Mannheimia haemolytica outer membrane vesicle as well as preparation method and application thereof

Abstract

The invention discloses mannheimia haemolytica outer membrane vesicles as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. The OMV is separated from Mh-5 strain culture supernatant through tangential flow ultrafiltration combined with an ultracentrifugation technology, and animal experiments prove that a high-level IgG / IgM antibody can be induced by intramuscular injection of 50 micrograms per mouse, expression of inflammatory factors such as IL-6 and TNF-alpha of the spleen is remarkably improved, the protection rate after challenge reaches 85.7%, and pathological damage is remarkably lighter than that of an inactivated vaccine group. According to the scheme provided by the invention, the problems of insufficient serotype coverage and antibiotic resistance in the prior art are solved, and a new strategy is provided for prevention and control of mannheimia haemolytica.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and in particular to an outer membrane vesicle of Mannheimia hemolytica and a preparation method and application thereof. Background Art

[0002] Mannheimia haemolytica, also known as "shipping fever," is a major respiratory infectious disease caused by Mannheimia haemotica (Mh), characterized by acute pneumonia, fibrinous necrotizing pneumonia, sheep mastitis, and young animal sepsis in cattle and sheep, causing significant economic losses to the livestock industry. There are 12 serotypes of Mh: A1, A2, A5-A9, A12-A14, A16, and A17. A1 and A2 are the predominant strains, while the prevalence of serotypes A5, A6, and A7 has been increasing annually. Currently, the primary control measure for Mh is antibiotics, but the infection rate and morbidity of Mh in sheep and goats remain high. The primary reasons for this are the overuse of antibiotics, the development of drug resistance, and the incomplete protection afforded by existing vaccines due to the diversity of serotypes. Vaccination is currently an effective preventive measure against Mannheimia haemolytica infection. Therefore, the development of new vaccines against Mh and the improvement of their protective efficacy are crucial. Summary of the Invention

[0003] The present invention aims to provide Mannheimia haemolytica outer membrane vesicles, their preparation method, and applications, to address the aforementioned problems of the prior art. The Mannheimia haemolytica outer membrane vesicles prepared by the present invention can effectively stimulate humoral immune responses in mice and provide good immune protection against Mannheimia haemolytica disease, demonstrating their potential as a subunit vaccine and providing a foundation for the application of Mannheimia haemolytica outer membrane vesicles.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The invention provides a method for preparing outer membrane vesicles of Mannheimia haemolytica. The method comprises the following steps: inoculating Mannheimia haemolytica into a BHI culture medium for shaking culture, removing bacteria by centrifugation, filtering the supernatant through a filter membrane, concentrating the supernatant using a tangential flow ultrafiltration system to obtain a concentrated solution, and resuspending the precipitate after ultracentrifugation with PBS to obtain the outer membrane vesicles of Mannheimia haemolytica.

[0006] Preferably, the Mannheimia haemolytica can be selected from the Mannheimia haemolytica Mh-5 strain.

[0007] Furthermore, the filtering of the supernatant through filter membranes is filtering the supernatant through 0.45 μm and 0.22 μm filter membranes in sequence.

[0008] Furthermore, the concentration is to 1 / 10 of the original volume.

[0009] Furthermore, the ultracentrifugation is carried out at 4°C and 31200 rpm for 1.5 h.

[0010] The present invention also provides the outer membrane vesicles of Mannheimia haemolytica prepared by the above preparation method.

[0011] The present invention also provides a vaccine composition comprising the above-mentioned Mannheimia haemolytica outer membrane vesicles and a pharmaceutically acceptable carrier.

[0012] The present invention also provides a use of the vaccine composition in preparing a medicine for preventing cattle and sheep transport fever.

[0013] The present invention also provides the use of the above-mentioned Mannheimia haemolytica outer membrane vesicles in preparing a vaccine for preventing Mannheimia haemolytica infection.

[0014] Furthermore, the vaccine is administered by intramuscular injection.

[0015] The present invention also provides a kit for detecting anti-haemolytic Mannheimia antibodies in animal serum, comprising the above-mentioned Mannheimia haemolytica outer membrane vesicles.

[0016] The present invention discloses the following technical effects:

[0017] The present invention cultivates Mannheimia haemolytica in BHI medium, obtains outer membrane vesicles of Mannheimia haemolytica after centrifugation, filtration, ultrafiltration and ultracentrifugation, and inoculates SPF-grade Balb / c mice with the outer membrane vesicles as subunit vaccines. The results show that the outer membrane vesicles can effectively stimulate the humoral immune response in mice and provide good immune protection against Mannheimia haemolytica, demonstrating its potential as a subunit vaccine. In addition, the outer membrane vesicles of Mannheimia haemolytica can be further prepared into a kit for detecting anti-Mannheimia haemolytica antibodies in animal serum, thereby expanding its scope of application. The Mannheimia haemolytica outer membrane vesicle (OMV) vaccine prepared by the present invention is a combination of various means to obtain high-purity vesicles (20-200 nm). In a mouse model, the following results were observed: serum IgG / IgM levels were significantly increased after 7 days of immunization (P<0.01); Th1 (IFN-γ, IL-18) and Th17 (IL-1β) cellular immune responses were activated; the protection rate after challenge (85.7%) was 28.6% higher than that of the inactivated vaccine, and lung and spleen pathological damage was significantly reduced; the preparation method is easy to operate and the product is pure (the present invention adopts tangential flow membrane filtration system concentration technology, which is more convenient to operate than the existing density gradient centrifugation method and can obtain a relatively pure outer membrane vesicle extract). The present invention provides a basis for the application of Mannheimia haemolytica outer membrane vesicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The SDS-PAGE results of the outer membrane vesicles of the hemolytic strain Mannheimia spp. Mh-5; wherein M is a marker; the whole-cell supernatant is the supernatant after the Mh-5 culture cells are resuspended in PBS and ultrasonically disrupted; the whole-cell pellet is the pellet after the Mh-5 culture cells are resuspended in PBS and ultrasonically disrupted; OMVs are the outer membrane vesicles of the hemolytic strain Mannheimia spp. Mh-5;

[0020] Figure 2 The results of scanning transmission electron microscopy observation of the outer membrane vesicles of Mannheimia haemolytica Mh-5 are shown. The scale bars in A and B are 200 μm and 100 μm, respectively.

[0021] Figure 3 This is the result of the serum IgG antibody level test after Mh-5 outer membrane vesicle immunization;

[0022] Figure 4 This is the result of the serum IgM antibody level test after Mh-5 outer membrane vesicle immunization;

[0023] Figure 5 The results are as follows: the mRNA transcription levels of inflammatory cytokines in the spleen of mice after immunization with Mh-5 outer membrane vesicles; A is the mRNA expression level of IL-6; B is the mRNA expression level of IL-1β; C is the mRNA expression level of TNF-α; D is the mRNA expression level of IFN-γ; E is the mRNA expression level of IL-18; F is the mRNA expression level of IL-10;

[0024] Figure 6 It is the evaluation standard of clinical symptoms after mice are infected with Mannheimia haemolytica;

[0025] Figure 7 The following are the pathological changes in the lungs and spleens of each immunization group after challenge with the virus. The magnification is 50 μm. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] For details of the Mannheimia haemolytica Mh-5 strain, please refer to the public article: Proteomic analysis of outermembrane vesicles derived from the type A5 strain of Mannheimia haemolytica.

[0032] Example 1 Preparation of outer membrane vesicles of Mannheimia hemolytica

[0033] 1. Strain recovery: Streak Mannheimia haemolytica Mh-5 strain stored at -70°C onto a BHI agar plate without antibiotics and incubate at 37°C for 24 h. Pick a single colony from the BHI plate and inoculate into 5 mL of BHI broth medium and incubate at 37°C with shaking at 180 rpm for 8 h.

[0034] The preparation of BHI broth medium is as follows: 10 g of proteose peptone, 12.5 g of dehydrated calf brain infusion powder, 5 g of dehydrated beef heart infusion powder, 5 g of NaCl, 2 g of glucose, and 2.5 g of disodium hydrogen phosphate are dissolved in 1000 mL of distilled water, the pH value is adjusted to 7.4±0.2 (25°C), high-pressure sterilization is performed at 121°C for 15 min; the BHI solid medium is prepared by adding 15 g of agar powder to 1000 mL of BHI broth medium, high-pressure sterilization is performed at 121°C for 15 min, and the medium is poured into a flat dish when the temperature is reduced to 50°C;

[0035] 2. Expansion culture: the hemolytic Mannheimia haemolytica bacterial solution cultured for 12 h is inoculated into 500 mL of BHI broth medium at a ratio of 1:100, and cultured at 37°C with 180 rpm shaking until the OD 600 is 0.4-0.6;

[0036] 3. Filtration and concentration: the hemolytic Mannheimia haemolytica culture solution is centrifuged at 4°C and 10,000 rpm for 30 min, the supernatant is filtered with 0.45 μm and 0.22 μm filters in sequence, and then the obtained cell-free supernatant is concentrated by a tangential flow ultrafiltration (TFF) system (100 KDa ultrafiltration tube) until the volume is concentrated to one-tenth of the original volume;

[0037] 4. Collection of outer membrane vesicles: the concentrated liquid is subjected to ultracentrifugation at 4°C and 31,200 rpm for 1 h 30 min, the supernatant is discarded, and the precipitate is resuspended with PBS buffer to prepare hemolytic Mannheimia haemolytica outer membrane vesicles.

[0038] The collected hemolytic Mannheimia haemolytica outer membrane vesicles are negatively stained with uranyl acetate and observed under a transmission electron microscope for the morphology of the outer membrane vesicles. Then the collected outer membrane vesicles are subjected to SDS-PAGE verification and the protein concentration of the hemolytic Mannheimia haemolytica outer membrane vesicles is calculated by the BCA method.

[0039] As shown in Figure 1 and Figure 2 , the main band of the outer membrane vesicles is about 50 KDa, and the structure of the outer membrane vesicles can be observed under a transmission electron microscope, with a size of 20-200 nm. The hemolytic Mannheimia haemolytica outer membrane vesicle sample is detected by a BCA protein concentration determination kit (Sevyl, G2026-200T), and the protein concentration is measured to be 1.2 mg / mL.

[0040] The present invention utilizes a tangential flow ultrafiltration system for concentration to prepare high-concentration OMVs in large quantities at one time. Using the method of the present invention, the yield of outer membrane vesicles of Mannheimia haemolytica can reach 1200 mg / L, which is approximately 100 times higher than the yield of the original strain cultured in BHI medium. The method is suitable for the large-scale production of outer membrane vesicles for biological products, significantly reducing production costs.

[0041] Example 2 Analysis of the immunological effects of Mannheimia haemolytica outer membrane vesicles

[0042] Twenty-seven 5-week-old SPF Balb / c mice were randomly divided into three groups and given free access to food and water for one week to adapt to the new environment. 600 =1.0), the cells were collected by centrifugation at 10,000 rpm for 10 min, inactivated with 0.2% formaldehyde solution at 37°C for 24 h, washed three times with PBS, and then resuspended to 10 9 CFU / mL, diluted to 10 7 CFU / 100μL / mouse) and outer membrane vesicles (Example 1) were inoculated into 6-week-old Balb / c mice by intramuscular injection, with an immunization dose of 50μg / mouse, and an unimmunized PBS control group was set up at the same time. Booster immunization was performed on the 14th day after the first immunization (8 weeks old) (Table 1). Blood was collected from the tail on the 7th, 14th, 21st, and 28th days after the first immunization. The collected blood was allowed to stand at 37°C for 2h, and then at 4°C for 2h to allow serum to precipitate. The serum was separated by centrifugation at 4°C, 2000rpm for 10min, and stored in a refrigerator at -80°C for subsequent indirect ELISA antibody detection. Two mice were randomly dissected on the 28th day after the first immunization, and the spleens were collected for subsequent cytokine detection by qPCR.

[0043] 2.1 Serum antibody IgG / IgM level detection

[0044] Serum samples stored in a -80°C refrigerator were taken out, and the titers of anti-hemolytic Mannheimia IgG and IgM in the serum were determined by indirect enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: Coating: Dilute Mh-5 outer membrane vesicles with coating solution (0.016M Na2CO3, 0.034M NaHCO3, pH 9.6) and coat 96-well plates at 1 μg / well, coating 100 μL per well and incubating at 4°C overnight; Washing: Aspirate the coating solution and add 200 μL of washing solution (TBS + 0.05% Tween20) to each well, soak for 2 minutes, discard the solution, and repeat washing three times; Blocking: Add 200 μL of blocking solution (TBST + 5% skim milk powder) to each well and incubate at 37°C for 2 hours; Washing: Aspirate the blocking solution and repeat the washing method as above for 3 times; Primary antibody incubation: Add 100 μL of serum sample diluted 1:1000 to each well and place at 37°C for 1 hour; Washing: Shake off the liquid in the well and repeat washing 5 times; Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG / IgM (dilution factor of 1:10,000) to each well and place at 37°C for 1 hour; Washing: Shake off the liquid in the well and repeat washing 5 times; Color development: Add 100 μL of TMB substrate color development solution to each well and react in the dark at room temperature for 10 minutes; Stop reaction: Add 50 μL of stop solution (4.5NH2SO4) to each well and measure its OD using a microplate reader. 450 nm value.

[0045] The results showed that compared with the control group (PBS), the antibodies in the mice immunized with outer membrane vesicles increased significantly on the 7th day after immunization (P<0.01), reached a peak on the 14th day, and remained stable ( Figure 3 ), indicating that the humoral immune response was enhanced. In addition, compared with the control group (PBS), the IgM antibody level of mice immunized with outer membrane vesicles increased significantly on the 14th day after immunization ( Figure 4 ).

[0046] 2.2 Inflammatory cytokine mRNA transcription levels

[0047] To better evaluate the immune response in mice after immunization with outer membrane vesicles, cytokine expression in the spleens of mice in different concentration groups was detected using real-time fluorescence quantitative PCR (qPCR). RNA was extracted from the supernatant of tissue samples according to the instructions of the AG RNAex Pro RNA extraction kit, and cDNA was synthesized using the M5 SuperFast qPCR RT kit. ① Weigh 100 mg of the tissue sample to be tested on an electronic balance; ② Transfer the sample to an EP tube containing 900 μL of tissue lysis buffer and 3 grinding beads and grind using a tissue grinder; ③ Then, centrifuge the EP tube at 12,000 rpm for 10 minutes at 4°C; ④ Extract RNA from the supernatant of the tissue sample according to the instructions of the RNA extraction kit; ⑤ Collect the eluate and store it at -80°C until use.

[0048] Compared with the control group (PBS), the expression levels of IL-6, IL-1β, and TNF-α mRNA in the spleen of mice immunized with outer membrane vesicles were significantly increased (P<0.01). This indicates that it can stimulate Th1 cell immune type (IL-6, TNF-α) and Th17 cell immune type (IL-1β) ( Figure 5 A- Figure 5 C). In addition, the expression levels of IFN-γ and IL-18 mRNA increased significantly after immunization with outer membrane vesicles (P<0.01). The results showed that each immunization group could stimulate Th1 cell immunity (IFN-γ, IL-18) ( Figure 5 D and Figure 5 IL-10 plays an important role as an anti-inflammatory factor in immune regulation, anti-inflammation and tissue repair. Compared with the control group, the expression level of IL-10 mRNA in each immune group increased significantly (P<0.01) ( Figure 5 F).

[0049] Example 3 Evaluation of the immune protection of Mannheimia haemolytica outer membrane vesicles

[0050] A single colony of Mh-5 was picked and inoculated into 5 mL of BHI broth medium, and cultured at 37°C, 180 rpm, and shaken for 12 h. Then, it was inoculated into 500 mL of BHI broth at a ratio of 1:100 and cultured at 37°C, 180 rpm, for 12 h. The bacteria were washed three times with sterile PBS to adjust the bacterial concentration to 9.33 × 10 10 CFU / mL(100LD 50 ); Each group of mice will be injected intraperitoneally on the 14th day after booster immunization. 50 As a result, the challenge dose was 10LD 50 (Expected lethality rate: 80%-100%), with an injection dose of 100 μL / mouse. Vital signs of the mice were observed and mortality was recorded. Dead mice were autopsied, and lung, liver, and spleen tissue samples were collected and fixed in 4% paraformaldehyde solution. The samples were then sent to Anhui Xinle Biotechnology Co., Ltd. for paraffin embedding, sectioning, staining, and photography. The experimental protocol for evaluating the immune protective capacity of Mannheimia haemolytica outer membrane vesicles is detailed in Table 1.

[0051] Table 1 Experimental scheme for evaluating the immune protection of Mannheimia haemolytica outer membrane vesicles

[0052]

[0053] The evaluation criteria for clinical symptoms of mice infected with Mannheimia haemolytica are detailed in the Figure 6The clinical symptoms of mice were evaluated using the clinical symptom index and compared with the non-immunized PBS group. The results are shown in Table 2. The mice in the control group (PBS) showed clinical symptoms such as depression, loss of appetite, weight loss, nasal secretions, chills and curling up, tremors and spasms, and all died 5 days after the infection. Except for the dead mice, most of the mice in the immunized group showed depression, chills and curling up after the infection, and gradually recovered after the acute phase. One mouse died in the outer membrane vesicle immunization group after the infection, and the vaccine protection rate was 85.7% (Table 3). All mice in the PBS control group died within 7 days after the infection, and the protection rate was 0. The results show that the OMV subunit vaccine has a good protective effect on mice.

[0054] Table 2 Clinical symptoms of mice after challenge with Mannheimia hemolytica

[0055]

[0056]

[0057] Table 3 Outer membrane vesicle immune protection rate

[0058]

[0059] Pathological sections of the lungs and spleens of mice that survived the challenge were observed. Figure 7 As shown, compared to the control group, the PBS group exhibited severe pathological changes in the lungs and spleen after Mh-5 inoculation. These included severe lung hemorrhage, loss of alveolar structure, fusion and dilation of alveoli, and thickening of the alveolar walls. Numerous red blood cells were present in the luminal and alveolar spaces, along with inflammatory cell infiltration (blue arrows). The normal structure of the spleen was lost, and the boundary between red and white pulp was unclear (yellow arrows). Compared to the Mh-5 challenge group, mice immunized with outer membrane vesicles showed no significant structural abnormalities in the lungs and spleen, and the severity of the lesions was minimal. These results demonstrate that outer membrane vesicle immunization provides a protective effect in mice.

[0060] Example 4 Determination of the cross-immune protection efficacy of Mannheimia haemolytica outer membrane vesicles

[0061] In order to evaluate the cross-immune protection of candidate antigen outer membrane vesicles, 15 5-week-old SPF Balb / c mice were randomly divided into 3 groups: blank control group, Mh-5 challenge group and Mh-1 challenge group. The mice were free to eat and drink water for 1 week to adapt to the new environment. During immunization, outer membrane vesicles (Example 1) were inoculated into 10 6-week-old Balb / c mice by intramuscular injection at an immunization dose of 50 μg / mouse. At the same time, an unimmunized PBS control group was established. A booster immunization was performed on the 14th day (8 weeks old) after the first immunization (Table 4). A single colony of Mh-1 (type A1) (provided by the Key Laboratory of Live Carrier Biomaterials and Animal Disease Prevention and Control in Luoyang City) was picked and inoculated into a 5mL BHI The bacteria were cultured in broth medium at 37°C with 180 rpm shaking for 12 h, then inoculated into 500 mL of BHI broth at a ratio of 1:100, and cultured at 37°C with 180 rpm for 12 h. The bacteria were washed with sterile PBS for 3 times, and the concentration of the bacteria was adjusted to 2.79 x 1010CFU / mL (100 LD 10 CFU / mL (100 LD 50 ) for challenge. The mice were intraperitoneally injected with the bacteria at the 14th day after the booster immunization. The injection dose was 100 μL per mouse, and the vital signs of the mice were observed and the death of the mice was counted.

[0062] Table 4 Test scheme for evaluating the cross-immunoprotection of outer membrane vesicles of Mannheimia haemolytica

[0063]

[0064]

[0065] After the mice were infected with Mannheimia haemolytica, the mice in the control group showed clinical symptoms such as depression, anorexia, weight loss, nasal discharge, shivering, tremor, and convulsions, and all of them died 5 days after the challenge. Two mice in the OMV / Mh-5 immunization group died after the challenge, and the vaccine protection rate was 60.0%; two mice in the OMV / Mh-1 immunization group died after the challenge, and the vaccine protection rate was 60.0% (Table 5). The results showed that OMV can be used as an efficient vaccine to induce cross-immunoprotection in mice against different serotypes of Mannheimia haemolytica infection, and provides a new vaccine development strategy for the control of Mannheimia haemolytica disease and even other pathogenic bacteria.

[0066] Table 5 Cross-immunoprotection rate of outer membrane vesicles

[0067]

[0068] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application defined by the claims.

Claims

1. A method for preparing outer membrane vesicles of Mannheimia haemolytica, characterized in that: The following steps are involved: Mannheimia haemolytica was inoculated into BHI culture medium and cultured by shaking. The bacteria were removed by centrifugation. The supernatant was filtered through a filter membrane and concentrated using a tangential flow ultrafiltration system to obtain a concentrate. The precipitate after ultracentrifugation was resuspended in PBS to obtain the outer membrane vesicles of Mannheimia haemolytica.

2. The preparation method according to claim 1, characterized in that The supernatant is filtered through filter membranes of 0.45 μm and 0.22 μm in sequence.

3. The preparation method according to claim 1, characterized in that The concentration is to concentrate to 1 / 10 of the original volume.

4. The preparation method according to claim 1, characterized in that The ultracentrifugation was carried out at 4°C and 31200 rpm for 1.5 h.

5. The outer membrane vesicles of Mannheimia haemolytica prepared by the preparation method according to any one of claims 1 to 4.

6. A vaccine composition, characterized in that The invention comprises the outer membrane vesicles of Mannheimia haemolytica according to claim 5 and a pharmaceutically acceptable carrier.

7. Use of the vaccine composition according to claim 6 in the preparation of a medicament for preventing transportation fever in cattle and sheep.

8. Use of the outer membrane vesicles of Mannheimia haemolytica according to claim 5 in the preparation of a vaccine for preventing Mannheimia haemolytica infection.

9. The use according to claim 8, characterized in that The vaccine is administered by intramuscular injection.

10. A kit for detecting antibodies against Mannheimia haemolytica in animal serum, characterized in that: The invention comprises the outer membrane vesicles of Mannheimia haemolytica according to claim 5.

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