A recombinant african swine fever virus polyantigen peptide, a coding gene thereof, a bacillus subtilis display engineering bacteria and an oral vaccine

Abstract

The present application relates to a kind of recombinant bacillus subtilis ASFV polyantigen peptide and the preparation method and application of IL-2 / IFN-alpha display engineering bacteria, belong to animal vaccine technical field.The P30 of the present application, P72, PP220 multi-antigen epitope of ASFV and immune enhancer factor IL-2, IFN-alpha are connected in series, using bacillus subtilis spore surface protein cotG as display carrier, construct recombinant engineering bacteria BSpDG1730-cotGASFV.ELISA and Western blot verification, target protein is efficiently expressed on spore surface and has good stability.Animal experiment shows that, after oral immunization, the engineering bacteria can significantly induce pig to produce specific IgG antibody, improve spleen and thymus index, promote IL-2, IFN-gamma and other cytokine secretion, and also specific fluid and cellular immune response.The preparation process of the present application is simple, spore is resistant to high temperature, easy to store, suitable for large-scale production, and provides a safe, efficient oral vaccine candidate for African swine fever prevention and control.

Description

Technical Field

[0001] This invention relates to the field of animal vaccine technology, specifically to a recombinant ASFV multiantigen peptide, its encoding gene, an engineered Bacillus subtilis spore surface display bacterium containing the gene, a specific antibody, its preparation method, and its application as an oral vaccine. Background Technology

[0002] African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV), which has had a catastrophic impact on the global pig farming industry. ASFV is a double-stranded DNA virus, and its structural proteins P30, P72, and PP220 play key roles in viral invasion and host immune response. However, the virus has a complex genotype and mutates frequently, resulting in limited protection from existing vaccines. Furthermore, traditional inactivated vaccines pose biosafety risks, and single-epitope antibody titers are only 1:1000, failing to provide durable protection.

[0003] Currently, multi-antigen peptide vaccines, by tandemly linking multiple conserved viral epitopes, can induce a broad-spectrum immune response, becoming an important direction for overcoming the bottlenecks in ASFV vaccine development. Bacillus subtilis spore surface display technology has advantages such as stable expression of exogenous proteins, storage resistance, and convenient oral administration. Its surface protein cotG can serve as a highly efficient display vector, anchoring antigenic peptides to the spore surface, enhancing antigen presentation efficiency and activating humoral and cellular immunity. However, existing cotG vectors exhibit low protein expression efficiency and insufficient epitope exposure. Therefore, how to screen for highly immunogenic epitopes, optimize the multi-antigen peptide linking method, and construct a stable spore display system remain pressing technical challenges. Summary of the Invention

[0004] Purpose of the invention:

[0005] This study provides a candidate oral vaccine for African swine fever that combines both fluid and cellular immune responses and exhibits high stability, in order to address the issues of incomplete protection and poor stability of existing vaccines.

[0006] Technical solution:

[0007] This invention provides a recombinant IBV multiantigen peptide, which is fused with the P30, P72, and PP220 antigenic epitopes of ASFV, linked by a flexible peptide sequence (APAPAPAPAPAPAPAP), the amino acid sequence of which is shown in SEQ ID NO.1. This design ensures that each epitope has an independent spatial structure, can be recognized by the immune system, and induces cross-immune responses against different strains.

[0008] The present invention also provides a gene encoding the recombinant ASFV multiantigen peptide and a biological material containing the gene.

[0009] Furthermore, the nucleotide sequence encoding the multiantigenic peptide was codon-optimized (SEQ ID NO.2) and inserted into the EcoRI site of the Bacillus subtilis vector pDG1730 to construct the recombinant expression vector pDG1730-cotGASFV.

[0010] Furthermore, biological materials containing this gene include recombinant bacteria BSPDG1730-cotGASFV (obtained by electroporation screening of Bacillus subtilis strain 168), which can achieve surface display of multiple antigenic peptides by inducing spore generation.

[0011] The present invention also provides a method for preparing the recombinant ASFV multiantigen peptide, which specifically includes the following steps:

[0013] Conserved epitopes from 31 prevalent ASFV strains were screened from GenBank and confirmed as transgenotype conserved sequences by ClustalX alignment. Flexible peptide ligation was used to avoid steric hindrance transformation under the conditions of 2.5 kV and 200 Ω. The resuscitation medium contained 0.5 M sorbitol and 0.38 M mannitol. Positive clones were verified by colony PCR (primers: F-cotG / P-rASFV), and the PCR product size was 2190 bp, consistent with expectations.

[0014] Furthermore, the recombinant bacteria with a target fragment size of 2190 bp were induced in DSM-2 medium (pH 9.0) at 40℃ for 72 h, with a spore formation rate >95%. The vegetative cells were removed by lysozyme (2 mg / mL, treated at 37℃ for 2 h), and the target protein with a purity ≥90% was obtained by purification using a Ni-NTA column (imidazolium gradient elution).

[0015] The present invention also provides an oral vaccine, comprising the aforementioned spore preparation (1×10⁻⁶). 10 The vaccine consists of CFU / mL and the target protein rCI2MKASFV, using recombinant B. subtilis-CI2MKASFV strain as a vector. The immunization procedure is two oral enemas (14 days apart), 1 mL each time, which can activate the intestinal mucosal IgA and systemic IgG responses.

[0016] Furthermore, immunogenicity assessment of the vaccine showed that it can induce humoral immunity in pigs, producing specific protective antibodies IgG; and may also induce cellular immunity in pigs.

[0017] The present invention also provides the application of the recombinant ASFV multiantigen peptide in the preparation of vaccines for the prevention of African swine fever or in the preparation of drugs for the treatment of diseases caused by African swine fever.

[0018] Beneficial effects:

[0019] 1. The recombinant ASFV multi-antigen peptide fusion provided by this invention fuses multiple transgenotype conserved antigenic epitopes such as P30, P72, and PP220, linked by a flexible peptide sequence APAPAPAPAPAPAPAP, ensuring that each epitope has an independent spatial structure and is fully exposed. Bioinformatics analysis shows an antigen index > 0.8 and a hydrophilicity coefficient -0.45, covering the main circulating ASFV strains (GI-GIX genotype), effectively reducing immune escape caused by antigenic variation, and solving the problem of narrow protection range of single-epitope vaccines.

[0020] 2. This invention uses the Bacillus subtilis spore capsid protein cotG as a display vector, inserting the multi-antigenic peptide encoding gene into the Nco I-Xho I site of the pDG1730 plasmid to construct the recombinant expression vector pDG1730-cotGASFV. This system utilizes the strong p43 promoter to drive the specific expression of the target gene at the spore stage. The target protein exists on the spore surface in the form of inclusion bodies, with an expression level exceeding 15% of the total bacterial protein. Furthermore, it eliminates the need for a complex renaturation process, simplifying the preparation process and reducing production costs.

[0021] 3. This invention is the first to activate humoral and cellular immunity simultaneously through the cotG protein tandem with IL-2 / IFN-α. Animal experiments showed that the titer of specific IgG antibodies in porcine serum reached its peak 28-35 days after immunization (P30 antibody ≥ 1:10159, P72 / PP220 antibody ≥ 1:6400), the spleen index increased by 25% compared with the control group, and the levels of IFN-γ and IL-2 cytokines increased by 1.8 times and 1.5 times, respectively, significantly enhancing the immune protection effect.

[0022] 4. This invention provides a recombinant bacterium, BSpDG1730-cotGASFV, whose spores, after purification with lysozyme, exhibit a survival rate of >90% after 6 months of storage at 4°C and are heat-resistant (activity retention rate >80% after 1 hour of treatment at 60°C). Oral immunization eliminates the need for injection, avoiding stress reactions and making it suitable for large-scale farming applications. Furthermore, the antigenic proteins displayed on the spore surface activate intestinal mucosal immunity without additional adjuvants, further enhancing vaccine safety and practicality.

[0023] 5. This invention removes endotoxins using urea gradient washing, native PAGE purification, and Triton X-114 liquid phase separation to obtain multi-antigen peptides with a purity ≥90%. The entire process cycle is shortened to 72 hours, and no complex chromatographic steps are required. The exogenous gene remains stable after 15 consecutive passages of the recombinant bacteria, ensuring the reproducibility and reliability of the production process and providing technical support for large-scale industrial production. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, they will not be difficult to understand.

[0025] Provided that creative effort is put in, other figures can be obtained from these figures.

[0026] Figure 1 Preparation for amplification of the target gene and vector; M: Marker; 1: p43+cotG (1046bp); 2: MKASFV (2190bp); 3: RCC (183bp); 4: plasmid pDG-CI2 (9875bp); 5: pDG-CI2 plasmid EcoRI digestion (9875bp)

[0027] Figure 2 Identification of recombinant plasmid pDG-CI2MKASFV; M: Marker; 1-2: Colony PCR product of MKASFV (2190bp); 3-4: Empty E. coli cells; 5-6: Recombinant plasmid pDG-CI2MKASFV (13300bp); 7: Hind III digestion (13300bp); 8: EcoRI digestion (3425bp, 9875bp); 9: Double digestion with Hind III and EcoRI (2288bp, 3425bp, 7587bp).

[0028] Figure 3 Identification of recombinant B. subtilis-CI2MKASFV; M: Marker; 1-2: MKASFV (2190bp); 3-4: amyE-front (1953bp); 5-6: amyE-back (1956bp); 7-9: empty B. subtilis cells; 10: empty B. subtilis cells (731bp); 11-12: amyE (7200bp).

[0029] Figure 4 Growth status of B. subtilis spores at 40℃ for different times

[0030] Figure 5 ELISA identification of protein display in B. subtilis-CI2MKASFV spores; ELISA results showing IL-2 protein on the spore surface indicate successful display of the target protein.

[0031] Figure 6Western blot identification of B. subtilis-CI2MKASFV spore display protein; 1: B. subtilis; 2: B. subtilis-CI2MKASFV

[0032] Figure 7 Stability assessment of recombinant B. subtilis-CI2MKASFV; M: marker; 1: MKASFV (2190bp); 2: amyE-front (1953bp); 3: amyE-back (1956bp); 4: amyE (7200bp); 5-8: B. subtilis (6 and 8 correspond to amyE 731bp)

[0033] Figure 8 The recombinant plasmid pCold-p30 was constructed

[0034] Figure 9 The recombinant plasmid pCold-p72 was constructed.

[0035] Figure 10 The recombinant plasmid pCold-pp220 was constructed.

[0036] Figure 11 For the amplification of the target gene; M: Marker; 1: pp220 (249bp); 2: p30 (645bp); 3: p72 (294bp)

[0037] Figure 12 For validation of recombinant bacterial PCR; M: Marker; 1-4: pCold-p72 colony PCR (407bp); 5-8: pCold-p30 colony PCR (788bp); 9-12: pCold-pp220 colony PCR (352bp).

[0038] Figure 13 Sequencing alignment results for p30, p72, and pp220 sequences

[0039] Figure 14 Chromatograms of recombinant protein purification; A: Chromatogram of rp30 purification; B: Chromatogram of rp72 purification; C: Chromatogram of rpp220 purification.

[0040] Figure 15 Electrophoresis images of rp30, rp72, and rp220 proteins; M: Marker; N: Uninduced protein; I: Flow-through sample; P: Protein purified by Ni-NTA chromatography; rp30: 24 kDa; rp72: 39.5 kDa; rpp220: 37.6 kDa

[0041] Figure 16 Standard curve for protein assay kit

[0042] Figure 17 Serum specific IgG antibody level

[0043] Figure 18 Effects of B. subtilis-CI2MKASFV vaccine on pig spleen

[0044] Figure 19 Standard curves and linear regression equations for IL-2 and IFN-γ

[0045] Figure 20 The values ​​represent the serum IL-2 and IFN-γ levels in pigs; rCI2MKASFV represents B. subtilis-CI2MKASFV. Compared with the PBS group, *p<0.05, **p<0.01 Detailed Implementation

[0046] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0047] Example 1: Construction and Identification of Recombinant Bacillus subtilis

[0048] I. Experimental Materials

[0049] 1. Plasmids and strains

[0050] (1) pDG1730 plasmid and Bacillus subtilis strain 168 were purchased from Kunshan Yuanmu Biotechnology Co., Ltd.

[0051] (2) The pET-28a-ASFV plasmid was preserved in our laboratory;

[0052] 2. Main Instruments

[0053] The following equipment was purchased: a clean bench (from Suzhou Clean Chemical Equipment Co., Ltd.); an electronic balance (from Shanghai Zhuojing Electronic Technology Co., Ltd.); a vertical pressure steam sterilizer (from Jiangyin Binjiang Medical Equipment Co., Ltd.); an electric thermostatic drying oven (from Tianjin Zhonghuan Experimental Furnace Co., Ltd.); a water-jacketed thermostatic incubator (from Shanghai Jinghong Experimental Equipment Co., Ltd.); a thermostatic water bath (from Jintan Baita Xinbao Instrument Factory); a Bio-Rad electrophoresis apparatus (from Bio-Rad Laboratories, USA); a high-speed benchtop centrifuge (from Eppendorf China Co., Ltd.); an ultrapure water preparation system (from Shanghai Hetai Instrument Co., Ltd.); a fully automatic snowflake ice maker (from Changshu Lingke Electric Appliance Co., Ltd.); a pH meter (from Ohaus Instruments (Changzhou) Co., Ltd.); a continuous wavelength microplate reader (from SPECTRA); a microwave oven (from Qingdao Haier Complete Household Appliance Service Co., Ltd.); and a metal bath (from Shanghai Huxi Industrial Co., Ltd.).

[0054] 3. Preparation of main solutions and culture media

[0055] (1) LB liquid culture medium: Tryptone 5g, Yeast Extract 2.5g, NaCl 5g, ddH2O to a final volume of 500mL, after complete dissolution, autoclave and use.

[0056] (2) LB solid medium: Tryptone 2.0g, Yeast Extract 1.0g, NaCl 2.0g, Agar 4g, ddH2O to a final volume of 200mL, autoclave and cool to about 60℃, pour into sterile culture dishes, and store upside down at 4℃ after cooling and solidification.

[0057] (3) Growth medium GM (2×YT+0.5M sorbitol): Tryptone 0.85g, Yeast Extract 0.5g, Nacl 0.5g, sorbitol 4.5g, add ddH2O to make up to 50mL, autoclave at 121℃ for 15min; store at 4℃ for later use.

[0058] (4) Electroporation medium ETM: sorbitol 9.0g, mannitol 9.0g, trehalose 9.0g, MgCl2 0.5mM, dipotassium hydrogen phosphate 0.5mM, potassium dihydrogen phosphate 0.5mM, add ddH2O to make up to 100mL, autoclave at 121℃ for 15min; store at 4℃ for later use.

[0059] (5) Electroporation recovery solution RM (2×YT+0.5M sorbitol+0.38M mannitol): Tryptone 0.17g, Yeast Extract 0.1g, NaCl 0.1g, sorbitol 0.9g, mannitol 0.7g, add ddH2O to make up to 10mL, autoclave at 121℃ for 20min; store at 4℃ for later use;

[0060] (6) Kan + Stock solution (50 mg / mL): Weigh 0.5 g kana powder, dissolve in ddH2O, and bring the volume to 10 mL. Vortex to dissolve completely, filter sterilize using a 0.22 μm filter, dispense into 1.5 mL EP tubes, and store at -20 °C. The concentration used is 50 μg / mL.

[0061] (7)Amp + Stock solution (100 mg / mL): Weigh 1 g of ampicillin powder, dissolve in ddH2O, and bring the volume to 10 mL. Vortex to dissolve completely, filter through a 0.22 μm filter for sterilization, dispense into 1.5 mL EP tubes, and store at -20℃. The concentration used is 100 μg / mL.

[0062] (8) Spe + Stock solution (100 mg / mL): Weigh 1 g of Magnoliopsin powder, dissolve in ddH2O, and bring the volume to 10 mL. Vortex to dissolve completely, filter sterilize using a 0.22 μm filter, dispense into 1.5 mL EP tubes, and store at -20 °C. The concentration used is 100 μg / mL.

[0063] (9) 1×TAE Buffer: Measure 10mL of 50×TAE Buffer, add ddH2O to make up to 500mL, and shake to mix well;

[0064] (10) 1% agarose gel: Weigh 0.2g agarose, add 20mL 1×TAE Buffer, microwave until completely dissolved, the solution is clear and transparent, add 1.5μL nucleic acid dye, shake to mix well and pour into the gel plate;

[0065] (11) 50% glycerol: Measure 50 mL of glycerol and 50 mL of ddH2O, stir thoroughly, and store at 4°C.

[0066] (12) Solution A (100ml): Weigh 12.12g of Tris base, dissolve it in 80ml of ddH2O, add 4mL of 10% SDS, adjust the pH to 6.8, and finally make up to 100mL. Store at 4℃ for later use.

[0067] (13) SDS electrophoresis buffer (1×): SDS 1.0g, Glycine 14.4g, Tris-base 3.3g, add 800mL ddH2O, wait for it to dissolve completely, and then make up to 1L with deionized water;

[0068] (14) Transfer solution (1×): SDS 1.0g, Tris base 3.3g, add 600mL ddH2O and stir thoroughly with a glass rod, add 200mL methanol, stir evenly and then dilute to 1L with deionized water;

[0069] (15) 1×TBST buffer (1L): NaCl 8.8g, Tris-base 2.4g, dissolved in 800mL ddH2O, mixed well, then add 500μL Tween-20, adjust the pH value to about 7.5 with concentrated HCl, and make up to 1L with deionized water.

[0070] (16) 10% ammonium persulfate (AP): Weigh 0.1g of ammonium persulfate and dissolve it completely in 1mL of ddH2O. Store at 4℃.

[0071] (17) 5× loading buffer: 0.1 g bromophenol blue, 2.5 g SDS, 5 mL solution A, and 10 mL autoclaved ultrapure water were added to a 50 mL centrifuge tube. The mixture was shaken for 2 h and then centrifuged. 2.5 mL β-mercaptoethanol was added, and the mixture was shaken for 10 min and then centrifuged. Finally, 25 mL glycerol was added, and the mixture was shaken for another 3 h and then centrifuged. The final volume was adjusted to 50 mL and stored at room temperature.

[0072] (18) Coating buffer: 50mM NaHCO3 buffer, pH 9.4;

[0073] (19) Blocking buffer: 10mM PBS + 5% skim milk powder, pH 7.4;

[0074] II. Experimental Methods

[0075] 1. Construction of recombinant expression plasmids

[0076] The IL-2 and IFN-α genes and the cotG gene of spore capsid protein were obtained from NCBI. First, a p43 promoter was introduced into the 5' end of the cotG gene. A linker sequence was then added between the cotG, IL-2, IFN-α protein, and targeting peptide genes. The sequence was optimized and synthesized by Shanghai Sangon Biotech Co., Ltd., and the resulting plasmid was named pDG1730-cotG-IT. Using the codon-optimized pDG1730-cotG-IT and the laboratory-preserved pet-28a-ASFV plasmid as templates, PCR amplification yielded p43+cotG, ASFV, and targeting gene fragments. pDG1730-cotG-IT was digested with EcoRI, and homologous recombination was performed on the amplified gene fragments and the digested vector.

[0077] 2. Construction and identification of recombinant Bacillus subtilis displaying spores

[0078] (1) Preparation of B. subtilis 168 electrocompetent cells

[0079] B. subtilis 168 strain was streaked onto antibiotic-free solid LB agar plates. A single colony was picked and inoculated into 3 mL of antibiotic-free 2×YT medium and incubated overnight at 37°C and 200 rpm.

[0080] Transfer 1.5 mL of overnight culture to 50 mL of GM solution and incubate at 37°C and 220 rpm for about 2.5-3 hours. When the bacterial culture grows to OD600 = 0.8-1.0, remove the bacterial culture and incubate on ice for 15 min.

[0081] Centrifuge at 5000 rpm for 10 min at 4℃, discard the supernatant and collect the bacterial cells.

[0082] Wash the bacterial cells with 50ml, 25ml, and 10ml of pre-cooled electroporation medium at 4℃, centrifuge at 5000rpm for 10min at 4℃ and discard the supernatant. Repeat the washing process three times.

[0083] The washed bacterial cells were resuspended in 500 μL of electroporation medium, aliquoted into 1.5 mL EP tubes (80 μL per tube), and stored at -80°C for later use.

[0084] (2) Extraction of pDG1730 plasmid from Escherichia coli

[0085] Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0086] Collect bacterial cells: Centrifuge 1-5 ml of overnight cultured bacterial solution, discard the supernatant, and collect the bacterial cell precipitate.

[0087] Bacterial cell lysis: Add 250 μL of solution P1 to the bacterial precipitate to suspend the bacteria, then add 250 μL of solution P2 and gently tumble to lyse the cells. Next, add 350 μL of solution P3, tumble to mix, and centrifuge to obtain a white flocculent precipitate. Supernatant treatment: Transfer the supernatant to the adsorption column CP3, centrifuge, and discard the waste liquid; add 500 μL of protein removal solution PD, centrifuge, and discard the waste liquid.

[0088] Washing the adsorption column: Add 600 μL of PW rinsing solution and centrifuge. Repeat once, then centrifuge again to remove residual rinsing solution.

[0089] Elution of plasmids: Place the adsorption column in a clean centrifuge tube, add 50-100 μL of elution buffer EB to the middle of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge to collect the plasmid solution.

[0090] (3) Plasmid linearization

[0091] Enzyme digestion reaction: Take 30 μL of pDG1730 plasmid, add 5 μL of 10×Buffer, 3 μL of NruⅠ and 12 μL of ddH2O, and incubate at 37℃ for 6 h for enzyme digestion.

[0092] Precipitate plasmids: After enzyme digestion, add 5 μl of 3M sodium acetate (pH 3.8) buffer, 2 μl of glycogen solution and 142 μl of -20℃ pre-cooled anhydrous ethanol, and precipitate overnight at -20℃.

[0093] Washing and drying: After centrifugation the next day, discard the supernatant, wash the precipitate twice with 75% pre-cooled ethanol, remove as much supernatant as possible after centrifugation, add 10 μl of deionized water to dissolve the precipitate, and store at 4℃ for later use.

[0094] (4) B. subtilis 168 electroconversion

[0095] Remove B. subtilis168 competent cells from the -80℃ freezer, allow them to thaw slowly in an ice bath, add 1 μg of plasmid (volume: 1-5 μL) to 80 μL of competent cells, and incubate on ice for 10 min.

[0096] Transfer the sample into a pre-cooled electric transfer cup (2mm) and perform one electric shock. Electric transfer instrument settings: 2.5KV.

[0097] After electroporation, remove the electroporation cup and immediately add 1 mL of RM electroporation recovery solution, transfer it to an EP tube, and incubate at 37°C and 180 rpm for 3 hours.

[0098] The first group had 0.1 ml of bacterial culture evenly spread on resistant solid LB agar plates and incubated at 37°C for 15 h. The second group had 0.1 ml of the revived bacterial culture added to 10 ml of resistant LB liquid medium and incubated overnight at 37°C with shaking at 220 rpm.

[0099] On the second day, a small amount of the second group of bacterial solution was diluted 10,000 times and then spread evenly on resistant solid LB plates at a dosage of 0.1 ml / plate. The plates were then incubated at 37°C for 15 hours.

[0100] (5) Identification of recombinant bacteria

[0101] After overnight culture, two single clones were randomly selected for colony PCR and sequencing identification. The PCR primer information is shown in Table 2.13, and the PCR reaction system is shown in Tables 2.14, 2.15, and 2.16. The obtained PCR products were verified by 1% agarose gel electrophoresis. The correctly identified positive recombinant strain was named BSPDG1730-cotGASFV.

[0102] 3. Optimization of culture conditions for Bacillus subtilis to induce spore production

[0103] (1) Six different culture media with different components and pH were designed and cultured at 30℃ and 40℃ respectively. Samples were taken at 24h, 48h and 72h, and the number and maturity of spores were observed by spore staining method.

[0104] (2) Culture medium formulations (6 types)

[0105] Nutritious broth + inorganic salts: contains calcium chloride, magnesium sulfate, manganese chloride, etc., pH=7.0.

[0106] Low-salt carbon source culture medium: containing sodium chloride, beef extract, yeast extract, and soluble starch, pH=9.0.

[0107] Nutritious broth + potassium dihydrogen phosphate + manganese sulfate: pH = 7.0.

[0108] Peptone + various inorganic salts: containing potassium chloride, magnesium sulfate, calcium chloride, manganese chloride, and ferrous sulfate.

[0109] Soluble starch + peptone + magnesium sulfate: pH = 7.0.

[0110] Low-salt carbon source + trace elements: Add manganese chloride and ferrous sulfate to the formula No. 2.

[0111] (3) Cultivation method:

[0112] B. subtilis 168 was removed from a -80℃ freezer and streaked in three zones on LB solid medium. After overnight incubation at 37℃ for 12-16 hours, single colonies were picked and cultured in LB liquid medium at 37℃ and 200 rpm until the logarithmic growth phase. Then, 10% of the colonies were transferred to spore-inducing medium and cultured at 200 rpm for 3 days. Cells were collected at 30℃ and 40℃ for spore staining at 24h, 48h, and 72h to select the optimal medium and conditions for maximizing spore numbers.

[0113] (4) Spore staining method (malachite green staining method):

[0114] Slide preparation: The cultured Bacillus subtilis is aseptically smeared, dried, and fixed;

[0115] Heating staining: Add 5% malachite green aqueous solution to the slide to cover the smeared area. Hold the slide with clips and heat it slightly above a flame. Start timing 5 minutes from when the staining solution starts to steam. Note that the staining solution should be added continuously during the heating process to prevent it from drying out.

[0116] Decolorization: Pour off the dye solution, and after the slide has cooled, rinse it with slow-flowing tap water until it is colorless;

[0117] Counterstain: Counterstain with a 0.5% safranin aqueous solution for 2 minutes;

[0118] Decolorization: Pour off the dye solution and rinse with slow-flowing tap water until colorless;

[0119] Microscopic examination: After the slide has dried, observe it under an oil immersion microscope. The bacterial cells appear red, and the spores appear green.

[0120] 4. Identification of the protein displayed by recombinant Bacillus subtilis BSpDG1730-cotGASFV spore display

[0121] (1) Preparation of spore protein

[0122] Induction of spore formation: Positive bacteria were inoculated onto LB plates containing 50 μg / mL spectinomycin and cultured overnight at 37°C. Then, they were transferred to spore-inducing medium at a ratio of 1:10 and cultured for 72 hours before collecting the bacterial cells.

[0123] Spore purification: Collect bacterial cells by centrifugation, wash with PBS, add lysozyme (2 mg / mL), incubate at 37°C for 2 h to destroy vegetative cells, centrifuge, discard the supernatant, and repeat washing to obtain pure spores.

[0124] Spore protein extraction: Add spore lysis buffer, incubate at 70℃ for 1 h to dissolve spores, centrifuge and take the supernatant as spore protein extract for subsequent Western blot identification.

[0125] (2) Detection of IL-2 and IFN-α antigens on spore surface by direct ELISA method

[0126] Sample preparation: The concentration was adjusted according to the A600 value of the spore suspension to prepare 8 gradients (A600 = 5 to 0) of test samples. After centrifugation, the samples were resuspended in coating buffer.

[0127] Coating and blocking: Add the sample to the ELISA plate and incubate at 37°C for 18-24 hours; block with PBS containing 5% skim milk powder for 1 hour and wash 3 times.

[0128] Antibody reaction and color development: Add HRP-labeled anti-pig IL-2 monoclonal antibody, incubate at 37°C for 1 hour, wash 4 times, add TMB color development solution, add stop solution after 15 minutes, and read the absorbance value at 450 nm using an ELISA reader.

[0129] (3) Western blot identification of ASFV protein

[0130] Sample preparation and electrophoresis: The spore protein was mixed with 5×SDS loading buffer, boiled to denature, and then subjected to 10% SDS-PAGE gel electrophoresis to separate the target protein.

[0131] Transfer and blocking: The protein was transferred to a PVDF membrane and blocked with 10% skim milk for 1 hour to block nonspecific binding.

[0132] (4) Antibody incubation:

[0133] The primary antibody was ASFV-P72 rabbit anti-antibody (1:3000), incubated overnight at 4℃; the secondary antibody was goat anti-rabbit IgG (1:5000), incubated at room temperature for 90 min, and the membrane was washed 3 times with TBST after each incubation.

[0134] Luminescence detection:

[0135] After adding ECL luminescent solution and detecting it with a chemiluminescence imaging system, a clear band corresponding to the size of the target protein was observed, confirming the successful expression of ASFV protein.

[0136] 5. Stability assessment of recombinant B. subtilis

[0137] The recombinant bacterial strain BSpDG1730-cotGASFV, which was correctly identified and stored at -80℃, was revived and continuously passaged for 15 generations. After each passage, PCR identification was performed to determine whether the exogenous gene in recombinant B. subtilis remained stable after multiple passages.

[0138] III. Experimental Results

[0139] 1. Amplification of the target gene and vector preparation

[0140] Using the optimized pDG1730-cotG-IT plasmid and the laboratory-preserved pet-28a-ASFV plasmid as templates, the target fragments were amplified by PCR, yielding p43+cotG, ASFV, and Targeting gene fragments with amplified sizes of approximately 1046 bp, 2190 bp, and 183 bp, respectively, consistent with the target gene fragment sizes, as shown in the figure. Linearization of the pDG1730-cotG-IT plasmid vector by EcoRI single enzyme digestion resulted in a target band of approximately 9875 bp, consistent with expectations, as shown in the electrophoresis results. Figure 1 As shown.

[0141] 2. Construction and transformation of plasmid pDG1730-cotGASFV

[0142] The target fragment amplified by PCR and the linearized plasmid vector were subjected to homologous recombination. The recombinant plasmid was then heat-shocked and transformed into E. coli Top10 competent cells. After overnight incubation at 37°C, colonies grew uniformly on plates containing Amp+ resistance. The colonies had smooth surfaces, regular edges, and were round, convex, and translucent. The colony morphology was uniform and there was no contamination.

[0143] 3. Identification of recombinant plasmid pDG1730-cotGASFV

[0144] Colonies incubated overnight were randomly selected, and transformants were identified by colony PCR. The ASFV gene fragment was amplified, and gel electrophoresis showed an amplified band of approximately 2190 bp, consistent with expectations. Positive colonies were then expanded and plasmids were extracted. The plasmids were digested with Hind III and EcoRI, respectively, using both single and double enzymes. Electrophoresis analysis after digestion showed that Hind III single digestion produced a target band of approximately 13300 bp, EcoRI single digestion produced two target bands of approximately 3425 bp and 9875 bp, and double digestion with Hind III and EcoRI produced three bands of approximately 2282 bp, 3425 bp, and 7593 bp, consistent with expectations. Electrophoresis results are as follows: Figure 2 As shown, the final comprehensive sequencing analysis results indicate that the spore display plasmid pDG1730-cotGASFV was successfully constructed.

[0145] 4. Identification results of recombinant BSpDG1730-cotGASFV

[0146] After linearizing the recombinant plasmid pDG1730-cotGASFV, it was electroporated into B. subtilis 168 competent cells. After overnight culture at 37°C, colonies grew uniformly on plates containing Spe+ resistance. The colonies had smooth surfaces, neat edges, were round and convex, and were translucent. The colony morphology was uniform and uncontaminated. A BSpDG1730-cotGASFV recombinant spore display expression strain was constructed.

[0147] Because *B. subtilis* is a Gram-positive bacterium with a thick cell wall, electroporation is challenging. When the recombinant plasmid pDG1730-cotGASFV is transformed into *B. subtilis*, it is crucial to verify whether the fused gene is fully integrated into the *B. subtilis* 168 genome. To this end, we performed PCR on randomly selected single colonies after shaking to verify this. The results are as follows... Figure 3 As shown, target bands of approximately 2190bp, 1953bp, 1956bp, and 7200bp appeared, consistent with expectations. Finally, the comprehensive sequencing analysis results showed that the recombinant plasmid pDG1730-cotGASFV was successfully integrated into the B. subtilis 168 genome.

[0148] 5. Results of Bacillus subtilis-induced spore production

[0149] To select the optimal culture medium and conditions for inducing B. subtilis spore production, a preliminary experiment determined the culture temperature to be 40℃. To determine the most suitable culture time, bacterial strains were collected at 24h, 48h, and 72h for staining and observation. The growth status of B. subtilis spores after culture in six DSM media at 40℃ was observed as follows: At 40℃, with the passage of time, except for DSM-1, the morphology of vegetative cells in the other media changed from slender to thick and short, and the number of spores also showed an increasing trend; at 24h, DSM-2, DSM-4, and DSM-6... Spore formation was observed in all samples, but DSM-2 had the highest number of spores. At 48 hours, significant spore formation was observed in all samples (DSM-2, DSM-3, DSM-4, DSM-5, and DSM-6), with DSM-2 and DSM-6 having the highest spore count. At 72 hours, many spores were still forming in DSM-2, DSM-4, and DSM-6, but DSM-2 had the highest spore count and almost no vegetative cells, with a spore ratio exceeding 95%. In contrast, DSM-3 and DSM-5 showed a decrease in both vegetative cells and spores. Figure 4Based on the above spore staining results, the following conclusions were drawn: DSM-2 medium is the most effective medium for inducing spore production, especially when cultured at 40℃ for 72 hours, resulting in the highest spore count. Therefore, we ultimately chose DSM-2 medium, with culture conditions of 40℃ for 72 hours, for subsequent experiments.

[0150] 6. ELISA test results

[0151] The recombinant plasmid pDG-CI2MKASFV was electroporated into B. subtilis 168 competent cells to construct a B. subtilis-CI2MKASFV recombinant spore-displaying strain. After inducing spore formation, samples were collected. The B. subtilis and B. subtilis-CI2MKASFV spore samples were processed, and ELISA was used to detect the expression of rCI2 protein on the spore surface. The results are as follows: Figure 5 As shown, with increasing spore concentration, the absorbance of B. subtilis-CI2MKASFV spores at 450 nm increased significantly, indicating that IL-2 protein was successfully displayed on B. subtilis-CI2MKASFV spores. Since cotG, IL-2, IFN-α, and RCC are linked together, the detection of IL-2 protein expression proves that this recombinant protein rCI2 was successfully expressed on the spore surface.

[0152] 7. Western blot identification results

[0153] To further verify rMKASFV protein expression, B. subtilis and B. subtilis-CI2MKASFV spore samples were processed and then identified by Western blot using an ASFV-p72 polyclonal antibody. The results are as follows: Figure 6 As shown, a clear band appears around 109 kDa, consistent with the size of the target protein, indicating successful expression of rMKASFV protein. In summary, these results demonstrate successful expression of rCI2MKASFV protein in recombinant B. subtilis-CI2MKASFV.

[0154] 8. Stability assessment of recombinant B. subtilis

[0155] B. subtilis168 and recombinant B. subtilis-CI2MKASFV strains were sequentially passaged for 15 generations, with colony PCR performed every 5 generations. Results are as follows: Figure 7 As shown, after 15 generations of continuous passage of the recombinant B. subtilis-CI2MKASFV strain, the recombinant exogenous gene was not lost and still existed, indicating that the exogenous gene can be stably present in B. subtilis168.

[0156] Example 2: ASFV epitope antigen design, prokaryotic expression, and preparation

[0157] I. Experimental Materials

[0158] 1. Bioinformatics analysis software / website

[0159] GenBank; IEDB; Detai Bio; Novopro; DNAMAN

[0160] 2. Plasmids and vectors

[0161] (1) The pCold2 expression plasmid was provided by Baori Biotechnology (Beijing) Co., Ltd.; the GST-pCold6 expression vector was obtained by inserting the GST coding sequence into the multiple cloning site of the pCold2 expression vector and was purchased from Suzhou Moquan Biotechnology Co., Ltd.

[0162] (2) E. coli Top10 strains were purchased from Shanghai Sangon Biotech Co., Ltd.

[0163] 3. Main Instruments

[0164] Electronic balance, Shanghai Zhuojing Electronic Technology Co., Ltd.; Ultrapure water preparation system, Shanghai Hetai Instrument Co., Ltd.; Constant temperature incubator shaker, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; Vertical pressure steam sterilizer, Jiangyin Binjiang Medical Equipment Co., Ltd.; Medical clean bench, Sujie Medical Device Co., Ltd.; Vortex shaker, VELPscientifica (Italy); Constant temperature incubator, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; High-speed low-temperature centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; Low-speed centrifuge, Keda Innovation Co., Ltd. Zhongjia Branch; Bio-Rad electrophoresis apparatus, Bio-Rad Inc. (USA); Constant temperature water bath, Jintan Baita Xinbao Instrument Factory; -80℃ ultra-low temperature freezer, Zhongke Meiling Co., Ltd.; Ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.

[0165] 4. Preparation of main reagents

[0166] (1) Preparation of culture medium-related reagents

[0167] LB liquid medium: Tryptone 10g, Yeast Extract 5g, NaCl 10g, ddH2O to a final volume of 1L, until completely dissolved, autoclave and set aside.

[0168] LB solid medium: Tryptone 2.0g, Yeast Extract 1.0g, NaCl 2.0g, Agar 4g, ddH2O to a final volume of 200mL, autoclave and cool to about 60℃, pour into sterile petri dishes, and after cooling and solidification, invert and store at 4℃ for later use.

[0169] IPTG stock solution (100mM): Weigh 0.24g of IPTG powder, dissolve in ddH2O, and bring the volume to 10mL. Filter through a 0.22μm bacterial filter, dispense, and store at -20℃.

[0170] Amp + Stock solution (100 mg / mL): Weigh 1 g of ampicillin powder, dissolve in ddH2O, and bring the volume to 10 mL. Vortex to dissolve completely, filter through a 0.22 μm filter for sterilization, dispense into 1.5 mL EP tubes, and store at -20 °C. The concentration used is 100 μg / mL.

[0171] (2) Polyacrylamide gel electrophoresis reagent

[0172] SDS electrophoresis buffer (1×): SDS 1.0g, Glycine 14.4g, Tris-base 3.3g, add 800mL ddH2O, wait for it to dissolve completely, and then make up to 1L with deionized water;

[0173] Solution A (100ml): Weigh 12.12g of Tris base, dissolve it in 80ml of ddH2O, add 4mL of 10% SDS, adjust the pH to 6.8, and finally bring the volume to 100mL. Store at 4℃ for later use.

[0174] 5× Loading buffer: 0.1 g bromophenol blue, 2.5 g SDS, 5 mL solution A, and 10 mL autoclaved ultrapure water were added to a 50 mL centrifuge tube. The mixture was shaken for 2 hours and then centrifuged. 2.5 mL β-mercaptoethanol was added, and the mixture was shaken for another 10 minutes and centrifuged. Finally, 25 mL glycerol was added, and the mixture was shaken for another 3 hours and centrifuged. The final volume was adjusted to 50 mL and stored at room temperature.

[0175] 10% Ammonium Persulfate (AP): Weigh 0.1g of ammonium persulfate and dissolve it completely in 1mL of ddH2O. Store at 4℃.

[0176] Coomassie Brilliant Blue Staining Solution: Weigh 0.25g of Coomassie Brilliant Blue R-250, measure 45mL of methanol, 10mL of glacial acetic acid, and 45mL of ultrapure water, mix thoroughly, and store at room temperature;

[0177] Decolorizing solution: Measure 125 mL of methanol and 40 mL of glacial acetic acid, and dilute to 500 mL with ddH2O.

[0178] (3) Protein purification related reagents

[0179] 10×PBS: Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4, dissolve them in 100mL ddH2O, adjust the pH to 7.4, and store at room temperature;

[0180] Disruption buffer: 1×PBS, 1mM PMSF;

[0181] 500mM imidazole solution: Weigh 34g of imidazole powder, dissolve it in 500mL of 1×PBS, and store at room temperature for later use;

[0182] Column equilibration buffer (30 mL): Mix 1.8 mL of 500 mM imidazole with 28.2 mL of 1×PBS.

[0183] Elution buffer (9 mL): Mix 5.4 mL of 500 mM imidazole with 3.6 mL of 1×PBS.

[0184] II. Experimental Methods

[0185] 1. ASFV-P30, ASFV-P72, and ASFV-P220 sequence design

[0186] ASFV antigenic epitope sequences were searched and analyzed in GenBank and IEBB (www.iedb.org), yielding the amino acid sequences of three ASFV-related epitope peptides: P30, P72, and PP220. Antigenic determinants of the ASFV amino acid sequences were predicted using Detai Bio (www.detaibio.com), and after passing the predictions, molecular weight, isoelectric point, hydrophilicity, and instability were analyzed using Expasy (web.expasy.org).

[0187] 2. Construction of expression vector

[0188] The P30 nucleotide sequence from the pDG1730-cotG-ASFV plasmid vector was inserted into the pCold2 vector linearized with NdeI restriction endonuclease using homologous recombination, aligning with the N-terminal 6×His tag reading frame to form the His-P30-pCold2 expression vector. The P72 and PP220 nucleotide sequences from the pDG1730-cotG-ASFV plasmid vector were then inserted into the GST-pCold6 plasmid digested with HindIIIh and XbaI, respectively, aligning with the N-terminal GST reading frame to obtain the GST-P72-pCold6 and GST-PP220pCold6 expression vectors.

[0189] 3. ASFV protein-induced expression

[0190] While prokaryotic expression systems offer advantages such as ease of operation, speed, and high yield, rapid expression of heterologous proteins in *E. coli* often results in the formation of insoluble inclusion bodies. These inclusion bodies require complex refolding and purification processes before practical application, posing significant inconvenience for large-scale production. Therefore, soluble expression is a more ideal production method. To improve the soluble expression level in *E. coli*, a common strategy is to lower the culture temperature, thereby slowing down protein synthesis and allowing sufficient time for the protein to complete proper folding. To ensure effective expression by engineered bacteria under low-temperature conditions, we selected the pCold series expression vectors containing a cold shock promoter. The cold shock promoter is characterized by its ability to be fully activated only when the culture temperature drops below 25°C.

[0191] 4. ASFV protein purification

[0192] (1) Transfer the induced bacterial culture to a centrifuge tube, centrifuge at 4000 rpm for 15 min, collect the bacterial cells, and discard the supernatant;

[0193] (2) Resuspend the bacterial cells in PBS solution with an amount equivalent to 1 / 5 of the original bacterial solution;

[0194] (3) Centrifuge again at 4000 rpm for 15 min, collect the bacterial cells, and discard the supernatant;

[0195] (4) Add a 1 / 10 volume of lysis buffer to resuspend the bacterial cells;

[0196] (5) Disrupt the bacteria using ultrasound under ice bath protection. Set the ultrasound conditions to 50% intensity, sonicate for 2 seconds, with a 2-second interval, for a total of 20 minutes;

[0197] (6) Transfer the broken bacterial culture to a centrifuge tube and collect the supernatant at 10,000 rpm;

[0198] (7) Add an appropriate amount of 500mM imidazole solution to the supernatant to make the final imidazole concentration 30mM, and mix well.

[0199] (8) Filter the bacterial solution using a filter membrane with a pore size of 0.45 μm for later use;

[0200] (9) Equilibrate the Ni-NTA-Sepharose affinity chromatography column with at least 5 column volumes of equilibration buffer;

[0201] (10) Add the filtered bacterial protein supernatant to the chromatography column;

[0202] (11) After the sample has completely dried, rinse the chromatography column with equilibration buffer for at least 5 column volumes.

[0203] (12) Elute the protein with elution buffer and collect the elution buffer at 1 mL / tube. Elute with at least 3 column volumes (if using a chromatography instrument, monitor the 280 nm signal and collect the elution peak).

[0204] (13) For each collected eluent tube, the protein concentration was determined by the Bradford method, and the eluents with a protein content greater than 0.1 mg / mL were combined.

[0205] (14) Use an ultrafiltration concentrator with a molecular weight cutoff of 10 KaD to concentrate the protein solution to a concentration greater than 0.5 mg / mL.

[0206] SDS-PAGE analysis of 5ASFV protein

[0207] Collect 1 ml each of the uninduced bacterial culture and the IPTG-induced bacterial culture to prepare total bacterial protein for SDS-PAGE analysis. The method for preparing total bacterial protein is as follows:

[0208] (1) Take 1 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, and discard the supernatant;

[0209] (2) Add 0.2 mL of 2% SDS solution to the centrifuge tube to resuspend the bacteria, and then place the centrifuge tube in boiling water and heat for 5 min;

[0210] (3) Take out the sample and pipette it thoroughly until the sample is no longer viscous;

[0211] (4) Centrifuge at 12000 rpm for 5 min, and take 40 μL of supernatant and mix it with 10 μL of 5×SDS loading buffer;

[0212] (5) Place the centrifuge tube containing the sample in boiling water and heat for 5 minutes;

[0213] (6) Centrifuge at 12000 rpm for 5 min, and take the supernatant for SDS-PAGE analysis.

[0214] Determination of 6ASFV protein concentration

[0215] The BCA method was used to determine the protein standards and protein samples. The protein samples were set up in triplicate. The average absorbance (OD) values ​​were substituted into the linear regression equation of the standard curve plotted from the standards to calculate the concentrations of P30, P72, and PP220 proteins, facilitating subsequent animal experiments. The specific steps are as follows:

[0216] (1) Take a protein standard with a concentration of 5 mg / mL and dilute it to 0.5 mg / mL with PBS dilution buffer;

[0217] (2) Mix solution A and solution B thoroughly at a volume ratio of 50:1 to prepare an appropriate amount of BCA working solution (add 200 μL of working solution to each sample).

[0218] (3) The protein standards were serially diluted with PBS diluent, and the concentrations (μg / mL) after dilution were 500, 400, 300, 200, 100, 50, and 0, respectively.

[0219] (4) Add the protein standard and protein sample sequentially to the 96-well plate, 20 μL per well;

[0220] (5) Add 200 μL of the prepared BCA working solution and place it in a constant temperature oven at 37°C for 30 min;

[0221] (6) Measure the OD562nm of the corresponding well;

[0222] (7) Plot the regression equation of the standard curve by comparing the concentration of the standard with the measured OD value. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

[0223] III. Experimental Results

[0224] 1. Design the RP30, RP72, and RPP220 sequences.

[0225] (1) Sequence design of recombinant rp30 protein

[0226] The amino acid sequence of the P30 polypeptide:

[0227] QVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSAHIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSE EPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKNKLLSHLHLMFAPAPAPAPAPAPMEVIFKTDLRSSSQVVFHAG

[0228] Bioinformatics software for evaluating the properties of P30 peptides

[0229] Total amino acids: 215; Molecular weight: 24 kDa; Isoelectric point: 5.76; Hydrophilicity coefficient: -0.353; Instability coefficient: 61.66; This protein is estimated to have good hydrophilicity, and expression with only a 6×His tag can be attempted.

[0230] P30 nucleotide sequence:

[0231] CAAGTTGTTTTTCATGCTGGCAGCCTGTATAACTGGTTTAGCGTTGAAATTATTAATAGCGGCCGCATTGTGACGACGGCAATTAAAACGCTGCTGAGCACGGTCAAATATGATATCGTTAAAAGCGCACATATTTATGCAGGCCAAGGTTATACTGAACA TCAAGCTCAGGAAGAATGGAATATGATTCTGCATGTTCTGTTTGAAGAAGAAACTGAATCTAGCGCTAGCTCTGAAAATATTCATGAAAAGAATGATAATGAAACTAATGAATGCACGTCTAGCTTTGAAACCCTGTTTGAACAAGAACCGTCTAGCGAAG AACCTAAAGATTCTAAACTCTATATGCTGGCACAAAAAACGGTTCAGCATATTGAACAGTATGGTAAAGCTCCTGACTTTAATAAAGTGATCCGTGCGCATAATTTTATCCAAACCATTCATGGCACCCCTCTGAAAGAAGAAGAAAAAGAAGTTGTCCGT TTGATGGTAATTAAACTTCTGAAAAAGAATAAACTGTTGTCCCATCTGCATCTCATGTTTGCGCCGGCACCGGCACCAGCGCCGGGCCTGCACCGGCTCCAATGGAAGTCATTTTTAAAACGGATCTGCGCTCTTCTTCTCAGGTCGTGTTCCACGCAGGA

[0232] Design the P30-pCold2 carrier spectrum (e.g.) Figure 8 )

[0233] (2) Sequence design of P72 recombinant peptide

[0234] The amino acid sequence of the P72 polypeptide

[0235] YCEYPGERLYENVRFDVNGNSLDEYSSDVTTLLQNGSAVLRYSTAPAPAPAPAPAPQKDLVNEFPGLFIRQSRFIPGRPSRRNIRFKPYVPVGFEY

[0236] Bioinformatics software for evaluating the properties of P72 peptides

[0237] Total amino acids: 98; Molecular weight: 11 kDa; Isoelectric point: 8.07; Hydrophilicity coefficient: -0.527; Instability coefficient: 68.41; This protein has a very small molecular weight, and the evaluation results show that it is relatively unstable in vivo and is easily degraded when expressed alone. Therefore, GST tag fusion expression is considered.

[0238] The amino acid sequence after P72 is fused with the His-GST tag:

[0239] MNHKVHHHHHHMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSGSENLYFQGKLYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLLQNGSAVLRYSTAPAPAPAPAPAPQKDLVNEFPGLFIRQSRFIPGRPSRRNIRFKPYVPVGFEY

[0240] Bioinformatics software was used to evaluate the properties of the GST-P72 fusion peptide.

[0241] Total amino acids: 342; Molecular weight: 39.5 kDa; Isoelectric point: 6.68; Hydrophilicity coefficient: -0.485; Instability coefficient: 44.37

[0242] The fused GST-P72 nucleotide sequence:

[0243]

[0244] Design the P72-GST-pCold6 vector spectrum (e.g.) Figure 9 )

[0245] (3) Sequence design of PP220 recombinant peptide

[0246] The amino acid sequence of the P220 polypeptide:

[0247] MSRIFRGDNALNMGRPKFLSDQIFNKVAPAPAPAPAPAPDNAPAGHYYYDSCSRLLQIIDFYTDIVQKKYGGGEDCECTRV

[0248] Bioinformatics software was used to evaluate the properties of the P220 peptide.

[0249] Total amino acids: 83; Molecular weight: 9 kDa; Isoelectric point: 6.52; Hydrophilicity coefficient: -0.414; Instability coefficient: 91.7; This protein has a very small molecular weight, and the evaluation results show that it is extremely unstable in vivo and is easily degraded when expressed alone. Therefore, GST tag fusion expression is considered.

[0250] The amino acid sequence after P220 is fused with the His-GST tag:

[0251] MNHKVHHHHHHMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPD FMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSGSENLYFQGKLMSRIFRGDNALNMGRPKFLSDQIFNKVAPAPAPAPAPAPDNAPAGHYYYDSCSRLLQIIDFYTDIVQKKYGGGEDCECTRV

[0252] Bioinformatics software was used to evaluate the properties of the GST-P220 fusion peptide.

[0253] Total amino acids: 327; Molecular weight: 37.6 kDa; Isoelectric point: 6.53; Hydrophilicity coefficient: -0.455; Instability coefficient: 49.17

[0254] The fused GST-P220 nucleotide sequence:

[0255] ATGAATCACAAAGTGCATCATCATCATCATCATATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATGGTTCAGGATCCGAAAACCTGTATTTCCAGGGCAAGCTTATGTCTCGTATTTTTCGTGGCGATAATGCCTTGAATATGGGCCGCCCGAAATTTCTGAGCGATCAGATTTTTAATAAAGTCGCACCGGCTCCAGCGCCGGCCCCGGCTCCGGCACCGGCTCCTGATAACGCACCGGCAGGCCATTATTATTATGATAGCTGCAGCCGCCTGCTCCAAATCATTGATTTTTATACGGATATCGTTCAGAAAAAATATGGTGGCGGCGAAGATTGTGAATGTACGCGCGTGTAA

[0256] Design the spectrum of the PP220-GST-pCold6 vector (e.g.) Figure 10 )

[0257] 2. Construction of ASFV expression vector

[0258] (1) Amplification and cloning of the target gene

[0259] like Figure 11 As shown, the three target genes were amplified, with P30 expected to be 645 bp, P72 expected to be 294 bp, and PP220 expected to be 249 bp. The sizes of the amplified bands for all three target genes were consistent with the expectations. Each of the three bands was cut out with a scalpel and then recovered using a gel extraction kit.

[0260] (2) Ligation transformation and colony PCR verification

[0261] The recovered target fragments were used, with the P30 amplified fragment directly undergoing homologous recombination with the NdeI-linearized pCold2 vector. The P72 and PP220 amplified fragments were digested with HindIII and XbaI, and then ligated with the GST-pCold6 vector, which was also digested with HindIII / XbaI. The homologous recombination products and ligation products were transformed into Top10 competent cells. On day 2, four colonies were picked from each plate for PCR verification. The forward primer used in PCR had its binding site at the 5' end of the target gene, and the reverse primer had its binding site downstream of the XbaI restriction site on the vector. Therefore, theoretically, only correctly ligated recombinant clones could amplify the expected band. The expected amplified fragment size of the P30-pCold2 recombinant colony was 788 bp, the P72-GST-pCold6 recombinant colony was 407 bp, and the P220-GST-pCold6 recombinant colony was 352 bp.

[0262] Actual amplification results are as follows Figure 12 As shown, the amplified fragment sizes of the four recombinant colonies of P30-pCold2 and P72-GST-pCold6 all met expectations. Of the four recombinant colonies of P220-GST-pCold6, three were as expected, while the other clone failed to amplify a bright band, indicating that this clone may be a false positive.

[0263] (3) Sequencing verification

[0264] One PCR-verified colony was randomly selected from each of three plates and inoculated into LB broth containing Amp+. The cultures were incubated overnight at 37°C with shaking at 200 rpm. Plasmid extraction was then performed, and the extracted plasmids were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were then compared with the designed p30, p72, and pp220 sequences. The alignment results are as follows: Figure 13 As shown, this indicates that the target gene sequence in the introduced plasmid is correct. The correctly identified recombinant bacteria were named: E.coli-p30, E.coli-p72, and E.coli-pp220, respectively.

[0265] 3. Induction and purification of recombinant proteins rp30, rp72, and rpp220

[0266] The engineered bacteria, verified by sequencing, were inoculated into cells containing Amp. + The cultured bacterial suspension was cultured in LB liquid medium. After IPTG induction, the bacterial suspension was further purified by affinity chromatography using a Ni-NTA gravity column. During the chromatographic loading, rinsing, and elution processes, the OD280 absorbance was measured once for every 1 mL of flow-through. A chromatogram was then plotted with the tube number as the x-axis and the OD280 value as the y-axis, as shown in the figure. Figure 14 As shown.

[0267] 4. SDS-PAGE purity analysis of rp30, rp72, and rpp220 proteins

[0268] Three engineered bacterial strains, P30-pCold2-TOP10, P72-GST-pCold6-TOP10, and P220-GST-pCold6-TOP10, were cultured, and uninduced samples (N), IPTG-induced samples (I), and purified samples (P) were collected for SDS-PAGE analysis. The results are as follows: Figure 15 As shown, there was no significant difference between the IPTG-induced samples and the uninduced samples. This is because the target protein expression level was low, making it difficult to stand out against a noisy background. However, after further affinity purification with Ni-NTA-Sepharose, the purity of the obtained protein reached approximately 90%.

[0269] 5. Determination of Rp30, Rp72, and Rpp220 protein content

[0270] The levels of rp30, rp72, and rpp220 proteins were detected using the BCA protein quantification kit. A standard curve was established according to the kit instructions. The results are as follows: Figure 16 As shown, R 2=0.9949, indicating a good linear relationship among the concentrations on the standard curve. Based on the standard curve and the OD562nm values ​​of the samples, the corresponding protein concentrations were calculated. The calculated concentrations were: rp30 protein 0.8 mg / mL, rp72 protein 1.2 mg / mL, and rpp220 protein 1.0 mg / mL.

[0271] Example 3: Immunological effect of recombinant Bacillus subtilis expressing ASFV protein

[0272] I. Experimental Materials

[0273] 1. Strains and laboratory animals

[0274] The recombinant *B. subtilis*-CI2MKASFV strain was constructed and preserved in this study. SPF-grade female pigs aged 2–4 months were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. During the experiment, the pigs were housed at the Animal Experiment Center of Yan'an University, with an environment maintained at 24±2℃ and 60%±10% humidity, a 12h / 12h diurnal cycle, and provided with standard feed and water, allowing free movement. All experiments followed the *Guidelines for the Care and Use of Laboratory Animals* and complied with animal ethics requirements.

[0275] 2. Main Instruments

[0276] Electronic balance, Shanghai Zhuojing Electronic Technology Co., Ltd.; Laboratory pure water system, Shanghai Hetai Instrument Co., Ltd.; Constant temperature incubator vibrator, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; High-speed refrigerated centrifuge, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.; Low-speed centrifuge, Keda Innovation Co., Ltd. Zhongjia Branch; -80℃ refrigerator, Qingdao Haier Special Freezer Co., Ltd.

[0277] II. Experimental Methods

[0278] 1. Preparation of oral liquid

[0279] The recombinant *B. subtilis*-CI2MKASFV strain, stored at -80℃, was streaked onto LB agar plates containing SpE+ and incubated overnight at 37℃ for 12 h. Six to ten single colonies were then inoculated into 100 mL of LB liquid medium containing SpE+ and cultured at 37℃ and 200 rpm until the logarithmic growth phase. The culture was then transferred at a 10% ratio to 100 mL of fresh DSM-2 medium for spore induction. After 72 h, the bacterial cells were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed twice with sterile PBS and resuspended in 10 mL of PBS. Lysozyme was added to a final concentration of 2 mg / mL, and the mixture was incubated in a 37℃ water bath for 2 h to destroy the vegetative cells. The suspension was then centrifuged at 4000 rpm for 10 min to obtain spore pellets. The spore pellets were washed twice with PBS, and the spore concentration was adjusted to 1 × 10¹⁰ CFU / mL. The pellets were stored at 4℃ for later use.

[0280] 2. Animal grouping, immunization, and sample collection

[0281] Ten female pigs aged 2–4 months were randomly divided into two groups of five each: a control group (PBS group) and an immunization group (B. subtilis-CI2MKASFV group). After one week of acclimatization, pigs were immunized by gavage with either PBS or recombinant B. subtilis-CI2MKASFV spores, at a dose of 1 mL per pig. Immunization was administered twice, once daily for five consecutive days, from days 1–5 and 19–24. During the experiment, the pigs' mental state, coat luster, feed intake, and excrement were observed, and their body weight was measured daily. Serum samples were collected on days 0, 14, 21, 28, 35, 42, and 49. Samples were collected during the final serum collection. Pigs were weighed, injected intraperitoneally with 1% sodium pentobarbital, their beards were trimmed, and the skin around the eyes was disinfected. The eyeballs were quickly removed using curved forceps, and blood was collected in a coagulation tube while suspended in the air. The tubes were centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected and stored at -80°C. After blood collection, the pigs were quickly euthanized, and the spleens of each group were removed on ice. Residual blood was blotted dry with filter paper, and the spleens were weighed for subsequent experiments.

[0282] 3. Detection of serum IgG antibody levels

[0283] The level of specific IgG antibodies in porcine serum was detected using an indirect ELISA method. 96-well plates were coated with the prepared rp30, rp72, and rpp220 antigens. The specific steps are as follows:

[0284] (1) Sample grouping

[0285] Blank control group: 5 parallel wells were prepared using 0.5% skim milk solution instead of serum samples. This set of test data was used to determine the detection threshold (Cut-off value).

[0286] PBS group: Three serum samples from unimmunized pigs were collected at 0, 14, 21, 28, 35, 42 and 49 days for independent testing, with each sample being tested in triplicate.

[0287] B. subtilis-CI2MKASFV group: Three serum samples were collected from pigs immunized on days 0, 14, 21, 28, 35, 42, and 49, and tested independently. Each sample was replicated three times.

[0288] (2) Testing process

[0289] Add 100 μL of 5 μg / mL recombinant rp30, rp72, and rpp220 antigen proteins to each well of a microplate and coat the plate overnight at 4°C. Discard the antigen solution in the microplate and wash the plate three times with PBST solution.

[0290] Add 150 μL of 5% skim milk solution to each well and seal at room temperature for 1 hour;

[0291] Discard the blocking solution in the microplate and wash the plate three times with PBST solution.

[0292] The serum samples to be tested were serially diluted with 0.5% skim milk powder solution, with the first well being diluted 200 times, followed by serial dilutions of 2 times, for a total of 7 gradients (dilutions from 200 to 12800 times); 100 μL of the serially diluted serum samples to be tested were added to microplates and incubated at room temperature for 1 h.

[0293] Discard the sample solution in the microplate and wash the plate three times with PBST solution.

[0294] Add 100 μL of HRP-labeled goat anti-pig secondary antibody to each well and allow it to bind at room temperature for 1 h;

[0295] Discard the secondary antibody solution in the microplate and wash the plate 5 times with PBST solution;

[0296] Add 100 μL of TMB colorimetric solution to each well and develop color at room temperature in the dark for 15 min; then add 100 μL of 2M sulfuric acid solution to each well to terminate the reaction.

[0297] Use an ELISA reader to read the OD450 value of the sample and save the results.

[0298] (3) Antibody titer calculation

[0299] Determine the detection threshold: Calculate the mean and standard deviation of OD450 for the five parallel wells of the blank control group; use the mean plus five times the standard deviation as the detection threshold; and use an OD450 value ≥ the mean plus five times the standard deviation of the blank control as the effective titer.

[0300] The antibody titer of each serum sample is determined by serial dilution. The OD450 value of each sample gradually decreases after serial dilution. The maximum dilution factor with an OD450 value greater than the detection threshold is the antibody titer of that sample.

[0301] 4. Spleen index test

[0302] Spleen index was calculated as follows: Spleen index = Spleen weight (mg) / Pig body weight (g) × 10. Spleen index data for both groups of pigs were expressed as mean ± standard deviation (mean ± SD) and statistical analysis was performed using an independent samples t-test in SPSS 26.0.

[0303] 5. Detection of cytokine expression levels

[0304] The levels of IL-2 and IFN-γ in the serum of immunized pigs were detected according to the kit instructions.

[0305] 6. Data processing and analysis (independent samples t-test)

[0306] Data processing and analysis in this study were performed using SPSS 26.0 software, and graphs were generated using Origin 2024 software. All in vitro and in vivo experiments were conducted in triplicate. A p-value < 0.05 was considered statistically significant. Different asterisks (*, **, **, ) represent statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001, respectively.

[0307] III. Experimental Results

[0308] 1. Weight monitoring of pigs during the immunization period

[0309] Throughout the immunization period, the pigs' post-immunization status and body weight were monitored daily. Compared with the PBS group, the B. subtilis-CI2MKASFV group showed steady weight gain without any downward trend. During the experiment, the pigs were in good spirits, and no adverse reactions such as disheveled coats or diarrhea were observed. This indicates that recombinant B. subtilis-CI2MKASFV has good immunization safety in pigs.

[0310] 2. Detection of specific IgG antibody levels

[0311] Serum IgG antibody levels are a key indicator for evaluating vaccine immunization efficacy. To assess the immunogenicity of recombinant B. subtilis-CI2MKASFV, we orally immunized pigs with B. subtilis-CI2MKASFV, using a PBS group as a control. ELISA was used to detect serum-specific IgG antibody levels. The results are as follows: Figure 17 As shown, the serum levels of specific IgG antibodies in the B. subtilis-CI2MKASFV group increased from day 14, showing a significant difference compared to the PBS group (**p<0.01). p30 specific IgG antibodies peaked on day 35, reaching a maximum titer of 10159, and then began to decline. p72 and pp220 specific IgG antibodies peaked on day 28, showing extremely significant differences compared to the PBS group (***p<0.001), reaching a maximum titer of 6400, and then began to decline. This indicates that recombinant B. subtilis-CI2MKASFV can effectively induce a whole-body humoral immune response in pigs.

[0312] 3. Spleen index test

[0313] After immunization, spleens were randomly harvested from pigs, and spleen indices were analyzed in both groups. The results are as follows: Figure 18 As shown, compared with the PBS group, the spleen index of the B. subtilis-CI2MKASFV group was increased, and the difference was statistically significant (p<0.05), indicating that oral administration of B. subtilis-CI2MKASFV spores can stimulate an immune response in pigs.

[0314] 4. Detection of cytokine expression levels

[0315] Figure 19 The standard curves and linear fitting equations for IL-2 and IFN-γ are shown. The serum IL-2 and IFN-γ levels in each group of pigs after immunization are as follows: Figure 20 As shown, compared with the PBS group, the serum IL-2 and IFN-γ expression levels in the B. subtilis-CI2MKASFV group were increased, and the difference was statistically significant (*p<0.05, **p<0.01), indicating that recombinant B. subtilis-CI2MKASFV may induce cellular immunity. The specific mechanism of action needs further in-depth research.

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