Brucella capable of generating lipoid A adjuvant and application

Abstract

The invention discloses a Brucella bacterium capable of generating a lipoid A vaccine adjuvant and an application of the Brucella bacterium. According to the invention, recombinant plasmids for expressing BacA and LpxL genes are constructed, and are transformed into Brucella through electric shock, so that a double-gene knockout strain is successfully obtained. By knocking out BacA, an ultra-long fatty acid chain of lipoid A is shortened, an organism is induced to generate an inflammatory reaction, and by knocking out LpxL, the inflammatory reaction is reduced, so that the effect of the immunologic adjuvant is achieved. LPS is extracted for in-vitro stimulation analysis, and it is found that the inflammatory response of double-gene deletion is reduced compared with that of single-gene deletion. Animal experiments show that double-gene deletion can induce an organism to generate good immune response and induce relatively high cellular immunity and humoral immunity, and a new thought is provided for research and development of gram-negative bacterium inactivated vaccines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology. Specifically, it relates to a Brucella bacterium capable of producing a lipid A vaccine adjuvant and application thereof. BACKGROUND

[0002] Brucella is a gram-negative, intracellular parasite, which belongs to the alpha-proteobacteria, rhizobiales, Brucella spp.; non-capsulated, non-spore, non-motile, with two different size circular chromosomes, but Brucella has all the genes related to flagellum synthesis. Brucella grows slowly, with very high nutritional requirements for the culture medium, and the optimum growth temperature is 37℃, and a certain concentration of carbon dioxide is required during the culture process. It usually takes 3-5 days to grow visible single colonies.

[0003] The transmission routes of Brucella are digestive tract, respiratory tract, skin and mucous membrane. The transmission of Brucella among livestock is mainly through contact with the body fluids, secretions, aborted fetuses or genital organs of infected animals during mating. Human infection with Brucella is mainly through direct contact, such as contact with pathogens through damaged skin or mucus, consumption of contaminated milk and dairy products, etc. Compared with other populations, people who are exposed to infected animals or carcasses, uterine secretions or aborted fetuses due to occupation are more likely to be infected with Brucella. In the laboratory, there are few cases of accidental infection with live vaccine strains or virulent Brucella. Currently, there is no report of human-to-human transmission.

[0004] Unlike other bacterial pathogens, Brucella does not have classic virulence factors such as capsules, plasmids, pili, exotoxins, and cytolysins. Its pathogenicity mainly manifests in its ability to escape immune system surveillance and successfully colonize in host cells after invading the host. Its pathogenicity mainly depends on the intracellular survival and proliferation ability of professional and non-professional phagocytes such as macrophages, dendritic cells, embryonic trophoblast cells, and epithelial cells. So far, the reported virulence factors of Brucella include lipopolysaccharide (LPS), type IV secretion system (T4SS), two-component regulatory system (TCS), and quorum sensing system (QS).

[0005] Lipopolysaccharide, also known as endotoxin, is a major component of the outer membrane of the cell wall of gram-negative bacteria, and plays an important role in resisting the invasion of external factors such as antibiotics and detergents. Lipopolysaccharide is located in the outer leaflet of the outer membrane of the cell wall of gram-negative bacteria, and is covalently linked by three parts, from inside to outside: lipid A, core polysaccharide and O-antigen. Lipid A is essential for the survival of gram-negative bacteria, which not only anchors LPS on the outer membrane of the bacterial cell wall, but also plays a core role in endotoxin. LPS is a typical pathogen-associated molecular pattern (PAMP), which can be recognized by a variety of receptor molecules, including the myeloid differentiation protein 2 / toll-like receptor 4 (MD2 / TLR4) signal receptor complex, cysteine aspartate specific protease (caspase)-4 and caspase-5 in humans, and caspase-11, transient receptor potential (TRP) channels and lectins in mice.

[0006] TLR4 is the most studied member of the TLR family, which recognizes lipopolysaccharide (LPS). TLR4 is located on the plasma membrane and is mainly expressed on myeloid cells, while pDCs and naive B cells do not express it. TLR4 recognizes LPS through its co-receptor myeloid differentiation factor-2 (MD-2) and CD14. Recent work on TLR4 agonists has focused on the development and evaluation of modified products such as monophosphoryl lipid A (MPLA) and glucopyranose lipid A (GLA), which are structurally related to LPS but are not highly pyrogenic and maintain strong immune-enhancing properties, thus increasing the feasibility of their clinical application. TLR agonists enhance vaccine adaptive immune responses using endogenous innate immune pathways. In the past two years, the field of adjuvants has been dominated by research on pneumonia vaccines for COVID-19 infection.

[0007] The purpose of the present study is to change the structure of lipid A by genetic modification, break the immune suppression reaction, and make lipid A act as an immune adjuvant, providing a new idea for the development of intracellular inactivated vaccines. SUMMARY

[0008] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the above problems, and to provide a construction method and application of a brucella capable of producing lipid A adjuvant.

[0009] The first object of the present application is to provide a method for constructing a Brucella deletion strain by scarless method.

[0010] The second object of the present application is to obtain a Brucella strain capable of producing a lipid A adjuvant effect.

[0011] The third object of the present application is to establish a method for extracting Brucella lipid A.

[0012] The fourth object of the present application is that the double-gene deletion strain can produce higher levels of cellular and humoral immunity, and has obvious bactericidal effect in vitro.

[0013] The above objects of the present application are achieved by the following technical solutions.

[0014] The present application discloses a Brucella bacteria capable of producing a lipid A vaccine adjuvant and application thereof. The present application constructs a recombinant plasmid expressing BacA and LpxL genes, performs electroporation into Brucella, and successfully obtains a double-gene knockout strain. By knocking out BacA, the ultra-long fatty acid chain of lipid A is shortened, and the body is induced to produce an inflammatory response. By knocking out LpxL, the inflammatory response is reduced, and the effect of an immune adjuvant is achieved. Through in vitro stimulation analysis of LPS extraction, it is found that the inflammatory response of the double-gene deletion is reduced compared with that of the single-gene deletion. Through animal experiments, it is found that the double-gene deletion can induce the body to produce a good immune response and induce higher cellular immunity and humoral immunity. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 : PCR diagram of double-gene deletion (Note: M: Marker; 1.2: positive control; 3.4: double-gene deletion)

[0016] Figure 2 : Flowchart of Brucella lipid A extraction method

[0017] Figure 3 : Establishment of lipid A thin layer chromatography method

[0018] Figure 4 : Mass spectrometry detection results

[0019] Figure 5 : In vitro stimulation of LPS inflammation results

[0020] Figure 6 : Safety evaluation of deletion strain

[0021] Figure 7 : Flow cytometry detection of CD4 and CD8, lymphocyte transformation rate

[0022] Figure 8 : Antibody in vitro bactericidal experiment

[0023] Figure 9 : Results of in vitro bactericidal rate DETAILED DESCRIPTION

[0024] The technical solutions described in the present application are conventional techniques in the art if not specifically stated; the reagents or materials described are from commercial channels if not specifically stated.

[0025] Example 1: Construction of deletion strain

[0026] 1. Construction of suicide plasmid

[0027] 1.1 Enzymatic digestion of suicide plasmid and fusion fragment

[0028] (1) Enzyme digestion of DNA using Thermo Fisher's endonuclease, reaction system as follows:

[0029]

[0030] (2) The reaction system was established according to the above table, and after fully mixing, it was placed on the enzyme cutter and incubated at 37°C for 30 min.

[0031] 1.2 Purification and recovery

[0032] Enzymatic digestion reaction system added with equal volume of Buffer PS, subsequent purification and recovery method same as 2.2.4 DNA purification and recovery.

[0033] 1.3 Ligation reaction

[0034] (1) The molar ratio of exogenous fragment and vector cloning fragment was controlled at 5:1 for optimal use of T4 DNA ligase, and the reaction system was as follows:

[0035]

[0036] The optimal molar ratio of the amount of vector to the inserted fragment was 3:1-10:1.

[0037] (2) The reaction system was prepared according to the above table. Incubate at 22°C for 1 h, and take out 5 μL of the mixture for chemical transformation.

[0038] 1.4 Preparation method of E. coli chemical sensitivity

[0039] Prepare the reagents and sterilized centrifuge tubes in advance: liquid LB, sterilized 100 mmol / L CaCl2 and 2 mol / L MgCl2, sterile glycerol, sterile bacteria bottles, 1.5 mL sterile centrifuge tubes (labelled and placed in -20°C for 24 h pre-cooling), 50 mL centrifuge tubes.

[0040] (1) Take the frozen E. coli DH5a strain from -80°C refrigerator, inoculate with sterilized loop, and streak on solid LB medium without antibiotics. Incubate at 37°C for 12-16 h. Pick a single colony that grows well and inoculate in a bacterial flask containing 5 mL liquid LB medium without antibiotics. Incubate at 37°C with shaking at 180 r / min for 12 h.

[0041] (2) Transfer the appropriate amount of activated bacterial liquid to a conical flask containing 50 mL LB on a sterile clean bench. Add 2 mol / L MgCl2 to a final concentration of 10 mmol / L, and continue to incubate at 37°C with shaking at 180 r / min until the OD 600 value reaches 0.4-0.6. Under sterile conditions, transfer the bacterial liquid to a 50 mL centrifuge tube pre-cooled with ice, and place on ice for 30 min.

[0042] (3) Centrifuge at 2 500 x g at 4°C for 5-10 min.

[0043] (4) Discard the supernatant, and add 1 / 5 volume of 100 mmol / L CaCl2 pre-cooled with ice to the precipitate. Gently shake to resuspend the bacterial cells. Centrifuge at 5 000 r / min at 4°C for 10 min.

[0044] (5) Discard the supernatant, and add 4 mL of 100 mmol / L CaCl2. Gently shake to mix the bacterial cells evenly. After dilution, centrifuge at 5 000 r / min at 4°C for 10 min.

[0045] (6) Discard the supernatant, and resuspend in 100 mmol / L CaCl2 solution containing 7% (v / v) DMSO. Quickly aliquot 50 μL per tube into 1.5 mL centrifuge tubes prepared in advance. Store at -80°C for later use.

[0046] 1.5 Chemical transformation of E. coli

[0047] (1) Take the E. coli competent cells from -80°C refrigerator, and place on ice until thawed. In a clean bench, add <10 μL ligation product to the centrifuge tube containing 50 μL competent cells, mix gently, and place on ice for 30 min.

[0048] (2) Place the centrifuge tube in a 42°C water bath for 43 sec, and quickly place the centrifuge tube in an ice bath for 2 min.

[0049] (3) In a clean bench, add 400 μL LB medium without antibiotics to the centrifuge tube, and incubate at 37°C with shaking at 180 r / min for 1 h.

[0050] (4) After the end of the recovery, the bacterial solution is carefully aspirated with a pipette in a clean bench, and a suitable volume of the solution is spread on a plate containing the desired antibiotic. After the solution has dried, the plate is inverted and incubated in a 37°C incubator for 12-16 hours.

[0051] (5) In a clean bench, a single colony is picked with a sterilized gun tip and spread on another plate to make a master plate. After incubation at 37°C for 12 hours, the bacterial lawn is picked with a sterilized gun tip and inoculated into 5 mL of LB medium containing the desired antibiotic. After incubation at 37°C and 180 r / min for 12 hours, the bacterial cells are collected and subjected to plasmid extraction.

[0052] 1.6 Extraction of E. coli plasmid

[0053] The plasmid is extracted using a plasmid extraction kit, and the steps are as follows:

[0054] (1) Column equilibration step: 500 μL of equilibration solution BL is added to the adsorption column CP3 (the adsorption column is placed in a collection tube), and centrifuged at 12,000 rpm (~ 13,400 x g) for 1 min. The waste liquid in the collection tube is discarded, and the adsorption column is placed back into the collection tube.

[0055] (2) 1-5 mL of bacterial solution from overnight culture is added to a centrifuge tube, and centrifuged at 12,000 rpm (~ 13,400 x g) for 1 min using a conventional benchtop centrifuge. The supernatant is carefully aspirated.

[0056] (3) 250 μL of solution P1 is added to the centrifuge tube containing the bacterial pellet, and the bacteria are thoroughly suspended using a pipette or vortex.

[0057] (4) 250 μL of solution P2 is added to the centrifuge tube, and the bacteria are gently inverted 6-8 times to fully lyse the bacteria.

[0058] (5) 350 μL of solution P3 is added to the centrifuge tube, and the mixture is immediately gently inverted 6-8 times to fully mix. At this time, a white flocculent precipitate will appear. Centrifuge at 12,000 rpm (~ 13,400 x g) for 10 min.

[0059] (6) The supernatant collected in the previous step is transferred to the adsorption column CP3 using a pipette, taking care not to aspirate the precipitate. Centrifuge at 12,000 rpm (~ 13,400 x g) for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube.

[0060] (7) Add 600 μL of rinse solution PW (check if anhydrous ethanol has been added first) to the adsorption column CP3, centrifuge at 12 000 rpm (~ 13 400 x g) for 30-60 sec, discard the waste in the collection tube, and place the adsorption column CP3 in the collection tube.

[0061] (8) Repeat operation step 7.

[0062] (9) Place the adsorption column CP3 in the collection tube, centrifuge at 12 000 rpm (~ 13 400 x g) for 2 min, in order to remove the residual rinse solution from the adsorption column.

[0063] (10) Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μL of elution buffer EB to the middle of the adsorption membrane, let stand at room temperature for 2 min, centrifuge at 12 000 rpm (~ 13 400 x g) for 2 min to collect the plasmid solution in the centrifuge tube, and store at -20 °C for later use.

[0064] 1.7 Enzymatic verification of the suicide plasmid

[0065] (1) Perform enzymatic verification of the suicide plasmid using the restriction enzymes from Thermo Fisher, with the following reaction system:

[0066]

[0067] (2) Set up the reaction system according to the table above, mix thoroughly, and place on the enzyme cutter, incubate at 37 °C for 30 min. After the reaction is completed, use agarose gel to detect and identify.

[0068] 1.8 Sequencing

[0069] Results: The upstream and downstream homologous fragments of the target gene obtained by fusion PCR were amplified by fusion PCR, and the agarose gel electrophoresis result showed that the size of the fusion product was about 1997 and 2320 bp, which met the expectation. After the fusion fragments were ligated with the pWF-SK2 suicide plasmid, the recombinant suicide plasmids pSK2-BacA and pSK2-LpxL were obtained, and the restriction enzyme was used for enzyme cutting plasmid identification, and the enzyme cutting result showed that the fragment size was correct. The correctly constructed suicide plasmid was sent to the company for sequencing to ensure that there was no gene mutation in the upstream and downstream homologous fragments of the constructed suicide plasmid.

[0070] 2. Electroporation of the plasmid and identification

[0071] The above plasmids were transformed into the Brucella competent cells, respectively, and the specific operation steps were as follows:

[0072] (1) Take the prepared competent cells from the -80 °C refrigerator, and melt them in an ice-water mixture;

[0073] (2) Add 5 μL of recombinant plasmid pSK2-BacA to each of the pre-prepared 1.5 mL centrifuge tubes containing competent cell lines. Mix thoroughly by tapping the bottom of the tube, and immediately place the tube on ice. Place the tube on an ice-water mixture for 10 minutes. Quickly transfer the competent cells and plasmid mixture to a 0.1 cm ice-warmed electroporation cuvette using a pipette tip. Cover the cuvette and keep the empty tube for later use. When adding the competent cells to the electroporation cuvette, avoid generating air bubbles, as air bubbles increase the risk of arc discharge.

[0074] (3) Set the parameters of the electroporator: voltage 2200V / cm, add 1mL of TSB liquid medium to mix the cells thoroughly (add TSB medium quickly after electroporation), and transfer to a 1.5mL EP tube. Note: The volume of plasmid added should not exceed 1 / 10 of the competent cell volume;

[0075] (4) After preheating at 37℃, transfer to a shaker at 200r / min for 8h of incubation and activation.

[0076] (5) Spread the bacterial suspension onto TSA agar plates containing Kan resistance, one plate for every 200 μL;

[0077] (6) Place the plate in a 37℃ constant temperature incubator until the liquid is completely absorbed, invert the plate, and observe the results after 72-96 hours of incubation.

[0078] (7) Once the bacteria have grown, pick a single colony for bacterial PCR;

[0079] (8) The bacterial solution with the correct test result is then spread on a sucrose plate for double exchange. The plate is then inverted and incubated for 72-96 hours before the results are observed.

[0080] (9) Select single colonies for BacA identification and gene deletion detection. Continuously passage gene-deleted strains and select colonies for gene detection and sequencing verification. After the sequencing results are verified to be correct, Brucella ΔBacA is obtained. Simultaneously, amplification culture is performed, and the bacteria are freeze-dried and stored at -80℃.

[0081] (10) Based on Brucella ΔBacA, LpxL was further deleted, competent cells were prepared, plasmids were transferred, and the deletion strain was identified.

[0082] (11) Select single colonies for lpxL identification and gene deletion detection. Continuously passage gene-deleted strains and select colonies for gene detection and sequencing verification. After the sequencing results are verified to be correct, Brucella ΔBacA-LpxL is obtained. Simultaneously, it is expanded into a large-scale culture and freeze-dried at -80℃.

[0083] The results showed that the PCR detection results of the Brucella double-gene deletion strain were as follows: Figure 1As shown, 1.2 is a positive control for detecting BacA and LpxL, 3.4 is a double gene deletion strain, and the target gene is detected by PCR. Finally, a strain of ΔBacA-LpxL Brucella is obtained.

[0084] Example 2: Lipid A extraction of Brucella

[0085] 1.1, LPS extraction

[0086] Brucella was inoculated in TSB medium and cultured at 37°C to the early exponential phase, and then heat-inactivated at 80°C for 2h. The bacterial body was collected and used to extract Brucella LPS according to the instructions of the bacterial LPS extraction kit.

[0087] (1) 50mL of the collected bacterial body precipitate was added with 1mL of lysis buffer and mixed well by vortexing.

[0088] (2) 200μL of chloroform was added and mixed well by vortexing for 10-20sec, and then incubated at room temperature for 5min.

[0089] (3) Centrifugation at 13 000rpm for 10min at 4°C, and then 400μL of supernatant was transferred to a new 1.5mL centrifuge tube.

[0090] (4) 800mL of purification buffer was added and mixed well. Incubation at -20°C for 10min.

[0091] (5) Centrifugation at 13 000rpm for 15min at 4°C.

[0092] (6) The LPS particles were washed with 1mL of 70% anhydrous ethanol and completely dried.

[0093] (7) The remaining ethanol was evaporated in a 56°C metal bath, and then stored at -80°C.

[0094] 1.2, Lipid A extraction

[0095] The extracted LPS was precipitated with 2 volumes of anhydrous ethanol, and then resuspended in 0.5mL of 12.5mM sodium acetate solution (pH 4.5). Ultrasonic oscillation was performed for 10min, and then treated in a 100°C water bath for 30min.

[0096] The volume of the aqueous solution was adjusted, and then chloroform and methanol were added in a ratio of water: chloroform: methanol = 1.8:2:2. Centrifugation was performed at 4000rpm for 10min, and then the lower phase was collected and dried by nitrogen blowing. Storage at -20°C.

[0097] 1.3, Thin layer chromatography

[0098] Lipid A samples can be observed by thin layer chromatography (TLC). The developing solvent is prepared in a chromatography jar according to the following formulation: chloroform:methanol:H20:ammonia = 40:25:4:1; v:v:v:v. After the jar is covered, it is allowed to stand for 6 h or more to allow the developing solvent to fill the jar. If the standing time is too short, it will affect the TLC results. The Lipid A sample is dissolved in chloroform / methanol (4:1; v:v) solution, and an appropriate amount of sample is taken up with a capillary tube and spotted on a silica gel TLC plate. After drying, the plate is placed in the developing jar, and the silica gel plate is removed when the plate reaches the top of the jar. The plate is dried, sprayed with 10% sulfuric acid in ethanol, and dried. The plate is heated at 180 °C until the silica gel plate develops color.

[0099] 1.4. MALDI-TOF / TOF-MS mass spectrometry method

[0100] The analysis of Lipid A is performed in MALDI-TOF / TOF MS (matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry) in negative ion mode. The Lipid A is reconstituted in 100 μL of chloroform / methanol solution (2:1, v / v)

[0101] and 1 μL of sample is spotted onto a MALDI sample plate, followed by 1 μL (20 mg / mL) of matrix solution dissolved in chloroform / methanol / water (3:1.5:0.25, v / v / v). The Bruker Autoflex Speed MALDI-TOF / TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) is used in this study. The intensity of the laser is 500 shots and 50% laser power. For MS / MS analysis, the LIFT mode is used. Based on the MS spectrum, the parent ion is selected, and MS / MS analysis is performed in the LIFT TOF / TOF mode. The ES Tuning Mix (Agilent, Palo Alto, CA, USA) standard is used for instrument calibration. In this study, de Norharman (9-hydro- pyrido[3,4-b]indole), CMBT, DHB, and THAP

[0102] are compared, and it is found that 9-hydro-pyrido[3,4-b]indole is the best, greatly improving the sensitivity and resolution of phospholipid and Lipid A analysis. In the following experiments, if not otherwise specified, the MALDI matrix used is 9-hydro-pyrido[3,4-b]indole.

[0103] The results show that:

[0104]

[0105] Figure 2 ​​The present experiment establishes a safe and rapid method for extracting Brucella lipids, which reduces bacterial culture and inactivation. By optimizing the resuspension concentration, resuspension system, and color development concentration, a thin-layer chromatography method for Brucella lipids A is established, as shown in Figure 3 . The mass spectrometry analysis results of lipid A are shown in Figure 4 . Mass spectrometry analysis confirms that the fifth fatty acid chain and part of the ultra-long fatty acid chain in Brucella are specifically removed, and the results show that the lipopolysaccharide structure of Brucella has been successfully modified.

[0106] Example 3: LPS in vitro stimulation experiment

[0107] 2. LPS in vitro stimulation experiment

[0108] (1) Quantitative analysis of extracted LPS, concentration is 100 ng / mL, 5 × 10 5 RAW264.7 cells are inoculated in a 12-well plate, 1 ml per well. The control group adds 200 μL of cell culture solution, and the experimental group adds 200 μL of LPS solution with different concentrations to make the final concentration of LPS 100 ng / ml. Incubate at 37°C, 5% CO2 for 24 h.

[0109] (2) Collect cell samples at 24 h of culture and detect cell inflammatory response.

[0110] The results show that: Figure 5 The TLR4 protein level produced by ΔBacA-LpxL deletion strain LPS compared to Brucella ΔBacA and E. coli LPS is significantly reduced, and QPCR detection of IL-6 and TNF-γ is also lower than that of Brucella ΔBacA and E. coli LPS. ΔBacA-LpxL deletion strain LPS significantly reduces inflammatory response.

[0111] Example 4: Safety evaluation

[0112] 1.1. Bacterial resuscitation and inactivation

[0113] (1) Take the gene deletion strain from the -80°C refrigerator, after thawing, inoculate in TSA plate with four zone lines, and incubate at 28°C, 5% CO2 for 96 h.

[0114] (2) Pick a single edge neat and round single colony and inoculate in a 50 mL centrifuge tube containing 20 mL TSB medium, and place it in a constant temperature incubator at 28°C, 200 r / min for 36 hours as a primary seed. Similarly, cultivate 80 mL of bacterial solution.

[0115] (3) Add β-propiolactone solution to the bacterial solution respectively, so that the final concentration is 0.2%, and inactivate at 37°C for 4 h. Take samples according to

[0116] (4) After inactivation is completed, 0.1 mL of the bacterial solution is sampled and inoculated into 3 TSB culture mediums, each with 0.2 mL; 3 TSA flat plates are coated with the inoculum using a disposable "L" aid, each with 0.1 mL, and placed in a 37°C incubator for 5-7 days, and whether there is growth of colonies is observed.

[0117] (5) Since Brucella is a gram-negative bacterium, its cell wall contains a lipopolysaccharide (LPS), known as endotoxin, which is released when the bacteria die or autolyze. In order to preserve the content of LPS, low-speed centrifugation is performed at 4000 r / min for 20 min.

[0118] 2. Animal experiment

[0119] (1) The safety of the gene deletion inactivated bacterial solution is evaluated using C57 mice inoculation, 4-6 weeks old, divided into 4 groups, 5 in each group.

[0120] (2) The 4 groups of mice are inoculated with wild type, ΔBacA-LpxL deletion strain, E. coli, and physiological saline, respectively, with a bacterial content of 1x10 10 CFU) before inactivation.

[0121] (3) All mice are inoculated subcutaneously on the neck, 200 μl per mouse.

[0122] (4) The injection site is observed for 7 consecutive days for redness, swelling, and ulceration.

[0123] (5) After 14 days of inoculation, all mice are dissected by CO2 asphyxiation, and the spleen is removed under sterile conditions.

[0124] (6) The spleen of all mice is observed for pathological changes and weighed, and the organ coefficient is calculated.

[0125] The results show that after guinea pigs were subcutaneously inoculated with ΔBacA-LpxL deletion antigen (containing 1x10 10 CFU), the injection site was observed for 7 consecutive days and no inflammatory reactions such as skin redness and edema were observed. After 14 days of inoculation, the mice were asphyxiated with CO2, and the spleen was observed and weighed.

[0126] The ΔBacA-LpxL deletion group, PBS group, and WT group showed no visible pathological changes in the spleen, as shown in Table 1. Figure 6

[0127] Example 5: Detection of mouse immune level

[0128] ​The immune level of mice was evaluated by inoculating C57 mice, 4-6 weeks old, divided into 4 groups, 5 in each group. The mice were divided into 5 groups, wild type (WT), wild type plus adjuvant (WT+), ΔBacA-LpxL (BL), ΔBacA-LpxL strain plus adjuvant (BL+), and negative control group. Immunization was performed at week 0, with a booster two weeks later, and the mice were killed for detection at week 6. Cell immunity and humoral immunity were detected.

[0129] 1. CD3+, CD4+, CD8+ T cell subpopulation count

[0130] (1) 14 days after the booster immunization, the guinea pig peripheral blood of the vaccine immunization group was collected, and the lymphocytes were separated and extracted using a peripheral blood lymphocyte separation kit, according to the instructions of the guinea pig peripheral blood lymphocyte separation kit.

[0131] (2) Centrifugation at 3000 r / min for 5 minutes, and washing the cells with sterile PBS.

[0132] (3) After washing, the cells were mixed with CD3, CD4, and CD8 monoclonal antibodies, respectively, and incubated at room temperature for 30 minutes in the dark, and then detected by flow cytometry.

[0133] 2. Lymphocyte transformation rate detection

[0134] The proliferation of lymphocytes was evaluated by the cck-8 method. Briefly, fresh anticoagulated peripheral blood samples of 5 mice in each group (1.0 mL / mouse) were collected, and mixed with 1.0 mL of 1640 culture solution, respectively. Then, 2 mL of the mixture was added to 5 mL of lymphocyte separation medium, and the lymphocytes in the middle layer of the white flocculent material were separated and collected by centrifugation at 1500 rpm / min and 4°C for 20 min, and then washed twice. The density of the lymphocytes was adjusted to 1×10 6 / m L, and before being added to the 96-well plate, 1640 cell culture solution was first added to the edge of the 96-well plate. To avoid edge effects and interfere with the test results, the adjusted lymphocyte suspension was then added to each well of the 96-well cell culture plate at 100 μL per well. Each sample was repeated in 6 wells in parallel, and 5 μg / mL of Con A solution was added to the first 3 wells, while 1640 culture solution was added to the last 3 wells as a control. The 96-well plate was sealed and labeled, and then placed in a incubator at 37°C in CO2 for 12 hours or more. The growth state of the cells was observed, and after the bottom layer of the cell culture plate was covered with cells, 10 was added to each well to make the final concentration of MTT 5 mg / mL. Incubation was performed for 4 hours, the culture solution was discarded, and 100 μL of DMSO was added to each well. After mixing, the OD value at 490 nm was detected by a microplate reader.

[0135] 3. Antibody killing test of Brucella

[0136] (1) Positive mouse serum (tuberculin test detected titer 1:400), negative mouse serum, positive complement inactivated mouse serum (56°C for 30 minutes), PBS buffer 200 μL were added to 1.5 mL sterile centrifuge tubes, respectively. 10 CFU / ml, vortexed, and placed in a 37°C constant temperature incubator for incubation.

[0137] (3) After 8 hours of culture, sample was taken for viable cell counting, and the bactericidal rate was calculated by comparing the number of viable cells between the serum treatment group and the PBS control group.

[0138] (4) The bactericidal rate calculation formula is: viable cell count after PBS incubation - viable cell count after serum incubation original bacterial liquid viable cell count x 1 / 2 x 100%. To study the changes of cell immunity caused by lipid A, the peripheral blood of all mice was collected after immunization, and the percentages of CD4+ T cells and CD8+ lymphocytes were detected by flow cytometry. Compared with the wild type, the proliferation ability of CD4+ and CD8+ T cells of the ΔBacA-LpxL strain was significantly enhanced (P<0.05). The lymphocyte transformation rate was also higher. To study the level of humoral immunity, the specific antibody in vitro bactericidal test was carried out. Under in vitro conditions, the ΔBacA-LpxL serum had strong bactericidal effect before and after complement inactivation. The bactericidal effect of wild type serum was not obvious, and there was a significant difference between ΔBacA-LpxL (containing positive serum after inactivation) and wild type as shown in Figure 8 、 9 .

Claims

1. A Brucella bacterium capable of producing adjuvanted lipid A, wherein, The bacteria include a deletion of expression of a gene encoding BacA and LpxL compared to a parental bacterial cell.

2. The bacterium of claim 1, wherein, The mutation is a deletion, and the deletion of the gene of interest is performed using scarless methods.

3. According to claim 2, BacA and LpxL double gene deletion.

4. The bacterium of claim 1, wherein, The bacteria produce adjuvanted lipid A without the need for induction of expression by expression of an inducer, expression of an inducer stimulus, or a combination thereof.

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