Animal castration vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal vaccines, and discloses an animal recombinant castration vaccine and a preparation method and application thereof, the animal recombinant castration vaccine comprises an animal castration vaccine antigen and an adjuvant, and the content of the animal castration vaccine antigen in the vaccine is 0.5-1 mg / mL. A gel type vaccine is prepared by adopting an animal castration vaccine antigen with an amino acid sequence as shown in SEQ ID No.2-4, and testis growth and development of rats are obviously inhibited after the rats are immunized. The animal castration vaccine antigen is also used for preparing vaccines in three dosage forms, namely an emulsion type vaccine, an aluminum salt type vaccine and a gel type vaccine, after the three vaccines are used for immunizing rats, the anti-GnRH antibody titer in the rats is obviously increased, the testosterone concentration is reduced (Plt; 0.05), and the testis growth and development are inhibited. The three dosage forms of vaccines all have good immune castration effect, and can be applied to castration or estrus inhibition of pets and economic animals in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of animal vaccines, in particular to an animal castration vaccine and a preparation method and application thereof. Background Art

[0002] Adjuvant refers to auxiliary substances used together with antigens in vaccines to enhance immune responses. They improve the immune effect of vaccines by promoting antigen presentation, prolonging the residence time of antigens in the body, or stimulating different components of the immune system. The mechanism of action of adjuvants generally includes improving the recognition of antigens by immune cells, promoting inflammatory responses, increasing antibody production, and enhancing T cell-mediated immune responses. Suitable adjuvants can synergize with antigens, significantly enhance the response of the immune system, and prompt it to produce high levels of antibodies. Since each adjuvant has its own advantages and disadvantages, and is suitable for different types of antigens, in order to maximize the immune effect of antigens, it is usually necessary to screen a variety of common adjuvants to determine the adjuvant that is most suitable for use with a specific antigen to achieve the best immune effect. In addition, the safety, cost, and toxicity of adjuvants will affect their use in vaccines.

[0003] Aluminum salt adjuvants were the earliest adjuvants used. They have good safety and stability and can effectively induce humoral immune responses, but are relatively weak in cell-mediated immune responses. Oil-emulsion adjuvants can enhance immune responses, but may cause local tissue damage in some cases. Gel adjuvants can slowly release antigens in vivo, prolong the release time of antigens in the body, and continuously stimulate the immune system. They can not only improve humoral immune responses, but also enhance cell-mediated immune responses and improve the overall immune effect of vaccines.

[0004] Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted by the hypothalamus and its surrounding nerve structures. As a key regulatory hormone at the top of the reproductive axis, it can regulate the activity of germ cells through neural and humoral pathways. GnRH can promote the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), thereby promoting sperm production or increasing ovulation. Therefore, by immunizing GnRH, the gonadal axis is regulated, the levels of LH and sex hormones in the blood are reduced, and then gonadal atrophy is induced, achieving the effect of castration. That is, after the animal is actively immunized with GnRH, the body will produce a large number of anti-GnRH antibodies, which specifically bind to endogenous GnRH. When the titer of anti-GnRH antibodies reaches a certain threshold, an immune barrier will be established between the hypothalamus and the pituitary gland, causing the endogenous GnRH to promote LH and FSH to disappear, thereby hindering the synthesis of sex hormones and the occurrence of gametes, thereby achieving the purpose of immune castration.

[0005] The preparation process of castration vaccine needs to be continuously improved to improve the production efficiency and quality stability of the vaccine. Three different dosage forms of castration vaccine were prepared using recombinant 2xGnRH-CRM197-383 protein as antigen. The present invention compared the effects of three castration vaccines and found that the gel adjuvant vaccine had the best castration effect, was simple to prepare, could achieve long-term sustained release of vaccine components, prolonged antigen exposure time, and thus continuously activated the immune response mechanism in the body, which was superior to aluminum salt adjuvant and emulsion adjuvant. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an animal castration vaccine and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: A recombinant animal castration vaccine, comprising an animal castration vaccine antigen and an adjuvant, wherein the content of the animal castration vaccine antigen in the vaccine is 0.5-1 mg / mL; the adjuvant is an emulsion, an aluminum salt or a gel; The amino acid sequence of the animal castration vaccine antigen (2×GnRH-CRM197) is shown as SEQ ID No.2, and the nucleotide sequence encoding the antigen is shown as SEQ ID No.6; or, the amino acid sequence of the animal castration vaccine antigen (2×GnRH-CRM197-190) is shown as SEQ ID No.3, and the nucleotide sequence encoding the antigen is shown as SEQ ID No.7; or, the amino acid sequence of the animal castration vaccine antigen (2×GnRH-CRM197-383) is shown as SEQ ID No.4, and the nucleotide sequence encoding the antigen is shown as SEQ ID No.8.

[0008] The above-mentioned animal castration vaccine antigen is designed using the diphtheria toxoid mutant CRM197 sequence (SEQ ID No. 1) and the GnRH-Ⅰ sequence (SEQ ID No. 5).

[0009] Preferably, a lytic tag is fused to the C-terminus of the animal castration vaccine antigen; preferably, the lytic tag is a SUMO and / or HIS tag.

[0010] Preferably, the recombinant castration vaccine for animals is an emulsion vaccine, which is prepared by mixing an oil phase preparation and an aqueous phase preparation in a volume ratio of 5:4, wherein the oil phase comprises Tween85, Span85, and liquid paraffin in a mass ratio of (5-6): (6-7): (88-89); the aqueous phase preparation comprises phosphate buffer and an animal castration vaccine antigen; Preferably, the vaccine further comprises chlorocresol, and the final mass percentage concentration of chlorocresol is 0.05-0.15%.

[0011] Preferably, the recombinant animal castration vaccine is an aluminum salt vaccine, which contains animal castration vaccine antigen, Al(OH) 3 Gel and phosphate buffer, animal castration vaccine antigen and Al(OH) 3 The gels were mixed in a volume ratio of 1:1-1:10, and the final Al content was 0.4-0.6 mg / mL; Preferably, the vaccine further comprises chlorocresol, and the final mass percentage concentration of chlorocresol is 0.05-0.15%.

[0012] Preferably, the recombinant castration vaccine for animals is a gel-type vaccine, which comprises an animal castration vaccine antigen, DEAE-dextran and phosphate buffer, wherein the final mass percentage concentration of DEAE-dextran is 14-16%; Preferably, the vaccine further comprises chlorocresol, and the final mass percentage concentration of chlorocresol is 0.05-0.15%.

[0013] Furthermore, the present invention also provides a method for preparing the recombinant castration vaccine for animals, comprising the following steps: 1) Mix Tween85, Span85 and liquid paraffin, filter and sterilize to obtain an oil phase preparation for later use; 2) adding the animal recombinant castration vaccine antigen into phosphate buffer, filtering and sterilizing to form an aqueous phase preparation for later use; 3) At 23-27℃, place the oil phase preparation in an emulsifier and mix well. After mixing well, gradually add the water phase preparation dropwise. After all the water phase is added, continue mixing for 25-35 minutes, then add chlorocresol and mix well.

[0014] Furthermore, the present invention also provides a method for preparing the recombinant castration vaccine for animals, comprising the following steps: 1) Combine animal castration vaccine antigen with Al(OH) 3 The gel was mixed, and diluted and mixed with phosphate buffer to obtain a protein-aluminum salt suspension; 2) Slowly stir the protein-aluminum salt suspension at 4-8°C for 3-24 h, then add chlorocresol and mix well.

[0015] Furthermore, the present invention also provides a method for preparing the recombinant castration vaccine for animals, comprising the following steps: 1) Dilute the animal castration vaccine antigen with phosphate buffer and adjust the pH to 7.4-7.7; 2) Add DEAE-dextran and mix well with an oscillator to dissolve, then add chlorocresol and mix well.

[0016] Furthermore, the present invention also provides the use of the recombinant animal castration vaccine in inhibiting estrus in male animals.

[0017] Preferably, the use of the recombinant animal castration vaccine for suppressing estrus in male animals is a male mouse, a boar or a male cat.

[0018] The present invention uses an animal castration vaccine antigen with an amino acid sequence such as SEQ ID No.2-4 to prepare a gel-type vaccine, and the testicular growth and development of rats are significantly inhibited after immunization. The animal castration vaccine antigen is also used to prepare three types of vaccines, namely, emulsion type, aluminum salt type, and gel type. After the three vaccines are immunized in rats, the anti-GnRH antibody titer in the body is significantly increased, the testosterone concentration is decreased (P<0.05), and the testicular growth and development is inhibited. The results of animal experiments show that the antibody titer level of the gel adjuvant group is higher than that of the oil emulsion adjuvant group and the aluminum salt adjuvant group, while the antibody titer level of the oil emulsion is slightly higher than that of the aluminum salt adjuvant group. The three immunization groups are significantly higher than the blank group, indicating that the three vaccines have good immune castration effects and can be used for castration or estrus inhibition of pets and economic animals in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are the testicular anatomical diagrams of each group in Example 4, wherein A: blank control group, B: aluminum salt adjuvant castration vaccine group, C: emulsion adjuvant castration vaccine group, and D: gel adjuvant castration vaccine group.

[0020] Figure 2 These are testicular pathological sections of each group in Example 4, wherein A: blank control group, B: aluminum salt adjuvant castration vaccine group, C: emulsion adjuvant castration vaccine group, and D: gel adjuvant castration vaccine group.

[0021] Figure 3 This is a graph showing the results of the rat anti-GnRH antibody titer determination in Example 4.

[0022] Figure 4 This is a comparison chart of the testicle sizes of rats in Example 5, wherein A: control group, B: 2×GnRH-CRM197 test group, C: 2×GnRH-CRM197-190 test group, and D: 2×GnRH-CRM197-383 test group. DETAILED DESCRIPTION

[0023] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with 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.

[0024] Preparation of recombinant 2xGnRH-CRM197-383 protein: GnRH-CRM197 (380aa) gene fragment and pET28a vector fragment were connected and transformed into E. coli DH5α, and then cultured overnight on LB solid medium containing 50 ug / ml kanamycin (Kana), and single clone colonies were selected for sequencing, and the bacteria with correct sequence were selected to extract plasmids, which were plasmids. The plasmids were transformed into E. coli expression strain BL21 (DE3) by chemical transformation method for expression and purification.

[0025] Example 1: Preparation of emulsion-type recombinant castration vaccine Materials: Tween85, Span 85, liquid paraffin, phosphate buffer, chlorocresol, recombinant 2xGnRH-CRM197-383 protein; phosphate buffer is 20 mM Tris HCl 20 mM urea pH 7.5 phosphate buffer.

[0026] Preparation of recombinant castration vaccine: 1) Mix Tween85, Span85 and liquid paraffin in a mass ratio of 5.24:6.16:88.6, filter and sterilize to obtain an oil phase preparation for later use; 2) Phosphate buffer and recombinant 2xGnRH-CRM197-383 protein were mixed to prepare an aqueous phase preparation, wherein the concentration of the recombinant 2xGnRH-CRM197-383 protein was 1.125 mg / mL; 3) Place 10 mL of the oil phase preparation in a homogenizer and mix at 7000 rpm. During the process, add 8 mL of the water phase preparation dropwise. After all the water phase is added, continue mixing for 30 minutes. Use a homogenizer to emulsify it into a slightly viscous two-phase oil emulsion vaccine. During the emulsification process, maintain the ambient temperature at 23-27 °C. After the emulsification is completed, the final concentration of the emulsion adjuvant vaccine is 0.5 mg / mL. Add chlorocresol and mix well. The final mass percentage concentration of chlorocresol is 0.1%.

[0027] Example 2: Preparation of aluminum salt-based recombinant castration vaccine Material: Al(OH) 3 Gel adjuvant, phosphate buffer, chlorocresol, recombinant 2xGnRH-CRM197-383 protein; phosphate buffer is 20 mM Tris HCl 20 mM urea pH 7.5 phosphate buffer.

[0028] Preparation of recombinant castration vaccine: 1) Mix 5 mL of 1 mg / mL recombinant 2xGnRH-CRM197-383 protein and 5 mL of Al(OH) 3The gel adjuvant was mixed in a ratio of 1:1, and diluted and mixed with urea phosphate buffer to obtain a protein-aluminum salt suspension with a final protein concentration of 0.5 mg / mL and an Al content of 0.5 mg / mL; 2) The protein-aluminum salt suspension was slowly stirred at 4-8 °C for 10 h using a magnetic stirrer, and chlorocresol was added and mixed. The final mass percentage concentration of chlorocresol was 0.1%.

[0029] Example 3: Preparation of gel-type recombinant castration vaccine Materials: DEAE-dextran gel, phosphate buffer, recombinant 2xGnRH-CRM197-383 protein, chlorocresol; phosphate buffer is 20 mM Tris HCl 20 mM urea pH 8.0 buffer salt.

[0030] Preparation of recombinant castration vaccine: 1) Dilute the recombinant 2xGnRH-CRM197-383 protein with phosphate buffer to a concentration of 0.5 mg / mL, and adjust the pH of the mixture to 7.5; 2) Add DEAE-dextran gel to a final mass percentage concentration of 15%, mix and dissolve with an oscillator, add chlorocresol and mix, the final mass percentage concentration of chlorocresol is 0.1%.

[0031] Example 4 (1) Vaccine immunization process Twenty three-week-old SD rats were selected and divided into four groups, 5 in each group, and immunized with 50 μg of recombinant castration vaccine on day 0 and day 28. A negative PBS group, an emulsion adjuvant group (vaccine in Example 1), an aluminum salt adjuvant group (vaccine in Example 2), and a gel adjuvant group (vaccine in Example 3) were set up. The anti-GnRH antibody titer and testosterone level of these rats were measured. On the 56th day after immunization, the rats were killed and the testicular weight was measured.

[0032] (2) Determination of rat anti-GnRH antibody titer At different time points during the experiment, serum was collected from immunized rats and control rats. Anti-GnRH IgG antibody titers were determined by ELISA as follows: 96-well plates were coated with 20 μg / mL gonadorelin overnight at 4 °C, 100 μL per well. The next day, the plates were washed 5 times with PBST, and 150 μL of 5% BSA-PBS was added for blocking at room temperature for 2 h. After that, the plates were washed 5 times with PBST. Rat serum was diluted in a gradient of 1:200 as the initial double dilution, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and the dilution was diluted with 1% casein diluent from high to low dilution, 100 μL per well, incubated at room temperature for 2 h, and the plates were washed 5 times with PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as the secondary antibody, with a dilution of 1:12000, diluted with 1% casein diluent, 100 μL per well, and incubated at room temperature for 2 h. Wash the plate 5 times with PBST. Add 150 μL of colorimetric solution to each sample well, color for 15 min in the dark, add 50 μL of stop solution, stop color development, and read directly at 450 nm. The optical density was measured with an ELISA reader at 450 nm. The average OD value of the serum of each group of rats after color development every week after immunization is shown in the following figure. Figure 3 shown.

[0033] (3) Determination of testosterone levels in rat blood A testosterone assay kit (Beijing North Biotechnology Institute) was used to determine the testosterone hormone levels in the serum of each group of rats 4 weeks after the second immunization. The enzyme-linked immunosorbent assay was used to detect the testosterone content in the serum. First, the microplate was coated with sheep anti-rabbit IgG to prepare a solid-phase secondary antibody, and then the serum to be tested, horseradish peroxidase-labeled testosterone, and anti-testosterone antibody were added to form a coated secondary antibody-anti-testosterone antibody-testosterone (HRP) complex. The amount of labeled testosterone bound is inversely proportional to the amount of testosterone in the serum. After color development, the absorbance value (OD value) was measured on an enzyme reader, and the testosterone content in the serum to be tested was calculated by computer or graphical fitting of the concentration-absorbance curve. The results are shown in Table 1. The macroscopic and histological changes of the testicles, as well as the changes in sperm count and deformity rate were observed to determine the effect of the castration vaccine. The results are shown in Table 1. Figure 1 , 2 And as shown in Table 2.

[0034] Table 1 Group Quantity (pcs) Average testosterone level (ng / mL) Gel adjuvant group 5 <![CDATA[3.79 ± 2.34 a ]]> Oil emulsion adjuvant group 5 <![CDATA[5.97 ± 4.26 a ]]> Aluminum salt adjuvant group 5 <![CDATA[11.52 ±16.67 a ]]> Blank control group 5 <![CDATA[104 ± 35.4 b ]]> The same letter in different groups (such as a): indicates that there is no significant difference between two or more groups; Different letters for different groups (such as a vs. b): Indicates that there is a significant difference between the two groups.

[0035] As can be seen from Table 1, the differences among the gel adjuvant group, oil emulsion adjuvant group, aluminum salt adjuvant group and the blank control group were statistically significant.

[0036] Table 2 Group Quantity (pcs) Castration rate Gel adjuvant group 5 100% Oil emulsion adjuvant group 5 100% Aluminum salt adjuvant group 5 80% Blank control group 5 0% Depend on Figure 1 It can be seen that the three different dosage forms of castration vaccines can significantly inhibit the development of rat testicles, eventually leading to testicular atrophy. The testicles of the immunized rats were smaller in size, which fully demonstrated that the three vaccines have extremely high immunogenicity.

[0037] from Figure 2 It can be observed that obvious vacuolar degeneration appeared in the testicular tissue of the three immunized rats, the number of spermatogenic cell layers decreased, and no sperm was produced. In contrast, the seminiferous tubules of the rats in the blank control group had normal structure, and the seminiferous tubules contained normal spermatogonia, spermatocytes, spermatids and sperm with clear layers, and were accompanied by a large amount of sperm production.

[0038] The castration vaccines prepared with the three vaccine adjuvants immunized rats and all achieved excellent castration effects ( Figure 1 , Table 2). Figure 3 It can be seen that 56 days after immunization, the anti-GnRH antibody titer increased significantly and the testosterone level decreased significantly. The increase in the anti-GnRH recombinant protein antibody titer was 50% to 90%, and the oil emulsion, aluminum salt and gel adjuvant were 50% to 70%, 60% to 80%, and 70% to 90%, respectively. In some tests, the testosterone level was reduced to 50% to 90% of the original level, and the oil emulsion, aluminum salt and gel adjuvant were 60% to 80%, 70% to 90%, and more specifically 50% to 60%, 60% to 70%, 70% to 80%, and 80% to 90%.

[0039] In summary, the oil emulsion, aluminum salt and gel adjuvant groups can induce rats to produce anti-GnRH antibodies well, causing a significant decrease in testosterone. Compared with the negative control group, the three immune groups can significantly atrophy the gonads. The results of animal experiments showed that the titer level of anti-GnRH recombinant protein antibodies in the gel adjuvant group was higher than that in the oil emulsion adjuvant group and the aluminum salt adjuvant group, while the titer level of antibodies in the oil emulsion group was slightly higher than that in the aluminum salt adjuvant group.

[0040] Example 5 Recombinant 2×GnRH-CRM197 protein, recombinant 2×GnRH-CRM197-190 protein and recombinant 2×GnRH-CRM197-383 protein were used to prepare gel vaccines, and the final concentration of antigen protein was 0.5 mg / mL.

[0041] Twenty 3-week-old male SD rats were selected and divided into four groups, each consisting of 5 rats. The rats were vaccinated with 100 μL of vaccine (containing 50 μg of antigen) on day 0 and day 28 respectively. The rats were killed and dissected on day 28 after the second immunization, and testicular samples were taken for measurement. The results are shown in Figure 4 shown.

[0042] The above is a description of the embodiments of the present invention. Through the above description of the disclosed embodiments, professionals and technicians in the field can implement or use the present invention. Various modifications to these embodiments will be apparent to professionals and technicians in the field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will conform to the widest range consistent with the principles and novelties disclosed herein.

Claims

1. A recombinant castration vaccine for animals, characterized in that: The recombinant animal castration vaccine comprises an animal castration vaccine antigen and an adjuvant, wherein the content of the animal castration vaccine antigen in the vaccine is 0.5-1 mg / mL; the adjuvant is an emulsion or an aluminum salt or a gel; The amino acid sequence of the animal castration vaccine antigen is shown as SEQ ID No.2, and the nucleotide sequence encoding the antigen is shown as SEQ ID No.6; or, the amino acid sequence of the animal castration vaccine antigen is shown as SEQ ID No.3, and the nucleotide sequence encoding the antigen is shown as SEQ ID No.7; or, the amino acid sequence of the animal castration vaccine antigen is shown as SEQ ID No.4, and the nucleotide sequence encoding the antigen is shown as SEQ ID No.

8.

2. The recombinant castration vaccine for animals according to claim 1, characterized in that: A solubility-promoting tag is fused to the C-terminus of the animal castration vaccine antigen.

3. The recombinant castration vaccine for animals according to claim 2, characterized in that: The lytic tag is SUMO and / or HIS tag.

4. A recombinant castration vaccine for animals according to claim 1, 2 or 3, characterized in that: The recombinant animal castration vaccine is an emulsion vaccine, which is prepared by mixing an oil phase preparation and an aqueous phase preparation in a volume ratio of 5:

4. The oil phase contains Tween85, Span85, and liquid paraffin in a mass ratio of (5-6): (6-7): (88-89); the aqueous phase preparation contains phosphate buffer and animal castration vaccine antigens.

5. The animal recombinant castration vaccine according to claim 4, characterized in that: The vaccine also contains chlorocresol, and the final mass percentage concentration of chlorocresol is 0.05-0.15%.

6. A recombinant castration vaccine for animals according to claim 1, 2 or 3, characterized in that: The recombinant animal castration vaccine is an aluminum salt vaccine, which comprises an animal castration vaccine antigen, Al(OH)3 gel and a phosphate buffer solution, wherein the animal castration vaccine antigen and the Al(OH)3 gel are mixed at a volume ratio of 1:1-1:10, and the final Al content is 0.4-0.6 mg / mL; Alternatively, the recombinant animal castration vaccine is a gel-type vaccine, which comprises an animal castration vaccine antigen, DEAE-dextran and phosphate buffer, and the final mass percentage concentration of DEAE-dextran is 14-16%.

7. The animal recombinant castration vaccine according to claim 6, characterized in that: The vaccine also contains chlorocresol, and the final mass percentage concentration of chlorocresol is 0.05-0.15%.

8. The method for preparing a recombinant castration vaccine for animals according to claim 6, characterized in that: The preparation method comprises the following steps: 1) Mix Tween85, Span85 and liquid paraffin, filter and sterilize to obtain an oil phase preparation for later use; 2) adding the animal recombinant castration vaccine antigen into phosphate buffer, filtering and sterilizing to form an aqueous phase preparation for standby use; 3) Place the oil phase preparation in an emulsifier and mix at 23-27°C. After mixing, gradually add the water phase preparation dropwise. After all the water phase is added, continue mixing for 25-35 minutes. Then add chlorocresol and mix well. Alternatively, the preparation method comprises the following steps: 1) Mix the animal castration vaccine antigen with Al(OH)3 gel, and dilute and mix with phosphate buffer to obtain a protein-aluminum salt suspension; 2) Slowly stir the protein-aluminum salt suspension at 4-8°C for 3-24 h, then add chlorocresol and mix well.

9. The method for preparing a recombinant castration vaccine for animals according to claim 8, characterized in that: The following steps are involved: 1) Dilute the animal castration vaccine antigen with phosphate buffer and adjust the pH to 7.4-7.7; 2) Add DEAE-dextran and mix well with an oscillator to dissolve, then add chlorocresol and mix well.

10. Use of the recombinant castration vaccine for animals according to any one of claims 1 to 7 in suppressing estrus in male animals.

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