Animal castration vaccine antigen as well as preparation method and application thereof

Abstract

The invention relates to the fields of molecular biology, immunology and vaccinology, and discloses an animal castration vaccine antigen as well as a preparation method and application thereof. The animal castration vaccine antigen is a recombinant protein of GnRH and CRM197 protein, and is a recombinant fusion expression protein divided and designed according to a diphtheria toxoid mutant CRM197 sequence (SEQ ID No.1) and a GnRH-I sequence (SEQ ID No.5) in combination with a CRM197 structural domain on Uniport, and the animal castration vaccine antigen contains 2 * GnRH sequences at the N end and the C end of a carrier protein. The animal castration vaccine antigen designed by the invention has strong immunogenicity, uses an Escherichia coli expression system, introduces a related dissolution promoting label, is safer and more convenient, and effectively increases the yield of protein.

Description

Technical Field

[0001] The invention relates to the fields of molecular biology, immunology and vaccinology, and in particular to an animal castration vaccine antigen and a preparation method and application thereof. Background Art

[0002] Traditional castration methods mainly rely on surgical castration, but this method causes great stress to animals, has a long recovery period, and is prone to wound infection and may even cause death. In addition, surgical castration has high technical requirements for operators, and the operation without anesthesia poses potential safety risks to both animals and humans. At the same time, surgical castration also faces ethical controversy over animal welfare. In some countries and regions, radical animal welfare advocates strongly oppose this method. In contrast, GnRH castration vaccine, as an alternative to surgical castration, effectively inhibits gonadotropin-releasing factor (GnRH) to prevent animals from sexual maturity. It has high safety, convenience and can effectively reduce the risk of infection. In addition, GnRH vaccine meets the needs of modern animal husbandry for large-scale and standardized production, and is therefore regarded as a more effective castration solution.

[0003] GnRH was originally isolated from the mammalian hypothalamus. As a neuroendocrine hormone, it participates in the reproductive regulation of the hypothalamic-pituitary-gonadal axis (HPG axis). The amino acid sequence of GnRH is highly conserved in mammals, and its structure is pyroglutamic acid-histidine-tryptophan-serine-tyrosine-glycine-leucine-arginine-proline-glycinamide. Its mechanism of action is to stimulate the pituitary gland to secrete hormones through periodic release, thereby regulating the development and function of the gonads, including spermatogenesis and ovulation. As a non-surgical castration method, the GnRH vaccine induces the body to produce antibodies against GnRH through active immunization. These antibodies bind to endogenous GnRH, weaken its biological activity, and block its stimulating effect on the pituitary gland, thereby reducing the secretion of gonadal hormones and gonadal function, ultimately achieving castration.

[0004] Since GnRH is composed of only 10 amino acids and has a molecular weight of 1.2 kDa, its immunogenicity is low and it is difficult to induce an effective immune response in the body. Therefore, the method of chemically coupling the GnRH small molecule peptide with a large molecule carrier protein is usually used to increase the overall molecular size of the antigen, thereby improving its immunogenicity.

[0005] The CRM197 protein is a mutant of diphtheria toxin in which the glycine at position 52 is replaced by glutamic acid. It is non-toxic, safe, and significantly enhances the immune response. This protein has been widely used in the field of small molecule conjugate vaccines and is one of the commonly used carrier proteins. The CRM197 protein is similar in structure to diphtheria toxin and consists of two fragments, A and B. The A fragment contains a catalytic domain, and the B fragment contains two domains, R and T, which are responsible for binding to the host cell surface receptor and promoting the transfer of fragment A to the cytoplasm, respectively.

[0006] The Improvac boar odor control vaccine launched by Zoetis in the United States is one of the few castration vaccines that can be commercially applied. The antigen of this vaccine is mainly prepared by chemically coupling the GnRH small molecule peptide with the CRM197 protein expressed by Corynebacterium diphtheriae. However, there are high safety risks and high costs in using Corynebacterium diphtheriae to express the CRM197 protein, and its application in actual production is subject to certain restrictions. Patent CN116162173A introduces the expression of the fusion protein of N-terminal hexalinked GnRH and CRM197 by Escherichia coli, thereby effectively improving the immune effect of GnRH. Patent CN103495161A uses the A chain protein of CRM197 as a carrier to couple 13 serotypes of pneumococcal capsular polysaccharides with the A chain protein, thereby significantly improving the immunogenicity of the pneumococcal capsular polysaccharide. Using the Escherichia coli expression system to replace the Corynebacterium diphtheriae expression system to produce the CRM197 protein has become the first choice for current biotechnology. However, due to the complex structure of the CRM197 protein, when expressed in Escherichia coli, the product is mostly in the form of inclusion bodies and the yield is low, which leads to higher subsequent production costs. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides an animal castration vaccine antigen and a preparation method and application thereof.

[0008] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The invention discloses an animal castration vaccine antigen, which is a recombinant protein of GnRH and CRM197 protein, and is a recombinant fusion expression protein designed based on the diphtheria toxoid mutant CRM197 sequence (SEQ ID No.1) and the GnRH-Ⅰ sequence (SEQ ID No.5) combined with the CRM197 domain division on Uniport. The animal castration vaccine antigen contains 2×GnRH sequences at the N-terminus and C-terminus of the carrier protein.

[0009] The amino acid sequence of the animal castration vaccine antigen (2×GnRH-CRM197) is shown in SEQ ID No.2, which contains all the amino acids of the CRM197 protein, and the nucleotide sequence encoding the antigen is shown in SEQ ID No.6; or, the amino acid sequence of the animal castration vaccine antigen (2×GnRH-CRM197-190) is shown in SEQ ID No.3, which contains the first 190 amino acids of the CRM197 protein, and the nucleotide sequence encoding the antigen is shown in SEQ ID No.7; or, the amino acid sequence of the animal castration vaccine antigen (2×GnRH-CRM197-383) is shown in SEQ ID No.4, which contains the first 383 amino acids of the CRM197 protein, and the nucleotide sequence encoding the antigen is shown in SEQ ID No.8.

[0010] An expression vector, the expression vector contains a gene encoding the animal castration vaccine antigen, the nucleotide sequence of the gene is shown as SEQ ID No.6 or SEQ ID No.7 or SEQ ID No.8.

[0011] Preferably, the expression vector comprises a lytic tag, which is fused to the C-terminus of the animal castration vaccine antigen.

[0012] Preferably, the lytic tag is a SUMO and / or HIS tag.

[0013] A host cell, the host cell contains the gene of the animal castration vaccine antigen, or contains the expression vector.

[0014] Preferably, the host cell is an Escherichia coli host cell containing the gene of the animal castration vaccine antigen or containing the expression vector.

[0015] Furthermore, the present invention also provides a method for preparing the animal castration vaccine antigen, the preparation method comprising the following steps: 1) inserting the gene fragment encoding the animal castration vaccine antigen into the expression vector pET28a through BamH I and Xho I double enzymes to construct a recombinant expression plasmid; 2) transforming the recombinant expression plasmid into DH5α competent cells, and extracting the plasmid after culture and resistance screening; 3) The recombinant plasmid obtained in step 2) is transfected into host cells, and the animal castration vaccine antigen is obtained after inducing expression, separation and purification.

[0016] Preferably, the antigen obtained in step 3) can be dialyzed and freeze-dried to obtain antigen freeze-dried powder that can be used to prepare a vaccine.

[0017] Furthermore, the present invention also provides the use of the animal castration vaccine antigen in preparing a castration vaccine for inhibiting the fertility of mammals.

[0018] Furthermore, the present invention also provides the use of the animal castration vaccine antigen in preparing a castration vaccine for inhibiting testicular growth of male animals.

[0019] The present invention designs a novel GnRH castration vaccine antigen, places 2×GnRH at the N-terminus and C-terminus of the CRM197 protein or its fragment, and fuses SUMO and HIS tags at the C-terminus. The specific technical effects include the following: 1) The animal castration vaccine antigen designed by the present invention has strong immunogenicity, and significantly improves the immunogenicity of GnRH compared with GnRH short peptide or simple tandem repeat GnRH protein.

[0020] 2) The present invention adopts gene recombination expression technology. Compared with the traditional method of chemical coupling of polypeptide and carrier protein, the method of the present invention is safer and more convenient.

[0021] 3. The present invention uses an E. coli expression system and introduces relevant solubility-promoting tags, which increases protein yield while reducing the difficulty of antigen protein purification and subsequent soluble processing, further reducing production costs.

[0022] In summary, the design and preparation method of the present invention not only improves the immunogenicity of the vaccine, but also effectively increases the protein yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a gel electrophoresis analysis diagram of the antigen protein in Example 1. In the figure, M refers to the Thermo 250kDa Merker band, 1 refers to the 2×GnRH-CRM197 band, 2 refers to the 2×GnRH-CRM197-190 band, and 3 refers to the 2×GnRH-CRM197-383 band.

[0024] Figure 2 This is a comparison chart of antibody concentrations in rat blood within 8 weeks in Example 2.

[0025] Figure 3 , 4 This is a comparison chart of the testicle and epididymis sizes of rats in Example 2, in which A: control group, B: 2×GnRH-CRM197 test group, C: 2×GnRH-CRM197-190 test group, and D: 2×GnRH-CRM197-383 test group.

[0026] Figure 5This is a histological observation picture of the testicles of rats in Example 2, the microscope magnification is 100×, in the figure, A: control group, B: 2×GnRH-CRM197 test group, C: 2×GnRH-CRM197-190 test group, D: 2×GnRH-CRM197-383 test group. DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1: Preparation of animal castration vaccine antigen 1. Construction of recombinant plasmid: The method for constructing a recombinant antigen expression plasmid for animal castration vaccine comprises the following steps: The lyophilized powder of DNA sequence (Seq 6-Seq 8) with BamH I and Xho I restriction sites synthesized by Beijing Qingke Biotechnology Co., Ltd. was dissolved in sterile ultrapure water according to the instructions. The dissolved DNA and pET28a plasmid were simultaneously digested with BamH I and Xho I endonucleases in a constant temperature water bath at 37°C for 2 h. The specific enzyme digestion system is shown in Table 1.

[0029] Table 1 Enzyme digestion system

[0030] Reagent name Volume (μL) Recycling products 10 Buffer 5 Endonuclease 1 2 Endonuclease 2 2 ddH2O 31

[0031] The target fragments were separated by nucleic acid electrophoresis, and the target bands were cut out with a scalpel and purified using a gel recovery kit. The purified enzyme-cut fragments and the vector were connected at a ratio of 6:1 at 25°C for 1 h using T4 ligase. The connection system is shown in Table 2.

[0032] Table 2 Connection system

[0033] Reagent name Volume (μL) Gene fragment 6 Carrier 1 Ligase 1 Buffer 2

[0034] Add the ligation product to 100 μL DH5α competent cells and place on ice for 30 min. Heat shock in a 42℃ water bath for 90 s and place on ice for 2 min. Add 600 μL of LB liquid medium without antibiotics and culture at 37℃ 200 r / min for 1 h. Centrifuge at 6000 rpm / min for 1 min and discard the supernatant. Keep about 100 μL of resuspended bacterial pellet and spread the bacterial suspension evenly on the kanamycin-resistant plate. Culture in a 37℃ incubator overnight until a single colony grows. Pick a larger single colony and culture it at 37℃, 200 rpm / min for 12-16 h, then culture it in a test tube and extract the recombinant plasmid.

[0035] 2. Induced expression and purification of recombinant proteins: 1. Induce expression: Transform the recombinant expression plasmid into BL21, spread on a kanamycin resistance plate, and culture in a 37°C incubator for 12-16 h. Pick a single colony and transfer it to 10 ml LB medium containing 50 μg / ml kanamycin, culture it at 37°C, 200 rpm / min for 16 h, then transfer it to 1 L LB medium containing 50 μg / ml kanamycin, and culture it at 37°C, 200 rpm / min until the bacterial solution OD reaches 0. 600 When the concentration of IPTG was 0.6, IPTG was added at a final concentration of 1.0 mmol / L, and the culture was continued at 37°C and 200 rpm / min for 10 h. After the culture was completed, the bacterial solution was collected and centrifuged at 4°C and 8000 rpm / min for 30 min, and the supernatant was discarded.

[0036] 2. Disrupt bacteria: resuspend the precipitate in a ratio of 20 ml buffer (20 mM Tris-HCl, pH 8.0) per gram of wet bacteria, add 1% Trition X-100, and ultrasonically disrupt on ice (260 W, ultrasonic for 3 seconds, stop for 2 seconds) for 10-30 minutes; after the ultrasonication, centrifuge at 4°C, 12000 rpm / min for 30 minutes, discard the supernatant, and collect the precipitate.

[0037] 3. Inclusion body washing: Add washing solution (2M Urea, 20mM Tris-HCl, pH8.0) to the precipitate in the same proportion to resuspend, add 1% Trition X-100 to wash once, centrifuge at 4℃, 12000 rpm / min for 30min, and discard the supernatant. Add an equal volume of buffer (20mM Tris-HCl, pH8.0) to resuspend, centrifuge at 4℃, 12000rpm / min for 30min, and discard the supernatant. Add inclusion body dissolution solution (8M Urea, 20mM Tris-HCl, pH8.0) to the precipitate at 1 g / 20 ml and dissolve at 4℃. Centrifuge the dissolved protein at 4℃, 12000rpm / min for 30min, discard the precipitate, and filter the supernatant through a 0.45 μM filter.

[0038] 4. Protein purification: The collected protein was purified by nickel affinity chromatography using AKTA. The purification buffer was 20 mM Tris-HCl / 4 M Urea / pH 8.0, and the eluent was 20 mM Tris-HCl / 4 M Urea / 500 mM Imidazole / pH 8.0.

[0039] 5. Protein desalting and concentration: The purified protein was concentrated using an ultrafiltration concentration system and dialyzed with PBS buffer at 4°C for 10 h. The protein solution was collected for vacuum freeze drying. The powder collected was the prepared antigen protein. A small amount of protein powder was weighed and re-dissolved with PBS buffer and then tested by SDS-PAGE electrophoresis (electrophoresis at 80V for 30min and electrophoresis at 120V for 70min). The electrophoresis showed that its molecular weight was in line with expectations ( Figure 1 ).

[0040] Example 2: Immunogenicity test of recombinant antigens The antigen protein was diluted with phosphate buffer, which was 20 mM Tris HCl 20 mM urea pH 8.0 buffer salt. The pH of the mixed solution was adjusted to 7.5, the final concentration of the antigen protein was 0.5 mg / mL, DEAE-dextran gel was added, and its final mass percentage concentration was 15%, and it was mixed and dissolved with an oscillator, and chlorocresol was added and mixed, and the final mass percentage concentration of chlorocresol was 0.1%, and it was set aside.

[0041] Twenty 3-week-old male SD rats were selected and divided into four groups, each consisting of 5 rats. 100 μL of vaccine (containing 50 μg antigen) was administered on day 0 and day 28, and blood was collected every week after immunization to detect GnRH antibodies in serum by indirect enzyme-linked immunosorbent assay ( Figure 2). On the 28th day after the secondary immunization, the rats were killed and dissected, and testicular samples were taken to prepare sections and stain to observe the histological structure.

[0042] Evaluation of the immune castration effect of the vaccine: After the first immunization, the level of GnRH antibodies in the serum of the rats in the test group showed a slow upward trend. After the second immunization, the GnRH antibodies in the serum of the rats in the test group increased significantly. After the autopsy, the morphology and size of the rat testicles and epididymis were observed, and it was found that the size of the testicles and epididymis of the rats in the test group was significantly smaller than that of the control group ( Figure 3 , 4 ). Microscopic observation of testicular tissue sections showed that the testicular tissue of the experimental group rats underwent obvious vacuolar degeneration. The testicular seminiferous tubules of rats with vacuolar degeneration showed obvious changes, the layered structure of spermatogonia, spermatocytes and spermatids was unclear, and no sperm production was observed. In contrast, the testicular seminiferous tubules of the control group rats had normal structure, clear layers in the seminiferous tubules, containing normal spermatogonia, spermatocytes, spermatids and sperm, and abundant sperm production ( Figure 5 ).

[0043] 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. An animal castration vaccine antigen, characterized in that: 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. An expression vector, characterized in that: The expression vector contains a gene encoding the animal castration vaccine antigen as claimed in claim 1, and the nucleotide sequence of the gene is shown as SEQ ID No.6 or SEQ ID No.7 or SEQ ID No.

8.

3. An expression vector according to claim 2, characterized in that: The expression vector comprises a lytic tag.

4. An expression vector according to claim 3, characterized in that: The lytic tag is SUMO and / or HIS tag.

5. A host cell, characterized in that The host cell contains the gene of the animal castration vaccine antigen according to claim 1, or contains the expression vector according to any one of claims 2-4.

6. A host cell according to claim 5, characterized in that The host cell is an Escherichia coli host cell containing the gene of the animal castration vaccine antigen according to claim 1 or containing the expression vector according to any one of claims 2-4.

7. The method for preparing an animal castration vaccine antigen according to claim 1, characterized in that: The preparation method comprises the following steps: 1) inserting the gene fragment encoding the animal castration vaccine antigen as claimed in claim 1 into the expression vector pET28a by using BamH I and Xho I double enzymes to construct a recombinant expression plasmid; 2) transforming the recombinant expression plasmid into DH5α competent cells, and extracting the plasmid after culture and resistance screening; 3) The recombinant plasmid obtained in step 2) is transfected into host cells, and the animal castration vaccine antigen is obtained after inducing expression, separation and purification.

8. The method for preparing an animal castration vaccine antigen according to claim 7, characterized in that: Step 3) The antigen obtained can be dialyzed and freeze-dried to obtain antigen freeze-dried powder that can be used to prepare a vaccine.

9. Use of the animal castration vaccine antigen according to claim 1 in the preparation of a castration vaccine for inhibiting the fertility of mammals.

10. Use of the animal castration vaccine antigen according to claim 1 in the preparation of a castration vaccine for inhibiting testicular growth in male animals.

Images