Use of a virus in the preparation of bacteriophage

Abstract

The application provides a virus in the preparation of bacteriophage, and belongs to the technical field of bacteriophage. The application provides PCV2, phPCV2, PCV3, phPCV3, HEV or phHEV in the preparation of bacteriophage. The application first proposes that eukaryotic viruses can infect bacteria and can replicate in the bacteria, and the viruses show the characteristics of lysogenic bacteriophage. The application has broad spectrum for the infection of the bacteria, can effectively inhibit the reproduction of the bacteria, and has a pioneering significance for developing broad-spectrum bacteriophage bactericides and developing vaccines by using the infection of the viruses on the bacteria. The application can stably amplify the viruses with high toxicity by using Escherichia coli, and the inactivated vaccines prepared by the viruses can significantly protect animals. The application also prepares a RecA-deficient Bacillus subtilis live vector vaccine, so that the virus infection is stably in a lysogenic state for a long time, and the animals are inoculated by oral administration, the animals are colonized in the intestinal tract, and the antigen is continuously expressed to induce a mucosal immune response.

Description

Technical Field

[0001] This invention belongs to the field of bacteriophage technology, specifically relating to the application of a virus in the preparation of bacteriophages. Background Technology

[0002] 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.

[0003] 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.

[0004] 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

[0005] In view of this, the purpose of the present invention is to provide an application of a virus in the preparation of bacteriophages.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of a virus in the preparation of bacteriophages, the virus including porcine circovirus and / or hepatitis E virus, wherein the porcine circovirus includes PCV2, phPCV2, PCV3 or phPCV3; and the hepatitis E virus includes HEV or phHEV.

[0008] In this invention, viruses from eukaryotic cells or organisms are represented by PCV2, PCV3, and HEV, and viruses from bacteria are represented by phPCV2, phPCV3, and phHEV.

[0009] The present invention also provides a bacteriophage bactericide, wherein the bacteriophage bactericide includes at least one of PCV2, phPCV2, PCV3, phPCV3, HEV and phHEV.

[0010] Preferably, the HEV includes HEV-2, HEV-3, or HEV-4; the phHEV includes phHEV-2, phHEV-3, or phHEV-4; 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.

[0011] The present invention 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.

[0012] Preferably, the bacteria include Escherichia coli, Streptococcus lactis, Citrobacter, Bacillus subtilis, Proteus mirabilis, Enterococcus, Escherichia fergusonii, Shigella flexneri, Kurtella, Citrobacter, Citrobacter flexneri, Leukemia, Enterobacter hominis, Klebsiella pneumoniae, Staphylococcus, Lactococcus gasseri, Klebsiella pneumoniae, lactic acid bacteria, Salmonella, or Clostridium difficile.

[0013] This invention also provides the application of the virus obtained by the above method in the preparation of vaccines.

[0014] Preferably, the vaccine includes inactivated vaccines and live vector vaccines.

[0015] The present invention also provides an inactivated vaccine comprising an inactivated virus obtained by the above method.

[0016] The present invention also provides a live vector vaccine comprising RecA-deficient Bacillus subtilis infected with a virus, wherein the RecA-deficient Bacillus subtilis infected with the virus obtained by the above method is obtained by infecting RecA-deficient Bacillus subtilis.

[0017] Preferably, the live vector vaccine also includes trehalose.

[0018] The beneficial effects of this invention are:

[0019] This invention 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 invention have 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 developing vaccines using bacteria to infect viruses. Prokaryotically cultured viruses phPCV2, phPCV3, and phHEV also possess broad-spectrum bacterial infectivity, similarly groundbreaking for the development of broad-spectrum phage bactericides and for developing vaccines using bacteria to infect viruses.

[0020] This invention utilizes Escherichia coli BL21(DE3) to stably amplify phPCV2, phPCV3 and phHEV with high toxicity, and uses these to prepare inactivated vaccines that have significant protective effects on animals.

[0021] This invention also prepared 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, allowing the virus infection to remain stably in a lysogenic state for a long period. 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 effect, and the ability to regulate the intestinal microbiota and improve overall host health. Attached Figure Description

[0022] Figure 1The results of nucleic acid extraction from bacteria isolated and purified from feces are shown, from top to bottom: phPCV2, phPCV3, and phHEV-3 detection results. In the phPCV2 detection results, lanes 1-12 contained the following bacteria: *Kurstella*, *Streptococcus lactis*, *Escherichia coli*, *Escherichia fentanyl*, *Citrobacter*, *Enterobacter homonas*, *Klebsiella pneumoniae*, *Bacillus subtilis*, *Staphylococcus*, *Proteus mirabilis*, *Klebsiella pneumoniae*, and *Enterococcus*. In the phPCV3 detection results, lanes 1-8 contained the following bacteria: *Escherichia coli*, *Kurstella*, *Streptococcus lactis*, *Escherichia fentanyl*, *Citrobacter*, and *Enterococcus*. The bacteria detected were *Enterobacter hamma*, *Klebsiella pneumoniae*, and *Bacillus subtilis*. In the PhHEV-3 test results, lanes 1-11 contained *Escherichia coli* 1, *Escherichia coli* 2, *Escherichia coli* 3 (representing different *E. coli* strains isolated from different positive pigs), *Kreutzer*, *Levitraceta*, *Staphylococcus*, *Enterococcus* 1, *Enterococcus* 2, *Enterococcus* 3, *Bacillus subtilis*, and *Clostridium* (representing different *Enterococcus* strains isolated from different positive pigs). Negative results were nucleic acids extracted from negative serum, and positive results were nucleic acids extracted from positive serum for each virus.

[0023] Figure 2 The results of the experiment on the inhibitory effect of wild-type bacterial flora isolated from pig feces infected with phPCV2, phPCV3, phHEV-3, phHEV-2 and phHEV-4 on bacteria were observed by streaking on blood plates.

[0024] Figure 3 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.

[0025] Figure 4 Images of E. coli adsorbed by phPCV2, phPCV3 and phHEV-3 under transmission electron microscopy;

[0026] Figure 5 To observe the virus release of phPCV2, phPCV3 and phHEV-3 ruptured BL21(DE3) Escherichia coli under a transmission electron microscope;

[0027] Figure 6 To observe negatively stained phPCV2, phPCV3 and phHEV-3 viral particles after PEG8000 precipitation under a transmission electron microscope;

[0028] Figure 7The study investigated the changes in serum specific IgG antibody levels in weaned piglets after vaccination with inactivated phPCV2, phPCV3, and phHEV-3 viruses prepared by propagation and concentration of BL21(DE3) Escherichia coli.

[0029] Figure 8 The viral copy number levels in the serum of weaned piglets in the control group and the inactivated vaccine immunization group were determined to be protective against challenge with phPCV2, phPCV3 and phHEV-3.

[0030] Figure 9 The viral copy number levels in the intestines, liver, and spleen of weaned piglets in the control group and the inactivated vaccine immunization group were measured 8 days after challenge protection experiments with phPCV2, phPCV3, and phHEV-3.

[0031] Figure 10 The results validate the expression of RecA-deficient WB800N Bacillus subtilis protein.

[0032] Figure 11 The results of identifying the lysogenic state of phPCV2, phPCV3 and phHEV-3 after infection with RecA-deficient WB800N Bacillus subtilis;

[0033] Figure 12 The changes in specific IgG antibody levels in the serum of weaned piglets in the wild mushroom feeding group and the phPCV2, phPCV3 and phHEV-3 live vector vaccine feeding groups were investigated.

[0034] Figure 13 To investigate 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 for protection against challenge with phPCV2, phPCV3, and phHEV-3;

[0035] Figure 14 Eight days after the challenge protection test of phPCV2, phPCV3 and phHEV-3, 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 were compared.

[0036] Figure 15 Eight days after the challenge protection experiment with phPCV2, phPCV3, and phHEV-3, the IgA secretion level in the small intestinal mucosal flushing fluid of weaned piglets in the wild-mushroom feeding group and the phPCV2, phPCV3, and phHEV-3 live vector vaccine feeding group was determined. Detailed Implementation

[0037] This invention provides the application of viruses in the preparation of bacteriophages, wherein the viruses include porcine circovirus and / or hepatitis E virus; the porcine circovirus includes PCV2, phPCV2, PCV3, or phPCV3; the hepatitis E virus includes HEV or phHEV. In this invention, the HEV preferably includes HEV-2, HEV-3, or HEV-4; the phHEV preferably includes phHEV-2, phHEV-3, or phHEV-4; the nucleotide sequence of phPCV2 is preferably as shown in SEQ ID NO.1, the nucleotide sequence of phPCV3 is preferably as shown in SEQ ID NO.2, the nucleotide sequence of phHEV-3 is preferably as shown in SEQ ID NO.3, the nucleotide sequence of phHEV-2 is as shown in SEQ ID NO.4, and the nucleotide sequence of phHEV-4 is as shown in SEQ ID NO.5. In this invention, ph in phPCV2, phPCV3, or phHEV-3 is a self-nominated designation used to indicate PCV2, PCV3, or HEV obtained from bacterial division.

[0038] This invention also provides a bacteriophage bactericide, the bacteriophage bactericide comprising at least one of PCV2, phPCV2, PCV3, phPCV3, HEV, and phHEV. In this invention, the HEV preferably comprises HEV-2, HEV-3, or HEV-4; the phHEV preferably comprises phHEV-2, phHEV-3, or phHEV-4; the nucleotide sequence of phPCV2 is preferably as shown in SEQ ID NO.1, the nucleotide sequence of phPCV3 is preferably as shown in SEQ ID NO.2, the nucleotide sequence of phHEV-3 is preferably as shown in SEQ ID NO.3, the nucleotide sequence of phHEV-2 is as shown in SEQ ID NO.4, and the nucleotide sequence of phHEV-4 is as shown in SEQ ID NO.5.

[0039] The present invention 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.

[0040] In this invention, the preferred method of infection is to inoculate the viral stock solution with bacteria, observe the virus adsorbing the bacteria, culture the adsorbed bacteria, select bacteria in a good lysogenic state for propagation, induction, removal of bacterial precipitate, concentration, filtration, centrifugation to precipitate the virus, and collection of the virus. In this invention, the bacteria preferably include *Escherichia coli*, *Streptococcus lactis*, *Citrobacter*, *Bacillus subtilis*, *Proteus mirabilis*, *Enterococcus*, *Escherichia fergusonian*, *Shigella flexneri*, *Kurstia*, *Citrobacter*, *Citrobacter flexneri*, *Lactobacillus*, *Enterobacter homunculus*, *Klebsiella pneumoniae*, lactic acid bacteria, *Salmonella*, or *Clostridium*. In this invention, the preferred nucleotide sequence of phPCV2 is shown in SEQ ID NO.1, the preferred nucleotide sequence of phPCV3 is shown in SEQ ID NO.2, the preferred nucleotide sequence of phHEV-3 is shown in SEQ ID NO.3, the preferred nucleotide sequence of phHEV-2 is shown in SEQ ID NO.4, and the preferred nucleotide sequence of phHEV-4 is shown in SEQ ID NO.5.

[0041] This invention also provides the application of the virus obtained by the above method in the preparation of vaccines. In this invention, the vaccines preferably include inactivated vaccines and live vector vaccines.

[0042] This invention also provides an inactivated vaccine comprising an inactivated virus obtained by the above-described method. In this invention, the inactivation is preferably performed by co-culturing the virus with diethyleneimine (BEI). Preferably, the inactivated vaccine also includes an immune adjuvant, preferably MONTANIDEIMISA206VG adjuvant, and the weight ratio of the immune adjuvant to the inactivated virus is preferably 1:1.

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

[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 superior viral tropism and gastric acid and bile salt tolerance compared to other bacteria. Therefore, this invention prioritizes the use of Bacillus subtilis for the preparation of bacterial vectors.

[0045] In this invention, the preferred method of infection is to co-culture the successfully constructed RecA-deficient Bacillus subtilis with the virus obtained by the above-mentioned virus culture method. After adsorption, positive colonies are screened and identified. In the live vector vaccine of this invention, an adjuvant is preferably also included, and the adjuvant preferably includes trehalose. Due to the hygroscopic nature of trehalose, the 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] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, the following embodiments are all conventional methods.

[0048] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] Example 1

[0050] Eukaryotic viruses PCV2, PCV3, and HEV were identified as lysogenic bacteriophages phPCV2, phPCV3, and phHEV.

[0051] 1.1 Detection of phPCV2, phPCV3 and phHEV in wild-type fungi

[0052] PCR amplification detection

[0053] Fecal samples from pigs infected with PCV2, PCV3, and HEV viruses were collected. The bacterial flora in the feces was isolated and purified using blood agar plates, and single colonies were selected for propagation and cryopreservation. The propagated bacteria were then sonicated. Nucleic acid was extracted from the sonicated 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 products were detected by 1% agarose gel electrophoresis. The results are shown below. Figure 1 As shown in Table 1, the primer design is shown in Table 2, the PCR amplification reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0054] Table 1 Primer sequences

[0055]

[0056] Table 2 PCR reaction system

[0057] Element Volume (μL) 2×TaqMasterMix 12.5 Forwardprimer 1 RorwardPrimer 1 sample 2 <![CDATA[ddH2O]]> Make up to 20

[0058] Table 3 PCR amplification reaction procedure

[0059] step temperature time Cycle number Pre-variation 95℃ 5min 1 transsexual 95℃ 30s 35 annealing 58℃ 30s 35 extend 72℃ 60s 35 Final extension 72℃ 5min 1 save 12℃ ∞ 1

[0060] 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.

[0061] Table 4 shows some of the positive test results for the strains.

[0062] strain phPCV2 phPCV3 phHEV-2 phHEV-3 phHEV-4 Streptococcus lactis √ √ E. coli √ √ √ √ √ Escherichia coli Fergusonii √ Shigella flexneri √ Kurtella √ √ Citrobacter √ √ Citrobacter √ Citrobacter freundii √ Leukemia √ Homeobacterium √ Klebsiella pneumoniae √ Bacillus subtilis √ √ √ √ √ staphylococcus √ Proteus mirabilis √ √ Lactococcus gasseri √ Klebsiella pneumoniae √ Enterococcus √ √ √ Lactic acid bacteria √ salmonella √ Clostridium √ √

[0063] 1.2 Infection with bacteria by eukaryotic PCV2, PCV3, HEV, and prokaryotic phPCV2, phPCV3, and phHEV-3 all inhibit bacterial growth.

[0064] To verify the bactericidal effects of the three viruses in a bacterial environment in the wild, 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 original solutions of phPCV2, phPCV3, phHEV-2, phHEV-3, and phHEV-4 viruses isolated from bacteria at an MOI of 1. After adsorption at 4℃ for 1 h, LB medium containing 0.1 μg / mL MMC was added and cultured for 8 h. 10 μL of the bacterial solution was streaked on blood agar plates to observe the number and types of colonies. The results are as follows: Figure 2 As shown. After viral infection, all three viruses exhibited inhibitory effects on bacteria. The various genotypes of phPCV2 and phHEV showed significant inhibitory effects on various bacteria. Among them, phPCV3 showed inhibition of the growth of some bacteria, while phHEV-3 showed complete inhibition of bacteria. Subsequently, phHEV-3 was used as the representative HEV strain for subsequent experiments.

[0065] 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), and HEV-3, 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 an MOI of 1 virus dose, the bacteria were washed three times with sterile PBS and added to LB medium containing 0.1 μg / mL mitomycin C (MMC, a lysogenous phage lysis inducer). A control group of bacteria with the same bacterial count was also 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 of 600nm. The results are shown below. Figure 3 As shown, BL21(DE3) Escherichia coli and WB800N Bacillus subtilis infected with phPCV2, phPCV3, and phHEV-3 all exhibited growth inhibition, and bacteria in the HEV-3-infected group began to die 8 hours 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.

[0066] Example 2

[0067] Inactivated vaccine prepared by culturing viruses from BL21(DE3) Escherichia coli

[0068] 1.1 Preparation and screening of lysogenic bacteria

[0069] (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.

[0070] (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.

[0071] (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℃ for 1 hour, the bacteria were subjected to ultrasonic sectioning. The virus adsorption could be observed using transmission electron microscopy. Figure 4 As shown in the figure. The bacteria that had been adsorbed were then added to LB medium and incubated at 16°C for 12 hours.

[0072] (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.

[0073] 1.2 Propagation and Induction of Lysogenic Bacteria

[0074] (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.

[0075] (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 rpm for 24 h. At this point, ultrathin sections of the bacteria were prepared, and transmission electron microscopy revealed the entire process of bacterial lysis by the virus and the release of the virus. The results are as follows... Figure 5 As shown.

[0076] 1.3 Virus Concentration

[0077] (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.

[0078] (2) Filtration: The supernatant is filtered through a 0.22μm or 0.45μm filter membrane to further remove bacterial debris and other impurities.

[0079] (3) Add PEG8000: Add PEG8000 to the filtered supernatant at a concentration of 5%. Stir well to ensure the PEG is fully dissolved.

[0080] (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.

[0081] (5) Centrifugation: Centrifuge at a high speed of 12000r / min for 30min at low temperature (4℃) to precipitate the virus.

[0082] (6) Remove the supernatant: Carefully remove the supernatant and retain the precipitate.

[0083] (7) Resuspending the virus: The precipitate was resuspended using sterile PBS to obtain a concentrated virus solution.

[0084] (8) Negative staining observation: Add an appropriate amount of virus sample to a copper grid, 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 virus particles. Typical virus particles can be observed, and the results are as follows. Figure 6 As shown.

[0085] 1.4 Preparation of inactivated vaccines

[0086] (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.

[0087] (2) Purity test of virus solution used for vaccine preparation

[0088] 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.

[0089] (3) Inactivation of virus solution used for vaccine preparation

[0090] 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.

[0091] (4) Preparation of inactivated vaccines

[0092] The inactivated virus stock solution and Seppic's MONTANIDEIMISA206VG 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 MONTANIDEIMISA206VG adjuvant. After completion, the stirring speed was rapidly increased to 2000 r / min and stirred thoroughly for 10 min. The solution was then quantitatively dispensed, sealed, and stored at 4°C.

[0093] Through the above operations, inactivated vaccines phPCV2, phPCV3 and phHEV-3 derived from BL21(DE3) Escherichia coli were obtained.

[0094] 1.5 Detection of Immunoprotective Effect

[0095] 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. The results are as follows: Figure 7 As shown in the figure. After immunization, the animals were observed for any adverse reactions. The results showed that the inactivated vaccine of this invention did not show any adverse symptoms after being injected into the animals, indicating good safety. Two weeks after the second immunization, a challenge experiment was conducted. Blood was collected every two days after the challenge to detect viremia. The results are shown in the figure. Figure 8 As shown. Eight days later, the experimental animals were euthanized, and liver and intestinal samples were collected for testing. The results are as follows. Figure 9 As shown.

[0096] 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.

[0097] Example 3

[0098] Live vector vaccines were prepared using RecA-deficient Bacillus subtilis in lysogenic states of phPCV2, phPCV3, and phHEV-3.

[0099] 1.1 Preparation of RecA-deficient Bacillus subtilis

[0100] (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.

[0101] Table 5. Homologous arms, RecA-loxp-kan sequences, and primer sequences.

[0102]

[0103] (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.

[0104] (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.

[0105] (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.

[0106] (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.

[0107] (6) Screen positive clones, culture them in kan resistance, and name them recA-kan-WB800N.

[0108] (7) The recA-kan-WB800N was passaged at 42℃ in Kan resistance until Amp resistance disappeared, i.e., the pkd46 plasmid was lost.

[0109] (8) Prepare electrocompetent states from recA-kan-WB800N with lost plasmid.

[0110] (9) Transfer pkd46-Cre plasmid into recAkan-WB800N (50 μL), add 1 mL of antibiotic-free LB medium, and revive at 30℃ for 30 min.

[0111] (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.

[0112] (11) Select clones and identify positive results by PCR.

[0113] (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.

[0114] (13) Streaking to purify the bacterial strain.

[0115] (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.

[0116] (15) Verify protein expression in RecA-deficient bacteria, such as Figure 10 As shown, RecA expression is defective.

[0117] 1.2 Preparation of RecA-deficient Bacillus subtilis live vector vaccines in lysogenic states of phPCV2, phPCV3, and phHEV-3

[0118] (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.

[0119] (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.

[0120] (3) Identification of viral lysogens: In the lysogenic state, the three viruses exhibited only viral capsid protein expression within the bacteria, with viral replication protein expression repressed. The viruses were unable to package live progeny viral 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. Results are as follows... Figure 11 As shown.

[0121] (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.

[0122] 1.3 Detection of the immunoprotective effect of live vector vaccines

[0123] 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 the different vector vaccines were thoroughly mixed with feed (5g of each live-type vaccine or wild-type vaccine 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 administered 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 as follows: Figure 12 As shown. One week after stopping immunization, a feeding challenge experiment was conducted. Blood was collected every two days after challenge to detect viremia. The results are as follows. Figure 13 As shown in the image. Eight days later, the experimental animals were euthanized, and liver, intestine, and spleen samples were collected for testing. The results are as follows. Figure 14 As shown in the figure. 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 using IgA antibody. The results are as follows. Figure 15 As shown.

[0124] The results showed that vaccination with the three live vector vaccines prepared in this invention induced pigs to produce sustained high levels of specific antibodies. The produced antibodies provided good protection for pigs, significantly reducing the incidence of viremia and viral replication within the body. Furthermore, as... Figure 15 As shown, this live vector vaccine can also induce the production of high levels of secretory IgA antibodies, greatly improving mucosal immunity.

[0125] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a virus in the preparation of bacteriophages, characterized in that, The viruses include porcine circovirus and / or hepatitis E virus; the porcine circovirus includes PCV2, phPCV2, PCV3 or phPCV3; the hepatitis E virus includes HEV or phHEV.

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

3. The application according to claim 1 or the bacteriophage bactericide according to claim 2, characterized in that, The HEV includes HEV-2, HEV-3, or HEV-4; the phHEV includes phHEV-2, phHEV-3, or phHEV-4; 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.

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

5. The method according to claim 4, characterized in that, The bacteria include Escherichia coli, Streptococcus lactis, Citrobacter, Bacillus subtilis, Proteus mirabilis, Enterococcus, Escherichia fergusonii, Shigella flexneri, Kurtella, Citrobacter, Citrobacter flexneri, Leukemia, Enterobacter hominis, Klebsiella pneumoniae, Staphylococcus, Lactococcus gasseri, Klebsiella pneumoniae, lactic acid bacteria, Salmonella, or Clostridium difficile.

6. The use of the virus obtained by the method of claim 4 or 5 in the preparation of a vaccine.

7. The application according to claim 6, characterized in that, The vaccines include inactivated vaccines and live vector vaccines.

8. An inactivated vaccine, characterized in that, This includes inactivated viruses obtained by the method described in claim 4 or 5.

9. A live vector vaccine, characterized in that, This 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 claim 4 or 5.

10. The live vector vaccine according to claim 9, characterized in that, It also includes trehalose.

Images