Preparation method of babesiella bovis vaccine

Abstract

The invention relates to a preparation method of a babesiella bovis vaccine, belongs to the technical field of vaccines, and particularly relates to a combined vaccine based on silver nanoparticles (AgNPs) and theileria bovis antigen, the vaccine comprises 10-50nm AgNPs, the surface of the AgNPs is modified with carboxyl, amino or PEG, and the AgNPs are used for covalently coupling RAP-1 protein or immunogenic fragments thereof and MSA-2 protein or B cell epitope peptide thereof, the RAP-1 protein comprises an amino acid sequence (nucleotide sequence is as shown in SEQ ID NO: 1), the MSA-2 protein comprises an amino acid sequence (nucleotide sequence is as shown in SEQ ID NO: 2), and the preparation method comprises the following steps: carrier construction, recombinant expression, protein purification and AgNPs coupling to form a silver nanoparticle antigen compound, and the silver nanoparticle antigen compound is coated with chitosan to prolong the in-vivo half-life period.

Description

Technical Field

[0001] The present application belongs to the field of vaccine technology, and specifically relates to a method for producing a bovine pyrophagia vaccine. Background Art

[0002] Bovine pyroxithosis is a disease caused by parasites of the genus Theileria of the family Theileriidae. After Theileria infects cattle, it first invades the reticuloendothelial cells and then invades the red blood cells. The disease is transmitted by ticks of the family Ixodidae by sucking the blood of cattle.

[0003] Bovine pyrophagoides can have a serious impact on the health of cattle. Infected cattle may experience symptoms such as high fever, anemia, jaundice, hemoglobinuria, etc. In severe cases, it may lead to the death of cattle, causing huge economic losses to farmers. In recent years, with the continuous expansion of the scale of breeding, the incidence of bovine pyrophagoides has been on the rise. The global infection rate is about 20% to 30%, and in some areas it is even as high as 70% to 80%. Traditional vaccine production has a long R&D cycle and low flexibility. It requires the isolation and cultivation of live pathogens. The R&D cycle is long and the flexibility is low. Some pathogens are difficult to effectively culture in vitro, which leads to research and development being hindered.

[0004] The prior art CN 85100848B discloses a method for manufacturing a bovine babesia vaccine. By changing the low-speed cell rotation culture production method to the high-speed cell rotation culture production method, the cell yield is improved, the seedling production procedures are simplified, the labor intensity is reduced, and chemical drugs are saved. However, during the high-speed cell rotation culture production process, the complexity of the equipment and the increase in operating links will increase the risk of cell contamination, and the equipment requirements are high. High-speed rotation will have the risk of cell damage, and it does not involve vector construction technology; the prior art US20210016012A1 discloses a DNA vaccine encoding Babesia bovisantigens, whose plasmid encodes RAP-1+MSA-2 multiple epitopes, but does not mention silver nanoparticles (AgNPs) as a method to improve antigen recognition.

[0005] Therefore, improving the ability of antigens to be more easily recognized by the immune system has become a direction worthy of research. MSA-2 silver nanoparticles, as an adjuvant or carrier, can change the way antigens are presented after combining with RAP-1, making antigens more easily recognized by the immune system, while improving the stability and sustained-release effect of the vaccine. Summary of the Invention

[0006] The present application provides a method for preparing a bovine Theileria vaccine, comprising a combination vaccine of silver nanoparticles (AgNPs) and bovine Theileria antigens. The vaccine comprises 10-50 nm AgNPs, whose surfaces are modified with carboxyl groups, amino groups, or PEG, for covalently coupling RAP-1 protein or an immunogenic fragment thereof and MSA-2 protein or a B cell epitope peptide thereof, wherein the RAP-1 protein comprises the amino acid sequence shown in SEQ ID NO:1 (the nucleotide sequence is shown in SEQ ID NO:3), and the MSA-2 protein comprises the amino acid sequence shown in SEQ ID NO:2 (the nucleotide sequence is shown in SEQ ID NO:4). The preparation method comprises: vector construction, recombinant expression, protein purification, coupling with AgNPs to form a silver nanoparticle-antigen complex, and coating with chitosan to increase the in vivo half-life.

[0007] In one aspect, the present application provides a bovine Theileria vaccine, characterized in that the vaccine comprises a combination of silver nanoparticles (AgNPs) and bovine Theileria antigen; Preferably, the silver nanoparticles have a particle size of 10 to 50 nm; the bovine Theileria antigen is derived from at least one of RAP-1 protein or an immunogenic fragment thereof, MSA-2 protein or a B cell epitope peptide thereof; Preferably, the surface of the silver nanoparticles is modified with any one of carboxyl groups, amino groups or PEG for covalently coupling antigens. The antigens are stably and directionally bound to the surface of the silver nanoparticles by covalently coupling the antigens, thereby optimizing the immunogenicity, delivery efficiency and biocompatibility of the antigens, and ultimately improving the effect of vaccines or immunotherapy. Preferably, the RAP-1 protein obtained from NCBI comprises the amino acid sequence shown in SEQ ID NO: 1, which is: MPLREFKIVVLGSGGVGKSALTVQFVQGIFVEKYDPTIEDSYRKQVEVDSNQCMLEILDTAGTEQFTAMRDLYMKNGQGFVLVYSIISNSTFNELPDLREQILRVKDCEDVPMVLVGNKCDLHDQRVISTEQGEELARKFGDCYFLEASAKNKVNVEQIFYNLIRQINRKNPVGPPSKAKSKCALL Its nucleotide sequence is shown in SEQ ID NO: 3, and its sequence is: 1 gaatatatat tataaaccag atgcctctta gagaattcaa aatcgtcgtt ttaggttcag 61 gtggtgtagg taaatctgct ttgactgtgc aatttgttca aggtattttt gttgaaaagt 121 acgatccaac catcgaagat tcctacagaa aacaagtcga agttgatagc aatcaatgca 181 tgttagaaat tttagataca gctggtactg aacaatttac tgcaatgaga gatctttaca 241 tgaaaaatgg tcaaggtttt gttttagtat attcaatcat ttcaaactcc acttttaacg 301 agttaccaga tctccgtgaa caaattctca gagttaagga ttgtgaagat gttccaatgg 361 ttcttgttgg taacaaatgc gatctccacg accaacgtgt tattagcaca gaacaaggtg 421 aagaactcgc tcgtaaattt ggtgattgtt actttttaga agcatctgcc aagaataaag 481 ttaatgttga acaaattttc tataacttaa tccgtcaaat caaccgtaaa aacccagttg 541 gtccaccaag caaagctaaa tcaaaatgtg ctttattgta aacaatccat caactctcca 601 acacccttcc atactcaccc acccatttca aatgtaacaa ttgaaaaaca gaaaaaaaaa 661 aaaaaaaaaa acagaaaaaa aaaaa The MSA-2 protein contains a B cell epitope as shown in SEQ ID NO: 2, the sequence of which is: MIGKIFLLTACCCASLLSVSASDETQETSLTTLYEEMKDVANYIKFLTKEEEGRYLEGKFTSVEMPSDSSLDALSAFVEILDVFGKNVPFETSLFDEAVFENLKYQDPDQIFKYLLLRIPLIKTKISAFNVFLNDNPPRMLANESGEMTDYYKKHICKEDSEVKDYNSLVKYCNDFLDSKSPFMRPYKHLNEYDELVKKKPAQESPPAPSSPQVNTTTQPSQDSAAPNTSAGNLNGQQGSPKPTGSSFTFGGLTVATLCYFVLSAF Its nucleotide sequence is as shown in SEQ ID NO: 4, and the sequence is: 1 atgatcggga aaatcttctt gttaacagca tgctgctgtg catccttact gtctgtgtct 61 gcttctgatg aaacacagga aacctcattg accacacttt atgaagaaat gaaagatgta 121 gctaattata ttaaattttt aacgaaagaa gaagaggggc gatatttaga gggaaagttt 181 acgagtgtcg agatgccttc cgatagctct cttgatgctc tcagtgcttt tgtagaaatt 241 ctggatgttt tcggtaaaaa cgtcccattt gaaacctcct tgtttgatga ggcagtattt 301 gaaaatttga agtatcagga tccagatcag atcttcaaat atcttcttct gagaataccc 361 ttaataaaaa caaaaattag tgcatttaac gtttttctca atgacaaccc tccacgtatg 421 ttggcgaatg aaagcggaga aatgactgat tattacaaaa aacatatatg caaggaagat 481 tctgaggtaa aggattacaa ctctttagtc aaatattgca acgacttttt agacagtaaa 541 tccccattca tgagaccata taagcatctc aatgaatatg atgagctagt gaagaagaag 601 ccagcacaag aatctccccc tgctccttca tccccgcaag taaacactac gacccaacca 661 tcccaggatt cagctgcacc aaacacatct gcaggaaacc tcaatggaca acagggttca 721 cccaagccta ccggatcttc tttcaccttt ggcggattga ccgtggctac tctctgctac 781 ttcgttctct ctgcatttta a Preferably, the bovine Theileria antigen is obtained by recombinant expression in CHO cells; On the other hand, the present application provides a method for producing a bovine pyrophagous vaccine, characterized in that the steps are: (1) Vector construction: RAP-1 and MSA-2 were constructed using pcDNA3.1(+) vectors. The plasmids were extracted and purified to an OD260 / 280 value of 1.8 to 2.0. RAP-1 and MSA-2 were detected by PCR and the results were sequenced. (2) Transformation of host protein expression: The constructed vector was transformed into the same CHO cells by electroporation, and cell culture was performed to express RAP-1 and MSA-2 recombinant cells. The specific steps are as follows: 1) CHO cell culture: CHO cells were cultured in a conventional culture medium containing leaf milk at a temperature of 36-38°C and a pH of 7.0-7.4 for 12-24 hours until the cells were in the logarithmic growth phase and had a density of 70-80%. 2) Remove the cell suspension using a pipette and reverse aspiration. Wash the cells 1-2 times with 37°C preheated PBS by gently pipetting to remove residual serum. Transfer the suspended cells to a centrifuge tube and centrifuge at 4°C, 1000 rpm / min for 5 minutes, discard the supernatant, resuspend the cells in 4°C precooled PBS, centrifuge again at 1000 rpm / min for 5 minutes, discard the supernatant, and collect the cells to completely remove proteins and ions in the serum to prevent interference with the electric field distribution during electroporation. 3) Pre-cool the dedicated electroporation buffer (Lonza Nucleofector Solution C), add 1-2 μg of RAP-1 and MSA-2 plasmid DNA (RAP-1:MSA-2 mass ratio is 1:2), mix well, resuspend the cells, determine the concentration by counting, and adjust to 1×10 7 , gently pipette during resuspending to avoid bubbles, and place on ice for 5-10 minutes to reduce cell membrane fluidity and mechanical damage during electric shock; 4) Set the electroporation cuvette parameters to voltage: 160-200 V, capacitance: 25-50, pulse time: 30-60 ms, pulse once, and introduce RAP-1 and MSA-2 plasmid DNA into CHO cells to obtain recombinant cells, which are then cultured and expressed in leaf emulsion medium for 24-72 h; (3) Lyse the recombinant cells and purify the protein. RAP-1 and MSA-2 recombinant proteins were obtained. The recombinant proteins were lysed and purified. The lysis buffer was 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM PMSF (added before use). The composite cells described in step (2) were directly centrifuged at 2000 rpm / min for 5 min to collect the cells. The cells were added with the above lysis buffer and incubated on ice for 15 to 30 min. Vortex oscillation was used to assist lysis. The lysate was centrifuged at 4°C and 12000 rpm / min for 30 min. The supernatant was collected and the precipitate (cell debris and membrane components) was discarded. The protein was purified by column to obtain an OD260 / 280 value of 0.6 to 0.8. (4) The purified protein was combined with silver nanoparticles with a particle size of 10-50 nm to obtain a silver nanoparticle-antigen complex: the mass ratio of the purified protein to the silver nanoparticles was 2:1; Preferably, the vector construction comprises the nucleotide sequence of claim 4; Preferably, the host culture in step (2) is leaf milk culture medium; Preferably, the leaf milk culture medium comprises 95% leaf milk nutrient solution and 5% calf serum; Preferably, in step (4), the surface of the silver nanoparticle-antigen complex is coated with chitosan to increase the in vivo half-life.

[0008] The beneficial effects of the embodiments of the present application are: (1) Vaccine components through the unique role of silver nanoparticles: Efficient antigen delivery: Silver nanoparticles with a particle size of 10-50 nm have a large specific surface area and can efficiently bind to bovine Theileria antigens (such as RAP-1, MSA-2 protein) through physical adsorption or chemical coupling (such as surface modification with -COOH, -NH2 or PEG), forming a stable antigen complex, significantly improving the delivery efficiency and immunogenicity of the antigen; (2) Silver nanoparticles themselves have immunostimulatory properties, enhance antigen presentation ability, and induce stronger humoral immunity (antibody secretion) and cellular immunity (T cell response); surface modification groups such as carboxyl, amino or PEG can reduce the immunogenicity of nanoparticles and prolong the circulation time in the body; carboxyl or amino modification facilitates the directional coupling of antigens and ensures the correct exposure of antigen epitopes; (3) Multi-antigen synergy and natural antigen characteristics: RAP-1 protein and MSA-2 protein are selected to cover different immune-dominant regions of Theileria calvescens, which can induce the host to produce broad-spectrum neutralizing antibodies and cellular immunity, avoiding immune escape caused by a single antigen; the antigen is recombinantly expressed in CHO cells, retaining the spatial conformation and immunogenicity of the natural protein. Compared with chemically synthesized peptides, it is easier to be recognized by the host immune system, thereby improving the protective efficacy of the vaccine; (4) Long-term effect of chitosan coating: Chitosan is coated on the surface of the silver nanoparticle-antigen complex, and its cationic properties are used to enhance the binding ability with the negative charge on the cell surface, thereby promoting antigen uptake; at the same time, the biodegradability and mucosal adhesion of chitosan can prolong the retention time of the vaccine in the body, continuously release antigens, and enhance immune memory. Silver nanoparticles and chitosan are both biocompatible materials with low toxicity and are degradable, reducing the local inflammatory response that may be caused by traditional adjuvants (such as aluminum adjuvants); the CHO cell expression system avoids the risk of contamination by pathogenic microorganisms and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the band diagram of agarose gel electrophoresis detection in Example 1 of the present application; Figure 2 This is a schematic diagram of antigen release in Example 5 of the present application; Figure 3 This is a pathological image of the intramuscular injection site in Group A of Experimental Example 1 of this application. DETAILED DESCRIPTION

[0010] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0011] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals expressing amounts, percentages, and other numerical values ​​used in this application should be understood as being modified by the word "about" in all cases. Each numerical parameter should at least be considered to be obtained in light of the number of reported significant digits and by conventional rounding methods.

[0012] The present invention is further described below by way of examples, which, however, do not limit the present invention in any way.

[0013] Preparation example: (1) Preparation of leaf milk solution: 10 mg folic acid, 6 g milk protein hydrolysate, add deionized water to 860 mL, mix until dissolved, sterilize by autoclave at 121°C for 30 min, and store in a refrigerator at 4°C. (2) Preparation of 0.5% phenol red solution: 5 g phenol red, 150 mg 0.1 N sodium hydroxide, add deionized water to 1000 mL, filter with filter paper, autoclave at 121°C for 30 min, and store in a refrigerator at 4°C. (3) Preparation of Group A salt solution: 68 g sodium chloride, 4 g potassium chloride, 1.5 g sodium dihydrogen phosphate, 40 mg 0.5% phenol red solution, add deionized water to 500 mL, mix well until dissolved, filter with filter paper, and sterilize by autoclaving at 121°C for 30 min; (4) Preparation of Group B salt solution: 2 g calcium chloride, 2 g magnesium chloride, 10 g glucose, add deionized water to 500 mL, filter with filter paper, and sterilize by autoclaving at 121°C for 30 min; (5) Preparation of double-antibody solution: 800,000 units of penicillin, 1 g of streptomycin, 160 mL of deionized water, and stored frozen at -20°C; (6) Preparation of 8% sodium bicarbonate solution: 8 g of sodium bicarbonate, 100 mL of deionized water, filter through a Cai filter, and store in a refrigerator at 4°C. (7) Preparation of leaf milk nutrient solution: 86% leaf milk solution, 5% group A salt solution, 5% group B salt solution, 2% double antibiotic solution, 2% 8% sodium bicarbonate solution, and adjust its pH to 7.4; (8) Cell culture medium: leaf milk nutrient solution 95%, calf serum solution 5%;

[0014] Example 1 Vector construction The RAP-1 and MSA-2 genes were synthesized and constructed by Shanghai Sangon Biotechnology Co., Ltd. on the pcDNA3.1 (+) vector. Plasmid DNA was extracted using a QIAprep Spin Miniprep Kit as template DNA. The OD260 / 280 value was 1.85 and the concentration was 500 ng / μL. PCR detection of RAP-1 and MSA-2 was performed using RAP-1 and MSA-2 as templates. Appropriate upstream / downstream amplification primers were designed. The primer sequences are as follows:

[0015] The amplification system is:

[0016] The PCR amplification procedure was as follows: pre-denaturation: 95°C for 1 min; 95°C for 10 s, 58°C for 20 s, 70°C for 30 s, 40 cycles of amplification, and the amplified products were detected by 1% agarose gel electrophoresis. There were bright bands in all of them. The results are as follows: Figure 1 As shown, A is: MSA-2, B is RAP-1, and then sequenced;

[0017] Example 2 CHO cell transfection and protein expression (1) The above plasmids were transformed into the same CHO cells by electroporation, and the cells were cultured and expressed to obtain RAP-1 and MSA-2 recombinant cells. The specific steps are as follows: 1) CHO cell culture: CHO cells were cultured in leaf milk medium at 36-38°C and pH 7.0-7.4 for 12-24 hours until the cells were in the logarithmic growth phase and the density reached 70-80%. 2) Remove the cell suspension using a pipette and reverse aspiration. Wash the cells 1-2 times with 37°C preheated PBS by gently pipetting to remove residual serum. Transfer the suspended cells to a centrifuge tube and centrifuge at 4°C, 1000 rpm / min for 5 minutes, discard the supernatant, resuspend the cells in 4°C precooled PBS, centrifuge again at 1000 rpm / min for 5 minutes, discard the supernatant, and collect the cells to completely remove proteins and ions in the serum to prevent interference with the electric field distribution during electroporation. 3) Pre-cool the dedicated electroporation buffer (Lonza Nucleofector Solution C), add 1-2 μg of RAP-1 and MSA-2 plasmid DNA (RAP-1:MSA-2 mass ratio is 1:2), mix well, resuspend the cells, determine the concentration by counting, and adjust to 1×10 7, gently pipette during resuspending to avoid bubbles, and place on ice for 10 minutes to reduce cell membrane fluidity and reduce mechanical damage during electric shock; 4) The electroporation cuvette parameters were set to voltage: 180 V, capacitance: 30, pulse time: 50 ms, and pulse once. RAP-1 and MSA-2 plasmid DNA were introduced into CHO cells to obtain recombinant cells. Flow cytometry analysis showed a transfection efficiency of 78%. The cells were then cultured and expressed in leaf emulsion medium for 72 h.

[0018] Example 3 Protein purification (1) After lysing the recombinant cells, protein purification was performed to obtain RAP-1 and MSA-2 recombinant proteins. The recombinant proteins were lysed and purified using the following lysis buffer: 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM PMSF (added just before use). The recombinant cells obtained by the above culture were directly centrifuged at 2000 rpm / min for 5 min to collect the cells, and the above lysis buffer was added. For every 1×10 7 Each CHO cell was incubated with 500 μL of lysis buffer on ice for 20 min, vortexed to assist lysis, and centrifuged at 4°C, 12,000 rpm / min for 30 min. The supernatant was collected and the precipitate (cell debris and membrane components) was discarded. The protein was purified by column. The protein purification steps were as follows: 1) Washing: Filter the lysate supernatant after centrifugation (0.45 μm) and load it onto Ni-NTA resin. Wash with equilibration buffer (50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole (to inhibit nonspecific binding)) to remove weakly bound proteins. The flow rate was 1 mL / min, and the amount of equilibration buffer was 5 column volumes. 2) Elution: Wash with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250 mM imidazole; desalt the column and collect the eluate for 3 column volumes. The recovery rate of the target protein should be ≥85%. 3) Protein concentration determination: The BCA method was used to determine the protein concentration after purification, which was 10 mg / mL. 280 The purity was 98% as determined by HPLC.

[0019] Example 4 Silver nanoparticle antigen complex The purified protein was combined with 30 nm silver nanoparticles at a mass ratio of 2:1 to obtain a silver nanoparticle-antigen complex. The surface of the silver nanoparticles was modified with -COOH. The specific steps were as follows: (1) Physical properties of silver nanoparticles: AgNPs with a particle size of 30 nm and modified with -COOH were purchased from Sigma-Aldrich. 390 is 1.2, the peak shape is symmetrical, and the half-peak width is 60nm; (2) Pretreatment of silver nanoparticles: 0.5 mg of silver nanoparticle solution was transferred to a centrifuge tube, centrifuged at 12000 rpm / min for 15 min, the supernatant was discarded, and the precipitate was resuspended in 0.5 mL of MES buffer (pH 5.5), vortexed to mix, and centrifuged and washed twice. Finally, the precipitate was resuspended in 0.25 mL of MES buffer to ensure complete dispersion and increase the particle concentration; (3) Carboxyl activation: Prepare EDC and NHS working solutions: EDC: 10 mg / mL, NHS: 10 mg / mL. Add 50 μL of EDC working solution and 50 μL of NHS working solution to the above silver nanoparticle suspension, gently vortex to mix, and react at room temperature in the dark for 25 min. (4) Protein coupling: The activated silver nanoparticle solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended with 0.5 mL PBS (pH 7.4). The protein solution was diluted to 2 mg / mL (0.2 mL concentrated solution + 0.8 mL PBS), 0.8 mL of protein solution was added, and the pH was adjusted to 7.4-7.6. The solution was incubated at room temperature in the dark at 120 rpm for 2 h (protein concentration calibration: the diluted protein solution (2 mg / mL) was taken and the A 280 =0.6, the calculated concentration is: ×Mw= × 38,000 = 1.27 mg / mL, so add 0.8 mL of protein solution, of which the initial protein solution A 280 =0.6 (corresponding to a concentration of 2 mg / mL, =18000M -1 CM -1 , Mw=38kDa)), is the molar extinction coefficient, The optical path of the cuvette is 1 cm; (5) Closure and purification Add 50 μL of ethanolamine (1 M, pH 8.0) and react at room temperature for 30 min to terminate the coupling. Transfer the solution to an ultrafiltration tube (molecular weight cutoff 100 kDa) and centrifuge at 4,000 rpm / min for 15 min. Discard the filtrate and add 1 mL of PBS to resuspend the concentrate. Repeat the centrifugation and washing three times. The final complex is diluted to 1 mL with PBS and stored at 4°C in the dark.

[0020] Example 5 Chitosan coating enhances in vivo stability (1) Preparation of chitosan solution: Weigh chitosan (molecular weight 50-150 kDa), dissolve it in 1% acetic acid solution to prepare a 1 mg / mL solution, and filter sterilize it with a 0.45 μm filter membrane; (2) Composite coating: The AgNPs-antigen complex prepared in Example 4 (1 mg / mL) was mixed with chitosan solution at a volume ratio of 1:1. The coating time was 40 min and the centrifugation speed was 5000 rpm for 10 min to allow chitosan to be adsorbed on the surface of AgNPs through electrostatic interaction (AgNPs are negatively charged and chitosan is positively charged). (3) Centrifugal purification: The mixture was centrifuged (5000 rpm, 10 min), the supernatant was discarded, and the solution was resuspended to the original volume with PBS and washed twice to obtain the chitosan-coated silver nanoparticle antigen complex, which was stored at 4°C for future use. (4) In vitro release: The above-mentioned coating was placed in PBS with a pH of 7.4 for in vitro release of the healthy side. The cumulative release of antigen was 65% in 48 hours and 89% in 7 days, which was consistent with the sustained release kinetic model. The release rate diagram is shown in the figure below. Figure 2 shown.

[0021] Example 6 Quality testing of vaccine semi-finished products (1) Protein purity: SDS-PAGE electrophoresis detection shows clear bands of target proteins (RAP-1 approximately 35 kDa, MSA-2 approximately 40 kDa), and the content of impurity proteins is less than 5%; (2) Nanoparticle size and potential: Dynamic light scattering (DLS) detection: the particle size should be 10-50 nm, and the surface potential (Zeta potential) should be +10-+20 mV (positively charged after chitosan coating); (3) Antigen coupling efficiency: ELISA method was used to detect the protein content in the complex, and the coupling efficiency was ≥80%; (4) Sterility test: Inoculate into thioglycollate fluid culture medium and trypticase soytone culture medium, culture at 37℃ for 7 days, and no microbial growth.

[0022] By implementing the vaccine semi-finished product quality inspection in step 6, the standardized production of the bovine pyrocaul vaccine can be achieved according to the vector construction, CHO cell expression, silver nanoparticle coupling and chitosan coating processes described in Examples 1 to 5.

[0023] Test Example 1 Mouse experimental design Experimental animals: 40 SPF-grade mice (half male and half female), weighing between 100-120 g, and 8 weeks old, were purchased from the Guangdong Medical Laboratory Animal Center. Each group consisted of 10 mice (half male and half female), ensuring that they were free of bovine Theileria infection and other diseases that could affect the experiment.

[0024] Test plan: Before the experiment, rats were housed in a standard animal room with a temperature controlled at 22±3°C, a humidity of 40%-60%, a 12-h light / dark cycle, free access to water and food, and were allowed to adapt to the environment for 3-5 days.

[0025] The experiment consisted of 4 groups: Group A: vaccine component chitosan-coated AgNPs-RAP-1 / MSA-2 complex, immunization dose of 50ug / animal (total antigen amount); Group B: free RAP-1 / MSA-2 protein (without adjuvant), immunization dose of 50ug / animal; Group C: commercially available bovine pyrocaul vaccine (control), dosage according to the instructions; Group D: normal saline, immunization dose of 100μL; all were intramuscularly injected, and the immunization times were 0 days and 14 days.

[0026] Test indicators and data results: (1) Humoral immune response (ELISA to detect serum antibodies) Antibody titer on day 21: Group A: 1:6400 (IgG-dominant, IgG1 / IgG2a ratio 1.8:1, Th1 / Th2 balance) Group B: 1:800 (simple protein has weak immunogenicity) Group C: 1:3200 (the commercially available vaccine adjuvant is aluminum salt) Neutralizing antibody titer: Group A was significantly higher than Group B and Group D; (2) Cellular immune response (ELISPOT detection of IFN-γ secretion) After spleen cell stimulation, the number of IFN-γ spots in group A was 280±25 spots / 10 6 cells, which were significantly higher than those in group B (50±10) and group D (10±5), indicating that Th1 cell immune activation was observed; (3) On the 21st day, the challenge protection experiment was performed on all mice: 1×10 6 The dose of infected red blood cells / cell is injected under aseptic conditions, with gentle and accurate movements. The protective effect is: Group A: 100% survival rate, peak bloodworm density <0.5%, fever reduction time 3 days and normal body temperature (37.5-38.5℃); Group B: survival rate 20%, peak bloodworm density 8%, all died 7 days after infection; Group C: survival rate 60%, peak bloodworm density 3%, fever reduction time 5 days; Group D: Survival rate 0%, peak bloodworm density 12%, and death time concentrated in 5-6 days.

[0027] The experimental data are shown in Table 1: Table 1 Comparison of experimental data

[0028] At the end of the experiment, the organs of group A were examined by histopathological examination, and no specific lesions were found. The pathological images of the intramuscular injection site of group A are as follows: Figure 3 As shown; There were no significant differences in liver and kidney function indicators (ALT, AST, and BUN) between Group A and Group D, indicating good biocompatibility between chitosan and AgNPs. Group A had the highest survival rate and returned to normal body temperature within 3 days. The vaccine was significantly safer than the other groups, and the cellular immune response was also higher than that of the other groups. The release kinetics of chitosan-coated antigens conformed to sustained-release kinetics. Test Example 2 Five healthy susceptible black and white calves were selected, with three in the experimental group and two in the control group. Each calf in the experimental group was injected with 1 million cells intramuscularly in the buttocks, while the control group was not injected. Thirty days after the injection, the calf was challenged with 50 Hyalomma anomala ticks carrying the spores of Theileria annularis in Ningxia. On the eighth day after the challenge, all the ticks were removed from the calves and killed. The calves in the experimental group were observed for 21 consecutive days. The calves in the experimental group had no clinical symptoms and normal body temperature, just like the calves in the normal group.

[0029] In summary, the vaccine of the present application significantly improves the antigen delivery efficiency and immunogenicity through the synergistic effect of silver nanoparticles and chitosan, and shows protective efficacy and safety superior to traditional vaccines in animal models. Chitosan is coated on the surface of the silver nanoparticle-antigen complex, and its cationic properties are used to enhance the binding ability with the negative charge on the cell surface, thereby promoting antigen uptake. At the same time, its biodegradability and mucosal adhesion can prolong the retention time of the vaccine in the body, continuously release antigens, and enhance immune memory. Silver nanoparticles and chitosan are both biocompatible materials with low toxicity and are degradable, reducing the local inflammatory response that may be caused by traditional adjuvants (such as aluminum adjuvants). In addition, the CHO cell expression system avoids the risk of contamination by pathogenic microorganisms and is suitable for large-scale industrial production.

[0030] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A bovine pyrocaul vaccine, characterized in that: The vaccine comprises a combination of silver nanoparticles (AgNPs) and bovine Theileria antigens.

2. A bovine pyrocaul vaccine according to claim 1, characterized in that The silver nanoparticles have a particle size of 10 to 50 nm; the bovine Theileria antigen is derived from at least one of RAP-1 protein or an immunogenic fragment thereof, MSA-2 protein or a B cell epitope peptide thereof.

3. A bovine pyrocaul vaccine as claimed in claim 1, characterized in that: The surface of the silver nanoparticles is modified with any one of carboxyl groups, amino groups or PEG for covalent coupling with antigens.

4. A bovine pyrocaul vaccine as claimed in claim 2, characterized in that: The RAP-1 protein comprises the amino acid sequence shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 3; the MSA-2 protein comprises the B cell epitope shown in SEQ ID NO: 2, and its nucleotide sequence is shown in SEQ ID NO:

4.

5. A bovine pyrocaul vaccine according to claim 1, characterized in that: The bovine Theileria antigen is obtained by recombinant expression in CHO cells.

6. A method for producing a bovine pyrocaul vaccine, characterized in that: The steps are: (1) Vector construction: RAP-1 and MSA-2 were constructed using pcDNA3.1(+) vectors. (2) Transformation of host protein expression: The constructed vector was transformed into the same CHO cells, cultured and expressed to obtain RAP-1 and MSA-2 recombinant cells; (3) Lysing the recombinant cells and purifying the proteins to obtain RAP-1 and MSA-2 recombinant proteins; (4) The purified protein is combined with silver nanoparticles to obtain a silver nanoparticle-antigen complex.

7. A method for producing a bovine pyrocaul vaccine according to claim 6, characterized in that: The vector construction comprises the nucleotide sequence of claim 4.

8. The method for producing a bovine pyrocaul vaccine according to claim 6, wherein: The host culture in step (2) is leaf milk culture medium.

9. A method for producing a bovine pyrocaul vaccine according to claim 8, characterized in that: The leaf milk culture medium contains 95% leaf milk nutrient solution and 5% calf serum.

10. The method for producing a bovine pyrocaul vaccine according to claim 6, wherein: In step (4), the surface of the silver nanoparticle antigen complex is coated with chitosan to increase the half-life in vivo.

Images