Outer membrane vesicle, preparation method thereof and application of outer membrane vesicle in vaccine preparation

Abstract

The invention provides an outer membrane vesicle as well as a preparation method and application thereof in preparation of vaccines, and belongs to the technical field of biology. According to the invention, the outer membrane vesicles in which ygeA and yhbB gene mutant strains in escherichia coli are knocked out are obtained, two endogenous proteins of YgeA and YhbB with higher abundance are reduced in the outer membrane vesicles of escherichia coli, and good immunological enhancement activity of OMV is maintained. After the outer membrane vesicle is applied to preparation of vaccines, especially preparation of swine foot-and-mouth disease vaccines, the immune effect can be remarkably enhanced, and higher specific immunogenicity is generated. The method can be further applied to preparation of swine foot-and-mouth disease vaccines in the pharmaceutical industry so as to achieve the purpose of improving quality and efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an outer membrane vesicle, a preparation method thereof, and an application thereof in preparing a vaccine. Background Art

[0002] Outer membrane vesicles (OMVs) are products of budding secretion from the surface of Gram-negative bacteria. They are formed by the bacterial outer membrane budding under a certain mechanism and forming a vesicle-like structure on the bacterial surface. The size of the vesicles ranges from about 20nm to 300nm. This structure includes lipopolysaccharide (LPS), outer membrane proteins, phospholipids, bacterial toxins, adhesins, DNA, and periplasmic components wrapped by the outer membrane during the formation process. Because outer membrane vesicles cannot replicate and contain a large number of bacterial antigens, they have good immunogenicity and immunoenhancing effects and can activate the host cell immune system. Therefore, outer membrane vesicles can be used as a new bacterial vaccine or immunoenhancer with great potential.

[0003] However, outer membrane vesicles contain a variety of highly abundant endogenous proteins. When biological products are prepared using outer membrane vesicles, specific antibodies against endogenous proteins (i.e., ineffective antigens) will be produced after immunization of animals, thus affecting the immune effect of the main antigens carried by OMVs used as carrier vaccines. This problem limits the use and promotion of outer membrane vesicles in vaccine preparation. Summary of the invention

[0004] One of the purposes of the present invention is to provide an outer membrane vesicle lacking two highly abundant endogenous proteins.

[0005] Another object of the present invention is to provide a method for preparing the outer membrane vesicles.

[0006] The present invention also provides the use of the outer membrane vesicles prepared by the above method in preparing vaccines, especially in preparing swine foot-and-mouth disease vaccines.

[0007] In order to achieve the above-mentioned purpose of the invention, the present invention discloses an outer membrane vesicle, wherein the outer membrane vesicle is from Gram-negative bacteria and lacks two endogenous proteins, wherein the endogenous proteins are YgeA and YhbB.

[0008] Preferably, the Gram-negative bacteria is Escherichia coli.

[0009] That is, the outer membrane vesicles are outer membrane vesicles of a mutant strain of Escherichia coli in which the ygeA and yhbB genes are knocked out.

[0010] Furthermore, the present invention also discloses a method for preparing the outer membrane vesicles, which comprises the step of knocking out the ygeA and yhbB genes of Gram-negative bacteria.

[0011] Specifically, a homologous recombination fragment with upstream and downstream homologous arms of the ygeA gene at both ends and a chloramphenicol resistance gene cat in the middle is introduced into the Escherichia coli J5 strain to perform homologous recombination, thereby replacing the ygeA gene on the chromosome of the Escherichia coli J5 strain with the chloramphenicol resistance gene, and then using the FLP site-specific recombinase of the temperature-sensitive plasmid pCP20 to eliminate the chloramphenicol resistance gene cat on the chromosome of the Escherichia coli J5 strain, thereby obtaining an Escherichia coli mutant strain with the ygeA gene knocked out;

[0012] Then, based on the E. coli mutant strain in which the ygeA gene was knocked out, the above method was used again to recombine and obtain a mutant strain in which the yhbB gene was knocked out, thereby obtaining an E. coli mutant strain in which both the ygeA gene and the yhbB gene were knocked out.

[0013] Specifically, a homologous recombination fragment with upstream and downstream homologous arms of the yhbB gene at both ends and the chloramphenicol resistance gene cat in the middle is introduced into the obtained Escherichia coli mutant strain with the ygeA gene knocked out for homologous recombination, and the yhbB gene on the Escherichia coli chromosome is replaced with the chloramphenicol resistance gene. Then, the FLP site-specific recombinase of the temperature-sensitive plasmid pCP20 is used to eliminate the chloramphenicol resistance gene cat on the Escherichia coli chromosome, thereby obtaining an Escherichia coli mutant strain with the ygeA gene and yhbB gene knocked out.

[0014] Preferably, the bacterial mutant is a strain lacking the chromosomal ygeA and yhbB genes through Red recombination technology.

[0015] Furthermore, the present invention also discloses the outer membrane vesicles prepared by the above method, that is, the outer membrane vesicles obtained by culturing a mutant strain of Gram-negative bacteria in which the ygeA and yhbB genes are knocked out.

[0016] The present invention further discloses the application of the outer membrane vesicles in preparing vaccines, especially in preparing swine foot-and-mouth disease vaccines.

[0017] At the same time, the present invention also discloses a method for preparing a vaccine, which includes the above-mentioned method for preparing outer membrane vesicles.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] (1) The method disclosed in the present invention can be used to obtain Escherichia coli outer membrane vesicles lacking two major endogenous proteins. This is a pure outer membrane vesicle that is more conducive to pharmaceutical industry production. The biological preparation prepared using the outer membrane vesicles can produce stronger specific immunogenicity for immunized animals.

[0020] (2) The bacterial outer membrane vesicles prepared by the method of the present invention have good immune enhancement function. Adding them to swine foot-and-mouth disease vaccine can significantly enhance the immune effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of agarose gel electrophoresis results for recombination identification of Escherichia coli J5 strain; "△ygeA::cat", "△yhbB::cat", "△ygeA", "△yhbB" and "M" respectively represent: amplification of ygeA gene by J5△ygeA::cat recombinant strain, amplification of yhbB gene by J5△ygeA△yhbB::cat recombinant strain, amplification of ygeA gene by J5△ygeA deletion strain, amplification of yhbB gene by J5△ygeA△yhbB deletion strain, and DNA marker Trans2K plus2.

[0022] Figure 2 Schematic diagram of the average antibody titer of mouse sera 1, 2, and 4 weeks after immunization; "J5 OMV" is the FMDV+J5 OMV+ISA206 immunization group (Group 1), "J5△" is the FMDV+J5△ygeA△yhbB OMV+ISA206 immunization group (Group 2), "ISA206" is the FMDV+ISA206 immunization group (Group 3), and PBS is the FMDV immunization group (Group 4).

[0023] Figure 3 This is a Tecnai 12 transmission electron microscope photograph of the outer membrane vesicles produced by J5△ygeA△yhbB. DETAILED DESCRIPTION

[0024] Example 1 Screening of two endogenous protein genes

[0025] (1) Materials

[0026] Escherichia coli J5 strain was purchased from ATCC, USA, with the strain code ATCC43745.

[0027] Preparation of LB medium: weigh 10g of tryptone, 5g of yeast powder and 10g of sodium chloride, dissolve them in distilled water and make up to 1000mL, adjust the pH to 7.4, and sterilize by autoclave at 121℃ for 15min. If solid plates are to be prepared, add 16g of agar before autoclaving.

[0028] (2) Extraction of bacterial OMVs

[0029] The constructed E. coli J5 strain was streaked and inoculated into LB solid medium and cultured at 37°C for 16 hours. A single colony was picked and inoculated into 10 mL of LB medium and cultured at 37°C and 180 rpm for 8 hours as seed liquid. The seed liquid was transferred to a conical flask (with a paddle at the bottom of the flask) containing 1000 mL of LB liquid medium at a volume ratio of 1:100 and cultured at 37°C and 180 rpm for 12 hours. At this time, the bacterial growth was in the plateau phase and the OD600 nm was about 4.0, and the culture was stopped.

[0030] Centrifuge 1000mL of bacterial culture at 4°C and 10000g for 15min, discard the precipitate, filter the supernatant through a 0.22μm filter membrane for sterilization, and obtain about 1000mL of cell-free supernatant. Concentrate the obtained cell-free supernatant 100 times through a 100KDa ultrafiltration membrane, and harvest a cell-free supernatant with a final volume of 10mL. Ultracentrifuge at 4°C and 120000g for 3h, discard the supernatant, and resuspend the precipitate in an appropriate amount of PBS buffer; ultracentrifuge again at 4°C and 120000g for 3h, discard the supernatant, and resuspend the precipitate in 5mL PBS buffer. Finally, filter through a 0.22μm filter membrane to obtain a bacterial outer membrane vesicle sample for use.

[0031] (3) Protein sequencing sample pretreatment

[0032] Add NH4HCO3 to the outer membrane vesicle sample obtained in (2) to a final concentration of 50mM. Add DTT solution to a final concentration of 10mmol / L and reduce in a 56℃ water bath for 1h. Add IAA solution to a final concentration of 50mmol / L and react in the dark for 40min. Add trypsin at a mass ratio of 1:100 to substrate and digest at 37℃ for 4h. Continue to add trypsin at a mass ratio of 1:100 and digest at 37℃ overnight (16h). After digestion, the peptides were desalted using a self-filled desalting column and the solvent was evaporated in a 45℃ vacuum centrifugal concentrator.

[0033] (4) Protein sequencing

[0034] The samples prepared in (3) were sent to the company for LC-MS / MS detection. After searching the database using the software MaxQuant (1.6.2.10), 19 proteins with high protein abundance in the outer membrane vesicles of the Escherichia coli J5 strain were obtained (Table 1). The obtained protein-coding genes were compared with the website Profiling of Escherichia coli chromosome (PEC) database (https: / / shigen.nig.ac.jp / ecoli / pec / index.jsp), and the target sites ygeA and yhbB genes that could be knocked out without affecting the growth of Escherichia coli were screened.

[0035] Table 1 Sequencing results of outer membrane vesicle proteome of Escherichia coli J5 strain

[0036]

[0037]

[0038] Note: Protein IDs: protein number from the uniprot database (Escherichia coli str. K-12 substr. MG1655). Example 2 Construction of Escherichia coli J5 strain lacking two endogenous proteins

[0039] (1) Materials

[0040] Escherichia coli J5 strain was purchased from ATCC, USA, with the strain code ATCC43745.

[0041] Preparation of LB medium: weigh 10g of tryptone, 5g of yeast powder and 10g of sodium chloride, dissolve them in distilled water and make up to 1000mL, adjust the pH to 7.4, and sterilize by autoclave at 121℃ for 15min. If solid plates are to be prepared, add 16g of agar before autoclaving.

[0042] Ampicillin, chloramphenicol and L-arabinose were purchased from Shanghai Biotechnology Co., Ltd.

[0043] rTaq PCR MIX was purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0044] The pKD3 plasmid, pKD46, and pCP20 plasmids were donated by Professor Zhu Guoqiang of Yangzhou University, but can all be purchased from commercial sources.

[0045] (2) Primer design

[0046] According to the upstream and downstream outer wings of the ygeA gene ORF sequence of Escherichia coli K-12MG1655 strain (accession number: NC_000913) published in GenBank, identification primers F1 and R1 were designed, and their nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. A pair of homologous recombination primers F2 and R2 were designed inside F1 and R1, and their 5' end sequences were homologous to the two wing sequences of ygeA, and the 3' end sequences were homologous to the cat gene sequence of the pKD plasmid.

[0047] As above, according to the upstream and downstream outer wings of the yhbB gene ORF sequence of Escherichia coli K-12 MG1655 strain (accession number: NC_000913) published in GenBank, identification primers F3 and R3 were designed, respectively, and their nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. A pair of homologous recombination primers F4 and R4 were designed inside F3 and R3, and their 5' end sequences were homologous to the two wing sequences of yhbB, and the 3' end sequences were homologous to the cat gene sequence of the pKD plasmid.

[0048] The sequences of the primers are as follows:

[0049] F1 (SEQ ID No. 1):5'-CTCGTTCCTTATTCAGCCTTGT-3';

[0050] R1 (SEQ ID No. 2): 5'-CGTCATCAGCGCTGGTTCAG-3'.

[0051] F2 (SEQ ID No.3):

[0052] 5'-TCTCGTTCCTTATTCAGCCTTGTTGCGGTAACACACATCAGGAGAGAGGATGTGTAGGCTGGAGCTGCTTCG-3';

[0053] R2 (SEQ ID No.4):

[0054] 5'-AGCGGGTTACCGAAACTGGTCACTTCTCTTGACGCTATTTTGTCGTCAGCATATGAATATCCTCCTTAG-3'.

[0055] F3 (SEQ ID No. 5):5'-ACAGCCCTTCCACCTGAAAT-3';

[0056] R3 (SEQ ID No. 6): 5'-GCATCGTTGCTGGACATACC-3'.

[0057] F4 (SEQ ID No.7):

[0058] 5'-TTAATTTTAAAAAGGGGCTAACAGCCCCTTTTTTTGTCAGGAGAATTTATTTGTGTAGGCTGGAGCTGCTTCG-3';

[0059] R4 (SEQ ID No.8):

[0060] 5'-AGCAACAAACGCCGTTAATGTCGCGACCGCGACGACGAGGACGACTCACATATGAATATCCTCCTTAG-3'.

[0061] (3) Preparation of recombinant fragments

[0062] Using pKD3 plasmid as template and F2 and R2 as primers, amplify the recombinant DNA fragment with upstream and downstream homology arms of ygeA gene at both ends and chloramphenicol resistance gene cat in the middle, with a fragment length of 1115bp. Take 25uL rTaq PCR MIX, 2uL primers F2 and R2, 2uL pKD3 plasmid (Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci US A 2000, 97: 6640-6645.), and add ultrapure water to a total volume of 50uL. PCR reaction program: 95℃ pre-denaturation for 5min; 94℃15s, 51℃ annealing for 15s, 72℃60s, 10 cycles; 94℃15s, 61℃ annealing for 15s, 72℃60s, 25 cycles; 72℃ extension for 5min. The amplified product was separated by 1% agarose gel electrophoresis, and the 1115bp recombinant ygeA gene fragment was recovered by gel excision.

[0063] According to the above method, the recombinant yhbB gene fragment was prepared with primers F4 and R4.

[0064] (4) Recombinant deletion of ygeA gene

[0065] Prepare competent cells of E. coli J5 strain as follows: pick a single colony of E. coli J5 strain and inoculate it into 4 mL LB liquid medium, culture it in a shaker at 37°C overnight, take 50 μL of the culture and add it into 5 mL LB liquid medium, culture it in a shaker at 37°C for 1.5-2 h, and make the culture OD 600 When the pH reaches 0.5-0.6, place the bacterial solution on ice at 4°C for 30 minutes, centrifuge at 5000r / min for 5 minutes, discard the supernatant, gently blow the precipitate with 0.1M CaCl2 solution, fully suspend it, ice-bathe for 10 minutes, and centrifuge to obtain the precipitate, gently blow the precipitate with 400μL pre-cooled 0.1MCaCl2 solution, fully suspend it, and ice-bathe for 30 minutes to obtain Escherichia coli J5 strain competent cells.

[0066] The plasmid pKD46 was introduced into the Escherichia coli J5 strain as follows: the plasmid pKD46 encoding the Red recombination system was mixed with 200 μL of competent cells of the Escherichia coli J5 strain, and the mixture was placed in an ice bath for 30 min. After heat shock at 42°C for 90 s, the mixture was immediately placed in an ice bath for 1-2 min. 800 μL of LB liquid culture medium without antibiotics was added, and the mixture was cultured at 30°C and 150 r / min with shaking for 45 min. 200 μL of the LB plate coated with ampicillin (100 μg / mL) resistance was taken and cultured at 30°C overnight.

[0067] A single colony of the transformed recombinant bacteria was picked and inoculated into 5 mL of fresh LB medium containing ampicillin resistance, and cultured at 30°C with shaking overnight. The next day, the colony was inoculated into 50 mL of LB medium at a ratio of 1:100 and cultured at 30°C until OD 600 When the concentration reaches 0.2-0.3, add L-arabinose to a final concentration of 30mmol / L and induce for 1h to fully express the three proteins Exo, Bet and Gam on pKD46. Precool the culture on ice for 30min, then centrifuge at 5000r / min for 5min, discard the culture medium, centrifuge and wash 3 times with precooled 10% glycerol aqueous solution, concentrate 100 times to make 500μL competent cells of recombinant bacteria carrying plasmid pKD46, and dispense 40μL into each tube.

[0068] Take about 100 ng of the purified recombinant ygeA gene amplified fragment in the title (3) of this example, mix it with 40 μL of competent cells of the recombinant bacteria carrying the plasmid pKD46, add the mixture to a 0.1 cm Bio-Rad electrode cup, and perform electric shock at a voltage of 1.8 KV, a pulse of 25 μF, and a resistance of 200 ohms. After electric shock, quickly add 1 ml of pre-cooled SOC culture medium, culture at 150 r / min and 30° C. for 1 hour, and spread all on LB plates containing chloramphenicol (34 ng / ml) and ampicillin (100 ng / mL) to screen for ygeA gene deletion mutants. Wherein, the SOC culture medium contains 2% (W / V) tryptone, 0.5% (W / V) yeast powder, 0.05% (W / V) sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, and 20 mM glucose.

[0069] Use PCR to identify the single colony on the plate in the previous step. Place the single colony on the plate in the previous step in 4mL LB medium containing ampicillin and chloramphenicol, place it in a 30℃ constant temperature incubator and shake it overnight (8-12h), take 1mL of the bacterial solution and use the boiling method to prepare the identification strain template. Establish a reaction system: take 10uL rTaq PCR MIX, 1uL each of F1 and R1 primers, 2uL of identification strain template, and add ultrapure water to a total volume of 25uL. The PCR reaction conditions are: 94℃ pre-denaturation for 4min; 94℃1min, 53℃30s, 72℃60s, 25 cycles; 72℃ extension for 10min. The amplification results were observed by 1.2% agarose gel electrophoresis, where "△ygeA::cat", "△yhbB::cat", "△ygeA", "△yhbB" and "M" respectively represent: J5△ygeA::cat recombinant strain amplifies ygeA gene, J5△ygeA△yhbB::cat recombinant strain amplifies yhbB gene, J5△ygeA deletion strain amplifies ygeA gene, J5△ygeA△yhbB deletion strain amplifies yhbB gene, and DNA marker Trans2K plus2. Figure 1 It can be seen that a band of about 1500 bp appeared in the amplification product of the ygeA gene deletion mutant, indicating that the ygeA gene ORF in the J5 strain genomic DNA was successfully knocked out and replaced with the chloramphenicol resistance gene.

[0070] The recombinant bacteria in which the ygeA gene was successfully knocked out by PCR were inoculated into LB medium containing chloramphenicol and cultured and subcultured at 42°C for three generations, with each generation culturing for no less than 6 hours, thereby eliminating the temperature-sensitive plasmid pKD46 in the strain. Finally, the bacteria were streaked on LB plates containing chloramphenicol and then cultured inverted at 37°C. Single colonies were randomly selected and streaked on ampicillin and chloramphenicol plates for resistance testing. Single colonies that were sensitive to ampicillin and resistant to chloramphenicol were screened, thereby obtaining the recombinant bacteria in which the plasmid pKD46 had been successfully eliminated and the ygeA gene ORF had been knocked out, and named J5△ygeA::cat.

[0071] (5) Elimination of cat gene

[0072] The competent cells of J5△ygeA::cat were prepared by the method in the title (4) of this example, and the pCP20 plasmid (Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000, 97: 6640-6645.) was introduced into the competent cells by electroporation. All of the cells were coated on LB plates containing chloramphenicol (34 ng / ml) and ampicillin (100 ng / mL), and cultured overnight (8-12 h) in a 30°C incubator. Since the pCP20 plasmid can express the FLP site-specific recombinase at 30°C, it recognizes the FRT sequences at both ends of the cat gene and cuts off the sequence in the middle, thereby achieving the purpose of eliminating the cat gene fragment in the primary recombinant bacteria.

[0073] Pick a single colony from the above plate and culture it in LB medium at 42°C for 3 generations, with each generation cultured for no less than 6 hours, and finally streak it on an LB plate without antibiotics. Pick a single colony of the secondary recombinant knockout strain (ygeA and cat genes knocked out) in 4mL LB medium, place it in a 37°C incubator with shaking and culture overnight (8-12h), take 1mL of the bacterial solution and use the boiling method to prepare the strain identification template. Establish a reaction system, take 10uL rTaq PCR MIX, 1uL each of primers F1 and R1, 2uL of identification strain template, and add ultrapure water to a total volume of 25uL. The PCR reaction conditions are: pre-denaturation at 94°C for 4min; 94°C for 1min, 53°C for 30s, 72°C for 60s, 25 cycles; extension at 72°C for 10min. Use 1.2% agarose gel electrophoresis to observe the results. From Figure 1 It can be seen that the secondary recombination knockout strain (knockout of ygeA and cat genes) produced a 220bp band by PCR, and the negative control (J5 strain) amplified a 700bp band, indicating that the cat gene was successfully eliminated, and the secondary recombination knockout strain was named J5△ygeA. The applicant also knocked out the yhbB gene based on the J5△ygeA strain according to the same method as above, and constructed the corresponding double gene deletion strain, named J5△ygeA△yhbB. Figure 3 This is a Tecnai 12 transmission electron microscope photograph of the outer membrane vesicles produced by J5△ygeA△yhbB.

[0074] Example 3 Immunoenhancing Effect of Outer Membrane Vesicles in Swine Foot-and-Mouth Disease Vaccine

[0075] The existing commercialized swine foot-and-mouth disease vaccine is mainly made by emulsifying inactivated FMDV antigen with Montanide ISA206 adjuvant of French Sybeck Company in a 1:1 ratio. The inactivated antigen of swine foot-and-mouth disease (abbreviated as FMDV) is provided by Jinyu Group. FMDV antigen + J5 strain OMVs, FMDV antigen + J5△ygeA△yhbB strain OMVs, and FMDV antigen are fully mixed and emulsified with Montanide ISA206 adjuvant in a mass ratio of 1:1 to form three groups of vaccines (Group 1, Group 2, and Group 3), of which the immunization dose of FMDV antigen is 24μg / mouse, and the immunization dose of OMVs added in Group 2 is 3.4μg / mouse. Another group immunized with FMDV antigen alone is set as Group 4. 3-week-old clean-grade female ICR mice were subcutaneously immunized with 200μL / mouse via the back of the neck, and boosted once after 14 days. Mouse sera were collected at 1 week, 2 weeks, and 4 weeks after the primary immunization. According to the instructions of the liquid-phase blocking enzyme-linked immunosorbent assay kit for swine foot-and-mouth disease type O antibodies, the serum to be tested, the positive and negative serum, and the antigen were incubated at 37°C for 90 minutes, and then transferred to the ELISA plate coated with the foot-and-mouth disease type O rabbit antibody in the kit. They were incubated at 37°C for 90 minutes, and then completely washed and patted dry. The O-type guinea pig antibody working solution was added and incubated at 37°C for 30 minutes. The rabbit anti-guinea pig IgG-HRP working solution and TMB color development solution in the kit were used for color development. After adding the stop solution, the OD450 value was detected using an enzyme reader. The positive and negative antibodies were determined according to the requirements of the kit and the antibody titer was calculated. The results are as follows Figure 2 As shown, Figure 2 The average antibody titers of mouse sera 1, 2, and 4 weeks after immunization are shown in the figure, where "J5 OMV" is the FMDV+J5OMV+ISA206 immunization group (Group 1), "J5△" is the FMDV+J5△ygeA△yhbB OMV+ISA206 immunization group (Group 2), "ISA206" is the FMDV+ISA206 immunization group (Group 3), and PBS is the FMDV immunization group (Group 4).

[0076] according to Figure 2 It can be seen that groups 1 and 2, in which OMVs were added to the vaccine, produced antibodies faster than the commercial vaccine formula group 3 and significantly increased the level of foot-and-mouth disease-specific antibodies in mouse serum (1.4-2.8 times). Among them, the J5△ygeA△yhbB strain OMV had the strongest antibody response ability, indicating that OMVs have a significant immune-enhancing effect.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An outer membrane vesicle, characterized in that: The outer membrane vesicles are from Gram-negative bacteria and lack two endogenous proteins, namely, YgeA and YhbB.

2. The outer membrane vesicle according to claim 1, characterized in that: The Gram-negative bacteria is Escherichia coli.

3. A method for preparing the outer membrane vesicles according to claim 1, characterized in that: The method comprises the steps of knocking out the ygeA and yhbB genes of Gram-negative bacteria.

4. The method according to claim 3, characterized in that: The homologous recombination fragment with upstream and downstream homologous arms of the ygeA gene at both ends and the chloramphenicol resistance gene cat in the middle is introduced into the Escherichia coli J5 strain for homologous recombination, and the ygeA gene on the chromosome of the Escherichia coli J5 strain is replaced with the chloramphenicol resistance gene. Then, the FLP site-specific recombinase of the temperature-sensitive plasmid pCP20 is used to eliminate the chloramphenicol resistance gene cat on the chromosome of the Escherichia coli J5 strain, thereby obtaining an Escherichia coli mutant strain with the ygeA gene knocked out. The homologous recombination fragment with upstream and downstream homologous arms of the yhbB gene at both ends and the chloramphenicol resistance gene cat in the middle was introduced into the obtained Escherichia coli mutant strain with the ygeA gene knocked out for homologous recombination, and the yhbB gene on the Escherichia coli chromosome was replaced with the chloramphenicol resistance gene. Then, the FLP site-specific recombinase of the temperature-sensitive plasmid pCP20 was used to eliminate the chloramphenicol resistance gene cat on the Escherichia coli chromosome, thereby obtaining the Escherichia coli mutant strain with the ygeA gene and yhbB gene knocked out.

5. The method according to claim 4, characterized in that: The bacterial mutant is a mutant lacking the chromosomal ygeA and yhbB genes through Red recombination technology.

6. The outer membrane vesicles prepared by the method according to claim 4.

7. Use of the outer membrane vesicles described in claim 1 and claim 6 in preparing vaccines.

8. The use according to claim 7, wherein the vaccine is a swine foot-and-mouth disease vaccine.

9. A method for preparing a vaccine, characterized in that: A method for preparing outer membrane vesicles comprising the method according to any one of claims 3 to 5.

10. A method for preparing a swine foot-and-mouth disease vaccine, characterized in that: Its characteristics are: A method for preparing outer membrane vesicles comprising the method according to any one of claims 3 to 5.

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