Proteins, nucleic acids, expression vectors, castration vaccines, methods and uses

Abstract

The application relates to the field of animal immunization castration vaccines, in particular to a protein, a nucleic acid, an expression vector, a castration vaccine, a method and application. The protein is shown as SEQ ID NO: 1. The nucleic acid is shown as SEQ ID NO: 2. The expression vector comprises the nucleic acid. The castration vaccine takes the protein, the nucleic acid or the expression vector as an effective component. The method comprises the steps of preparing the nucleic acid and / or the expression vector. The application comprises the preparation of the castration vaccine. The protein, the nucleic acid, the expression vector and / or the castration vaccine can produce an immune response in mice, reduce the testosterone and estrogen levels in mice and produce obvious trend effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal immunocastration vaccines, in particular to proteins, nucleic acids, expression vectors, castration vaccines, methods and applications. BACKGROUND

[0002] Immunocastration of animals is a method with many technical advantages compared with physical and chemical castration. The definition of immunocastration in the Veterinary Dictionary is: a technology that uses immunization to inhibit the development of sexual organs to achieve the purpose of castration. It mainly controls fertility by inducing high enough levels of antibodies against antigens that play an important role in the reproductive process, blocking the reproductive hormone regulation pathway through antigen-antibody reaction, thereby achieving the purpose of controlling fertility. Immunocastration has the advantages of safety, convenience, mild effect, low stress on animals, and reversibility, and has great advantages compared with surgical and chemical castration. However, the main problem is how to select suitable antigens with sufficient immunogenicity to obtain contraceptive products.

[0003] The core of immunocastration is to select one or a combination of hormones or receptors related to reproduction as a target to express exogenous castration genes in the animal body to produce antibodies and neutralize endogenous hormones, thereby achieving significant immunization effect. The main antigens for immunization at present are the zona pellucida (ZP) around the oocyte, gonadotropin-releasing hormone (GnRH), prolactin receptor (LH), follicle-stimulating hormone (FSH), etc. that play an important role in fertilization. The above antigens can induce high antibody levels through the action of appropriate coupling and adjuvants, thereby achieving the effect of castration. However, the zona pellucida antigen (ZP) can only be used for female animals, and its castration effect on cats is uncertain. The immunization duration of the prolactin receptor antigen (LH) and the follicle-stimulating hormone antigen (FSH) is short. SUMMARY

[0004] The present application embodiment constructs a protein, which is used as an effective component of a castration vaccine, and can obtain a protein vaccine with high activity, good immunogenicity and high biological safety. The protein can produce an immune response in mice, reduce the levels of testosterone and estrogen in mice, and produce a significant castration effect.

[0005] Therefore, the present application embodiment discloses 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 uses a protein having SEQ ID NO: 1 as an effective component.

[0009] In a fourth aspect, the embodiments disclose a castration vaccine, which uses a nucleic acid having SEQ ID NO: 2 as an effective component.

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

[0011] In a sixth aspect, the embodiments disclose a castration vaccine, which uses the expression vector of the fifth aspect as an effective component.

[0012] In a seventh aspect, the embodiments disclose a preparation method of an expression vector, which comprises: 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 by 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; transferring the connection product into E. coli; screening positive clones from the transformants; and extracting a second expression vector from the positive clones or culture thereof.

[0013] In an eighth aspect, the embodiments disclose a preparation method of a protein as shown in SEQ ID NO: 1, which comprises: transferring the expression vector of the fifth aspect into Pichia pastoris; screening positive clones from the transformants, i.e., obtaining an expression strain; inoculating the expression strain into a culture medium for induced expression; and collecting the protein as shown in SEQ ID NO: 1 from the culture.

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

[0015] Figure 1 Western-Blot detection results of the supernatant containing the target protein of the embodiments under different fermentation times are provided. Figure 1 A is a detection chart, M is a protein maker, lanes 1-6 are the supernatant samples of the expression strain fermented for 24h, 48h, 72h, 96h, 120h and 144h respectively, lane 7 is the supernatant sample of the control strain fermented for 48h, and lane 8 is the supernatant sample of the empty plasmid strain fermented for 48h. Figure 1 B is a gray value statistical chart of lanes 1-6.

[0016] Figure 2Western-Blot detection results of supernatant containing target protein obtained from different methanol induction conditions for the examples. Figure 2 A is a detection map, M is a protein maker, lanes 1-6 are supernatant samples obtained from the expression strains respectively fermented under conditions containing 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0% methanol, lane 7 is a supernatant sample obtained from the control strain fermented under conditions containing 1.5% methanol for 48 h, and lane 8 is a supernatant sample obtained from the empty plasmid strain fermented under conditions containing 1.5% methanol for 48 h. Figure 2 B is a gray value statistical chart of lanes 1-6.

[0017] Figure 3 Western-Blot detection results of supernatant containing target protein obtained from different fermentation temperatures for the examples. Figure 3 A is a detection map, M is a protein maker, lanes 1-6 are supernatant samples obtained from the expression strains respectively fermented under conditions at 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, lane 7 is a supernatant sample obtained from the control strain fermented under conditions at 30°C, and lane 8 is a supernatant sample obtained from the empty plasmid strain fermented under conditions at 30°C. Figure 3 B is a gray value statistical chart of lanes 1-6.

[0018] Figure 4 Western-Blot detection results of supernatant containing target protein obtained from different fermentation pHs for the examples. Figure 4 A is a detection map, M is a protein maker, lanes 1-7 are supernatant samples obtained from the expression strains respectively fermented under conditions at pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, lane 8 is a supernatant sample obtained from the control strain fermented under conditions at pH 7.0, and lane 9 is a supernatant sample obtained from the empty plasmid fermented under conditions at pH 7.0. Figure 4 B is a gray value statistical chart of lanes 1-7.

[0019] Figure 5 Protein atlas obtained from strong anion exchange chromatography for purifying supernatant of the expression strain fermentation for the examples. Figure 5 A is a protein absorption peak atlas, and the number of tubes to be collected is counted from the start of the peak; Figure 5 B is a protein SDS-PAGE atlas, Figure 5 C is a protein WB result, M is a protein maker, and lanes 1-13 are proteins eluted from the even-numbered tubes from the 8th to the 32nd tubes in turn.

[0020] Figure 6The statistical results of testosterone (T) and estradiol (E2) in blood collected every 2W after the priming of the examples.

[0021] Figure 7 The results of mouse testis and epididymal tissue taken every 2W after the priming of the examples. Figure 7 A The pictures of mouse testis of the control group (C) and the test group (T) at 2W, 4W and 6W. Figure 7 B The results of mouse testis weight. Figure 7 A The results of mouse testis weight. Figure 7 C The results of mouse testis organ index. Figure 7 A The results of mouse testis organ index. Figure 7 D The pictures of mouse epididymal tissue of the control group (C) and the test group (T) at 2W, 4W and 6W. Figure 7 E The results of mouse epididymal tissue weight. Figure 7 A The results of mouse epididymal tissue weight. Figure 7 F The results of mouse epididymal tissue organ index. Figure 7 A The results of mouse epididymal tissue organ index.

[0022] Figure 8 The HE staining pictures of testis tissue section taken every 2W after the priming of the examples. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. The reagents not described 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 known from the prior art.

[0024] The term "castration" used herein, also known as emasculation, refers to the removal of the gonads of an animal or the 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 capacity of livestock or poultry, making the temperament of livestock or poultry docile and hardworking, which is convenient for management; it can also improve the meat quality and yield of meat animals; it can also control the mating behavior in the herd, which is beneficial to the breeding and selection of good breeds.

[0025] In one aspect, the examples disclose 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 multimer. When the protein is used to immunize animals, it can effectively stimulate the immune response of the animals, improve the immune effect, enhance the neutralizing antibody level, and is safer, has a shorter safety evaluation period, and has a wide application prospect in production.

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

[0027] The term "coding sequence" or "nucleotide sequence encoding a particular protein" is a DNA sequence which is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate control sequences. The boundaries of the coding sequence are determined by a start codon at the 5' terminus and a translation stop codon at the 3' terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0028] In another aspect, the embodiments also disclose an expression vector which is a plasmid having a nucleotide sequence as set forth in SEQ ID NO: 2. The nucleic acid as set forth in SEQ ID NO: 2 can be operably linked to a base plasmid. The base plasmid can be a plasmid which can replicate, transcribe, translate and express a protein in an animal or a microorganism, such as pPICZ alpha A, pCAG series plasmids or pCIG plasmids.

[0029] The coding sequences are "operably linked" when the RNA polymerase will transcribe both coding sequences into mRNA and then translate them into the chimeric polypeptide encoded by the two coding sequences. The coding sequences need not be contiguous, as long as the transcribed sequences are ultimately processed into the desired chimeric protein. A control sequence "controls" a coding sequence if it controls the transcription or the translation of the coding sequence.

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

[0031] A "host cell" is a cell that has been or can be transformed with an exogenous DNA sequence and has the ability to be transformed or has been transformed.

[0032] When such foreign DNA is introduced into a cell, the foreign DNA "transforms" the cell when the foreign DNA can or can not integrate (covalently attach) into the chromosomal DNA making up the genome of the cell. In prokaryotes and yeast, for example, the foreign DNA can remain as an episomal element, such as a plasmid. As for eukaryotic cells, a stably transformed cell is one in which the foreign DNA has become integrated into a chromosome so as to be inherited by daughter cells following cell division. The ability of eukaryotic cells to establish cell lines from a single transformed cell by clonal propagation makes stable transformation the preferred technique for introduction of foreign DNA.

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

[0034] In the formula, the "effective component" is a component capable of producing an immune response in an animal.

[0035] An "immune response" to an antigen or vaccine is the development of a cell and / or antibody-mediated immune response in a host to a 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 specific to an antigen or antigens in a desired composition or vaccine, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma delta T cells.

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

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

[0038] The embodiments of the present application will be described in more detail below, but do not constitute limitations on the embodiments of the present application.

[0039] 1. Synthesis of a first expression vector

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

[0041] 2. Synthesis of a second expression vector

[0042] The second expression vector is a plasmid carrying a nucleotide sequence as shown in SEQ ID NO: 2. The step of synthesizing the second expression vector comprises: inserting a nucleotide sequence as shown in SEQ ID NO: 4 into the 5' end of the nucleotide sequence as 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 comprises:

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

[0045] 2) performing PCR amplification on the nucleic acid as 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 as shown in SEQ ID NO: 4 with the linearized fragment of the first expression vector;

[0048] 5) transforming the connection product obtained in step 4) into E. coli;

[0049] 6) screening positive clones from the transformants;

[0050] 7) extracting the second expression vector from the positive clones or their cultures.

[0051] In some step 2), the PCR amplification system comprises, in 20 μL: plasmid vector 1 μL, 2x Rapid Taq MasterMix 10 μL, F1 1 μL, R1 1 μL, and the rest of ddH2O. The PCR amplification step comprises: 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 step 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 comprises, in 20 μL: plasmid vector 1 μL, 2x Rapid Taq Master Mix 10 μL, F2 1 μL, R2 1 μL, and the rest of ddH2O. The PCR amplification step comprises: 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 step 4), the ligation reaction system was counted as 20 μL, including: 2 μL of the linearized vector of the first expression vector, 1 μL of the PCR amplification product of the nucleic acid as shown in SEQ ID NO: 4, 4 μL of 5x CE II Buffer, 2 μL of Exnase II, and the rest of ddH2O. The ligation reaction conditions included: 37°C for 30 min, and then cooling on ice immediately.

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

[0055] In some step 6), the positive clones were screened from the transformants by broth PCR. The broth PCR reaction system was counted as 20 μL, including: 1 μL of the broth, 10 μL of 2x Rapid Taq Master Mix, 1 μL of F3 (SEQ ID NO: 9), 1 μL of R3 (SEQ ID NO: 10), and the rest of ddH2O. The broth PCR reaction steps included: 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. The broth PCR verification step also included: electrophoresis detection of the broth PCR reaction product, and if the target band at about 2182 bp was detected, it was confirmed as a positive clone.

[0056] Referring to the above steps, the 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 was transformed into P. pastoris X33 competent cells, and positive clones were screened from the transformants, which were the expression strain. Specifically, the second expression vector was digested to obtain a linearized fragment; the linearized fragment was electroporated into P. pastoris X33 competent cells; immediately after the electroporation, sterile and pre-cooled 1 mol / L sorbitol solution was added to the P. pastoris X33 cells, and the cells were incubated in a 30°C constant temperature incubator for 1 h; the incubated broth was plated on a YPDS plate containing 100 μg / mL Zeocin in a clean bench, and the plate was cultured at 30°C for 2-10 days until colonies were formed. The grown colonies were cultured to extract the yeast genome, and colony PCR was detected using F4 (SEQ ID NO: 11) and R4 (SEQ ID NO: 12) primers, and the detection conditions were the same as above.

[0059] Referring to the above steps, the pPICZ alpha A plasmid and the pPICZ alpha A plasmid containing only SEQ ID NO: 4 are also transformed into P. pastoris X33, respectively, 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 induction expression, and the protein as shown in SEQ ID NO: 1 is collected from the culture.

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

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

[0064] 2) 2 ml of bacterial solution is taken and added to 100 mL of BMGY medium, and 100 μL of Bolemycin (100 mg / mL) is added, and cultured at 30°C, 250 rpm / min for 16-18 h until the OD value is 2-4;

[0065] 3) Centrifuged at 1500g for 8 min at room temperature, and the supernatant was discarded.

[0066] 4) Resuspend the bacterial body with 100 mL of BMMY medium, and start induction at 30°C, 250 rpm / min;

[0067] 5) Every 12 h, 1.5 mL of bacterial solution is taken out, and 1.5 mL of 100% sterile methanol is added to a final methanol concentration of 1.5%, and the bacterial solution is stored at -20°C, and the induction is performed for a total of 144 h; at the same time, the empty plasmid yeast strain (negative control) and the MBP yeast strain (positive control) are induced;

[0068] 6) The 1.5 mL of bacterial solution is left for sampling, and the protein supernatant is collected by centrifugation at 12000 r / min for 10 min at 4°C;

[0069] 7) 80 μL of protein is taken and mixed with 20 μL of 5x SDS loading buffer, boiled at 100°C for 10 min, and centrifuged at 12000 rpm for 4 min, and the supernatant is the solution containing the protein as shown in SEQ ID NO: 1.

[0070] In some test examples, the supernatant obtained by the above method is subjected to Western-Blot detection. As Figure 1As shown, the protein expression level of the target protein reached the maximum value at 48h of induction. Figure 1 As shown, the protein expression level of the target protein reached the maximum value 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 step 5) above is prepared as 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, and the corresponding concentration of methanol is added every 12h, and the protein supernatant is induced for 48h, centrifuged at 15000g for 15min at 4°C, 80μL of which is mixed with 20μL of 5×SDS loading buffer, boiled at 100°C for 10min, centrifuged at 12000rpm for 4min, and the supernatant is used for Western-blot test.

[0072] Figure 2 As shown, the protein expression level of the target protein reached the maximum value at 48h of induction. Figure 2 As shown, the protein expression level of the target protein reached the maximum value at 48h of induction.

[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, respectively, and 1.5% methanol is added every 12h, and the protein supernatant is induced for 48h, centrifuged at 15000g for 15min at 4°C, 80μL of which is mixed with 20μL of 5×SDS loading buffer, boiled at 100°C for 10min, centrifuged at 12000rpm for 4min, and the supernatant is used for Western-blot test. The conditions are the same as the above embodiments.

[0074] Figure 3 As shown, the protein expression level of the target protein reached the maximum value at 48h of induction. Figure 3 As shown, the protein expression level of the target protein reached the maximum value at 48h of induction.

[0075] In some embodiments, the fermentation pH of the expression strain is also optimized. For example, the expression strain is subjected to protein fermentation testing 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, with temperature control at 30°C, 1.5% methanol added every 12 hours, induction for 48 hours, centrifugation at 4°C, 15000g for 15 minutes, 80 μL of protein supernatant mixed with 20 μL of 5x SDS loading buffer, boiling at 100°C for 10 minutes, centrifugation at 12000 rpm for 4 minutes, and then the supernatant is used for Western-blot testing.

[0076] Figure 4 The protein expression level after 48 hours of induction at different fermentation medium pH is shown. As shown in Figure 4 The protein expression level reaches the maximum when the medium pH is 7.0.

[0077] Therefore, the optimal induction expression conditions for 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 of 7.0.

[0078] 5. Protein purification

[0079] In order to separate the target protein from the protein supernatant, ion exchange chromatography is used to purify the protein supernatant after induction expression using strong anion exchange resin. ① Preparation: filter the protein supernatant after induction expression using a 0.45 micron filter membrane; and replace the sample with 3 times the amount of equilibration buffer to reduce the conductivity. ② Washing: wash the storage solution in the chromatography column with 3-5 times the column volume of deionized water. ③ Equilibration: equilibrate the chromatography column with 5 times the column volume of equilibration buffer to place the filler in the same buffer system as the protein, which protects the protein. ④ Sample loading: load the sample into the equilibrated chromatography column at a flow rate of 1.5 mL / min to ensure that the target protein is in full contact with the filler and to improve the recovery rate of the target protein. ⑤ Washing: wash with 5-10 times the column volume of equilibration buffer until the ultraviolet absorption reaches a stable baseline. Remove non-specifically adsorbed impurity proteins. ⑥ Elution: use a linear gradient elution with elution buffer to separate proteins with different binding strengths, and collect the sample from the protein absorption peak. ⑦ Cleaning and storage: use 6 times the column volume of 0.2M NaOH and 6 times the column volume of deionized water to equilibrate the filler, and finally store at 4°C to prevent bacterial contamination of the filler. ⑧ Detection: use SDS-PAGE and WB to detect the sample obtained during the purification process to determine the purification effect.

[0080] Figure 5 The protein map obtained by strong anion exchange chromatography is shown. As shown in Figure 5As shown, the protein of interest was contained in the 22-26 tubes and the bands were correct, and the rest of the protein bands were less.

[0081] 6. Immune effect test

[0082] (1) Animal feeding management

[0083] 60 SPF male Kunming mice aged 3 weeks were purchased from the Experimental Animal Center of Central China Agricultural University. After 1 week of pre-feeding, they were randomly divided into groups and entered the experimental period. They were raised in a special animal room in the laboratory, with the feeding temperature controlled at about 25°C. The animal room maintained a 12h / 12h light-dark cycle change, with the light period from 8:00 am to 8:00 pm and the dark period from 8:00 pm to 8:00 am the next day. All test mice were given free access to food and water. The animal room was equipped with air conditioning and ventilation equipment, and the temperature and humidity were strictly controlled. Cage breeding was implemented, with 5 mice per cage. The animals were cleaned once a week, and the normality of food and water intake and the death due to muscle injection or blood sampling stress were observed daily. The body weight of each mouse was recorded every week, and the normality of mouse growth and development was observed.

[0084] (2) Immune grouping test

[0085] 60 female Kunming mice aged 4 weeks were randomly divided into 2 groups (control group C and experimental group T), 30 mice / group. The mice were immunized according to the grouping. At the first immunization, 90 μL (0.5 mg) of the protein shown as SEQ ID NO: 1 was added to 10 μL of aluminum hydroxide adjuvant; two weeks after the first immunization, booster immunization was performed every week, with a total of four times. At the booster immunization, 90 μL (1 mg) of the protein shown as SEQ ID NO: 1 was added to 10 μL of aluminum hydroxide adjuvant; 90 μL of normal 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) Test of testosterone and estradiol levels in mouse serum

[0087] The operation method refers to the kit instruction, and the levels of T and E2 in the serum of different treated mice are detected by competitive ELISA method. The specific operation steps are as follows: ①Take out the required board from the aluminum foil bag after equilibration at room temperature for 20 min, and put the remaining board back into 4℃ with a self-sealing bag. ②Set standard hole and sample hole, add different concentrations of standard 50uL to standard hole; add 10uL of sample to be tested to sample hole, then add sample diluent 40uL; blank hole is not added. ③Except blank hole, add 100uL of horseradish peroxidase (HRP) labeled detection antibody to each well of standard hole and sample hole, seal the reaction hole with sealing film, and incubate in 37℃ constant temperature incubator for 60 min. ④Discard the liquid, pat dry on the blotting paper, add enough washing solution to each well, stand for 1 min, shake off the washing solution, pat dry on the blotting paper, and repeat the plate washing for 5 times. ⑤Add 100uL of single-component TMB color developing liquid per well, and react for 15 min at 37℃ in the dark. ⑥Add 50uL of 2mol / L H2SO4 termination liquid per well, and measure the OD value of each well at 450nm within 15 min. ⑦Draw the standard curve, and calculate the concentration of estradiol and progesterone in the sample (the calculated value is multiplied by the final concentration value of 5 digits).

[0088] As Figure 6 mentioned, the levels of testosterone (T) and estradiol (E2) in blood collected every 2W after priming were found to be significantly lower than those of the control group, with a very significant difference (p<0.0001), indicating that the immunization experiment was successful.

[0089] (4) Effect of mouse gonadal tissue

[0090] The body weight data was recorded by weighing at the same time period every week during the immunization period, and the testis and epididymis of mice were collected by dissection, the weights of various tissues were recorded, and the organ index (organ index = organ weight / body weight x 100) was calculated.

[0091] As Figure 7 shown, it was found by comparison that the testicular tissue of the experimental group decreased, and there was no significant difference in the testicular weight and organ index of mice after immunization injection for 2W and 4W, and the testicular weight (p<0.01) and organ index (p<0.05) of mice after immunization injection for 6W were significantly lower than those of the control group. The epididymis is the organ for sperm storage and transport, and it was found by comparison that there was no obvious difference in the size of the epididymis of mice between the experimental group and the control group; statistics found that there was no significant difference in the epididymal weight and organ index of mice.

[0092] (5) Observation of testicular tissue

[0093] Testicular tissue sections and hematoxylin-eosin staining were performed every two weeks after priming. As Figure 8As shown, the immune testis appeared to shed spermatogenic cells (blue arrow), vacuoles appeared in the seminiferous tubules (red box) and spermatogenic cells arranged loosely (red arrow) and other phenomena. It showed that after immunization, the ability of male mice to produce sperm decreased.

[0094] (4) Safety detection of mice

[0095] During the immunization period, the body weight data was recorded at the same time every week, and after 6 weeks of immunization, the heart, liver, spleen, lung and kidney of the male mice were collected, the weight was recorded, and the organ index of each tissue was calculated.

[0096] As shown in Table 1, the organ index results showed that the organ index of the immunization group had no significant difference compared with the control group, which proved that the vaccine did not cause damage to the internal organs of the mice. From the beginning of the first immunization, the body weight of the mice was measured every other week, and the results are shown in Table 2. From the beginning of the first immunization, the body weight of the mice in the control group and the experimental group did not have significant difference (p>0.05) with the increase of age, and remained stable growth, but at 10W, the body weight of 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 change 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 as shown in SEQ ID NO: 1 was used to immunize mice by the method of the present application, which could effectively stimulate the immune response of the animals, the levels of testosterone and estrogen in the mice were extremely significantly reduced, the effects on testis weight and organ index after immunization were more obvious, the testis HE section showed that the ability of male mice to produce sperm decreased after immunization; and the protein as shown in SEQ ID NO: 1 did not affect the body weight and organ index of the mice, indicating that the protein vaccine as shown in SEQ ID NO: 1 for active immunization could reduce the reproductive ability of male mice and had reliable biological safety.

[0102] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be covered in the protection scope of the present application.

Claims

1. A protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO:

2.

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

2.

4. A castration vaccine, comprising a protein having an amino acid sequence as shown in SEQ ID NO: 1 as an active ingredient.

5. A castration vaccine, comprising a nucleic acid having a nucleotide sequence as 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; PCR amplification of the nucleic acid shown in SEQ ID NO: 4 using the primer pair; Obtaining a linearized fragment of a first expression vector, wherein the first expression vector is a plasmid carrying the nucleotide sequence shown in SEQ ID NO: 3; ligating 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 according to claim 3 into Pichia pastoris; Screen the positive clones from the transformants to obtain the expression strain; The expression strain is inoculated into a culture medium for induction of expression, and a protein with an amino acid sequence as shown in SEQ ID NO: 1 is collected from the culture. The preparation 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 preparing a castration vaccine for male mice.

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