Use of virus in preparation of phage

Abstract

Provided in the present application is use of a virus in the preparation of a phage, pertaining to the technical field of phages. Provided in the present application is use of PCV2, phPCV2, PCV3, phPCV3, HEV, or phHEV in the preparation of a phage. The present application also prepares and obtains a RecA-deficient Bacillus subtilis live vector vaccine.

Description

Application of a virus in the preparation of bacteriophages

[0001] This application claims priority to Chinese Patent Application No. 202411806951.8, filed on December 9, 2024, entitled "Application of a Virus in the Preparation of Bacteriophages", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of phage technology, specifically relating to the application of a virus in the preparation of phages. Background Technology

[0003] Currently, antibiotic resistance makes many bacterial diseases difficult to treat. In livestock and poultry farming, gradually reducing reliance on antibiotics and replacing them with other effective antibacterial agents can help reduce the development of bacterial resistance and protect public health. Phage therapy offers a novel solution. However, the high host specificity of bacteriophages severely limits their infectious range, necessitating the development of bacteriophages with broad-spectrum bactericidal capabilities.

[0004] Porcine circovirus type 2 (PCV2) is a major pathogen causing various symptoms in piglets, including post-weaning multisystemic wasting syndrome, dermatitis and nephropathy syndrome, reproductive disorders, enteritis, and respiratory diseases, resulting in significant economic losses to the pig industry. PK-15 cells are the primary cell line for PCV2 propagation; however, the permissibility of this cell line for PCV2 infection is limited, with only about 20% of the cell population susceptible, and viral titers rarely exceeding 10. 5 TCID 50 / ml. Porcine circovirus type 3 (PCV3) infection in pigs can cause a variety of clinical manifestations, including slow growth, respiratory distress, and diarrhea. Currently, there is no known method for mass propagation of PCV3 virus. Hepatitis E virus (HEV) is a zoonotic disease with several subtypes. HEV-1 and HEV-2 primarily circulate in humans, while HEV-3 and HEV-4 can circulate in animals and infect humans, with the highest positive infection rate in pigs. Infection in humans can lead to acute hepatitis, with symptoms including fatigue, loss of appetite, jaundice, fever, and upper abdominal discomfort. Pregnant women infected with HEV may face a higher risk of complications and death. Currently, there are no effective methods for isolation and culture. Isolation and propagation of PCV2, PCV3, and HEV viruses are crucial for studying their biological characteristics, developing vaccines, and controlling the disease. Currently, apart from PCV2, which has a relatively mature subunit vaccine, there are no effective vaccines for PCV3 and HEV viruses.

[0005] Previous researchers believed that eukaryotic viruses and prokaryotic viruses had strong host specificity, and that their origins and evolutionary pathways were relatively independent. Eukaryotic viruses cannot infect bacteria like bacteriophages, and it is even more impossible to isolate and culture eukaryotic viruses through bacteria. Summary of the Invention

[0006] In view of this, the purpose of this application is to provide an application of a virus in the preparation of bacteriophages.

[0007] To achieve the above-mentioned objectives, this application provides the following technical solutions:

[0008] This application provides the use of a virus in the preparation of bacteriophages, the virus including porcine circovirus and / or hepatitis E virus, the porcine circovirus including PCV2, phPCV2, PCV3 or phPCV3; the hepatitis E virus including HEV or phHEV.

[0009] This application also provides a bacteriophage bactericide, which includes at least one of PCV2, phPCV2, PCV3, phPCV3, HEV, and phHEV.

[0010] This application also provides a method for culturing viruses, wherein the virus infects bacteria and then the bacteria are cultured; the virus includes PCV2, phPCV2, PCV3, phPCV3, HEV, or phHEV.

[0011] This application also provides the application of the virus obtained by the above method in the preparation of vaccines.

[0012] This application also provides an inactivated vaccine comprising an inactivated virus obtained by the above method.

[0013] This application also provides a live vector vaccine comprising virus-infected RecA-deficient Bacillus subtilis, wherein the virus-infected RecA-deficient Bacillus subtilis is obtained by infecting RecA-deficient Bacillus subtilis with the virus obtained by the above method. Attached Figure Description

[0014] Figure 1 shows the nucleic acid extraction results of bacteria isolated and purified from feces. From top to bottom, the results are for phPCV2, phPCV3, and phHEV-3. In the phPCV2 results, lanes 1-12 contained *Kurstia*, *Streptococcus lactis*, *Escherichia coli*, *Escherichia fentanyl*, *Citrobacter*, *Enterobacter homonas*, *Klebsiella pneumoniae*, *Bacillus subtilis*, *Staphylococcus*, *Proteus mirabilis*, *Klebsiella pneumoniae*, and *Enterococcus*. In the phPCV3 results, lanes 1-8 contained *Escherichia coli*, *Kurstia*, *Streptococcus lactis*, *Escherichia fentanyl*, *Citrobacter*, and *Citrobacter*. The results showed that the bacteria in lanes 1-11 of the PhHEV-3 assay were Escherichia coli 1, Escherichia coli 2, Escherichia coli 3 (E. coli 1-E. coli 3 represent different E. coli isolated from different positive pigs), Curtaella foenum-graecum, Leukemia ulmoides, Staphylococcus aureus, Enterococcus 1, Enterococcus 2, Enterococcus 3, Bacillus subtilis, and Clostridium difficile (Enterococcus 1-Enterococcus 3 represent different Enterococcus isolated from different positive pigs); negative results were nucleic acids extracted from negative serum, and positive results were nucleic acids extracted from positive serum of various viruses.

[0015] Figure 2 shows the results of the experiment on the inhibitory effect of blood agar plates on wild-type bacterial flora isolated from pig feces infected with phPCV2, phPCV3, phHEV-3, phHEV-2 and phHEV-4.

[0016] Figure 3 shows the effects of PCV2, PCV3 and HEV-3 derived from eukaryotic cell propagation and phPCV2, phPCV3 and phHEV-3 derived from bacterial amplification on the bacterial growth curves of Escherichia coli (BL21(DE3)) and Bacillus subtilis (WB800N), respectively.

[0017] Figure 4 shows transmission electron microscopy observations of BL21(DE3) Escherichia coli adsorbed by phPCV2, phPCV3 and phHEV-3.

[0018] Figure 5 shows the release of viruses from phPCV2, phPCV3, and phHEV-3 ruptured BL21(DE3) Escherichia coli observed by transmission electron microscopy.

[0019] Figure 6 shows the phPCV2, phPCV3 and phHEV-3 viral particles after negative staining following PEG8000 precipitation, observed by transmission electron microscopy.

[0020] Figure 7 shows the changes in the serum specific IgG antibody levels of weaned piglets after vaccination with inactivated phPCV2, phPCV3 and phHEV-3 viruses prepared by propagation and concentration of BL21(DE3) Escherichia coli.

[0021] Figure 8 shows the viral copy number levels in the serum of weaned piglets in the control group and the inactivated vaccine immunization group during the protection experiments against phPCV2, phPCV3 and phHEV-3 challenges.

[0022] Figure 9 shows the viral copy number levels in the intestines, liver, and spleen of weaned piglets in the control group and the inactivated vaccine immunization group 8 days after challenge protection experiments with phPCV2, phPCV3, and phHEV-3.

[0023] Figure 10 shows the validation results of RecA-deficient Bacillus subtilis protein expression in WB800N;

[0024] Figure 11 shows the identification results of phPCV2, phPCV3 and phHEV-3 in the lysogenic state after infection with RecA-deficient Bacillus subtilis WB800N;

[0025] Figure 12 shows the changes in the serum specific IgG antibody levels of weaned piglets in the wild mushroom feeding group and the phPCV2, phPCV3 and phHEV-3 live vector vaccine feeding groups.

[0026] Figure 13 shows the viral copy number levels in the serum of weaned piglets in the wild-mushroom-fed group and the phPCV2, phPCV3, and phHEV-3 live vector vaccine-fed group during the challenge protection test of phPCV2, phPCV3, and phHEV-3.

[0027] Figure 14 shows the viral copy number levels in the intestine, liver, and spleen of weaned piglets in the wild-mushroom feeding group and the phPCV2, phPCV3, and phHEV-3 live vector vaccine feeding group after 8 days of challenge protection experiment with phPCV2, phPCV3, and phHEV-3.

[0028] Figure 15 shows the IgA secretion levels in the small intestinal mucosal rinsing fluid of weaned piglets 8 days after challenge protection experiments with wild-mushrooms and live vector vaccines (phPCV2, phPCV3, and phHEV-3). Detailed Implementation

[0029] This application provides the use of a virus in the preparation of bacteriophages, the virus including porcine circovirus and / or hepatitis E virus; the porcine circovirus including PCV2, phPCV2, PCV3 or phPCV3.

[0030] In some embodiments of this application, the hepatitis E virus includes HEV or phHEV. In one embodiment of this application, the HEV includes HEV-2, HEV-3, or HEV-4; the phHEV includes phHEV-2, phHEV-3, or phHEV-4.

[0031] In one embodiment of this application, the nucleotide sequence of phPCV2 is shown in SEQ ID NO.1, the nucleotide sequence of phPCV3 is shown in SEQ ID NO.2, the nucleotide sequence of phHEV-3 is shown in SEQ ID NO.3, the nucleotide sequence of phHEV-2 is shown in SEQ ID NO.4, and the nucleotide sequence of phHEV-4 is shown in SEQ ID NO.5. In this application, ph in phPCV2, phPCV3, or phHEV-3 is a self-named designation used to represent PCV2, PCV3, or HEV obtained from bacterial division.

[0032] In this application, viruses from eukaryotic cells or organisms are represented by PCV2, PCV3, and HEV, and viruses from bacteria are represented by phPCV2, phPCV3, and phHEV.

[0033] This application is the first to propose that eukaryotic viruses PCV2, PCV3, and HEV can infect bacteria and replicate within them, exhibiting lysogenic phage characteristics. Bacterial viruses phPCV2, phPCV3, and phHEV also exhibit lysogenic phage characteristics. Furthermore, the eukaryotic viruses PCV2, PCV3, and HEV proposed in this application possess broad-spectrum infectivity against bacteria, effectively inhibiting bacterial reproduction, including Gram-positive and Gram-negative bacteria. This is groundbreaking for the development of broad-spectrum phage bactericides and for the development of vaccines using bacteria to infect viruses. Similarly, prokaryotically cultured viruses phPCV2, phPCV3, and phHEV also possess broad-spectrum bacterial infectivity, making them groundbreaking for the development of broad-spectrum phage bactericides and for the development of vaccines using bacteria to infect viruses.

[0034] This application also provides a bacteriophage bactericide, which includes at least one of PCV2, phPCV2, PCV3, phPCV3, HEV, and phHEV.

[0035] In some embodiments of this application, the HEV includes HEV-2, HEV-3, or HEV-4; the phHEV includes phHEV-2, phHEV-3, or phHEV-4. In one embodiment of this application, the nucleotide sequence of phPCV2 is shown in SEQ ID NO.1, the nucleotide sequence of phPCV3 is shown in SEQ ID NO.2, the nucleotide sequence of phHEV-3 is shown in SEQ ID NO.3, the nucleotide sequence of phHEV-2 is shown in SEQ ID NO.4, and the nucleotide sequence of phHEV-4 is shown in SEQ ID NO.5.

[0036] This application also provides a method for culturing viruses, wherein the virus infects bacteria and then the bacteria are cultured; the virus includes PCV2, phPCV2, PCV3, phPCV3, HEV, or phHEV.

[0037] In some embodiments of this application, the HEV includes HEV-2, HEV-3, or HEV-4; the phHEV includes phHEV-2, phHEV-3, or phHEV-4. In one embodiment of this application, the nucleotide sequence of phPCV2 is shown in SEQ ID NO.1, the nucleotide sequence of phPCV3 is shown in SEQ ID NO.2, the nucleotide sequence of phHEV-3 is shown in SEQ ID NO.3, the nucleotide sequence of phHEV-2 is shown in SEQ ID NO.4, and the nucleotide sequence of phHEV-4 is shown in SEQ ID NO.5.

[0038] In one embodiment of this application, the infection method involves inoculating the viral stock solution with bacteria, observing the virus adsorbing onto the bacteria, culturing the adsorbed bacteria, selecting bacteria in a good lysogenic state for propagation, induction, removal of bacterial precipitate, concentration, filtration, centrifugation to precipitate the virus, and collecting the virus. In some embodiments of this application, the bacteria include *Escherichia coli*, *Streptococcus lactis*, *Citrobacter*, *Bacillus subtilis*, *Proteus mirabilis*, *Enterococcus*, *Escherichia fergusonian*, *Shigella flexneri*, *Kurstia*, *Citrobacter* flexneri, *Lactobacillus*, *Enterobacter homunculus*, *Klebsiella pneumoniae*, *Lactobacillus*, *Salmonella*, or *Clostridium*. In one embodiment of this application, the nucleotide sequence of phPCV2 is shown in SEQ ID NO.1, the nucleotide sequence of phPCV3 is shown in SEQ ID NO.2, the nucleotide sequence of phHEV-3 is shown in SEQ ID NO.3, the nucleotide sequence of phHEV-2 is shown in SEQ ID NO.4, and the nucleotide sequence of phHEV-4 is shown in SEQ ID NO.5.

[0039] This application also provides the application of the virus obtained by the above method in the preparation of vaccines.

[0040] In one embodiment of this application, the vaccine includes an inactivated vaccine and a live vector vaccine.

[0041] This application also provides an inactivated vaccine comprising an inactivated virus obtained by the above method.

[0042] In one embodiment of this application, the inactivation is performed by co-culturing the virus with diethyleneimine (BEI). In another embodiment, the inactivated vaccine further includes an immune adjuvant. In one embodiment, the immune adjuvant is MONTANIDEIMISA 206VG adjuvant, and the weight ratio of the immune adjuvant to the inactivated virus is 1:1. This application utilizes the ability of *E. coli* BL21(DE3) to stably amplify phPCV2, phPCV3, and phHEV with high viral titers, thereby preparing an inactivated vaccine that exhibits significant protective effects against animals.

[0043] This application also provides a live vector vaccine comprising virus-infected RecA-deficient Bacillus subtilis, wherein the virus-infected RecA-deficient Bacillus subtilis is obtained by infecting RecA-deficient Bacillus subtilis with the virus obtained by the above method.

[0044] Lysogenic phages are a specific type of bacteriophage that, after infecting bacteria, does not immediately induce bacterial lysis but instead integrates its genetic material into the host bacterial genome. This latent state is called the "lysogenic state." The transition from the lysogenic state to lysis and the release of viral particles depends entirely on RecA activation, which in turn regulates the hydrolysis of LexA to release viral replication-related genes and complete the viral replication cycle. However, RecA-deficient bacteria will prevent viral lysis, only exhibiting a lysogenic state. Bacteria in this state colonize the gut, and the viral genes multiply along with the bacteria. Furthermore, the virus continuously expresses proteins that maintain the lysogenic state. When the bacteria die, they release viral antigens into the intestinal mucosa, thereby stimulating further mucosal immunity. In terms of bacterial selection, Salmonella, Escherichia coli, Lactobacillus, and Bacillus subtilis are widely used as bacterial vectors for animal disease control. After screening, Bacillus subtilis shows significantly better viral tropism and gastric acid and bile salt tolerance than other bacteria. Therefore, in one embodiment of this application, Bacillus subtilis is used to prepare the bacterial vector.

[0045] In one embodiment of this application, the infection method involves co-culturing the successfully constructed RecA-deficient Bacillus subtilis with the virus obtained by the above-described virus culture method. After adsorption, positive colonies are screened and identified. In one embodiment of this application, the live vector vaccine further includes an adjuvant. In one embodiment of this application, the adjuvant further includes trehalose. Due to the hygroscopic nature of trehalose, bacteria are adsorbed onto the trehalose and fully encapsulated, effectively resisting adverse environments such as gastric acid and intestinal fluid, allowing the bacterial solution to smoothly enter the intestines after oral administration.

[0046] This application describes the preparation of a RecA-deficient Bacillus subtilis live vector vaccine. Utilizing a lysogen-phage-dependent RecA-LexA-regulated SOS response mechanism, RecA-deficient Bacillus subtilis was constructed, maintaining a long-term stable lysogenic state during viral infection. After oral inoculation into animals, the vaccine colonizes the intestines and continuously expresses the antigen, inducing a mucosal immune response. This vaccine offers multiple advantages, including convenient administration, safety and reliability, large-scale production capability, low cost, good disease control efficacy, and the ability to regulate the gut microbiota and improve overall host health.

[0047] The technical solutions provided in this application will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this application.

[0048] Unless otherwise specified, the following embodiments are all conventional methods.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] Example 1

[0051] Eukaryotic viruses PCV2, PCV3, and HEV were identified as lysogenic bacteriophages phPCV2, phPCV3, and phHEV.

[0052] 1.1 Detection of phPCV2, phPCV3 and phHEV in wild-type fungi

[0053] PCR amplification detection

[0054] Fecal samples from pigs infected with PCV2, PCV3, and HEV viruses were collected. The bacterial flora in the feces was isolated, purified, and cultured using blood agar plates. Single colonies were selected for propagation and cryopreservation. The propagated bacteria were then disrupted using an ultrasonic homogenizer. Nucleic acid was extracted from the disrupted bacterial culture using a viral total DNA / RNA extraction kit from Meiji Biotechnology Co., Ltd. The bacterial species were identified using 16S rRNA sequencing. Subsequently, the extracted nucleic acid was amplified by PCR to detect the phPCV2, phPCV3, and phHEV genes. The PCR amplification products were detected by 1% agarose gel electrophoresis, and the results are shown in Figure 1. Primer design is shown in Table 1, the PCR amplification reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0055] Table 1 Primer sequences

[0056] Table 2 PCR reaction system

[0057] Table 3 PCR amplification reaction procedure

[0058] The results are shown in Table 4. Viral nucleic acid was present in many different bacterial species, including Gram-negative bacteria represented by *Escherichia coli* and Gram-positive bacteria represented by *Bacillus*. Table 4 shows the detection of phPCV2, phPCV3, and three genotypes of HEV (phHEV-2, phHEV-3, and phHEV-4) in some strains. (Due to limited sample collection, the absence of detected strains does not necessarily mean they are not infected with the virus. The genotypes can be identified by sequencing and alignment after obtaining the sequences amplified using the phHEV primer sequences in Table 1.) The detection results indicate that phPCV2, phPCV3, and phHEV have a broad-spectrum ability to infect bacteria.

[0059] Table 4 shows some of the positive test results for the strains.

[0060] 1.2 Infection with bacteria by eukaryotic PCV2, PCV3, HEV, and prokaryotic phPCV2, phPCV3, and phHEV-3 all inhibit bacterial growth.

[0061] To verify the bactericidal effects of the three viruses in a field environment with bacteria, fresh pig feces were collected and diluted with sterile PBS to obtain the original bacterial solution. A control group (original bacterial solution, NC) and a virus infection group were set up. The virus infection group was treated with stock solutions of phPCV2, phPCV3, phHEV-2, phHEV-3, and phHEV-4 viruses with an MOI of 1, respectively. After adsorption at 4℃ for 1 h, the solutions were incubated in LB medium containing 0.1 μg / mL MMC for 8 h. 10 μL of the bacterial solution was then streaked onto blood agar plates to observe the number and types of colonies. The results are shown in Figure 2. After viral infection, all three viruses showed inhibitory effects on bacteria. phPCV2 and phHEV genotypes showed significant inhibitory effects on various bacteria. phPCV3 showed inhibition of some bacterial growth, while phHEV-3 showed complete inhibition. phHEV-3 was subsequently used as the representative HEV strain for further experiments.

[0062] To further verify whether infection with eukaryotic PCV2, PCV3, and HEV-3, as well as prokaryotic phPCV2, phPCV3, and phHEV-3, affects bacterial growth, three viruses were selected for viral infection experiments: one of the Gram-negative bacteria Escherichia coli (BL21(DE3)) and the other of the Gram-positive bacteria Bacillus subtilis (WB800N). (The viruses were isolated from Escherichia coli BL21(DE3) and Bacillus subtilis WB800N.) PCV2, phPCV2 (SEQ ID NO.1), PCV3, phPCV3 (SEQ ID NO.2), HEV-3, and phHEV-3 (SEQ ID NO.3) were used to infect Escherichia coli (BL21(DE3)) and Bacillus subtilis (WB800N), respectively. After adsorption at 4°C for 1 hour using a virus dose of MOI=1, the bacterial cells were washed three times with sterile PBS and added to LB medium containing 0.1 μg / mL mitomycin C (MMC, lysogen phage lysis inducer). At the same time, a control group of bacteria with the same number of bacteria was cultured under the same conditions. Bacterial cultures were collected at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 20h, and the bacterial count was measured using a spectrophotometer at an OD wavelength of 600nm. The results are shown in Figure 3. BL21(DE3) *E. coli* and WB800N *Bacillus subtilis* infected with phPCV2, phPCV3, and phHEV-3 all showed growth inhibition. Furthermore, bacteria in the HEV-3-infected group began to die 8h after infection. These results indicate that infection with eukaryotic PCV2, PCV3, HEV-3, and prokaryotic phPCV2, phPCV3, and phHEV-3 inhibits bacterial growth and reproduction and induces bacterial death.

[0063] Example 2

[0064] Inactivated vaccine prepared by culturing viruses from BL21(DE3) Escherichia coli

[0065] 1.1 Preparation and screening of lysogenic bacteria

[0066] (1) Resuscitate the bacterial strain: Add BL21(DE3) Escherichia coli strain to 200 mL of LB medium and incubate at 37°C for 1 h.

[0067] (2) Spreading: Spread the revived bacterial culture onto a plate, invert it in a 37℃ constant temperature incubator, and after 12 hours, pick out a single colony for propagation.

[0068] (3) Infection: Virus stock solutions of phPCV2 (SEQ ID NO.1), phPCV3 (SEQ ID NO.2), and phHEV-3 (SEQ ID NO.3) with an MOI of 1 were inoculated into bacteria. After adsorption at 4°C for 1 hour, the bacteria were sectioned using ultrasound, and the virus adsorption was observed by transmission electron microscopy, as shown in Figure 4. Subsequently, the adsorbed bacteria were added to LB medium and cultured at 16°C for 12 hours.

[0069] (4) Bacterial identification: Subsequently, bacteria infected with the virus were plated, single colonies were selected and cultured for propagation, and then a portion of the bacterial solution was ultrasonically broken to extract bacterial nucleic acid. PCR was performed according to the method in Example 1 to verify whether the virus was lysed in the bacteria. Bacteria in good lysed state were selected for propagation.

[0070] 1.2 Propagation and Induction of Lysogenic Bacteria

[0071] (1) Propagation: Select bacteria in good lysogenic state and inoculate them into 200 mL of sterile LB culture medium and culture them at 16℃ and 60 r / min for 24-48 h.

[0072] (2) Induction: When bacterial OD 600 When the absorbance value reached approximately 1, 1 μg / mL of MMC was added to the bacterial culture for induction, and the culture was continued at 16℃ and 60 r / min for 24 h. At this point, ultrathin sections of the bacteria were prepared, and the entire process of bacterial lysis and viral release was observed using transmission electron microscopy. The results are shown in Figure 5.

[0073] 1.3 Virus Concentration

[0074] (1) Removal of bacterial precipitate: Pre-cool the centrifuge to 4°C, centrifuge the above culture medium at 10000r / min for 30min to remove bacterial precipitate, and store the supernatant.

[0075] (2) Filtration: The supernatant is filtered through a 0.22μm or 0.45μm filter membrane to further remove bacterial debris and other impurities.

[0076] (3) Add PEG8000: Add PEG8000 to the filtered supernatant at a concentration of 5%. Stir well to ensure the PEG is fully dissolved.

[0077] (4) Low temperature standing: Place the mixture at a low temperature (e.g., 4°C) for several hours to overnight to promote the precipitation of the virus.

[0078] (5) Centrifugation: Centrifuge at a high speed of 12000r / min for 30min at low temperature (4℃) to precipitate the virus.

[0079] (6) Remove the supernatant: Carefully remove the supernatant and retain the precipitate.

[0080] (7) Resuspending the virus: The precipitate was resuspended using sterile PBS to obtain a concentrated virus solution.

[0081] (8) Negative staining observation: Add an appropriate amount of virus sample to a copper grid, and then add 1% phosphotungstic acid to the sample. Allow the sample to react with the staining solution for about 1-5 minutes. Gently absorb excess negative staining agent with absorbent paper to avoid leaving excessive dye on the sample surface. Allow the sample to air dry at room temperature or dry it at low temperature for a few minutes. Place the dried sample in a transmission electron microscope and adjust the microscope to observe the virus particles. Typical virus particles can be observed, as shown in Figure 6.

[0082] 1.4 Preparation of inactivated vaccines

[0083] (1) Virus titer determination: Concentrated virus was inoculated into suitable cell lines (PK-15 cells for phPCV2 and phPCV3, and HepG2 cells for phHEV-3). The virus was serially diluted 10-fold. Different dilutions of virus were inoculated into suitable cell lines and cultured. After 72 hours, the culture medium was discarded, and the cells were fixed with 4% neutral paraformaldehyde. The cells were then subjected to penetration, blocking, primary antibody incubation, washing, fluorescent secondary antibody incubation, washing, and fluorescence microscopy to observe cell infection. The TCID of the virus was calculated based on the infection rate. 50 value.

[0084] (2) Purity test of virus solution used for vaccine preparation

[0085] According to the current appendix of the Chinese Veterinary Pharmacopoeia, the test results showed that the basic strain was free from bacterial, mycoplasma, and exogenous viral contamination.

[0086] (3) Inactivation of virus solution used for vaccine preparation

[0087] Mix 2 mol / L NaOH solution and 2 mol / L 2-bromoethylamine hydrobromide (BEA) in a 1:1 ratio, shake well, and place in a 37°C water bath. Shake well every 10-15 minutes during this process. After 1 hour, cyclization will produce diethyleneimine (BEI), with a final concentration of 1 mol / L. Take a 10% dilution... 5 TCID 50 The virus solution was inactivated by adding the prepared BEI solution to a concentration of 2 mmol / L, and then placed in a constant temperature shaker at 37℃ for 24 h at 120 rpm / min. Finally, sodium thiosulfate with a final concentration of 2 mmol / L was added to terminate the inactivation.

[0088] (4) Preparation of inactivated vaccines

[0089] The inactivated virus stock solution and Seppic's MONTANIDEIMISA 206VG adjuvant were preheated to 30°C and prepared at a weight ratio of 1:1. While stirring at low speed, the preheated virus solution was slowly and uniformly added to the isothermal MONTANIDEIMISA 206VG adjuvant. After completion, the stirring speed was quickly increased to 2000 r / min and stirred thoroughly for 10 min. The solution was then quantitatively dispensed, sealed, and stored at 4°C.

[0090] Through the above operations, inactivated vaccines phPCV2, phPCV3 and phHEV-3 derived from BL21(DE3) Escherichia coli were obtained.

[0091] 1.5 Detection of Immunoprotective Effect

[0092] To verify the immunoprotective efficacy of these three inactivated vaccines, an animal immunoprotective experiment was conducted. Thirty weaned piglets were purchased and randomly divided into six groups (phPCV2 control group, phPCV2 immunization group, phPCV3 control group, phPCV3 immunization group, phHEV-3 control group, and phHEV-3 immunization group). Pigs in different groups were intramuscularly injected with 2 mL of the prepared inactivated vaccine, while the control group was injected with 2 mL of PBS buffer. The first immunization was recorded as day one, and a second immunization was administered two weeks later. Blood samples were collected from the pigs during this period to detect antibody levels, and the results are shown in Figure 7. After immunization, the animals were observed for any adverse reactions. The results showed that the inactivated vaccine applied for was safe and did not cause any adverse symptoms in the animals. Two weeks after the second immunization, a challenge experiment was conducted. Blood samples were collected every two days after challenge to detect viremia, and the results are shown in Figure 8. Eight days later, the experimental animals were euthanized, and liver and intestinal samples were collected for testing, and the results are shown in Figure 9.

[0093] The results showed that vaccination with all three inactivated vaccines induced high levels of specific antibodies in pigs, reaching the highest level on day 10 after two vaccinations. The resulting antibodies provided good protection for pigs, significantly reducing the incidence of viremia and viral replication within the body.

[0094] Example 3

[0095] Live vector vaccines were prepared using RecA-deficient Bacillus subtilis in lysogenic states of phPCV2, phPCV3, and phHEV-3.

[0096] 1.1 Preparation of RecA-deficient Bacillus subtilis

[0097] (1) Recombinant arms and primers were designed at the 5' and 3' ends of the recA gene. The recombinant fragment RecA-loxp-kan was amplified by PCR. The primers are shown in Table 5. The fragment was constructed on the puc57 vector and sequenced to verify that the sequence was correct.

[0098] Table 5. Homologous arms, RecA-loxp-kan sequences, and primer sequences.

[0099] (2) The recA-loxp-kan recombinant template fragment was amplified by PCR using primers RecA-5HR-F1 and RecA-3HR-R1 and recovered by ethanol precipitation.

[0100] (3) The λRed recombinase plasmid pkd46 was transformed into Bacillus subtilis strain WB800N, clones were selected, and incubated at 30℃ until OD600=0.3. When an inducer was added, λRed recombinase expression was induced. The culture was continued until OD=0.5 to prepare electrotransfer competent cells WB800N-pkd46.

[0101] (4) Transfer an appropriate amount of the recombinant fragment recA-loxp-kan into WB800N-pkd46 (50 μL) under the conditions recommended by the electroporator, add 1 mL of antibiotic-free LB medium, and revive at 30℃ for 1 h.

[0102] (5) After 1 hour of recovery, centrifuge at 4500 rpm for 5 minutes to remove a large amount of supernatant. Gently resuspend the bacterial cells with the remaining supernatant and spread them evenly on Kans LB plates (the plates are pre-coated with an appropriate amount of inducer to induce λRed recombinase expression). Incubate at 30°C upside down for 24 hours.

[0103] (6) Screen positive clones, culture them in kan resistance, and name them recA-kan-WB800N.

[0104] (7) The recA-kan-WB800N was passaged at 42℃ in Kan resistance until Amp resistance disappeared, i.e., the pkd46 plasmid was lost.

[0105] (8) Prepare electrocompetent states from recA-kan-WB800N with lost plasmid.

[0106] (9) Transfer pkd46-Cre plasmid into recA-kan-WB800N (50 μL), add 1 mL of antibiotic-free LB medium, and revive at 30℃ for 30 min.

[0107] (10) After recovery, take 20 μL of the transformation product and spread it on an Amp LB plate (the plate is pre-coated with an appropriate amount of inducer to induce Cre enzyme expression), and incubate at 30°C upside down for 18 h.

[0108] (11) Select clones and identify positive results by PCR.

[0109] (12) RecA-del-WB800N was passaged at 42℃ in a non-resistant environment until the Amp resistance disappeared, i.e., the pkd46-cre plasmid was lost.

[0110] (13) Streaking to purify the bacterial strain.

[0111] (14) The purified strain was tested again for resistance to Amp, kan, etc. After confirming that there were no errors, the glycerol strain was retained.

[0112] (15) The protein expression status of RecA-deficient bacteria is shown in Figure 10. RecA expression is defective.

[0113] 1.2 Preparation of RecA-deficient Bacillus subtilis live vector vaccines in lysogenic states of phPCV2, phPCV3, and phHEV-3

[0114] (1) Infection: The successfully constructed RecA-deficient Bacillus subtilis WB800N was infected with viruses of phPCV2, phPCV3 and phHEV-3 with MOI=1. The virus was adsorbed at 4℃ for 1h, and then cultured in sterile LB medium with 1mL added at 16℃ and 60r / min for 24h.

[0115] (2) Identification of positive colonies: The streak plate method was used to streak on a solid culture plate, and a single colony was picked out for propagation. Positive colonies of phPCV2, phPCV3 and phHEV-3 were detected by PCR.

[0116] (3) Identification of viral lysogens: The three viruses in a lysogenic state exhibited only viral capsid protein expression within the bacteria, with viral replication protein expression being repressed. The viruses were unable to package live progeny virus particles, but viral nucleic acid replicated with bacterial division. Western blotting revealed that RecA-deficient Bacillus subtilis, after infection with the three viruses, only expressed the capsid protein, and its replication protein was not expressed. The results are shown in Figure 11.

[0117] (4) The successfully identified Bacillus subtilis was propagated, centrifuged, the supernatant was discarded, sterile PBS buffer was added, and after full resuspension, the same weight of trehalose was added and mixed 1:1 to prepare a vector live bacterial vaccine. Due to the water absorption of trehalose, the bacteria will be adsorbed on the trehalose and fully wrapped, which can effectively resist adverse environments such as gastric acid and intestinal fluid, so that the bacterial solution can be smoothly entered into the intestines by oral administration.

[0118] 1.3 Detection of the immunoprotective effect of live vector vaccines

[0119] To verify the immunoprotective effects of these three live vector vaccines, an animal immunoprotective experiment was conducted. Thirty weaned piglets were purchased and randomly divided into six groups (phPCV2 wild-type group, phPCV2 vector vaccine immunization group, phPCV3 wild-type group, phPCV3 vector vaccine immunization group, phHEV-3 wild-type group, and phHEV-3 vector vaccine immunization group). Equal amounts of different vector vaccines were thoroughly mixed with feed (5g of each different vector live-type vaccine or wild-type fungus was weighed and mixed thoroughly into 500g of feed). The mixture was fed to the corresponding groups of pigs. The first immunization was recorded as day one, and immunizations were performed every two days for a total of seven immunizations. Blood samples were collected from the pigs during this period to test antibody levels. The results are shown in Figure 12. One week after cessation of immunization, a feeding challenge experiment was conducted. Blood samples were collected every two days after challenge to test for viremia. The results are shown in Figure 13. Eight days later, the experimental animals were euthanized, and liver, intestine, and spleen samples were collected for testing. The results are shown in Figure 14. A segment of the colon without feces was taken and rinsed with PBS buffer. The rinsing solution was used to detect the intestinal mucosal immune level by IgA antibody detection. The results are shown in Figure 15.

[0120] The results showed that vaccination with the three live vector vaccines prepared in this application induced a sustained high level of specific antibodies in pigs. The generated antibodies provided good protection for pigs, significantly reducing the incidence of viremia and viral replication. Furthermore, as shown in Figure 15, this live vector vaccine also induced a high level of secretory IgA antibodies, greatly enhancing mucosal immunity.

[0121] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. Use of a virus in the manufacture of a bacteriophage, characterized in that, The viruses include porcine circovirus and / or hepatitis E virus; the porcine circovirus includes PCV2, phPCV2, PCV3 or phPCV3.

2. The application according to claim 1, characterized in that, The hepatitis E virus includes HEV or phHEV.

3. A bacteriophage bactericide, characterized in that, The bacteriophage bactericide includes at least one of PCV2, phPCV2, PCV3, phPCV3, HEV, and phHEV.

4. The bacteriophage bactericide according to claim 3, characterized in that, The HEV includes HEV-2, HEV-3, or HEV-4.

5. The bacteriophage bactericide according to claim 3, characterized in that, The phHEV includes phHEV-2, phHEV-3, or phHEV-4.

6. The bacteriophage bactericide according to claim 3, characterized in that, The nucleotide sequence of phPCV2 is shown in SEQ ID NO.

1.

7. The bacteriophage bactericide according to claim 3, characterized in that, The nucleotide sequence of phPCV3 is shown in SEQ ID NO.

2.

8. The bacteriophage bactericide according to claim 5, characterized in that, The nucleotide sequence of phHEV-3 is shown in SEQ ID NO.

3.

9. The bacteriophage bactericide according to claim 5, characterized in that, The nucleotide sequence of phHEV-2 is shown in SEQ ID NO.

4.

10. The bacteriophage bactericide according to claim 5, characterized in that, The nucleotide sequence of phHEV-4 is shown in SEQ ID NO.

5.

11. A method for culturing a virus, characterized in that, After the virus infects the bacteria, bacterial culture is performed; the virus includes PCV2, phPCV2, PCV3, phPCV3, HEV, or phHEV.

12. The method according to claim 11, characterized in that, The nucleotide sequence of phPCV2 is shown in SEQ ID NO.

1.

13. The method according to claim 11, characterized in that, The nucleotide sequence of phPCV3 is shown in SEQ ID NO.

2.

14. The method according to claim 11, characterized in that, The phHEV includes phHEV-2, phHEV-3, or phHEV-4; the nucleotide sequence of phHEV-3 is shown in SEQ ID NO.3; the nucleotide sequence of phHEV-2 is shown in SEQ ID NO.4; and the nucleotide sequence of phHEV-4 is shown in SEQ ID NO.

5.

15. The method according to claim 11, characterized in that, The bacteria include Escherichia coli, Streptococcus lactis, Citrobacter, Bacillus subtilis, Proteus mirabilis, Enterococcus, Escherichia fergusonii, Shigella flexneri, Korotchenko, Citrobacter, Citrobacter flexneri, Leukemia, Enterobacter hominis, Klebsiella pneumoniae, Staphylococcus, Lactococcus gasseri, Klebsiella pneumoniae, lactic acid bacteria, Salmonella, or Clostridium difficile.

16. The use of the virus obtained by the method according to any one of claims 11 to 15 in the preparation of a vaccine.

17. The application according to claim 16, characterized in that, The vaccines include inactivated vaccines and live vector vaccines.

18. An inactivated vaccine, characterized in that, This includes inactivated viruses obtained by the method described in any one of claims 11 to 15.

19. A live vector vaccine, characterized in that, Includes virus-infected RecA-deficient Bacillus subtilis, wherein the virus-infected RecA-deficient Bacillus subtilis is obtained by infecting RecA-deficient Bacillus subtilis with the virus obtained by the method of any one of claims 11 to 15.

20. The live vector vaccine according to claim 19, characterized in that, It also includes trehalose.

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