Antigen carrier strain and its application in preparing oral Staphylococcus aureus vaccine

Abstract

The present invention discloses an antigen carrier strain, a recombinant strain and their application in the preparation of a Staphylococcus aureus oral vaccine. The strain is LR076, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 27, 2022, with the deposit number CGMCC No. 25845, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Limosilactobacillus reuteri; the recombinant strain is formed by transforming a recombinant plasmid carrying an exogenous Staphylococcus aureus antigen fragment into the LR076 strain; the antigen fragment includes Hla H35L , IsdB, and MntC, which are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively. Applying the recombinant strain to the preparation of a Staphylococcus aureus oral vaccine can effectively alleviate the lesions of the lungs after infection and reduce the colonization amount of Staphylococcus aureus in the lungs.

Description

Technical Field

[0001] The invention relates to the technical field of oral Staphylococcus aureus vaccines, and more specifically to antigen carrier strains and applications thereof in the preparation of oral Staphylococcus aureus vaccines. Background Art

[0002] Staphylococcus aureus (S. aureus) is one of the most common zoonotic pathogens worldwide. It causes a wide range of diseases in humans, including invasive infections such as pneumonia, which are the main causes of serious diseases and deaths worldwide. With the emergence of infections caused by multidrug-resistant bacteria and the increasing mortality rate of diseases caused by them, the development of safe and effective S. aureus vaccines is urgent, but its vaccine development has failed in the clinical trial stage. Based on the understanding of the immune escape mechanism of S. aureus, the reason for the failure of S. aureus vaccines in the clinical stage may be that the focus is not on the activation of cellular immunity and mucosal immunity by vaccines, but on the development of vaccines based on humoral immune responses. More and more studies have shown that mucosal vaccines based on live bacterial carriers and nano-delivery particles may be the ideal direction for the future development of S. aureus vaccines.

[0003] The mucosa is the largest tissue organ in the human and animal body, including the digestive tract, respiratory tract, urogenital tract, etc. The mucosal surface is in direct contact with the outside world (such as the environment, food, symbiotic bacteria and pathogens, etc.), so it plays an extremely important role in anti-infection as the first line of defense. At present, most pathogenic microorganisms come into contact with the mucosal surface to invade the body. Traditional systemic vaccines are usually injected subcutaneously, which cannot induce antigen-specific mucosal responses and cannot prevent pathogenic invasion of epithelial surfaces. Mucosal vaccines can not only induce mucosal immune responses, but also induce systemic immune responses, and can play an immune protective role in the early stages of pathogen invasion, effectively overcoming the limitations of existing vaccines. Using a suitable delivery vehicle to stably deliver antigens to specific mucosal immune induction sites can simultaneously induce mucosal and systemic immune responses, thereby achieving a double-layer protection effect against mucosal invasive pathogens.

[0004] As a presentation carrier with great development potential, lactic acid bacteria have shown many advantages in presenting various therapeutic and preventive molecules to the mucosa. First, it is immunized through the oral route, which is convenient to use, safe, minimizes the side effects and pain caused by intramuscular or intravenous injection, and can also reduce pollution. Secondly, there is no need to purify the expressed protein, the production capacity is rapid, and the protein product will not be mixed with endotoxins. In addition, many lactic acid bacteria strains, especially lactobacilli, can tolerate extreme gastrointestinal environments (such as degrading enzymes and extreme pH environments) in animals or humans, and have strong adhesion and colonization capabilities, which is conducive to the long-term delivery of drug molecules. Most importantly, lactic acid bacteria themselves have probiotic properties and adjuvant properties. While being safer, they can protect antigens from degradation, prolong the retention time of antigens at mucosal sites, and promote the presentation of exogenous proteins to phagocytes. The new S. aureus mucosal vaccine developed with lactic acid bacteria as live bacterial carriers is a field that researchers have just started to explore and is receiving more and more attention.

[0005] Therefore, how to provide a lactobacillus that can be used as a live bacteria carrier is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0006] In view of this, the present invention provides an antigen carrier strain and its application in preparing an oral Staphylococcus aureus vaccine.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A antigen carrier strain, the strain is LR076, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 27, 2022, with the deposit number CGMCC No.25845, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is Limosilactob acillus reuteri.

[0009] As an inventive concept identical to the above technical solution, the present invention also requests protection for a recombinant strain, characterized in that the recombinant strain is formed by transforming a recombinant plasmid carrying an exogenous Staphylococcus aureus antigen fragment into the LR076 strain; the antigen fragment includes Hla H35L , IsdB, and MntC are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively.

[0010] As a preferred technical solution of the above technical solution, the recombinant plasmid includes pNZ8148-sp-His-hla H35L , pNZ8148-sp-His-isdB or pNZ8148-sp-His-mntC.

[0011] As a preferred technical solution of the above technical solution, the method for constructing the recombinant plasmid includes:

[0012] 1) Select the S-layer signal peptide Sp and His tag of Lactobacillus brevis, and obtain Staphylococcus aureus hla through GenBank H35L , isdB, and mntC full sequences were optimized according to the codon preference of L. reuteri, and the optimized sequences of Sp, His tag, and antigen were synthesized and ligated into the cloning vector pUC57-Simple;

[0013] 2) After double enzyme digestion, gel recovery, ligation, and transformation, hla H35L , isdB or mntC were connected to the expression vector pNZ8148 to construct a recombinant plasmid.

[0014] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of the strain in the preparation of an oral Staphylococcus aureus vaccine.

[0015] As an inventive concept identical to the above technical solution, the present invention also requests protection for the use of the recombinant strain in the preparation of an oral Staphylococcus aureus vaccine.

[0016] As an inventive concept identical to the above-mentioned technical solution, the present invention also requests protection for a method for immunizing mice with a vaccine prepared from the recombinant strain, characterized in that the process comprises: activating the recombinant strain to obtain a bacterial solution, and then gavaging the mice with the bacterial solution on days 1, 15 and 29.

[0017] It can be seen from the above technical scheme that, compared with the prior art, the present invention uses L. reuteri LR076 as the receptor strain, utilizes the lactobacillus surface display system and the inducible promoter PnisA, and constructs a delivery system for Staphylococcus aureus antigen Hla H35L , IsdB, MntC recombinant L. reuteri LR076 (pNZ8148-sp-His-hla H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mnt C). The physiological characteristics of recombinant L. reuteri were not significantly different from those of wild strains. The recombinant plasmids could be stably inherited and foreign proteins could be successfully expressed. H35LAfter oral immunization of mice with LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC), the levels of IgG in serum and sIgA in mucosa were significantly increased, and the cytokines IL-17 and IFN-γ in mucosal tissue were significantly upregulated. The three recombinant bacteria had different degrees of protective effects against Staphylococcus aureus pneumonia infection in mice, and could effectively alleviate the lesions in the lungs after infection and reduce the fixed amount of Staphylococcus aureus in the lungs. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 The attached figure is a diagram showing the results of PCR identification of recombinant L. reuteri; A: M: Trans2K DNA Marker; 1: LR076 (pNZ8148-sp-His-hla H35L ) Colony PCR results; B: M: Trans2K DNA Marker; 1: LR076 (pNZ8148-sp-His-isdB) colony PCR results; C: M: Trans2K DNA Marker; 1: LR076 (pNZ8148-sp-His-mntC) colony PCR results;

[0020] Figure 2 The attached figure is a Western blotting identification diagram of recombinant L. reuteri;

[0021] Figure 3 The accompanying figure is a growth curve of recombinant L. reuteri;

[0022] Figure 4 The accompanying figure is a graph showing the results of the determination of the resistance of recombinant L. reuteri to artificial gastric juice;

[0023] Figure 5 The accompanying figure is a graph showing the results of the determination of the tolerance of recombinant L. reuteri to artificial intestinal fluid containing bile salts;

[0024] Figure 6 The attached figure shows the results of genetic stability test of recombinant L. reuteri; A: M: Trans8K DNA Marker; 1-5: LR076 (pNZ8148-sp-His-hla H35L) The results of plasmid extraction at the 1st, 5th, 10th, 15th and 20th generations; 6-10: The results of plasmid extraction at the 1st, 5th, 10th, 15th and 20th generations of LR076 (pNZ8148-sp-His-mntC); B: M: Trans8K DNA Marker; 1-5: The results of plasmid extraction at the 1st, 5th, 10th, 15th and 20th generations of LR076 (pNZ8148-sp-His-isdB);

[0025] Figure 7 The attached figure is a quantitative result diagram of the specific antibody level in the bronchoalveolar lavage fluid of mice after immunization with the recombinant bacteria;

[0026] Figure 8 The attached figure is a quantitative result of the specific antibody level in the small intestinal mucosa of mice after immunization with the recombinant bacteria;

[0027] Fig. 9 The attached figure is a quantitative result diagram of the specific antibody level in the small intestinal feces of mice after immunization with the recombinant bacteria;

[0028] Fig.10 The attached figures are quantitative results of IL-17 levels in the lung, spleen and Peyer's Patches tissues of mice after immunization with recombinant bacteria; A: IL-17 levels in the lungs of mice; B: IL-17 levels in the spleen of mice;

[0029] Fig.11 The attached figures are the quantitative results of IFN-γ levels in the lung, spleen and Peyer's Patches of mice after immunization with recombinant bacteria; A: IFN-γ levels in the lung; B: IFN-γ levels in the spleen; C: IFN-γ levels in the Peyer's Patches of mice;

[0030] Fig.12 The attached figure is a statistical chart of the survival rate of mice in the Staphylococcus aureus pneumonia model after oral immunization with recombinant bacteria;

[0031] Fig.13 The attached figure is a pathological section of mouse lung tissue after Staphylococcus aureus infection; A: A: Lung pathological sections of group A, B: Lung pathological sections of group B, C: Lung pathological sections of group B, D: Lung pathological sections of group B, E: Lung pathological sections of group B, F: Lung pathological sections of group B, LR076(pNZ8148-sp-His-isdB, LR076(pNZ8148-sp-His-mntC, LR076(pNZ8148-sp-His-hla H35L ) group lung pathological sections;

[0032] Fig.14The attached figure shows the results of measuring the bacterial load in the lungs of mice after infection with Staphylococcus aureus. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The strain used in the embodiment is LR076, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 27, 2022, with a deposit number of CGMCC No. 25845, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Limosilactobacillus reuteri.

[0035] Example 1 Construction of recombinant lactobacillus

[0036] The signal peptide Sp and His tag of the S layer of Lactobacillus brevis were selected, and the hla of Staphylococcus aureus was obtained through GenBank. H35L , isdB, mntC, the sequence was optimized according to the codon preference of L. reuteri LR076, the sequence of Sp, His tag and antigen after optimization hla H35L , as shown in SEQ ID NO.1, isdB, as shown in SEQ ID NO.2, and mntC, as shown in SEQ ID NO.3, were synthesized and connected to the cloning vector pUC57-Simple. After double enzyme digestion, gel recovery, connection, and transformation, hla H35L , isdB, and mntC were connected to the expression vector pNZ8148 to construct recombinant plasmids, and then the recombinant plasmids were electroporated into L. reuteri LR076 to construct recombinant L. reuteri, and the expression was induced by NisinA to obtain recombinant L. reuteri that could stably express hla H35L , isdB, mntC of L. reuteri LR076 (pNZ8148-sp-His-hla H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC).

[0037] Strains and plasmids required during the experiment: Top10 (pUC57-hla H35L), Top10(pUC57-isdB), Top10(pUC57-mntC), pNZ8148

[0038] The specific process is:

[0039] 1) Contains codon-optimized recombinant plasmid pUC57-hla H35L Extraction of pUC57-isdB, pUC57-mntC: E. coli Top10 (pUC57-hla H35L. After the strains were activated, the recombinant plasmid pUC57-hla was extracted according to the instructions of Beijing All-Gold Plasmid Mini-Extraction Kit. H35L , pUC57-isdB, pUC57-mntC. After completion, the product concentration was determined by spectrophotometry and the plasmid DNA was stored at -20°C.

[0040] 2) Recombinant plasmids pNZ8148-sp-His-isdB, pNZ8148-sp-His-hla H35L , Construction of pNZ8148-sp-His-mntC

[0041] The lactic acid Lactococcus expression vector pNZ8148 and the recombinant plasmid were subjected to Pst I and Kpn I / Hind III double restriction digestion reactions respectively. Enzyme digestion system: recombinant plasmid ≤ 2 μg, 10×FlyCut Buffer 5 μL, FlyC ut Pst I 1.5 μL, FlyCut Kpn I / Hind III 1.5 μL, ultrapure water to 50 μL, incubate at 37°C for 20 min, and after the reaction, use loading buffer (10×DNA Loading Buffer) to precipitate the enzyme digestion product.

[0042] Electrophoresis analysis and gel recovery of enzyme digestion products: Analyze the enzyme digestion products by 1% agarose gel electrophoresis. Place the gel after electrophoresis under ultraviolet light, cut the gel at the location of the target band, and use EasyPure Quick Gel Extraction Kit to recover the double enzyme digestion products.

[0043] Ligation reaction: The pNZ8148 double enzyme digestion gel recovery product was respectively connected with pUC57-hla H35L , pUC57-isdB, pUC57-mntC gel recovery products were connected. The connection operation was carried out according to the instructions of the T4 DNA Ligase Kit. Connection system: pNZ8148 double enzyme digestion gel recovery product 1μL, pUC57-hla H35L6 μL of / isdB / mntC gel recovery product, 2 μL of 5×T4 DNA Ligase Buffer, and 1 μL of T4 DNA Ligase were mixed, placed in a PCR instrument with temperature control and reacted at 25°C for 2 h.

[0044] Transformation of ligation product: Transform the above ligation product into 50 μL E.coli DH5α competent cells and place on ice for 30 min. Place in 42°C water bath for 45 s and continue to place on ice for 2 min. Add 940 μL LB liquid culture medium, resuscitate at 37°C, 200 r / min for 1 h, take 100 μL and spread on LB (Cm+) plate, and culture at 37°C overnight.

[0045] Screening and identification of positive clones: Pick a single white colony on the plate for culture and screen and identify the positive clones. Extract the recombinant plasmid pNZ8148-sp-His-hla according to the instructions of Beijing All-Gold Plasmid Extraction Kit. H35L , pNZ8148-sp-His-isdB, and pNZ8148-sp-His-mntC were double-digested with Pst I and Kpn I / Hind III, and the double-digestion system and reaction were the same as above. After the double-digestion of the recombinant plasmid was completed, the digestion products were analyzed by 1% agarose gel electrophoresis.

[0046] 3) Construction of recombinant L. reuteri

[0047] Extraction of recombinant plasmid: Lactobacillus cell wall is thick, and the efficiency of exogenous DNA entering the cell during electroporation is very low. In addition to optimizing the conditions of electroporation to improve the transformation efficiency, it is also necessary to extract a large amount of plasmids to repeat the transformation experiment. Therefore, the operation was performed according to the instructions of the Omega Plasmid Extraction Kit. After the completion, the product concentration was determined by spectrophotometry, and the plasmid DNA was stored at -20°C.

[0048] Preparation of competent cells of L. reuteri LR076: Pick a single colony of wild-type L. reuteri LR076 from each MRS plate, inoculate it into MRS liquid medium, and culture it at 37°C overnight; inoculate 1% of the overnight culture of L. reuteri LR076 into weakened cell wall structure medium R (1% glycine is added to MRS broth, completely dissolved, and sterilized by filtration with a 0.22 μm filter membrane) and dilute it to an optical density of 0.2A (OD 600 =0.2A); the above culture medium was statically cultured at 37°C until OD 600=0.5A, 4°C, centrifuge for 10 min, 5000r / min to collect cells; wash the cells twice with 3mL pre-cooled sterile ultrapure water, and then wash the cells once with 3mL washing buffer (10% glycerol); resuspend the cells with washing buffer to one percent of the initial volume; dispense the cell suspension into sterile cooled cryovials at 200μL each, and store at -80°C for later use.

[0049] Recombinant plasmid was electroporated into L. reuteri LR076 competent cells: Take L. reuteri LR076 competent cells from -80℃ refrigerator and melt on ice; add 2-3μg recombinant plasmid or empty vector to the cell suspension and place on ice for 10min; transfer the solution to a pre-cooled 2mm electric shock cup and place it in the electric shock tank; adjust the electrode instrument and set the click conditions to 1.9-2.2kV, 25μF, 200Ω, and the electric pulse time to 4-5ms; after the electric shock, add 1mL regeneration liquid culture medium R (MRS broth with 0.5mol / L sucrose, 2mmol / L CaCl2 and 20mmol / L MgCl2, after it is completely dissolved, filter it through a 0.22 μm filter membrane for sterilization), tap it gently to mix it evenly, transfer it to a 1.5 mL sterile centrifuge tube, and culture it at 37°C for 2.5-3 h; take 200 μL of the resuscitated bacterial solution, spread it on an MRS medium plate, and culture it upside down at 37°C for 3 days to screen the transformants.

[0050] Screening of transformants: From the transformed plate, randomly pick multiple transformants and culture them in MRS liquid medium. Take a small amount of bacterial liquid for centrifugation to collect bacteria, take a small amount of bacteria and add 10μL of the best partner, treat it in a 100℃ metal bath for 10min, centrifuge and take the supernatant as a template, use the bacterial liquid to PCR amplify the target gene for detection, the PCR system is Template DNA 1-2μL, 2×M5 Hiper ultralight speed mix (with blue dye) 10μL, Primer 1 (10μM) 0.5μL, Primer 2 (10μM) 0.5μL, Nuclease-free ddH2O is added to 20μL. Reaction conditions: 95℃3min, 32-36cycles of (94℃25sec, 55-64℃25sec, 72℃30-60sec / kb DNA), 72℃5min, 4℃forever, primers are P15'-GTCGATAACGCGAGCATAATAAA-3' and P2

[0051] 5'-TTTGGCTATCAATCAAAGCAACA-3'. After PCR, the PCR product was analyzed by 1% agarose gel electrophoresis. Figure 1 .

[0052] Induced expression of recombinant L. reuteri: Pick a single clone colony on a solid plate and add it to MRS liquid medium, and culture it statically at 37°C overnight; transfer the overnight culture of recombinant L. reuteri to 45 mL of MRS liquid medium containing 25 μg / mL chloramphenicol at a 2% inoculum, and culture it statically at 37°C until OD 600 Reach 0.7A; add Nisin A with a final concentration of 5-10 ng / mL to the recombinant L. reuteri and continue static culture for 6 hours.

[0053] Sample preparation: Take 40mL of bacterial solution and centrifuge at 6000r / min and 4℃ for 8min, aspirate the supernatant into another tube, add 4g of trichloroacetic acid to it, mix well, and place in a 4℃ refrigerator overnight; wash the expression bacteria three times with ultrapure water; resuspend the bacteria with PBS (containing Lysozyme) with 1 / 20 volume of culture medium, incubate at 37℃ for 40min; place on ice and ultrasonicate for 20min; aspirate appropriate amount of lysate suspension and lysate supernatant respectively, add equal volume of 6×Protein Loading Buffer, and boil in a metal bath for 10min; perform SDS-PAGE gel electrophoresis on protein samples, and use the lysate supernatant as a control.

[0054] SDS-PAGE protein electrophoresis: Mix 7.5mL of acrylamide gel solution (12% separation gel) and transfer it between two glass plates on the gel plate, drip distilled water on the upper layer of the separation gel and let it stand for 1.5h; use filter paper to absorb the distilled water, mix 2.5mL of acrylamide gel solution (5% concentrated gel) and transfer it to the upper layer of the separation gel, gently insert the comb horizontally, let it stand for 1h, and take out the comb horizontally; take 35μL of the prepared protein sample and add it to the gel well; connect the electrode, set the electrophoresis instrument voltage to 90V and run for 2.5h.

[0055] Protein identification by Western blotting: remove the gel between the glass plates, transfer the whole to the electrophoresis transfer buffer (1×TransferBuffer) and soak for 40 min; treat the PVDF membrane in methanol solution for 15 s, transfer to double distilled water and rinse for 10 min, then soak the PVDF membrane and two sponges in the electrophoresis transfer buffer for 30 min; according to the semi-dry transfer device, i.e., (-)-sponge-PVDF membrane-gel-sponge-(+) combination, set the transfer device voltage to 25 V and run for 45 min; transfer the transferred PVDF membrane to 5% blocking solution, incubate at 25°C with shaking for 1-2 h, and rinse the PVDF membrane with 1×TBST Buffer; transfer the transferred PVDF membrane to the primary antibody solution, incubate at 4°C with shaking overnight, and rinse the PVDF membrane with 1×TBST Buffer; transfer the transferred PVDF membrane to the secondary antibody solution, incubate at 25°C with shaking for 1 h, and rinse the PVDF membrane with 1×TBST Buffer; detect the target protein band with ECL mixed solution, see Figure 2 .

[0056] Example 2 Analysis of Physiological Characteristics and Genetic Stability of Recombinant L. reuteri

[0057] Growth curve of recombinant L. reuteri

[0058] Activate recombinant L. reuteri and wild strains: dilute the activated bacterial solution to OD 600 =0.02, inoculated in a 96-well plate at a 1% (v / v) inoculum, and added 200 μL MRS liquid culture medium (Cm + / - ). Use growth curve measuring instrument to draw growth curve, see Figure 3 .

[0059] Determination of the resistance of recombinant L. reuteri to artificial gastric fluid

[0060] Activate recombinant L. reuteri and wild strains. Collect bacteria by centrifugation of culture medium, resuspend in sterile PBS to prepare bacterial suspension, add to artificial gastric juice at a ratio of 1:9, mix well, and culture at 37°C anaerobically. Pipette culture medium at 0h and 3h and spread on MRS plate (Cm + / - ) were set up in three parallel groups and cultured at 37°C for 48 hours. The total number of lactic acid bacteria colonies was counted and the survival rate was calculated. Figure 4 .

[0061] Survival rate (%) = (number of colonies on the 3h plate / number of colonies on the 0h plate) × 100%

[0062] Determination of the tolerance of recombinant L. reuteri to artificial intestinal fluid containing bile salts

[0063] Activate recombinant L. reuteri and wild strains. Add the culture medium to artificial intestinal fluid containing bile salts at a ratio of 1:25 and culture at 37°C. Apply the culture medium to MRS plates (Cm + / - ) were set up in three parallel groups and cultured at 37°C for 48 hours. The total number of lactic acid bacteria colonies was counted and the survival rate was calculated. Figure 5 .

[0064] Survival rate (%) = (number of colonies on the 24h plate / number of colonies on the 0h plate) × 100%

[0065] Genetic stability

[0066] Activate the recombinant L. reuteri and wild strains. Add the recombinant L. reuteri and wild strains to 10 mL MRS liquid medium (Cm + / - ) for 20 days, with one generation per day, and plasmids were extracted every day to detect whether the recombinant plasmids in each recombinant strain were stably inherited by PCR. The PCR identification results of the recombinant bacterial plasmids on the 10th and 20th days were used as representatives for plotting, see Figure 6 .

[0067] Example 3 Detection of immunogenicity of recombinant L. reuteri mucosal vaccine

[0068] L.reuteri LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-hla H35L ) and LR076(pNZ8148-sp-His-mntC) were experimental groups, and empty vector bacteria LR076(pNZ8148) was the negative control group;

[0069] Preparation of vaccine and determination of oral dose: recombinant L. reuteri LR076 (pNZ8148-sp-His-isdB), LR076 (pNZ8148-sp-His-hla H35L ), LR076(pNZ8148-sp-His-mntC) and empty vector LR076(pNZ8148) were plated on MRS plates (Cm + ) and activated by streaking, picking single clones and adding them into liquid culture medium (Cm + ), the culture solution was added to 10 mL MRS liquid medium (Cm +) was cultured in an anaerobic static culture until the optical density at 600 nm was between 0.6 and 0.8, and NisinA was added at a final concentration of 5-10 ng / mL. The culture was continued for 6 h, and the bacteria were collected by centrifugation. The bacterial concentration was adjusted with sterile PBS and administered to mice by gavage. The culture solution was diluted to 10 -7 -10 -9 , take 100 μL and spread it on MRS plate (Cm + ) and cultured anaerobically at 37°C for 48 h, and the total number of lactic acid bacteria colonies was counted.

[0070] Immunization strategy and sample collection: All mice were immunized orally with 200 μL of recombinant lactobacillus vaccine three times on days 1, 15, and 29, with an intragastric dose of 1.0 × 10 9 -10 11 CFU / mL. The mice in the control group were given PBS alone in the same way. Blood was collected from the mouse orbits on the 7th day after the second and third immunizations, centrifuged at 3000r / min for 15min, and the antibody level in the serum was quantified by ELISA. The mice were killed on the 7th day after the second and third immunizations, and the alveolar lavage fluid, small intestinal mucosa and small intestinal feces of the mice were taken. 0.1g of the sample was resuspended in 1mL PBS and centrifuged at 3000r / min for 10min. The antibody level in the sample was quantified by ELISA. On the 7th day after the third immunization, the mice were killed, and the lungs, spleens and Peyer's Patches of the mice were taken. The supernatant was obtained by grinding and centrifugation, and the levels of cytokines IL-17 and IFN-γ in the samples were quantified by ELISA.

[0071] Quantification of Staphylococcus aureus antigen-specific sIgA antibodies in mouse bronchoalveolar lavage fluid: Indirect ELISA was used to measure the Staphylococcus aureus protein antigens IsdB, MntC and Hla in the supernatant of mouse bronchoalveolar lavage fluid on the 7th day after the second and third immunizations. H35L Specific sIgA antibody levels were quantified. The bronchoalveolar lavage fluid supernatant was diluted at a ratio of 1:80 when adding samples. Antibody levels were expressed as OD values. Figure 7 The results showed that the recombinant bacteria induced antigen-specific sIgA antibody levels in the bronchoalveolar lavage fluid of mice. H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC) 7 days after the mice bronchoalveolar lavage fluid was collected to measure the sIgA level. Figure A shows the hla H35LFigure 1 shows the level of isdB-specific sIgA antibodies in bronchoalveolar lavage fluid; Figure 2 shows the level of mntC-specific sIgA antibodies in bronchoalveolar lavage fluid.

[0072] Quantification of Staphylococcus aureus antigen-specific sIgA antibodies in the small intestinal mucosa of mice: Indirect ELISA was used to measure the Staphylococcus aureus protein antigens isdB, mntC and Hla in the supernatant of the small intestinal mucosa of mice 7 days after the second and third immunizations. H35L The specific sIgA antibody level was quantified. The small intestinal mucosal supernatant was diluted at a ratio of 1:80 when adding samples. Figure 8 The results showed that the recombinant bacteria induced antigen-specific sIgA antibody levels in the small intestinal mucosa of mice. Mice were orally immunized with PBS, LR076 (pNZ8148) and the recombinant strain LR076 (pNZ8148-sp-His-hla H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC) 7 days after the mouse small intestinal mucosa was taken to measure the sIgA level. Figure A shows the hla H35L Figure 1 shows the level of isdB-specific sIgA antibodies in the small intestinal mucosa; Figure 2 shows the level of mntC-specific sIgA antibodies in the small intestinal mucosa.

[0073] Quantification of Staphylococcus aureus antigen-specific sIgA antibodies in mouse small intestinal feces: Indirect ELISA was used to measure the Staphylococcus aureus protein antigens IsdB, MntC and Hla in the supernatant of mouse small intestinal feces on the 7th day after the second and third immunizations. H35L Specific sIgA antibody levels were quantified. Small intestinal fecal supernatant was diluted at a ratio of 1:80 when adding samples. Fig. 9 The results showed that the recombinant bacteria induced antigen-specific sIgA antibody levels in the small intestinal feces of mice. H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC) 7 days after the mice were treated with the sIgA level in the small intestine. H35L Figure 1 shows the level of isdB-specific sIgA antibodies in small intestinal feces; Figure B shows the level of isdB-specific sIgA antibodies in small intestinal feces; Figure C shows the level of mntC-specific sIgA antibodies in small intestinal feces.

[0074] Quantification of IL-17 levels in mouse lung, spleen and Peyer's Patches tissues: Measure the IL-17 levels in the supernatant of mouse lung, spleen and Peyer's Patches tissues on day 7 after the third immunization. Quantify the IL-17 levels in tissues according to the instructions of the mouse IL-17 ELISA kit. Measure the optical density of IL-17 in tissues at 450nm using an ELISA automatic reader. Fig.10 The results showed that the recombinant bacteria induced IL-17 levels in the lungs, spleens and Peyer's Patches of mice. Mice were orally immunized with PBS, LR076 (pNZ8148) and the recombinant strain LR076 (pNZ8148-sp-His-hla H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC), the lungs, spleens and Peyer's Patches of the mice were taken out on the 7th day to measure the IL-17 level. Figure A shows the IL-17 level in the lung; Figure B shows the IL-17 level in the spleen; Figure C shows the IL-17 level in Peyer's Patches.

[0075] Quantification of IFN-γ levels in mouse lung, spleen and Peyer's Patches tissues: Measure the IFN-γ levels in the supernatant of mouse lung, spleen and Peyer's Patches tissues on day 7 after the third immunization. Quantify the IFN-γ levels in tissues according to the kit instructions. Measure the IFN-γ optical density value in tissues at 450nm using an ELISA automatic reader. Fig.11 The results showed that the recombinant bacteria induced IFN-γ levels in the lungs, spleens and Peyer's Patches of mice. H35L ), LR076(pNZ8148-sp-His-isdB), LR076(pNZ8148-sp-His-mntC), the lungs, spleens and Peyer's Patches of mice were taken out on the 7th day to measure the IFN-γ level. Figure A shows the IFN-γ level in the lung; Figure B shows the IFN-γ level in the spleen; Figure C shows the IFN-γ level in Peyer's Patches.

[0076] Example 4 Study on the protective effect of recombinant L. reuteri in a mouse pneumonia model caused by Staphylococcus aureus

[0077] Establishment of mouse pneumonia model: Immunize mice according to the strategy described above, and on day 36, intranasally infect mice with Staphylococcus aureus to establish a pneumonia model. The specific steps are as follows:

[0078] Staphylococcus aureus was cultured in LB liquid medium and the bacterial concentration was adjusted to 5 × 10 10 -10 11 CFU / mL. 4% chloral hydrate was used as an anesthetic, 160 μL of which was injected intraperitoneally into the mouse, and then 40 μL of the Staphylococcus aureus suspension was slowly dripped into the left nostril of the mouse, and the mouse was placed back in the cage in a supine position.

[0079] Statistics of mouse survival rate: 72 hours after each group of mice (10 mice / group) were infected with Staphylococcus aureus, the death of all mice was observed and continuously recorded, and the survival rate was calculated. Fig.12 The recombinant bacteria were shown to effectively protect mice from pneumonia caused by Staphylococcus aureus.

[0080] LR076(pNZ8148-sp-His-hla H35L The survival rates of mice in the LR076(pNZ8148-sp-His-isdB) and LR076(pNZ8148-sp-His-mntC) groups were 70%, 60% and 50%, respectively, while the survival rates of the empty bacteria LR076(pNZ8148) and PBS groups were 10% and 0%.

[0081] Histopathological analysis of mouse lungs: 24 h after infection, mice in each group (3 mice / group) were killed, and the intact left lung was taken out and placed in 4% paraformaldehyde for histopathological analysis. Fig.13 The results showed that the recombinant bacteria can effectively alleviate lung lesions after Staphylococcus aureus infection. 24 hours after the mice were infected with Staphylococcus aureus, the left lungs of the mice were taken and HE stained. Group B is the PBS group, Group C is the LR076 (pNZ8148) group, Group D is the LR076 (pNZ8148-sp-His-isdB) group, Group E is the LR076 (pNZ8148-sp-His-mntC) group, and Group F is the LR076 (pNZ8148-sp-His-hla H35L )Group.

[0082] Determination of bacterial load in mouse lungs: 24 hours after infection, mouse lungs were taken and sterile PBS was added at a ratio of 1:10 (m / v). The lungs were broken up and ground into a uniform paste using a tissue homogenizer. The homogenate was spread on an LB plate, and the total number of Staphylococcus aureus colonies was counted the next day to determine the number of Staphylococcus aureus in the lung tissue of infected mice. Fig.14 It showed that the recombinant bacteria could significantly reduce the colonization of Staphylococcus aureus in the lungs.

[0083] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0084] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An antigen carrier strain, characterized in that: The strain is LR076, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 27, 2022, with the deposit number CGMCC No.25845, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is Limosilactobacillus reuteri .

2. A recombinant strain, characterized in that: The recombinant strain is formed by transforming a recombinant plasmid carrying an exogenous Staphylococcus aureus antigen fragment into the LR076 strain described in claim 1; the antigen fragment includes Hla H35L , IsdB or MntC, as shown in SEQ ID NO.1 to SEQ ID NO.3, respectively.

3. A recombinant strain according to claim 2, characterized in that: The recombinant plasmids include pNZ8148-sp-His- hla H35L 、pNZ8148-sp-His- iSdB or pNZ8148-sp-His- mntC .

4. A recombinant strain according to claim 3, characterized in that: The construction method of the recombinant plasmid comprises: 1) Select the S-layer signal peptide Sp and His tag of Lactobacillus brevis and obtain Staphylococcus aureus through GenBank hla H35L 、 isdB、mntC The whole sequence, according to L. reuteri The codon preference of the sequence was optimized, and the optimized sequences of Sp, His tag and antigen were synthesized and ligated into the cloning vector pUC57-Simple; 2) After double enzyme digestion, gel recovery, ligation, and transformation hla H35L , isdB or mntC The recombinant plasmid was constructed by connecting to the expression vector pNZ8148.

5. Use of the strain according to claim 1 in preparing an oral Staphylococcus aureus vaccine.

6. Use of the recombinant strain according to any one of claims 2 to 4 in the preparation of an oral Staphylococcus aureus vaccine.

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