A method for preparing a reproductive vaccine based on a novel carrier protein coupled GnRH polypeptide and products thereof

Abstract

The application discloses a method for preparing a reproductive vaccine based on a novel carrier protein coupled GnRH polypeptide and a product, wherein the amino acid sequence of the carrier protein is shown as SEQ ID NO.1, the gene sequence for coding the carrier protein is shown as SEQ ID NO.2, the gene for coding the carrier protein is subjected to prokaryotic expression, and then subjected to affinity chromatography, anion exchange chromatography purification, TEV enzyme cutting to remove a tag protein, and then subjected to affinity chromatography and molecular sieve to obtain the purified carrier protein, and finally covalently coupled with a GnRH polypeptide to obtain the reproductive vaccine, wherein the reproductive vaccine prepared by the application has a contraceptive effect, and high-quality, low-cost recombinant protein vaccines can be obtained on a large scale for commercialization.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing a reproductive vaccine based on a novel carrier protein coupled with GnRH peptides, and also to the product prepared by the method. Background Technology

[0002] In today's society, people's demand for pet ownership is increasing. To avoid the troubles caused to owners by the related behaviors of pets during mating season, such as prolonged barking by females and urinating everywhere due to increased territorial marking by males, and the significantly stronger odor of urine caused by hormonal changes during courtship, as well as the financial burden on owners due to unintentional successful mating leading to puppies and reproductive diseases caused by aging pets, many owners choose to spay or neuter their pets, surgically removing their gonads. However, this method permanently renders the pet infertile. Furthermore, for highly intelligent pets like dogs, this procedure can potentially lead to psychological problems, thereby affecting the relationship between owner and pet.

[0003] Gonadotropin-releasing hormone (GnRH) is a polypeptide hormone synthesized by the hypothalamus that promotes the maturation of animal gonads, and its main functions are related to reproduction and sexual behavior. Studies have shown that inhibiting GnRH release has a contraceptive effect in both male and female animals. Furthermore, GnRH deficiency can be used to treat malignant tumors such as prostate cancer. Therefore, GnRH is currently being developed as a reproductive vaccine.

[0004] Recombinant protein vaccines are synthetic vaccines obtained by covalently linking pathogen-specific polysaccharides or peptides to a carrier protein. The carrier protein is crucial to the success of a recombinant protein vaccine, stimulating a T-cell-dependent immune response, thereby stimulating antigen presentation in the germinal center and promoting the differentiation and selection of memory B cells. Currently, recombinant protein vaccines mainly rely on five carrier proteins: CRM197 (diphtheria toxin), DT (artificially prepared inactivated diphtheria toxin), TT (formaldehyde-treated Clostridium tetani preparation), OMP (outer membrane protein complex), and PD (surface lipoprotein derived from Haemophilus influenzae). Among these, only CRM197 and PD are single-component recombinant proteins.

[0005] CRM197 is a non-toxic mutant strain of diphtheria toxin (DT), possessing a single mutation in which glycine at position 52 is replaced by glutamic acid. While losing its toxicity, it retains the same immunostimulatory properties as DT. CRM197 was initially produced using Corynebacterium diphtheriae, but yields were low, and complex laboratory conditions were required to culture the strain. Currently, CRM197 can be produced in heterologous recombinant systems such as Escherichia coli, with higher yields. The resulting CRM197 is structurally and immunologically similar to the Corynebacterium diphtheriae product and can be used as a vector for conjugate vaccine development. However, due to its single-component advantage, most commercially available vaccines based on vector proteins currently use either CRM197 or PD, thus limiting vaccine development to the choice of vector protein. Therefore, screening for novel vector proteins and successfully developing related vaccines is particularly important.

[0006] Currently, while there are reports on the preparation of GnRH-based reproductive vaccines, no reproductive vaccines based on recombinant proteins have emerged, especially those based on novel carrier proteins. These vaccines possess a competitive advantage in the vaccine industry due to their large-scale, high-quality, and low-cost production. The successful development and deployment of reproductive vaccines based on novel carrier proteins could also make it possible to construct multivalent vaccines with other vaccines based on the CRM197 carrier protein. Furthermore, the availability of such vaccines would reduce the price of pet reproductive vaccines, allowing pet owners to achieve contraception without surgery, and enabling them to restore their pets' reproductive capacity at any time when owners are able and wish to do so. Summary of the Invention

[0007] In view of the above, the present invention aims to provide a carrier protein; a second objective of the present invention is to provide a gene encoding the carrier protein of claim 1; a third objective of the present invention is to provide a method for preparing a reproductive vaccine based on the carrier protein coupled with a GnRH polypeptide; a fourth objective of the present invention is to provide a reproductive vaccine prepared by the method; and a fifth objective of the present invention is to provide the application of the reproductive vaccine in the castration of male animals.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] 1. A carrier protein, said carrier protein being derived from CRM197, said carrier protein having the amino acid sequence shown in SEQ ID NO.1.

[0010] 2. A gene encoding the vector protein of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0011] 3. A method for preparing a reproductive vaccine based on the carrier protein coupled with GnRH peptide, comprising the following preparation steps:

[0012] The gene encoding the carrier protein of claim 1 is expressed in prokaryotes, then purified by affinity chromatography and anion exchange chromatography, the tag protein is removed by TEV enzyme digestion, and then the purified carrier protein is obtained by affinity chromatography and molecular sieve. It is then covalently coupled with GnRH peptide to obtain the reproductive vaccine.

[0013] Preferably, the prokaryotic expression vector is pET-28a, and the prokaryotic expression strain is BL21(DE3).

[0014] Preferably, the covalent coupling of the present invention is using glutaraldehyde coupling.

[0015] Preferably, the final concentration of glutaraldehyde in this invention is 1% by volume.

[0016] Preferably, the molar ratio of the GnRH polypeptide to the carrier protein is 30:1.

[0017] 4. The reproductive vaccine prepared by the method described above.

[0018] 5. The application of the reproductive vaccine in the castration of male animals.

[0019] The beneficial effects of this invention are as follows:

[0020] The recombinant protein vaccine prepared in this invention is mainly derived from the CRM197 recombinant protein vector, which has lost its toxicity but retains the same immunostimulatory properties as DT, and was redesigned using the EvoDesign algorithm. In this invention, five different novel vector protein sequences and related structures were first obtained. Subsequently, the antibody cross-reactivity and corresponding titers of these five novel vector proteins in mice were verified. Finally, D3 and D5 were determined to be suitable for subsequent research. Through redesign of D3 and D5, this invention obtained four mutants based on D3 and D5. After cross-reactivity testing with CRM197, D3M1 was ultimately selected as the novel vector protein for subsequent use in this invention. By covalently linking a GnRH peptide that plays a key regulatory role in reproduction and sexual behavior, a novel vaccine was finally obtained.

[0021] To obtain large-scale, high-purity D3M1 recombinant protein, this invention utilizes a prokaryotic expression system to prepare the D3M1 recombinant protein. BL21(DE3) strain was selected as the *E. coli* strain expressing the D3M1 recombinant protein, and pET-28a was used as the prokaryotic expression vector. After inducing expression to obtain a large number of strains, high-pressure lysis was performed to obtain a bacterial lysate mixture containing the D3M1 recombinant protein. This mixture was then purified by affinity chromatography and anion exchange chromatography to obtain the D3M1 recombinant protein carrying an affinity chromatography purification tag. Subsequently, the affinity chromatography purification tag was cleaved using TEV Protease, resulting in a mixture of the D3M1 recombinant protein and the affinity chromatography purification tag. Next, affinity chromatography was used to remove the tag from the mixture, and gel filtration chromatography was used to finally obtain the structurally correct, high-purity D3M1 recombinant protein.

[0022] The next step is to screen for efficient conjugation methods between the D3M1 recombinant protein and the GnRH peptide. In this invention, the SMCC method was initially used for conjugation; however, this method still resulted in a large number of uncrosslinked peptides, leading to reduced vaccine yield and making it unsuitable for this invention. Subsequently, the EDC method was used for conjugation; however, EDC conjugation requires adjusting the solution pH to between 4.5 and 5.0, but D3M1 protein precipitation occurs under these pH conditions, therefore this conjugation method is also unsuitable for this invention. Finally, this invention attempted a glutaraldehyde conjugation method, which showed that this method could obtain crosslinked products in a mixed state, therefore this conjugation method is suitable for obtaining the vaccine in this invention.

[0023] Finally, this invention evaluated the D3M1-GnRH peptide-conjugated reproductive vaccine obtained through the above steps at the animal level. Four-week-old male mice were injected three times every two weeks with the D3M1-GnRH peptide-conjugated reproductive vaccine (D3M1 peptide) dissolved in SDA and MF59 adjuvants, respectively. Two weeks after the third injection (D42), the total IgG titer in the serum of the immunized mice and the reproductive capacity of the male mice in the experimental group were tested. The results showed that, compared with the negative control PBS group, the D3M1 peptide, whether dissolved in MF59 or SDA adjuvants, produced a high concentration of total IgG titer in the immunized mice. Furthermore, male mice treated with the D3M1 peptide completely lost their reproductive capacity compared to wild-type mice, suggesting that the vaccine can suppress reproductive capacity and achieve a contraceptive effect.

[0024] In summary, the reproductive vaccine conjugated with the novel carrier protein D3M1 and GnRH peptide obtained by the method described in this invention has contraceptive effect and can be used to obtain high-quality, low-cost recombinant protein vaccines on a large scale for commercialization. Attached Figure Description

[0025] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0026] Figure 1 Schematic diagrams of different designed carrier protein structures (purple represents the nitrogen terminus, and red represents the carbon terminus).

[0027] Figure 2 Cross-reactivity and titer determination of antibodies against mice immunized with different designed carrier proteins (A, time axis of mouse immunization and sample collection; BF, bar charts of cross-reactivity and titers of antibodies against mice immunized with carrier proteins from D1 to D5. The red boxes in D and F show the cross-reactivity and titers between antibodies against D3 and D5, and the results show that the cross-reactivity between these two carrier proteins and CRM197 is weak).

[0028] Figure 3 Cross-reactivity tests of CRM197 with different mutants of D3 and D5 (A, timeline of mouse immunization and sample collection; BF, bar charts of cross-reactivity between CRM197, D3M1, D3M2, D5M1, D5M2 and CRM197. The red boxes in B and C show that there is almost no cross-reactivity between the D3M1 carrier protein and CRM197).

[0029] Figure 4 The complete map of the modified pET-28a plasmid;

[0030] Figure 5 This is a gel image showing the protein content of each component of D3M1 after affinity chromatography purification (arrows indicate the target protein).

[0031] Figure 6 The images show the elution regions of the target protein purified by anion exchange chromatography (A, gel images of proteins in different elution regions of anion exchange chromatography; B, protein peak images of proteins in different elution regions of anion exchange chromatography, with the area shown in the red box indicating the elution region of the target protein).

[0032] Figure 7 Gel images of proteins before and after TEV Protease treatment and affinity chromatography purification (arrows indicate the target protein).

[0033] Figure 8 Display of elution regions for target protein purification by gel filtration chromatography (A, Gel graphs of proteins in different elution regions during gel filtration chromatography; B, Peak graphs of proteins in different elution regions during gel filtration chromatography).

[0034] Figure 9 Peak diagram for Ellman's Reagent detection of D3M1 and peptide products using the SMCC method (red arrows indicate the positions of uncrosslinked peptides).

[0035] Figure 10 The precipitation of D3M1 protein under pH 4.5-5.0 conditions (the area shown in the red box is the precipitated D3M1 protein).

[0036] Figure 11 The image shows the electrophoretic gel images of D3M1 and GnRH products after cross-linking with glutaraldehyde (the red arrow on the left indicates the D3M1 protein, and the red arrows in the middle column indicate the cross-linked products of D3M1 and GnRH in different states).

[0037] Figure 12 A schematic diagram showing the grouping and timing of animal-level immunization testing of the D3M1 and GnRH peptide-conjugated reproductive vaccine (A, a simplified table of injected substances in different injection groups; B, a schematic diagram of the immunization time for the three injections).

[0038] Figure 13 The total IgG titer in the serum of each group of D42 immunized mouse models;

[0039] Figure 14 Male mice treated with a reproductive vaccine conjugated with D3M1 and GnRH peptide lost their reproductive capacity. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] The specific embodiments of the present invention mainly include the design and screening of novel carrier proteins, the expression and purification of D3M1 recombinant protein, the efficient covalent conjugation of D3M1 with GnRH peptide, and the evaluation of the immunization effect of the vaccine at the animal level.

[0042] Example 1: Design and Screening of Novel Carrier Protein D3M1

[0043] This invention utilizes the protein redesign product of CRM197 based on the structural framework of our laboratory. The redesigned CRM197 variant exhibits loss of toxicity while retaining the same immunostimulatory properties as DT, with improved stability and solubility, and a richer array of T-cell epitopes. The design was performed using the EvoDesign algorithm, ultimately selecting 5 design sequences and related structures (…). Figure 1 Subsequent experiments were conducted. Different designed proteins were obtained through heterologous expression in *E. coli*, and then animal-level antibody cross-reactivity and titer detection were performed on the five designed proteins. Referring to published literature, this invention injected mice with a total amount of 20 µg of the five different designed proteins on Day 1 and Day 14, and blood was collected on Day 21 for downstream detection. The results showed that among the five designs, Design 3 and 5 (D3 and D5) showed relatively weak cross-reactivity with CRM197. Figure 2 ).

[0044] Subsequently, D3 and D5 were further modified to obtain four mutants based on D3 and D5: D3M1, D3M2, D5M1, and D5M2. Related animal tests were then conducted. The results showed that there was almost no cross-reactivity between D3M1 and CRM197. Figure 3 Ultimately, a novel carrier protein was determined for subsequent use in this invention. Therefore, the carrier protein D3M1 used in this invention is a novel carrier protein obtained through screening. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleic acid sequence encoding D3M1 is shown in SEQ ID NO.2. This protein is derived from CRM197 diffraction and exhibits almost no cross-reactivity with CRM197. Vaccines based on this carrier protein can be used in combination with vaccines constructed based on the CRM197 protein to prepare multivalent vaccines, thus enabling further applications.

[0045] Example 2: Prokaryotic expression and purification of carrier protein D3M1

[0046] To obtain large-scale, high-purity D3M1 recombinant protein, this invention selected a prokaryotic expression system to express and prepare the D3M1 recombinant protein. The BL21(DE3) strain was selected as the strain expressing the D3M1 recombinant protein, and the modified pET-28a was used as the prokaryotic expression vector sequence. Specifically, the pET-28a expression vector was modified by inserting a 6xHis-MBP tag and TEV site sequence after the T7 promoter using XbaI and BamHI restriction sites (see complete image of the modified plasmid). Figure 4 The nucleic acid sequence of D3M1 shown in SEQ ID NO.2 was cloned into the modified pET-28a plasmid via BamHI and NheI restriction sites, transformed into BL21(DE3) strain, and after a large number of strains were obtained through induced expression, the bacterial lysate was subjected to high pressure to obtain a bacterial lysate mixture containing recombinant D3M1 protein. After affinity chromatography and anion exchange chromatography purification, the recombinant D3M1 protein carrying the affinity chromatography purification tag was obtained. Subsequently, the affinity chromatography purification tag was cleaved using TEV Protease to obtain a mixture of recombinant D3M1 protein and affinity chromatography purification tag. Next, affinity chromatography was used to remove the tag from the mixture, and gel filtration chromatography was used to finally obtain the structurally correct and high-purity recombinant D3M1 protein.

[0047] 1. D3M1 carrier protein induced expression

[0048] (1) Take 50 μL of E.coli BL21(DE3) competent cells, thaw them on ice, add 50 ng of pET-28a plasmid containing the nucleic acid sequence of D3M1 (SEQ ID NO.2), mix gently, and place on ice for 30 minutes;

[0049] (2) After heat shock in a 42℃ water bath for 45 seconds, place the centrifuge tube on ice for 2 minutes. Do not shake the centrifuge tube during this process.

[0050] (3) Add 400 μL of sterile LB medium (without antibiotics) to the centrifuge tube, mix well, and incubate in a shaker at 37°C and 220 rpm for 45 min to revive the bacteria;

[0051] (4) After centrifuging at 2000 rpm for 2 min at room temperature, remove 300 μL of supernatant, gently pipette to mix the remaining culture medium and bacterial precipitate, drop it onto LB plate (Kanamycin 50 μg / mL) and spread it evenly, and after drying, invert it in a 37℃ oven overnight.

[0052] (5) Pick a single colony from the plate and put it into 5 mL of LB medium (Kanamycin 50 μg / mL). For glycerol bacteria, take 10 μL into the medium and incubate overnight at 37°C and 220 rpm for 14-16 h.

[0053] (6) Preheat 1L LB medium (Kanamycin 50μg / mL) in a shaker at 37℃, add 5 mL of Day 2 bacterial culture to expand the culture, and incubate at 37℃ and 220rpm until the bacterial culture reaches OD. 600 Induction begins around 1.0-1.2;

[0054] (7) When the temperature of the shaker drops to 16℃, add IPTG to a final concentration of 0.2mM (1L is added to 200μL of 1M IPTG stock solution) to induce protein expression, and incubate overnight at 16℃ and 220rpm for 14-16h.

[0055] 2. Sample collection and high-pressure crushing

[0056] (1) Pre-cool the large-capacity centrifuge to 4°C, use a 1L collection tank to collect the bacterial solution, and centrifuge at 3800rpm for 15min;

[0057] (2) After centrifugation, discard the supernatant, resuspend the precipitate in 20 mL of lysis buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM TCEP), and then add PMSF to a final concentration of 1 mM.

[0058] (3) Set the working temperature of the high pressure crusher to 4℃, rinse the sample cup and pipeline with ddH2O, then rinse the pipeline with the above-mentioned lysisbuffer, and put the sample outlet tube into the sample cup after rinsing it clean.

[0059] (4) Pour the bacterial solution into the injection cup, turn on the circulation system, and the circulation flow rate is 45-50 mL / min;

[0060] (5) Pressurize to 800 bar (each pressurization should not exceed 100 bar) to lyse the bacterial culture, and generally circulate for 2-4 minutes for lysis;

[0061] (6) After lysis is complete, reduce the system pressure to 0 and transfer the lysis buffer from the injection cup. At this time, the bacterial solution will become clear.

[0062] (7) Transfer the lysed bacterial culture to a high-speed centrifuge tube and centrifuge at 17,000 rpm and 4°C for 30 min.

[0063] 3. Affinity chromatography purification (Ni-NTA)

[0064] (1) Ni-NTA pillars were rinsed with the above-mentioned lysis buffer;

[0065] (2) Transfer the supernatant after high-speed centrifugation to a clean centrifuge tube, add 4 mL (appropriate amount) of rinsed Ni-NTA column to it, and incubate at 4℃ for 40 min. During incubation, place the centrifuge tube on a rotary mixer and use low speed and no bubbles in the supernatant as the standard during the rotation process.

[0066] (3) After incubation, centrifuge at 3000 rpm and 4℃ for 5 min, remove most of the supernatant, leave 10 mL of supernatant and mix it with the lower column material, then transfer it to the gravity column. After all the supernatant flows out of the gravity column, prepare to elute.

[0067] (4) Add wash buffer (25mM Tris-HCl pH 8.0, 150mM NaCl, 20mM Mimidazole pH 8.0, 0.5mM TCEP) to the gravity column to elute the impurities bound to the Ni-NTA column. Add 10mL of wash buffer each time and elute a total of 3 times.

[0068] (5) Add elution buffer (25mM Tris-HCl pH 8.0, 150mM NaCl, 250mM Mimidazole pH 8.0, 0.5mM TCEP) to the gravity column to elute the target protein. Add 2 mL of elution buffer each time.

[0069] (6) Monitor the elution with Bradford 1× Dye Reagent. Take 5 μL of elution buffer flowing from the gravity column and add it to 100 μL of 1× Reagent. If the Reagent turns blue, continue elution. When the Reagent no longer turns blue, it indicates that the target protein has been basically eluted.

[0070] 4. Sampling and testing

[0071] After affinity chromatography, samples need to be taken for gel electrophoresis. The samples taken are: the precipitate (P) and supernatant (S) after centrifugation; the supernatant eluted from the gravity column after incubation with the Ni-NTA column (Fl, Flow); the target protein eluted from the wash buffer (W, Wash); and the target protein eluted from the elution buffer (E, Elution); and the eluted Ni-NTA column (R, Resin). To determine if the eluted protein is the target protein, 20 μL of elution buffer E can be added and incubated with 2 μL of 2 mg / mL TEV Protease at room temperature for 15 min (+T), with a TEV Protease control (T). The prepared samples are then added to loading buffer, incubated at 95°C for 3 min, briefly centrifuged, and then run on a gel. The specific steps are as follows:

[0072] (1) Required reagents: 5× loading buffer: 250mM Tris-HCl (pH 6.8), 10% (W / V) SDS, 0.5% (W / V) bromophenol blue, 50% (V / V) glycerol, 5% (W / V) DTT; 10× SDS-PAGE running buffer (Tris 30.2g, Glycine 144g, SDS 10g, add ddH2O to make up to 1L);

[0073] (2) After loading the sample, run the gel at 250V for 30 minutes and then stain.

[0074] (3) Staining solution formula: 420 mL of 95% ethanol, 80 mL of glacial acetic acid, 500 mL of ddH2O, 2 g of R250. After decolorization with water, observe the protein gel to determine the target protein.

[0075] Experimental results show that heterologous expression of recombinant D3M1 protein can be obtained in this invention, and the protein content obtained by elution buffer is the highest. Figure 5 ).

[0076] 5. Anion exchange chromatography purification

[0077] (1) All D3M1 proteins after affinity chromatography were purified by ion exchange chromatography.

[0078] (2) The eluted protein was diluted with anion exchange chromatography buffer (25 mM Tris-HCl pH 8.0, 0.5 mM TECP) to 4 times its original volume, and then filtered through a 0.22 μM filter membrane before loading.

[0079] (3) Install the HiTrap Q anion exchange column (5 mL) (be careful to avoid air bubbles), place the purification instrument A pump into 1M NaOH, and set the parameters as follows:

[0080] Manual → Execute → Pumps → Set System flow to 5 mL / min → Insert

[0081] Alarm → Precolumn pressure → Set to 0.5 MPa → Insert

[0082] Other → timer → Acc.volumn → 20ml → End→ Insert →Execute

[0083] (4) Clean column A with 20 mL of 1M NaOH;

[0084] (5) Place the A pump and B pump of the purifier into buffer A and buffer B (25 mM Tris-HCl pH 8.0, 0.5 mM TECP, 1M NaCl) respectively, and select the program "Q 5ml High low salt" to complete the Q column equilibration;

[0085] (6) At this time, the conductivity (ms / cm) should be less than 2. If the conductivity is high, the program settings are as follows:

[0086] Manual → Execute → Pumps → Set System flow to 5 mL / min → Insert

[0087] Alarm → Precolumn pressure → Set to 0.5 MPa → Insert

[0088] Other → timer → Acc.volumn → 15ml → Pause→ Insert →Execute

[0089] (7) Rinse with buffer A for another 15 mL and then pause;

[0090] (8) Place pump A into the diluted and filtered sample and load the sample onto the HiTrap Q ion exchange column at a flow rate of 5 mL / min. Collect the effluent (waste) from the Q column. The specific parameters are as follows:

[0091] Continue → Other → timer → Acc.volumn → 45 mL (slightly less than the sample volume) → insert

[0092] (9) After pausing, manually load the sample (press the start and end buttons on the instrument) until the end, being careful not to inhale air bubbles. Rinse pump A with ddH2O water and return it to buffer A, then stop the program at this point;

[0093] (10) After arranging the EP tubes (usually 60) on the sample receiving tray of the purifier, place them on the sample receiver at the bottom of the purifier. Select the "Q 5ml Elution" program set by the purifier (100 mL elution volume, B buffer increases linearly from 0 to 100%, 1.5 mL / tube) → Name file → Start;

[0094] (11) When the conductivity exceeds 60 ms / cm (at which point most of the target protein has been eluted), set Pump→Gradient→Target:100%B, length:0CV→Execute, that is, directly rinse the Q column with 100% B buffer high salt solution.

[0095] Anion exchange chromatography was used to further obtain high-purity target protein, and the results showed that high concentrations of target protein could be obtained in the 31-36 range. Figure 6 ).

[0096] 6. TEV enzyme digestion

[0097] Transfer the ion-exchanged fraction to a 50 mL centrifuge tube, add 100 μL of TEV Protease (2.0 mg / mL) according to the peak value of the 10 mL 100 mAU ion-exchange chromatography (the volume of enzyme added should be scaled up proportionally according to the mAU value), and digest at 4 °C for 4-8 h.

[0098] 7. Affinity chromatography to remove His-MBP tag protein

[0099] (1) Take two gravity columns of Ni-NTA material that have been rinsed with lysisi buffer, add the protein solution after enzyme digestion to the gravity column and let it flow out (Fl, flow), and then pass it through the second column after passing through the first column;

[0100] (2) Add 4-8 mL of wash buffer to the gravity column to allow it to flow out (W, wash), and take a sample after elution is complete;

[0101] (3) Take 10 μL of the pre-digestion mixture (Mix), TEV Protease treated sample (+T), and Fl and W samples, add 10 μL of 2× loading buffer, and run gel analysis.

[0102] Experiments show that high-purity target proteins with affinity tags removed can be obtained by affinity chromatography purification. Figure 7 ).

[0103] 8. Gel filtration chromatography

[0104] (1) Combine the Fl and W samples from the above steps and add them to a 30 kD ultrafiltration tube. Centrifuge at 3200-3800 rpm at 4℃ to concentrate to 2 mL. Transfer the sample in the concentration tube to a 2 mL EP tube and centrifuge at 15000 rpm at 4℃ for 5 min to remove air bubbles from the sample.

[0105] (2) Molecular sieve equilibration: Use a Superdex Increase 200 separation column. Click "A pump wash" to flush pump A, then reinstall the separation column. Before use, equilibrate the separation column with the molecular sieve buffer (lysis buffer) as follows:

[0106] Manual → Execute → Pumps → System flow setting 0.5 ml / min → Insert

[0107] Alarm → Precolumn pressure → Set to 1.8 MPa → Insert

[0108] Other → timer → Acc.volumn → 30ml → End→ Insert →Execute.

[0109] (3) After the separation column is equilibrated, load the sample. First, rinse the adapter with ddH2O, then use a 2 mL syringe to draw in molecular sieve buffer to rinse the 2 mL loop (try to avoid air bubbles from entering).

[0110] (4) Draw 2 ml of the centrifuged sample into the syringe and gently inject it into the loop, avoiding air bubbles. Place the EP tubes (usually 35) on the sample receiving tray of the purifier and place it on the sample receiver at the bottom of the purifier. Select the "Superdex200 Increase" program (0.5 mL / min flow rate, 0.5 mL / tube) set by the purifier → Name file → Start;

[0111] (5) After the program stops, take a sample and run the gel.

[0112] (6) Transfer the molecular sieve sample to a 30 kD ultrafiltration tube, centrifuge at 3200-3800 rpm at 4℃ to concentrate to 500 μL, transfer the sample in the concentration tube to a 1.5 mL EP tube, centrifuge at 15000 rpm at 4℃ for 5 min to remove air bubbles from the sample.

[0113] (7) After determining the protein concentration using the BCA method, the protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80℃.

[0114] The TEV Protease-treated protein purified by affinity chromatography still needs to undergo gel filtration chromatography, i.e., it needs to pass through a molecular sieve, to obtain the high-purity D3M1 protein finally used for vaccine production. Experimental results show that the purification method used in this invention can obtain high concentrations of the target protein in the 6-9 range. Figure 8 ).

[0115] Example 3: Synthesis of GnRH peptide

[0116] The GnRH peptide sequence pEHWSYGLRPGGC (SEQ ID NO.3) was synthesized by a commercial synthesis company for use in subsequent vaccine production.

[0117] Example 4: Coupling of carrier protein D3M1 with GnRH peptide

[0118] 1. D3M1 conjugation with peptides using the SMCC method

[0119] (1) Use buffer 1 (25mM Tris 8.0, 150mM NaCl, 0.5mM TCEP) to open disulfide bonds;

[0120] (2) Replace gel filtration with buffer 2 (PBS 7.2, 0.5mM EDTA);

[0121] (3) Add 5.0 mg / ml D3M1 protein, 800 µl; 2.5 mg / ml Sulfo-SMCC, 200 µl; and 15 times the mass ratio of GnRH peptide. Incubate the mixture at room temperature (25 °C) for 60 minutes.

[0122] (4) Overnight dialysis to remove Sulfo-SMCC;

[0123] (5) Use Ellman's Reagent to detect uncrosslinked -SH.

[0124] 2. D3M1 conjugation with peptides using the EDC method.

[0125] (1) Dissolve D3M1 in coupling buffer (0.1M MES, pH 4.5-5.0) to a final concentration of 5 mg / mL;

[0126] (2) Dissolve the polypeptide to be coupled in coupling buffer (0.1M MES, pH 4.5-5.0) to a final concentration of 2mg / ml;

[0127] (3) D3M1 and peptide solution are mixed, with a peptide to carrier protein molar ratio of 10:1;

[0128] (4) Prepare EDC in coupling buffer to a concentration of 2 mg / mL;

[0129] (5) Slowly add EDC solution (the molar amount of EDC added is the same as that of the polypeptide), and react at 25°C for 2 hours.

[0130] 3. D3M1 conjugation with peptides using the glutaraldehyde method.

[0131] (1) Dissolve D3M1 in coupling buffer (0.1M carbonate, 0.15M NaCl, pH 8.5) to a final concentration of 2 mg / mL;

[0132] (2) Dissolve the polypeptide to be coupled in the coupling buffer to a final concentration of 2 mg / mL;

[0133] (3) Mix D3M1 and the polypeptide solution, with a polypeptide to carrier protein molar ratio of 30:1;

[0134] (4) Add glutaraldehyde to a final concentration of 1% and react at 4°C overnight.

[0135] This embodiment screened for efficient conjugation methods between D3M1 recombinant protein and GnRH peptide. Initially, the SMCC method was used for conjugation; however, Ellman's Reagent assay showed that a large number of uncrosslinked peptides remained. Figure 9This resulted in reduced vaccine production, making it unsuitable for this invention. Subsequently, EDC (Extended Direct Conjugation) was used for coupling; however, EDC coupling requires adjusting the solution pH to between 4.5 and 5.0, but D3M1 exhibits protein precipitation under these pH conditions. Figure 10 Therefore, this coupling method is also not applicable to this invention; finally, this invention attempts to use glutaraldehyde coupling, and the results show that this method can obtain cross-linked products in a mixed state ( Figure 11 Therefore, this coupling method is suitable for obtaining vaccines using this invention.

[0136] Example 5: Evaluation of the immunization effect of the vaccine at the animal level

[0137] This invention evaluates the D3M1-GnRH peptide-conjugated reproductive vaccine obtained through the above steps at the animal level. Four-week-old male mice were intraperitoneally injected three times every two weeks with 20 µg of the D3M1-GnRH peptide-conjugated reproductive vaccine dissolved in SDA and MF59 adjuvants, respectively. Two weeks after the third injection (D42), the total IgG titer in the serum of the immunized mice and the reproductive capacity of the male mice in the experimental group were tested. Figure 12 The experimental results showed that, compared with the negative control PBS group, both D3M1 peptides dissolved in MF59 adjuvant and SDA adjuvant could produce high concentrations of total IgG titers in immunized mice. Figure 13 The results of this experiment demonstrate that the reproductive vaccine conjugated with D3M1 and GnRH peptides can achieve high levels of immunization at the animal level.

[0138] Four-week-old male mice were given their third injection on day 28 (D42) and then co-cultured with untreated wild-type female mice aged 8-10 weeks at a 1:1 density. After 3-4 weeks of co-culture, litter size was observed and calculated in each group, and relevant experimental analyses were performed. Results showed that male mice treated with the D3M1-conjugated GnRH peptide reproductive vaccine were less reproductively capable than wild-type mice that completely lost their reproductive capacity. Figure 14 This suggests that the vaccine can suppress reproductive ability and thus have a contraceptive effect.

[0139] In summary, the reproductive vaccine conjugated with the novel carrier protein D3M1 and GnRH peptide obtained by the method described in this invention has contraceptive effect and can be used to obtain high-quality, low-cost recombinant protein vaccines on a large scale for commercialization.

[0140] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A carrier protein, characterized in that: The carrier protein is derived from CRM197, and the amino acid sequence of the carrier protein is shown in SEQ ID NO.

1.

2. The gene encoding the vector protein of claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A method for preparing a castration vaccine for male animals based on the carrier protein conjugated with GnRH peptide according to claim 1, characterized in that: The preparation process includes the following steps: expressing the gene encoding the carrier protein of claim 1 in prokaryotes, purifying it by affinity chromatography and anion exchange chromatography, removing the tag protein by TEV enzyme digestion, obtaining the purified carrier protein by affinity chromatography and molecular sieve, and finally covalently coupling it with the GnRH polypeptide to obtain the castration vaccine for male animals; the amino acid sequence of the GnRH polypeptide is pEHWSYGLRPGGC.

4. The method according to claim 3, characterized in that: The prokaryotic expression vector is pET-28a, and the prokaryotic expression strain is BL21(DE3).

5. The method according to claim 3, characterized in that: The covalent coupling is performed using glutaraldehyde.

6. The method according to claim 5, characterized in that: The final concentration of glutaraldehyde is 1% by volume.

7. The method according to claim 3, characterized in that: The molar ratio of the GnRH polypeptide to the carrier protein is 30:

1.

8. The castration vaccine for male animals prepared by the method according to any one of claims 3 to 7.

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