CN116162173B - A GnRH6-CRM197 recombinant protein castration vaccine and preparation method thereof - Google Patents

Abstract

The present invention discloses a GnRH6-CRM197 recombinant protein castration vaccine and a preparation method thereof. The mammalian castration recombinant protein vaccine contains a GnRH recombinant protein antigen, and the amino acid sequence of the GnRH recombinant protein antigen is shown in the sequence table Seq_1. The GnRH6-CRM197 recombinant protein castration vaccine contains a mammalian GnRH sequence, and the mammals include dogs, cats, pet pigs, guinea pigs, and mice. The mammalian castration recombinant protein vaccine contains the canine GnRH sequence of the sequence table Seq_2. The mammalian castration recombinant protein vaccine contains the goat CRM197 sequence of the sequence table Seq_3. The present invention discloses the construction, transformation expression and purification of a recombinant GnRH6-CRM197 protein vaccine, and the acquisition of an engineered bacterium BL21(DE3)-PET21a-GnRH6-CRM197. The vaccine causes testicular atrophy after immunization of male animals, thereby achieving the purpose of controlling animal fertility.

Description

A GnRH6-CRM197 recombinant protein castration vaccine and preparation method thereof

Technical Field

The present invention relates to the field of molecular vaccinology, and in particular to a GnRH6-CRM197 recombinant protein castration vaccine and a preparation method thereof.

Background technique

The more commonly used method of traditional castration is surgical castration. This method has high requirements for the site, environment, and operation. At the same time, the operation will have side effects, such as a high infection rate leading to a certain degree of death, and it brings certain difficulties to China, which has a huge annual output of livestock. In addition, the cost of surgery is high, and it is not conducive to animal welfare. This surgical castration that has been passed down for thousands of years is facing challenges from the needs of large-scale and standardized livestock production. Studies have shown that immunocastration has the advantages of convenience, efficiency, reversibility, and safety. Therefore, immunocastration technology is most likely to become a substitute for traditional castration.

Gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus plays a decisive role in reproductive endocrine regulation. Its main biological function is to control the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the anterior pituitary cells, that is, to regulate the reproductive axis. Studies have shown that in male animals, FSH and LH are closely related to reproductive activities such as estrus and mating, as well as testicular development and sperm production. Therefore, by immunizing GnRH, inducing the production of GnRH antibodies, neutralizing GnRH in the body and reducing GnRH levels, resulting in reduced LH and FSH synthesis, accompanied by the destruction of estrus, testicular development and sperm production, ultimately achieving the purpose of sterilization. In the research of GnRH immune castration vaccine, the early chemically synthesized peptide vaccine was made by connecting GnRH monomers to carrier proteins. However, the vaccines constructed with GnRH monomers were unstable in almost all immune animals and had weak immunogenicity. Even in the same batch of animals of the same species, the responses to vaccine immunization were very different, and some even had no response to the vaccine. Fang Fugui et al. conducted a study on the immunogenicity of GnRH hexamer and found that it had certain immunogenicity. In order to further improve the active immune effect of GnRH, it is necessary to select a carrier protein that enhances its immunogenicity. Studies have confirmed that diphtheria toxin mutant CRM197 can enhance the immune effect as a carrier protein and is a good carrier of weak antigens. We connected diphtheria toxin mutant CRM197 on the basis of GnRH hexamer to enhance its immunogenicity. At present, there have been many reports on GnRH vaccines at home and abroad, but none of them has reached commercial application. The reason is that the immunogenicity is not strong and the cost is high. And there are no reports on the combined use of GnRH hexamer and CRM197 protein. Therefore, it is necessary to develop a fusion protein vaccine of GnRH hexamer and CRM197 with strong immunogenicity and low cost.

Summary of the invention

The object of the present invention is to provide a GnRH6-CRM197 recombinant protein castration vaccine and a preparation method thereof, which improves the immunogenicity of low molecular weight antigens, improves the immune effect of antigens, and avoids the effect of polypeptide instability.

In one aspect of the present invention, the present invention provides a GnRH recombinant protein antigen. According to an embodiment of the present invention, the amino acid sequence of the GnRH recombinant protein antigen is shown in Sequence Listing Seq_1.

In another aspect of the present invention, the present invention provides a GnRH6-CRM197 recombinant protein castration vaccine. According to an embodiment of the present invention, the mammalian castration recombinant protein vaccine contains the GnRH recombinant protein antigen.

In addition, the GnRH6-CRM197 recombinant protein castration vaccine according to the above embodiment of the present invention may also have the following additional technical features:

In some embodiments of the present invention, the GnRH6-CRM197 recombinant protein castration vaccine contains mammalian GnRH sequences, and the mammals include dogs, cats, pet pigs, guinea pigs, and mice.

In some embodiments of the present invention, the mammalian castration recombinant protein vaccine contains the canine GnRH sequence of Sequence Listing Seq_2.

In some embodiments of the present invention, the mammalian castration recombinant protein vaccine contains the goat CRM197 sequence of Sequence Listing Seq_3.

In some embodiments of the present invention, the GnRH6-CRM197 recombinant protein castration vaccine contains the recombinant protein expressed by Escherichia coli transformed with GnRH6-CRM197 of Sequence Listing Seq_1.

In another aspect of the present invention, the present invention proposes a GnRH6-CRM197 transformed Escherichia coli. According to an embodiment of the present invention, the GnRH6-CRM197 transformed Escherichia coli was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 19, 2022, with a deposit number of CGMCC NO.25027, classified and named Escherichiacoli, specifically named BL21(DE3)-PET21a-GnRH6-CRM197, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a zip code of 100101. The GnRH6-CRM197 transformed Escherichia coli expresses a recombinant protein containing the amino acids in Sequence Listing Seq_1.

In another aspect of the present invention, the present invention proposes a method for constructing GnRH6-CRM197 transformed Escherichia coli. According to an embodiment of the present invention, the method comprises the following steps:

(1) designing a recombinant CRM197 sequence containing the selected GnRH hexamer based on the mammalian GnRH sequence and the goat CRM197 sequence;

(2) using a codon optimization system to design and synthesize a DNA sequence with BamHI and XhoI restriction sites, and after double digestion with BamHI and XhoI, ligating the DNA product with the vector PET21a that was also digested with the same enzymes to obtain a ligation solution;

(3) The ligation solution was then transferred into Escherichia coli DH5α, and the plasmid PET21a-GnRH6-CRM197 was screened and obtained. The plasmid GnRH6-CRM197 was transferred into the Escherichia coli BL21 (DE3) strain to obtain the engineered bacteria BL21 (DE3)-PET21a-GnRH6-CRM197, and finally the recombinant protein containing the amino acids in the sequence table Seq_1 was expressed, that is, the GnRH6-CRM197 transformed Escherichia coli.

In another aspect of the present invention, the present invention provides a method for preparing a GnRH6-CRM197 recombinant protein castration vaccine. According to an embodiment of the present invention, the GnRH6-CRM197 is transformed into Escherichia coli and the bacteria are broken and purified to obtain a recombinant protein containing the amino acids of Sequence Table Seq_1, which is the GnRH6-CRM197 recombinant protein castration vaccine.

In another aspect of the present invention, the present invention provides an application of a GnRH6-CRM197 recombinant protein castration vaccine. According to an embodiment of the present invention, the GnRH6-CRM197 recombinant protein castration vaccine is used for controlling fertility of mammals.

Compared with the prior art, the present invention has the following beneficial effects:

1) The present invention adopts the method of recombining GnRH hexamer and CRM197 protein, which improves the immunogenicity of low molecular weight antigens and the immune effect of antigens, and avoids the effect of polypeptide instability; at the same time, the present invention uses recombinant GnRH6-CRM197 protein, which reduces the cost of polypeptide synthesis preparations and is more convenient to use.

2) The BL21(DE3)-PET21a-GnRH6-CRM197 engineered bacterium prepared by the present invention is different from other engineered bacteria in the preparation process. The culture time, culture temperature, number of revolutions, time and temperature for inducing expression, and conditions such as the bacterium breaking and purification process of the engineered bacteria are unique to the engineered bacteria and are only suitable for the preparation conditions of the engineered bacteria. The research and development of this new engineered bacterium has a shorter preparation time, simpler preparation conditions, and lower preparation cost during the preparation process, and is suitable for large-scale production, with further research and development value and good industrialization prospects. The castration vaccine prepared by the new engineered bacteria has stronger immunogenicity. Compared with other castration vaccines, it reduces the stress of animals and is more convenient to use. It also has the advantages of safety, convenience, and mild effect. It can be directly used for animal muscle injection, is simple and easy to operate, achieves more ideal effects in controlling the development of animal reproductive organs and reproductive function, and better promotes the growth of meat animals, further promoting the development of animal castration technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of the plasmid PET21a-GnRH6-CRM197 in Example 1 of the present invention;

FIG2 is a gel electrophoresis analysis diagram of the recombinant protein GnRH6-CRM197 (71.3 KDa) in Example 1 of the present invention, in which 1 refers to the Merker band, 2 refers to the band before GnRH6-CRM197 purification, and 3 refers to the band after GnRH6-CRM197 purification;

FIG3 is a Western blot analysis of the purified recombinant protein GnRH6-CRM197 (71.3 KDa) in Example 1 of the present invention, wherein 1 refers to the Mark band, and 2 refers to the GnRH6-CRM197 (71.3 KDa) and GnRH antibody reaction band;

Fig. 4 is a testis image of the testis of male rats in the control group (left) and the immunization group (right) in Example 2 of the present invention;

Figure 5 is a histological observation diagram of the testis of male rats in the immunization group and the control group provided in an embodiment of the present invention, wherein the microscope magnification of Figure A and Figure C is 100×, the microscope magnification of Figure B and Figure D is 400×, A and B are the control group, C and D are the experimental group, a is spermatogonia, b is spermatocyte, c is spermatid, d is sperm, e is vacuolar degeneration, and f is seminiferous tubule.

Detailed ways

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

A method for constructing a GnRH6-CRM197 recombinant protein castration vaccine comprises the following steps:

(1) Construction of plasmid PET21a-GnRH6-CRM197

According to the canine GnRH sequence (Sequence Listing Seq_2) and the goat CRM197 sequence (Sequence Listing Seq_3), a recombinant CRM197 sequence containing the selected GnRH hexamer was designed. The lyophilized powder of the DNA sequence with BamHI and XhoI restriction sites synthesized by Beijing Qingke Biotechnology Co., Ltd. (as shown in Seq_4) was dissolved in sterile ultrapure water according to the instructions. The dissolved DNA and PET21a plasmid were double-digested with BamHI and XhoI respectively. The double-digestion system was 40μL of GnRH6-CRM197 gene fragment (100ng/μL) or PET21a (100ng/μL), and the other systems were 10μL of 10×CutSmart Buffer, 1μL of BamHI and XhoI, and 48μL of sterile ultrapure water. The enzyme digestion was carried out at 37℃ for 10h, and the target fragment was separated by nucleic acid electrophoresis. The target band was cut out with a scalpel and purified with a gel recovery kit.

The GnRH6-CRM197 gene fragment and the PET21a vector fragment were ligated at a molar ratio of 5:1 using T4 ligase at 4°C overnight. The ligation mixture was transformed into Escherichia coli DH5α and then cultured overnight on LB solid medium containing 100 μg/mL ampicillin (Amp+). Several single clones were selected for sequencing, and the bacteria with the correct sequence were selected to extract the plasmid, which was the plasmid PET21a-GnRH6-CRM197.

(2) Induced expression and purification of GnRH6-CRM197 vaccine protein

① Preparation of bacteria: The above-mentioned plasmid PET21a-GnRH6-CRM197 was transformed into Escherichia coli BL21 (DE3) by conventional methods, and cultured on LB solid medium containing Amp+ (100 μg/mL) for 12-15 h, and a transformant was picked and placed in LB liquid medium (10 mL) containing Amp+ (100 μg/mL), and cultured at 37°C with shaking at 200 rpm for 16 h; 1 mL of the bacterial solution after 16 h of culture was taken, added to 100 mL of fresh LB liquid medium containing Amp+ (100 μg/mL), and cultured at 37°C with shaking at 200 rpm for 3 h. When OD600 was 1.0, IPTG was added at a final concentration of 1.0 mmol/L, and the culture was continued at 20°C with shaking at 200 rpm for 12 h, and centrifuged at 4000g and 4°C for 45 min to collect the bacterial precipitate.

② Bacterial disruption: 100 mL of bacterial precipitate was added with 8 mL (accounting for 8% of the total volume of the bacterial cells) of non-denaturing lysis buffer for resuspending, and the resuspended bacterial cells were frozen at -20°C or directly used for ultrasound. During ultrasound, the protease inhibitor PMSF (diluted by volume ratio of 1:100-1:1000, with a working concentration of 17-174 μg/mL) could be added; the ultrasound power was 200 W, ultrasound was performed for 4 s, with an interval of 2 s, and ultrasound was performed for 20 min; after the ultrasound was completed, the bacterial solution was centrifuged at 10000 g at 4°C for 30 min, the supernatant was discarded, urea solution (8 mol/L) was added to the precipitate, 5 to 10 mL of urea (8 mol/L) solution was added to 100 mL of bacterial precipitate for dissolution, and the solution was dissolved at 4°C for 12 h. After the solution was completely dissolved, it was centrifuged at 4°C at a speed of 5000 g for 10 min, the supernatant was taken, and the supernatant was purified by a nickel column to obtain the GnRH6-CRM197 recombinant protein castration vaccine.

Among them, plasmid extraction and double enzyme digestion electrophoresis recovery both used the corresponding kits of "Beijing Aidelai Life Science Technology Co., Ltd."; plasmid and DNA ligation liquid were transformed into Escherichia coli using the competent cell method; all restriction endonucleases and ligases were purchased from "Beijing New England Biotechnology Co., Ltd."

FIG. 1 is a diagram of the plasmid PET21a-GnRH6-CRM197 in Example 1 of the present invention. A DNA sequence with BamH I and Xho I restriction sites was designed and synthesized using a codon optimization system. After double digestion by BamH I and Xho I, the DNA product was connected to the carrier PET21a that was also digested. The purified protein was detected by SDS-PAGE electrophoresis (electrophoresis at 80V for 30min, followed by electrophoresis at 120V for 60min), and its molecular weight was determined. As shown in FIG. 2 , the purified protein was analyzed by WB, confirming that it was an antigen protein GnRH6-CRM197 having an amino acid sequence in the sequence table Seq_1. The non-denaturing lysate was a final concentration of 50mmol/LTris>500mmol/LNaCl, pH7.5. As shown in FIG. 3 , the developed GnRH6-CRM197 has good reactivity with the GnRH antibody.

Sequence Listing Seq_1

QHWSGG1RPGGGSEHWSYG1RPGGGSEHWSYG1RPGGGSEDWSYG1RPGGGSEHWSYG1RPGGGSEHWSYG1RPGGGGSGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENP1SGKAGGVVKVTYPG1TKV1A1KVDNAETIKKE1G1S1TEP1MEQVGTEEFIKRFGDGASRVV1S1PFAEGSSSVEYINNWEQAKA1SVE1EINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSS1SCIN1DWDVIRDKTKTKIES1KEHGPIKN KMSESPNKTVSEEKAKQY1EEFHQTA1EHPE1SE1KTVTGTNPVFAGANYAAWAVNVAQVIDSETADN1EKTTAA1SI1PGIGSVMGIADGAVHHNTEEIVAQSIA1SS1MVAQAIP1VGE1VDIGFAAYNFVESIIN1FQVVHNSYNRPAYSPGHKTQPF1HDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTP1PIAGV11PTIPGK1DVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHAN1HVAFHRSSSEKIHSNEISSDSIGV1GYQKTVDHTKVNSK1S1FFEIKS

Sequence Listing Seq_2

MEPIPK1VAG1111TFCVVSCSGQHWSYG1RPGGKRNAEH1IDSFQEMAKE1DQPAEPQH1ECTIHKPRPP1RD1RGA1ES1IEEETGQKRI

Sequence Listing Seq_3

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENP1SGKAGGVVKVTYPG1TKV1A1KVDNAETIKKE1G1S1TEP1MEQVGTEEFIKRFGDGASRVV1S1PFAEGSSSVEYINNWEQAKA1SVE1EINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSS1SCIN1DWDVIRDKTKTKIES1KEHGPIKNKMSESPNKTVSEEKAKQY1EEFHQTA1EHPE1SE1KTVT GTNPVFAGANYAAWAVNVAQVIDSETADN1EKTTAA1SI1PGIGSVMGIADGAVHHNTEEIVAQSIA1SS1MVAQAIP1VGE1VDIGFAAYNFVESIIN1FQVVHNSYNRPAYSPGHKTQPF1HDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTP1PIAGV11PTIPGK1DVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHAN1HVAFHRSSSEKIHSNEISSDSIGV1GYQKTVDHTKVNSK1S1FFEIKS

Sequence Listing Seq_4

GGATCCCAGCATTGGAGCGGTGGTCTGCGTCCTGGTGGTGGTAGTGAACATTGGAGCTATGGTCTGCGTCCGGGTGGTGGTTCTGAACATTGGAGTTATGGTCTGCGCCCTGGTGGTGGCTCTGAAGATTGGAGTTATGGCCTGCGTCCGGGCGGTGGTAGTGAACATTGGTCATACGGTCTGCGTCCAGGTGGTGGTAGCGAACATTGGTCTTATGGTCTGCGTCCGGGTGGCGGTGGTAGCGGTGCAGATGATGTTGTTGATAGCAGTAAAAGTTTTGTGATGGAAAATTTTAGTAGTTATCATGGTACGAAACCGGGTTATGTTGATAGCATTCAGAAAGGTATTCAGAAACCGAAAAGCGGTACCCAGGGTAATTATGATGATGATTGGAAAGAATTTTATAGTACCGATAACAAATATGATGCCGCAGGTTATTCCGTGGATAATGAAAATCCGCTGA GCGGCAAAGCAGGTGGTGTTGTTAAAGTGACATATCCGGGTCTGACGAAAGTGCTGGCCCTGAAAGTTGATAATGCGGAAACCATTAAAAAGGAACTGGGTCTGAGCCTGACCGAACCGCTGATGGAACAGGTGGGTACAGAAGAATTTATTAAACGTTTTGGCGATGGTGCTAGCCGTGTTGTTCTGAGCCTGCCTTTTGCCGAAGGTAGCTCTTCAGTTGAATATATTAATAACTGGGAACAGGCAAAAGCCCTGAGCGTGGAACTGGAAATTAATTTTGAAACACGTGGTAAACGTGGTCAGGATGCAATGTATGAATATATGGCACAGGCATGTGCAGGTAACCGTGTTCGTCGTAGCGTAGGTAGTAGTCTGAGTTGTATTAATCTGGATTGGGATGTTATTCGTGATAAAACAAAAACAAAAATCGAAAGCCTGAAAGAACATGGTCCGATTAAAAAT AAAATGAGCGAAAGCCCGAATAAAACCGTTAGCGAAGAAAAAGCAAAACAGTATCTGGAAGAATTTCATCAGACCGCACTGGAACATCCGGAACTGAGCGAACTGAAAACAGTGACAGGTACCAACCCGGTGTTTGCCGGTGCAAATTATGCAGCATGGGCAGTGAATGTTGCACAGGTGATTGATAGCGAAACCGCAGATAACCTGGAAAAAACCACCGCAGCACTGAGCATTCTGCCGGGTATTGGTAGCGTTATGGGTATTGCAGATGGTGCAGTTCATCATAATACCGAAGAAATTGTTGCACAGAGCATTGCACTGAGCAGCCTGATGGTTGCACAGGCAATTCCGCTGGTTGGTGAACTGGTTGATATTGGTTTTGCAGCATATAATTTTGTTGAAAGCATTATTAATCTGTTTCAGGTTGTTCATAATAGCTATAATCGTCCGGCATATAGCCCGG GTCATAAAACCCAGCCGTTTCTGCATGATGGTTATGCAGTTAGCTGGAATACCGTTGAAGATAGCATTATTCGTACCGGTTTTCAGGGTGAAAGCGGTCATGATATTAAAATTACCGCAGAAAATACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACCATTCCGGGTAAACTGGATGTTAATAAAAGCAAAACCCATATTAGCGTTAATGGTCGTAAAATTCGTATGCGTTGTCGTGCAATTGATGGTGATGTTACCTTTTGTCGTCCGAAAAGCCCGGTTTATGTTGGTAATGGTGTTCATGCAAATCTGCATGTTGCATTTCATCGTAGCAGCAGCGAAAAAATTCATAGCAATGAAATTAGCAGCGATAGCATTGGTGTTCTGGGTTATCAGAAAACCGTTGATCATACCAAAGTTAATAGCAAACTGAGCCTGTTTTTTGAAATTAAAAGCCTCGAG

Example 2

Application of GnRH6-CRM197 recombinant protein castration vaccine:

Twenty adult male rats were selected, with 10 in each control group and test group. The rats were first immunized at 5 months of age, and then boosted immunizations at intervals of 2 weeks. Each immunization dose was 300 micrograms, and the rats were killed 2 months after the last immunization. Testicular samples were prepared for sectioning and stained with hematoxylin-eosin to observe the histological structure.

Evaluation of immunocastration outcomes with GnRH6-CRM197 vaccine:

The testicular tissue of the rats in the experimental group underwent obvious vacuolar degeneration. The testicular seminiferous tubules of rats with vacuolar degeneration changed significantly, the spermatogonia, spermatocytes and spermatids were not clearly layered, and no sperm was produced. The testicular seminiferous tubules of the rats in the control group had normal structure, normal spermatogonia, spermatocytes, spermatids and sperm in the seminiferous tubules, and the layers were clear, and a large number of sperm were produced. It can be seen that GnRH6-CRM197 inhibited the development of rat testicles and eventually atrophied. As shown in Figure 4, the testicles of the rats in the immunization group were smaller, indicating that the developed GnRH6-CRM197 had strong immunogenicity. As shown in Figure 5, the testicular tissue of the rats in the immunization group underwent obvious vacuolar degeneration, the number of spermatogenic cell layers decreased, and no sperm was produced. It can be seen that GnRH6-CRM197 inhibited the development of rat testicles and eventually atrophied, indicating that the developed GnRH6-CRM197 had strong immunogenicity.

The above contents are merely examples and explanations of the structure of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims (4)

1. A GnRH recombinant protein antigen, characterized in that: the amino acid sequence of the GnRH recombinant protein antigen is shown in the sequence listing SEQ ID No. 1. 2. A GnRH6-CRM197 recombinant protein castration vaccine, characterized in that the recombinant protein vaccine contains the GnRH recombinant protein antigen according to claim 1. 3. A GnRH6-CRM197-transformed Escherichia coli, characterized in that: the GnRH6-CRM197-transformed Escherichia coli was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on June 19, 2022, with a deposit number of CGMCCNO.25027, classified and named Escherichia coli, specifically named BL21 (DE3) -PET21a-GnRH6-CRM197, and the GnRH6-CRM197-transformed Escherichia coli expresses a recombinant protein containing the amino acids of SEQ ID No. 1 in the sequence list. 4. A method for preparing a GnRH6-CRM197 recombinant protein castration vaccine, characterized in that: the GnRH6-CRM197 transformed Escherichia coli described in claim 3 is broken and purified to obtain a recombinant protein containing the amino acids of SEQ ID No. 1 in the sequence list, namely the GnRH6-CRM197 recombinant protein castration vaccine.