Brucella abortus S19 strain outer membrane vesicle as well as preparation method and vaccine thereof
By adding horse serum to bovine Brucella cultures and subjecting them to specific treatments, the problem of low OMV production was solved, and a highly effective Brucella vaccine was prepared for the prevention of brucellosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-13
- Publication Date
- 2026-04-14
AI Technical Summary
The production of existing bovine Brucella OMVs is low, and they are susceptible to infection during growth, making it difficult to prepare highly effective Brucella vaccines.
By adding horse serum during Brucella culture, combined with specific centrifugation and filtration steps, outer membrane vesicles of Brucella bovis S19 strain were extracted and prepared into a vaccine, thereby improving the yield and purity of OMVs.
The efficient extraction and concentration of outer membrane vesicles of Brucella S19 strain were achieved, and a safe and effective vaccine was prepared, which can significantly prevent brucellosis and has good safety and application value.
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Figure CN121852230A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of vaccine preparation technology, specifically relating to an outer membrane vesicle of Brucella bovis S19 strain, its preparation method, and a vaccine. Background Technology
[0002] Brucellosis is a chronic zoonotic infectious disease caused by Brucella bacteria. It is prevalent in more than 170 countries and regions worldwide and is one of China's major public health problems. Susceptible animals include more than 60 species of livestock and wild animals. When animals suffer from brucellosis, female animals exhibit decreased milk production, abortion, arthritis, and endometritis; male animals mainly exhibit epididymitis and orchitis. In short, brucellosis has a significant impact on social and economic development. Currently, our understanding of the pathogenesis of brucellosis infection is limited, and preventive measures cannot completely control the occurrence of the disease, making brucellosis a major problem in the pig farming industry. Developing safe and effective vaccines is currently the primary task in controlling brucellosis. Currently reported types of brucellosis vaccines include inactivated vaccines, subunit vaccines, DNA vaccines, and live attenuated vaccines. The vaccines currently in use are mainly live attenuated vaccines, which pose a risk of infection to humans and animals during use, becoming a major obstacle to the widespread adoption of vaccines. These vaccines also have drawbacks such as high virulence, a tendency to cause abortion, and relatively low protective efficacy. In light of the successful development of Neisseria meningitidis outer membrane vesicle (OMV) vaccines, this invention explores methods for preparing Brucella OMVs.
[0003] Commonly used brucellosis vaccines in my country include M5-90Δ26, S2, Rev.1, S19, and A19, all of which are live attenuated vaccines. Although existing vaccines can control the spread of brucellosis, the high virulence of live vaccines, which can easily cause abortions in vaccinated animals and even infection of related personnel, means that a low-virulence, highly effective vaccine is still lacking. Neisseria meningitidis outer membrane vesicles (OMVs) have been successfully developed into an officially approved vaccine for human use, which has inspired research on outer membrane vesicle vaccines for many pathogenic Gram-negative bacteria. Furthermore, OMVs have high thermal stability and are relatively safe for immunization, making their vaccine development possible.
[0004] However, while there is a theoretical basis for the efficacy of bovine Brucella OMVs in the current industry, there are significant shortcomings in their actual synthesis and production. OMVs have a slow growth rate and are susceptible to infection during the growth process, and it is still not possible to obtain high-performance bovine Brucella OMVs that are easy to prepare.
[0005] Therefore, overcoming the low yield of bovine Brucella OMVs is a technical problem that urgently needs to be solved in this field.
[0006] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention
[0007] This disclosure provides at least one embodiment of Brucella bovis S19 outer membrane vesicle, its preparation method, and a vaccine. The invention aims to increase the yield of Brucella oMVs by adding horse serum during culturing, while simultaneously concentrating the vesicles to formulate a vaccine. This allows for convenient, rapid, and large-scale extraction of Brucella S19 outer membrane vesicles, thereby enabling the industrialization of Brucella S19 OMV vaccines and achieving effective prevention of brucellosis.
[0008] In a first aspect, the present disclosure provides a method for preparing outer membrane vesicles of Brucella bovis strain S19, characterized by comprising the following steps: S1, inoculating Brucella bovis strain S19 into TSB medium and adding 3-5% horse serum by weight, culturing on a shaker for at least 48 hours, then inoculating the entire bacterial culture into fresh TSB medium and culturing on a shaker for at least 72 hours, as a primary working seed for production; S2, inoculating the primary working seed for production onto the medium and culturing until pure. Add peptone water to the culture medium, wash off the bacterial growth, and inoculate it into a seed tank containing TSB medium. Aerate and culture to obtain secondary working seed for production. S3: Add antifoaming agent to the culture medium, sterilize, and add 1-2% of the secondary working seed culture medium volume. Ferment and culture, gradually increasing the aeration rate, and harvest the culture medium. S4: Centrifuge the cultured culture medium at 2-8℃, take the supernatant, filter it through a 0.45µm filter membrane, and then ultracentrifuge to obtain a precipitate. S5: Resuspend the precipitate in PBS buffer and filter it through a 0.22µm filter membrane to obtain Brucella outer membrane vesicles, namely Brucella bovis S19 strain outer membrane vesicles.
[0009] In one optional embodiment, the primary working seed used in production in S1 is inoculated onto TSA medium plates and incubated at 36–38°C for 48–72 hours. The plates are then subjected to heat agglutination test, acridinium agglutination test, and crystal violet staining. The colonies should exhibit smooth colony characteristics, without agglutination or staining.
[0010] In one optional embodiment, the temperature of the shaker culture in step S1 is 36-37°C.
[0011] In an optional embodiment, step S3 further includes adding a 50% glucose solution and adjusting the pH value to 6.8–7.2.
[0012] In one optional embodiment, in step S4, the centrifugation speed is not less than 10,000 rpm and the duration is not less than 15 minutes; the ultracentrifugation speed is not less than 45,000 rpm and the duration is not less than 2 hours.
[0013] Secondly, this disclosure also provides an outer membrane vesicle of Brucella bovis S19 strain, which is secreted by Brucella bovis S19 strain and obtained by culturing, activating, expanding culture and extraction as described above.
[0014] In one alternative embodiment, the outer membrane vesicles are vesicle-like bodies with a double-membrane structure, carrying bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens, and pathogen-associated molecular patterns.
[0015] In one optional embodiment, the outer membrane vesicles have a particle size of no more than 200 nm, and their protein band sizes are concentrated in the range of 15–200 kDa.
[0016] Thirdly, embodiments of this disclosure also provide a vaccine for the prevention of brucellosis, comprising outer membrane vesicles of Brucella bovis S19 strain as described above, wherein the concentration of the outer membrane vesicles is 0.05–20 mg / ml.
[0017] Fourthly, this disclosure also provides the application of the outer membrane vesicles of Brucella bovis S19 strain as described above in the preparation of a medicament for the prevention / treatment of brucellosis.
[0018] The beneficial effects of this invention are that the outer membrane vesicles of Brucella S19 strain of bovine breed and their preparation method, and the vaccine, for the first time, utilize the outer membrane vesicles extracted from Brucella cultured with horse serum as a vaccine for animal immunization. Animal experiments have demonstrated that the Brucella outer membrane vesicles extracted by this invention have a significant effect in preventing brucellosis. They can be used to prepare products for the prevention of brucellosis, and thus have good safety and practical application value.
[0019] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.
[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description
[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0022] Figure 1 This is a flowchart illustrating the preparation process of outer membrane vesicles (OMVs) provided in embodiments of this disclosure. Figure 2 Transmission electron microscopy image of Brucella S19 OMVs in Example 3 provided for embodiments of this disclosure; Figure 3 A standard curve of protein concentration-absorbance of Brucella S19 strain OMVs provided in Example 3 of this disclosure; Figure 4 SDS-PAGE electrophoresis image of Brucella S19 strain OMVs in Example 3 provided in this disclosure; Figure 5 The particle size distribution of Brucella S19 OMVs in Example 3 provided in this disclosure is shown in the figure. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] As used herein, the phrases “in one embodiment,” “according to one embodiment,” “in some embodiments,” etc., generally refer to the fact that a particular feature, structure, or characteristic following the phrase can be included in at least one embodiment of this disclosure. Therefore, a particular feature, structure, or characteristic can be included in more than one embodiment of this disclosure, such that these phrases do not necessarily refer to the same embodiment. As used herein, the terms “example,” “exemplary,” etc., are used to “serve as an example, instance, or illustration.” Any implementation, aspect, or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or superior to other implementations, aspects, or designs. Rather, the use of the terms “example,” “exemplary,” etc., is intended to present concepts in a specific manner.
[0025] In this document, exemplary embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings. As used herein, expressions such as “at least one of…” modify the entire list of elements when following a list of elements, rather than individual elements in the list. For example, the expression “at least one of a, b, and c” should be understood to include only a, only b, only c, both a and b, both a and c, both b and c, or all of a, b, and c.
[0026] The terminology used herein is for the purpose of describing specific exemplary configurations only and is not intended to be limiting. As used herein, the singular articles “a,” “an,” and “the” may also be intended to include plural forms unless otherwise clearly stated herein. The terms “comprising,” “including,” and “having” are inclusive and thus specify the presence of features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein should not be construed as requiring them to be performed in the specific order discussed or shown, unless specifically identified as such. Additional or alternative steps may be employed.
[0027] Horse serum is composed of water, proteins, carbohydrates, amino acids, cytokines, and inorganic salts. Proteins are a abundant component of horse serum, playing a crucial role in cell and bacterial growth and differentiation, and thus potentially indirectly affecting OMV (Oxygen-Modified Viruses) production. Horse serum also contains various growth factors, such as insulin-like growth factor (IGF) and transforming growth factor-β (TGF-β), which promote growth and division. Furthermore, horse serum contains antioxidants and antibacterial substances that protect cells and bacteria from damage and infection. Simultaneously, certain amounts of lipids and cholesterol also play important roles in cell membrane construction and function. Compared to sheep and bovine serum, culture media supplemented with horse serum are easier to dispense and store.
[0028] The conclusions drawn from the above concepts are all results obtained by the inventors after practice and careful research. Therefore, the discovery process of the above problems and the solutions proposed in this disclosure below should be considered as contributions made by the inventors to this disclosure.
[0029] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.
[0030] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.
[0031] The Brucella abortus strain S19 used in this application was produced and tested in Ames by the National Veterinary Services Laboratory (NVSL) of the U.S. Department of Agriculture (USDA). It meets the USDA's proven quality control standards (lowa, USAin), reagent catalog code 15, and the full name of the reagent is Brucella culture - Brucella abortusstrain 19 original seed. Culture used for the production of Brucella abortusstrain 19 vaccine.
[0032] This disclosure provides a method for preparing outer membrane vesicles of Brucella bovis strain S19, characterized by the following steps: S1, inoculating Brucella bovis strain S19 into TSB medium and adding 3-5% horse serum by weight, culturing on a shaker for at least 48 hours, then inoculating the entire bacterial culture into fresh TSB medium and culturing on a shaker for at least 72 hours, as a primary working seed for production; S2, inoculating the primary working seed for production onto the medium and culturing until purified. Add peptone water to wash off the bacterial growth and inoculate it into a seed tank containing TSB medium. Aerate and culture to obtain secondary working seed for production. S3: Add antifoaming agent to the medium, sterilize, and add 1-2% of the secondary working seed bacterial solution to the medium volume. Ferment and culture, gradually increasing the aeration rate, and harvest the bacterial solution. S4: Centrifuge the cultured bacterial solution at 2-8℃, take the supernatant and filter it through a 0.45µm filter membrane, and then ultracentrifuge to obtain the precipitate. S5: Resuspend the precipitate in PBS buffer and filter it through a 0.22µm filter membrane to obtain Brucella outer membrane vesicles, namely Brucella bovis S19 strain outer membrane vesicles.
[0033] In some embodiments, specifically, in step S1, the primary working seed used for production is inoculated onto TSA medium plates and incubated at 36–38°C for 48–72 hours. The plates are then subjected to heat agglutination tests, acridinium agglutination tests, and crystal violet staining. The colonies should exhibit smooth colony characteristics, without agglutination or staining.
[0034] Specifically, the heat agglutination test is performed as follows: Brucella is inoculated onto TSA slant agar and incubated at 36–38°C for 48–72 hours. The culture is then transferred to a test tube containing approximately 10 ml of physiological saline, shaken well, and turbidified to obtain a bacterial suspension containing 1.0 × 10⁹ CFU of viable bacteria per ml. The suspension is then divided into two tubes, each containing 4–5 ml, and heated in a 90°C water bath for 1 hour. The results are determined at 30 minutes and 60 minutes. If significant agglutination occurs at the bottom of the tube, it indicates rough-type Brucella; if no agglutination occurs, it indicates smooth-type Brucella.
[0035] Specifically, the acridine yellow agglutination test: Place one drop of a 1:500 acridine yellow aqueous solution on a clean glass slide, then add one drop of the above bacterial suspension containing 1.0 × 10⁹ CFU of viable bacteria per ml (or use a platinum ear to extract a small amount of 48-hour Brucella culture and gently grind it evenly). Mix the two thoroughly and observe the results immediately. If agglutination occurs within 2–3 minutes, it indicates rough Brucella; if no agglutination occurs, it indicates smooth Brucella.
[0036] Specifically, for crystal violet staining: Cover the entire surface of an agar plate with a diluted staining solution, staining for 15-20 seconds. Then pour the staining solution into a disinfectant solution and immediately examine the colonies using a magnifying glass or microscope. Smooth colonies do not stain, have neat, rounded edges, and are yellowish-green; rough colonies are stained red, blue, purple, or other colors, with irregular, rough edges, sometimes with cracks. The crystal violet stock solution is prepared as follows: Solution A: 2.0g of crystal violet dissolved in 20ml of anhydrous ethanol; Solution B: 0.8g of ammonium oxalate dissolved in 80ml of water for injection. Mix the two solutions to obtain the stock solution. Before use, dilute the stock solution 40 times with water for injection.
[0037] In some embodiments, specifically, the temperature of the shaker culture in step S1 is 36-37°C.
[0038] In some embodiments, specifically, step S3 further includes adding a 50% glucose solution and adjusting the pH value to 6.8–7.2.
[0039] In some embodiments, specifically, in step S4, the centrifugation speed is not less than 10,000 rpm and the duration is not less than 15 minutes; the ultracentrifugation speed is not less than 45,000 rpm and the duration is not less than 2 hours.
[0040] This disclosure also provides an outer membrane vesicle of Brucella bovis S19 strain, which is secreted by Brucella bovis S19 strain and obtained by culturing, activating, expanding culture and extraction as described above.
[0041] In some embodiments, specifically, the outer membrane vesicles are vesicle-like bodies with a double-membrane structure, carrying bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens, and pathogen-associated molecular patterns; wherein the outer membrane components can stimulate the body to produce adaptive immune memory, the contained LPS can act as an adjuvant, and it has a certain degree of safety as a non-replicating vaccine.
[0042] In some embodiments, specifically, the outer membrane vesicles have a particle size of no more than 200 nm, and their protein band sizes are concentrated in the range of 15–200 kDa.
[0043] Specifically, the composition of OMVs makes them important factors in activating the host's innate and adaptive immune response pathways. In addition to the powerful immunomodulatory molecule LPS, vesicles also contain OM porins and other important innate immune activating ligands.
[0044] This disclosure also provides a vaccine for the prevention of brucellosis, comprising outer membrane vesicles of Brucella bovis S19 strain as described above, wherein the concentration of the outer membrane vesicles is 0.05–20 mg / ml.
[0045] This disclosure also provides an application of the outer membrane vesicles of Brucella bovis S19 strain as described above in the preparation of a medicament for the prevention / treatment of brucellosis.
[0046] Example 1: Selection and Cultivation of Strains Strains selection and activation: Strain S19 (for preparing outer membrane vesicles), identified, stored, and supplied by Jinyu Baoling Biological Pharmaceutical Co., Ltd., was selected. The bacterial strains used for testing were highly virulent Brucella mesenteriae strain M28 and Brucella abortus strain 2308, identified, stored, and supplied by the China Institute of Veterinary Drug Control. The lyophilized Brucella S19 strain was dissolved in PBS and streaked onto TSA agar plates, incubated at 37°C for 2–6 days. At least 10 single colonies meeting the above-mentioned characteristics in terms of purity, morphology, biochemical characteristics, culture characteristics, variation examination, and serological characteristics were inoculated onto TSA agar slant tubes and incubated at 37°C for 2–6 days as primary working seed for production.
[0047] Large-scale culture of the strain: 1) Inoculate the primary working seed culture onto TSA medium flasks or other suitable solid media and incubate at 37°C for 2–6 days. After visually inspecting for purity, add an appropriate amount of peptone water (pH 6.8–7.0) to each flask to wash off the bacterial growth. Inoculate this mixture into a seed tank containing TSB medium and incubate at 36–37°C with continuous aeration for 48–50 hours. This will serve as the secondary working seed culture for production. 2) Add an appropriate amount of antifoaming agent according to the volume of the culture medium. After sterilization, inoculate the secondary working seed culture at a ratio of 1%–2% of the culture medium volume in the fermenter. Ferment at 36–37°C, gradually increasing the aeration rate. During the culture process, 50% glucose solution can be added as needed to adjust the pH to between 6.8 and 7.2. After 48–50 hours of incubation, harvest the culture.
[0048] Example 2, Extraction of OMVs Outer membrane vesicles (OMVs) are vesicle-like bodies with a double membrane structure that detach from the cell membrane or are secreted by the cell. They carry bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens, and various pathogen-associated molecular patterns.
[0049] Brucella S19 strain culture (culture medium prepared according to the method in Example 1) was obtained by aeration fermentation. 5 L of the culture was centrifuged at 10000 rpm, 4 °C for 20 min, and the supernatant was collected, discarding the precipitate. The supernatant was concentrated using a 100 KD hollow fiber system to retain OMVs in the concentrate, finally obtaining 200 ml of concentrate. The concentrate was then centrifuged at 45000 rpm, 4 °C for 2 hours, and the supernatant was discarded. The precipitate was resuspended in PBS buffer and filtered through a 0.22 μm filter membrane to obtain Brucella S19 outer membrane vesicles, which were stored at -80 °C for later use (see [link to example]). Figure 1 ).
[0050] Example 3, Characterization of OMVs TEM characterization: The uniformly dispersed OMVs solution in Example 2 was diluted to 500-800 μg / ml. 10 μL of the diluted solution was dropped onto a copper grid and allowed to adsorb for half an hour. Then, it was blotted dry with filter paper. Subsequently, 10 μL of 1% phosphotungstic acid was dropped on the grid for staining for 3-5 minutes. The OMVs were then blotted dry with filter paper. The size and morphology of the OMVs were observed by TEM.
[0051] The TEM exhibits a complete shape and a regular spherical structure, with a particle size within 200 nm, such as... Figure 2 Transmission electron microscopy image of Brucella S19 strain OMVs shown.
[0052] OMVs protein quantification: OMVs protein concentration was determined using the BCA method. Following the standard procedures outlined in the instruction manual, a protein concentration-absorbance standard curve was established using protein standards (e.g., ...). Figure 3 The protein concentration-absorbance standard curve shown is R. 2 =0.9993>0.99, showing a good linear relationship.
[0053] Take 25 μL of OMVs sample diluted 10-fold and add it to two sub-wells. Then add 200 μL of BCA working solution, incubate at 37℃ for 30 min, and measure the absorbance at 562 nm. The OMVs protein concentration was calculated to be 8.011 mg / mL.
[0054] OMVs protein types: 80 μL of OMVs solution diluted to 500-800 μg / ml was mixed with 20 μL of 5× protein loading buffer and boiled for 10 min. 5 μL and 10 μL of the boiled mixed sample were loaded onto 12% SDS-PAGE and electrophoresed at 80 V for 20 min, followed by electrophoresis at 100 V for 30 min. Coomassie brilliant blue staining was then performed, followed by destaining with destaining solution.
[0055] Electrophoresis results as follows Figure 4 As shown, OMVs contain a variety of protein bands, with the size of the protein bands mainly ranging from 25 kDa to 200 kDa.
[0056] Particle size detection: After the nanoparticle size and zeta potential meter is powered on and preheated for 30 min, add 1 mL of OMVs sample to the sample cup. The sample concentration should not be less than 25 μg / mL. Place the sample cup into the sample cell, select the "size" mode to enter the particle size measurement interface, and perform the detection.
[0057] See results Figure 5 Its average particle size is 306 nm.
[0058] Example 4: Preparation of live brucellosis vaccine (S19 strain) Fermentation culture: In a fermenter, add an appropriate amount of antifoaming agent according to the volume of the culture medium. After sterilization, inoculate with secondary working seed culture at a ratio of 1% to 2% of the culture medium volume in the fermenter. Ferment and culture at 36-37℃, gradually increasing the aeration rate. During the culture process, 50% glucose solution can be added as needed to adjust the pH to between 6.8 and 7.2. After culturing for 48-50 hours, harvest the culture and perform purity testing. It should be pure, and viable cell counts should be performed simultaneously.
[0059] (2) Concentration of bacterial solution: Add sodium carboxymethyl cellulose solution to the pure bacterial solution at a total concentration of 0.2% to 0.4% to precipitate the bacterial cells, discard the supernatant, or use other physical methods to concentrate the solution to a concentration factor of 5 to 10 times, and store at 2 to 8°C for later use.
[0060] (3) Viable cell count: Take 1.0 ml of concentrated bacteria and serially dilute it 10-fold with PBS buffer (pH 7.2) or peptone water. Inoculate it onto TSA medium plates and incubate at 36-38°C for 96-120 hours. Perform viable cell count according to the appendix of the current Chinese Veterinary Pharmacopoeia. The viable cell count per 1.0 ml of concentrated bacterial solution should not be less than 1.0 x 10⁻⁶. 10 CFU can be used for seedling preparation.
[0061] (4) Vaccine preparation and packaging: Mix 10% gelatin, 20% sucrose freeze-drying protectant, and bacterial solution at a ratio of 1:6, then add thiourea to a final concentration of approximately 1%. Each dose of vaccine contains 2.0 x 10^9 live bacteria. 9 CFU quantitative dispensing.
[0062] (5) After freeze-drying and packaging, the product is quickly freeze-dried under vacuum.
[0063] Example 5, Immunogenicity assay in guinea pigs Each guinea pig was injected subcutaneously with 1 ml of the prepared OMVs vaccine (50 ug / ml) in the groin. After 21 days, a second immunization was administered at the same dose. At the same time, a live brucellosis vaccine (S19 strain) was used as a vaccine control group, with 1 ml injected subcutaneously in the groin of each guinea pig at a dose of 2.5 billion CFU / ml. A blank control group was also set up, which was injected with physiological saline. Clinical symptoms were continuously observed.
[0064] Twenty-one days after the second immunization, the animal was challenged with a subcutaneous injection of 60 CFU / ml per animal of the virulent strain M28, and its clinical symptoms were continuously observed.
[0065] Thirty days after the guinea pigs were challenged with the virus, their spleens were aseptically collected. The spleens were weighed and a certain amount of PBS buffer was added according to their weight so that the grinding volume was equivalent to the weight of the spleen. The spleens were then ground into a suspension.
[0066] The bacterial suspension was serially diluted 10-fold to 10. -2 Select 3 dilutions: original, 10, and 10 times. -1 10 -2 Inoculate 3 TSA medium plates for each dilution, 100 μL per plate, and use an L-shaped stick to distribute the bacterial solution evenly on the surface of the medium. Incubate at 36-38℃ for 96-120 hours.
[0067] Visually inspect for bacterial colonies; if bacteria grow on any petri dish, it proves that the guinea pig has not been protected.
[0068] The results, as shown in Table 1, indicate that both the OMVs vaccine and the S19 live vaccine provide protection for guinea pigs, with no significant difference between them.
[0069] Table 1. Immunogenicity assay in guinea pigs
[0070] Example 6 This animal efficacy evaluation test (1) Group immunization: Thirty healthy brucellosis-negative cattle aged 3-8 months were randomly divided into experimental group 1, experimental group 2, live vaccine control, and blank control group, with 10 cattle in each experimental group and 5 cattle in each control group. Each cattle in experimental group 1 received a subcutaneous injection of 1 ml of vaccine (50 ug / ml) in the neck, and each cattle in experimental group 2 received a subcutaneous injection of 1 ml of vaccine (100 ug / ml) in the neck. A booster immunization was performed 21 days later using the same dose and method. The challenge control was immunized with live brucellosis vaccine (S19 strain), and each cattle in the control group received the same dose of sterile saline.
[0071] (2) Antibody fluctuation pattern: RBT test was performed on blood samples before and after immunization. The time to antibody seroconversion, positive rate and time to seroconversion were statistically analyzed, as shown in Table 2.
[0072] (3) The pattern of antibody fluctuations is shown in Table 2:
[0073] Table 2
[0074] (4) Challenge: The virulent Brucella strain 2308 was cultured to the logarithmic growth phase and diluted with PBS buffer to 1.0 × 10⁻⁶. 7 CFU / ml, 1ml subcutaneously injected per cow.
[0075] (5) Dissection and bacterial identification: After the experimental cattle were eviscerated, the spleen, submandibular lymph nodes (left and right), anterior shoulder lymph nodes (left and right), and inguinal lymph nodes (left and right) were aseptically collected and placed in sterile self-sealing bags for later use. An appropriate amount (about 0.20 g) of each tissue sample was aseptically taken and an appropriate amount (about 2.0 ml) of PBS buffer was added and ground to prepare a suspension. The ground bacterial suspension was aspirated and inoculated into three Brucella selective culture medium plates, 100 μl per plate. The bacterial suspension was evenly distributed on the surface of the culture medium with an L rod and incubated at 36-38℃ for 96-120 hours.
[0076] (6) Judgment: The presence of bacteria in any part of the body indicates that the cow is either infected or unprotected. The infection rate of the blank challenge control was 100%, proving that the challenge was successful; the protection rates of experimental group 1 and experimental group 2 were 60% and 80%, respectively, and the protection rate of the live vaccine S19 control was 80%.
[0077] In conclusion, Brucella S19 OMVs have a protective effect on animals and can indeed be developed and applied as a novel vaccine.
[0078] In summary, this invention relates to the outer membrane vesicles of Brucella S19 strain from cattle and their preparation method. The vaccine utilizes the outer membrane vesicles extracted from Brucella cultured with horse serum for the first time as a vaccine for animal immunization. Animal experiments have demonstrated that the Brucella outer membrane vesicles extracted by this invention have a significant effect in preventing brucellosis. They can be used to prepare products for the prevention of brucellosis, thus demonstrating good safety and practical application value.
[0079] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.
Claims
1. A method for preparing outer membrane vesicles of Brucella bovis strain S19, characterized in that, Includes the following steps: S1. Take bovine Brucella S19 strain and inoculate it into TSB medium, add 3-5% horse serum by weight, and incubate on a shaker for no less than 48 hours. Inoculate all the bacterial culture into a new TSB medium and incubate on a shaker for no less than 72 hours to use as the primary working seed for production. S2, the primary working seed for production is inoculated onto the culture medium, and after it is cultured until pure, peptone water is added. The bacterial growth is washed off and inoculated into a seed tank containing TSB culture medium. It is then cultured with aeration to serve as the secondary working seed for production. S3, add defoamer to the culture medium, sterilize, add 1-2% of the secondary working seed culture for production, ferment and culture, and gradually increase the aeration rate, and harvest the culture liquid; S4. After the cultured bacterial solution is centrifuged at 2-8℃, the supernatant is filtered through a 0.45µm filter membrane and then ultracentrifuged to obtain the precipitate. S5. The precipitate was resuspended in PBS buffer and filtered through a 0.22 μm filter membrane to obtain Brucella outer membrane vesicles, namely Brucella bovis S19 outer membrane vesicles.
2. The preparation method according to claim 1, characterized in that, The primary working seed used in S1 is inoculated onto TSA medium plates and incubated at 36–38°C for 48–72 hours. Heat agglutination test, acrid yellow agglutination test, and crystal violet staining are then performed. The colonies should exhibit smooth colony characteristics, without agglutination or staining.
3. The preparation method according to claim 1, characterized in that, The temperature of the shaker culture in step S1 is 36-37℃.
4. The preparation method according to claim 1, characterized in that, Step S3 also includes adding a 50% glucose solution and adjusting the pH value to 6.8-7.
2.
5. The preparation method according to claim 1, characterized in that, In step S4, the centrifugation speed shall not be less than 10,000 rpm and the duration shall not be less than 15 minutes; The speed of ultracentrifugation should not be less than 45,000 rpm and the duration should not be less than 2 hours.
6. An outer membrane vesicle of Brucella bovis strain S19, characterized in that, The outer membrane vesicles are secreted by Brucella bovis S19 strain and obtained by culturing, activating, expanding culture, and extracting as described in any one of claims 1-5.
7. The outer membrane vesicles of Brucella bovis S19 strain as described in claim 6, characterized in that, The outer membrane vesicles are vesicle-like bodies with a double membrane structure, carrying bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens, and pathogen-associated molecular patterns.
8. The outer membrane vesicles of Brucella bovis S19 strain as described in claim 6, characterized in that, The outer membrane vesicles have a particle size of no more than 200 nm, and their protein band sizes are concentrated in the range of 15–200 kDa.
9. A vaccine for the prevention of brucellosis, characterized in that, Including the outer membrane vesicles of Brucella bovis S19 strain as described in any one of claims 6-8; The concentration of the outer membrane vesicles is 0.05–20 mg / ml.
10. The use of the outer membrane vesicles of Brucella bovis S19 strain as described in any one of claims 6-8 in the preparation of a medicament for the prevention / treatment of brucellosis.