cleaning was performed once a week. Whether the feeding and drinking were normal was observed every day, and whether there was death due to intramuscular injection or blood sampling stress. The weight of each mouse was recorded every week to observe whether the growth and development of the mice were normal.

[0084] (2) Immunity group test

[0085] 60 4-week-old Kunming female mice were randomly divided into 2 groups (control group C and experimental group T), 30 mice / group. The mice were immunized according to the grouping. For the first immunization, 90 μL (0.5 mg) of the protein shown in SEQ ID NO: 1 was added with 10 μL of aluminum hydroxide adjuvant; two weeks after the first immunization, booster immunization was performed weekly for a total of four times. During the booster immunization, 90 μL (1 mg) of the protein shown in SEQ ID NO: 1 was added with 10 μL of aluminum hydroxide adjuvant; 90 μL of physiological saline was mixed with 10 μL of aluminum hydroxide adjuvant as the treatment method for the control group. The mental and physical conditions of the mice were observed for one week after immunization.

[0086] (3) Detection of testosterone and estradiol levels in mouse serum

[0087] The operation method refers to the instructions of the kit, and the competitive ELISA method is used to detect the levels of T and E2 in the serum of mice with different treatments. The specific operation steps are as follows: ① Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a self-sealing bag and return them to 4℃. ② Set up standard wells and sample wells, add 50uL of different concentrations of standard wells to each standard well; add 10uL of the sample to be tested to the sample well first, and then add 40uL of sample diluent; do not add blank wells. ③ Except for the blank wells, add 100uL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate in a 37℃ constant temperature box for 60min. ④ Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing plate 5 times. ⑤ Add 100uL / well of single-component TMB colorimetric solution, and react at 37℃ in the dark for 15min. ⑥ Add 2mol / L H2SO4 stop solution 50uL / well, and measure the OD value of each well at a wavelength of 450nm within 15min. ⑦ Draw a standard curve and calculate the concentration of estradiol and progesterone in the sample based on the standard curve (the calculated value multiplied by the 5-digit final concentration value).

[0088] like Figure 6 As described above, the levels of testosterone (T) and estradiol (E2) in the blood were collected every 2 weeks after the initial immunization, and it was found that they were significantly lower than those in the control group and the differences were extremely significant (p<0.0001), indicating that the immunization experiment was relatively successful.

[0089] (4) Effects of mouse gonadal tissue

[0090] The mice were weighed and recorded at the same time each week during the immunization period. The testicles and epididymis of each group were collected by dissection, the weights of various tissues were recorded, and the organ index was calculated (organ index = organ weight / body weight × 100).

[0091] like Figure 7 As shown in the figure, the experimental group had a smaller testicular tissue, and there was no significant difference in the testicular weight and organ index of mice 2 and 4 weeks after immunization injection. After 6 weeks of immunization injection, the testicular weight (p<0.01) and organ index (p<0.05) of mice were significantly lower than those of the control group. The epididymis is an organ for sperm storage and transportation. The size of the epididymis of mice in the experimental group and the control group was not significantly different; statistics showed that there was no significant difference in the epididymis weight and organ index of mice.

[0092] (5) Observation of testicular tissue

[0093] After the initial immunization, testicular tissue sections and hematoxylin-eosin staining were performed every two weeks. Figure 8As shown in the figure, spermatogenic cells fell off (blue arrows), vacuoles appeared in the seminiferous tubules (red boxes), and spermatogenic cells were loosely arranged (red arrows) in the testicles of the immunized group. This indicates that immunization can lead to a decrease in the spermatogenic ability of male mice.

[0094] (4) Safety testing in mice

[0095] The mice were weighed and the weight data were recorded at the same time each week during the immunization period. Six weeks after the male mice were immunized, their hearts, livers, spleens, lungs, and kidneys were collected, their weights were recorded, and the organ indexes of each tissue were calculated.

[0096] As shown in Table 1, the organ index results showed that the organ index of the immunized group was not significantly different from that of the control group, proving that the vaccine did not cause damage to the internal organs of the mice. Starting from the first immunization, the mice were weighed every week. The results are shown in Table 2. Starting from the first immunization, as the age increased, there was no significant difference in the weight of the mice in the control group and the experimental group (p>0.05), and they maintained a steady growth, but at 10 weeks, the weight of both the control group and the experimental group decreased.

[0097] Table 1 Organ index of different organs of mice

[0098] Group Heart liver spleen lung kidney C 0.56±0.08 3.67±0.20 0.24±0.05 0.50±0.06 1.35±0.12 T 0.62±0.08 3.58±0.19 0.25±0.03 0.51±0.05 1.39±0.11

[0099] Table 2 Body weight changes after immunization

[0100] age 4W 5W 6W 7W 8W 9W 10W C 31.70±2.00 40.45±2.31 42.09±3.22 45.72±3.56 47.71±3.96 50.76±4.87 47.44±4.60 T 32.03±2.71 40.13±2.64 42.64±3.68 47.80±3.57 48.63±3.10 50.30±2.94 47.32±2.24

[0101] In summary, the protein shown in SEQ ID NO:1 adopted in the present application was used to immunize mice, which can effectively activate the immune response of animals, and the testosterone and estrogen levels in mice were significantly reduced. The effects on testicular mass and organ index after immunization injection were more obvious. Testicular HE sections showed that immunization can lead to a decrease in spermatogenesis in male mice; and the protein shown in SEQ ID NO:1 does not affect the weight and organ index of mice, indicating that the use of the protein vaccine shown in SEQ ID NO:1 for active immunization can reduce the reproductive capacity of male mice and has reliable biological safety.

[0102] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A protein having the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid having the nucleotide sequence shown in SEQ ID NO:

2.

3. An expression vector, which is a plasmid having the nucleotide sequence shown in SEQ ID NO:

2.

4. A castration vaccine, comprising the protein shown in SEQ ID NO: 1 as an active ingredient.

5. A castration vaccine, comprising the nucleic acid shown in SEQ ID NO: 2 as an active ingredient.

6. A castration vaccine, comprising the expression vector according to claim 3 as an active ingredient.

7. A method for preparing the expression vector according to claim 3, comprising: Synthesize the nucleic acid shown in SEQ ID NO:4; Performing PCR amplification on the nucleic acid shown in SEQ ID NO:4 using the primer pair; Obtaining a linearized fragment of the first expression vector; Connecting the PCR amplification product of the nucleic acid shown in SEQ ID NO:4 to the linearized fragment of the first expression vector; The resulting ligation product was transformed into Escherichia coli; Screening positive clones from transformants; The second expression vector is extracted from the positive clone or its culture.

8. A method for preparing the protein according to claim 1, comprising: Transforming the expression vector as claimed in claim 3 into Pichia pastoris; Screen the positive clones from the transformants to obtain the expression strains; The expression strain is inoculated into a culture medium for inducing expression, and the protein shown in SEQ ID NO: 1 is collected from the culture.

9. The production method according to claim 8, wherein the culture medium contains methanol.

10. Use of the protein according to claim 1 or the nucleic acid according to claim 2 in the preparation of a castration vaccine.

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