Bacterial holoantigen-loaded PLGA microsphere vaccine as well as preparation method and application thereof

Abstract

The invention discloses a bacterial holoantigen-loaded PLGA (poly (lactic-co-glycolic acid)) microsphere vaccine as well as a preparation method and application thereof. The method comprises the following steps: cracking target bacteria by adopting a bacterial cell protein lysis solution to obtain whole-bacterial antigens, and preparing whole-bacterial antigen particles in a nanoparticle form in a high-pressure co-extrusion manner; the antigen particles and a PLGA microsphere carrier are mixed through a multiple emulsion method, and a microsphere vaccine (MSV) with antigens loaded inside and outside the carrier can be obtained. The antigen on the surface of the MSV is released in a burst release manner, and the antigen loaded in the MSV can achieve a long-acting slow release effect, so that the programmed step-by-step release control of the antigen release behavior is realized, that is, the dual effects of primary immunization and enhanced immunization can be achieved by single-dose vaccine injection. In addition, the MSV can effectively avoid damage of bacterial toxins to immune cells of the body, and the biological safety of bacterial vaccines is improved. The design is reasonable, and the introduction of the microsphere preparation provides a safe and reliable carrier platform for a bacterial inactivated vaccine.

Description

Technical Field

[0001] The present invention relates to the pharmaceutical field, and in particular to a PLGA microsphere vaccine carrying complete bacterial antigens and a preparation method and application thereof. Background Art

[0002] It is estimated that by 2050, drug-resistant bacteria may cause about 10 million deaths per year, making antibiotic resistance (AMR) one of the most serious threats to global public health. Among them, superbug infection is an important cause of antibiotic resistance. Clinical practice shows that limiting the use of antibiotics can restore bacterial sensitivity (Front Antibiot.2023;2).

[0003] Currently, intervention strategies to combat superbugs that have been developed and applied include new antibiotics, vaccines, diagnostic tools, monoclonal antibodies, and microbiome intervention. Among them, bacterial vaccines are not affected by bacterial resistance and can provide long-term protection to the body by actively activating the immune defense system. They are the most promising method to combat superbug infections. In addition, vaccines can also assist in antibiotic treatment and are a favorable weapon to reduce antibiotic use and alleviate AMR. However, most of the world's super-resistant bacterial vaccines are currently in the preclinical stage, and no products have been launched on the market. Moreover, most of the vaccines currently in the clinical stage are single antigen components, which are not enough to block the multi-pathway pathogenic pathways of superbugs. Although the inactivated bacterial vaccine contains the full antigen components of the bacteria, the bacterial toxin antigens that spread rapidly after injection pose a major safety hazard to the body. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a PLGA (poly(lactic-co-glycolic acid) poly(lactic-co-glycolic acid)) microsphere vaccine carrying a complete bacterial antigen and a preparation method and application thereof. The vaccine has high biological safety and comprehensive immune induction.

[0005] In order to solve the technical problem, the present invention provides a method for preparing a PLGA microsphere vaccine carrying a complete bacterial antigen, which comprises the following steps:

[0006] 1) using bacterial cell protein lysis solution to lyse the target bacteria to obtain whole bacterial antigens, and preparing whole bacterial antigen particles in the form of nanoparticles by probe ultrasound and high-pressure co-extrusion;

[0007] 2) dissolving PLGA in an organic solvent as an oil phase, suspending bacterial full antigen particles in water as an inner aqueous phase, and mixing the two to obtain colostrum; using a PVA solution as an outer aqueous phase, placing the colostrum in the outer aqueous phase, and obtaining a double emulsion after high-speed shearing; using the PVA aqueous solution as a solidifying liquid, adding the double emulsion to the solidifying liquid for solidification, and then volatilizing the organic solvent, washing, and centrifuging to enrich, to obtain PLGA microspheres with uniform particle size, namely the PLGA microsphere vaccine carrying bacterial full antigens.

[0008] As an optional solution of the present invention, the target bacteria include but are not limited to Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa.

[0009] Furthermore, the step 1) is specifically as follows: culturing the target bacteria to the logarithmic growth phase, and collecting the live bacterial precipitate by centrifugation; after the bacteria are repeatedly frozen and thawed by liquid nitrogen / room temperature, adding bacterial cell protein lysis solution, and lysing the bacterial cells on ice to obtain bacterial lysate; after diluting the bacterial lysate with pure water, subjecting it to probe ultrasonic fragmentation treatment, and then rearranging the hydrophilic and hydrophobic antigen components by high-pressure co-extrusion to obtain nano-sized whole bacterial antigen particles.

[0010] Furthermore, in step 1), the particle size of the whole bacterial antigen particles is 20-200 nm.

[0011] Furthermore, in step 2), the organic solvent is dichloromethane; the colostrum is a water-in-oil emulsion; and the double emulsion is a W / O / W water-in-oil-in-water emulsion.

[0012] Furthermore, the mass percentage concentration of PVA in the external water phase is 2%, and the mass percentage concentration of PVA in the solidifying liquid is 0.2%.

[0013] Furthermore, the particle size of the PLGA microsphere vaccine is 1-10 μm, and the bacterial antigen loading on the PLGA microsphere vaccine is 4wt%-10wt%.

[0014] In a second aspect, the present invention provides a PLGA microsphere vaccine loaded with complete bacterial antigens prepared by the method. Furthermore, the microspheres in the microsphere vaccine perform surface adsorption and internal loading of nano-scale antigen particles, and the antigen particles adsorbed on the surface of the preparation can achieve a burst release, and the antigen particles loaded inside can achieve a long-term release.

[0015] In a third aspect, the present invention provides the use of the PLGA microsphere vaccine carrying complete bacterial antigens in the preparation of vaccine products.

[0016] The PLGA microsphere vaccine and vaccine product provided by the present invention have strong antigen loading stability, high biosafety, and can induce a comprehensive immune response.

[0017] The induction of a comprehensive immune response includes humoral immunity and cellular immunity. Immune indicators include serum antibody titer, types and abundance of immune factors, DC activation and the generation of B cell germinal centers, CD4 + and CD8 + Differentiation of T cells, generation of memory T cells, etc.

[0018] The PLGA microsphere vaccine and vaccine product provided by the present invention can be used to prevent bacterial infection and improve the body's sensitivity to antibiotic treatment.

[0019] The bacterial infection can be super-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa, and the infection model can be bloodstream infection, lung infection, skin infection, etc. After infection, a bacterial vaccine is prepared, and mice are immunized by subcutaneous injection of a single dose to construct a mouse model of homologous bacterial infection.

[0020] The innovation of the present invention lies in the preparation of a PLGA microsphere vaccine loaded with all bacterial antigen epitopes. Compared with ordinary inactivated vaccines, the antigen of the microsphere vaccine exists in the form of nanoparticles, which can not only promote the antigen abundance in a single cell of dendritic cells (DC) with the strongest professional antigen presentation ability in the human body, enhance antigen transfer between different subpopulations of DC, but also play the role of natural adjuvant and induce immune responses of multiple cells. In addition, the vaccine is loaded with PLGA to form a PLGA microsphere antigen reservoir. On the one hand, the reservoir can adsorb toxins, thereby greatly improving the safety of bacterial vaccines. On the other hand, the present invention adopts a pharmaceutical method to load and deliver bacterial antigens, which can effectively control the release behavior of antigens: the antigen particles on the surface of the microsphere can achieve sudden release, and the antigens loaded inside can achieve long-term release. Therefore, the vaccine can be released in a programmed step-by-step manner after a one-time injection, achieving the dual effects of primary immunity and booster immunity, thereby improving the patient's immune response and effectively resisting drug-resistant bacteria in the body. The present invention has a reasonable design, simple antigen preparation, and the introduction of microsphere preparations provides a safe and reliable carrier platform for bacterial inactivated vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A transmission electron microscopy image of a Staphylococcus aureus vaccine prepared according to an embodiment of the present application is shown.

[0022] Figure 2 The scanning electron micrographs of PLGA microspheres loaded with Staphylococcus aureus antigen particles inside and outside are shown, where A is a cross-sectional view of the microspheres, with many voids and antigen particles visible inside; B is a picture of microspheres adsorbed with a large number of antigen particles; and C is a picture of looser microspheres formed after increasing the antigen dose.

[0023] Figure 3Shown is SDS-PAGE silver-stained protein analysis of SPA, Free Ag, and MSV.

[0024] Figure 4 The release behavior of antigens in PLGA microspheres is shown.

[0025] Figure 5 Shown are the cytotoxicity of different free concentrations of Ag and MSV on RAW264.7 and DC2.4 cells.

[0026] Figure 6 The hemolysis rate of rat erythrocytes by bacterial antigens at different concentrations (A) and the hemolysis rate of erythrocytes by antigen-loaded microspheres (B) are shown.

[0027] Figure 7 Shown are fluorescent images of DC2.4 cells taking up antigen-loaded PLGA microspheres.

[0028] Figure 8 Shown are flow cytometry images of bone marrow-derived BMDCs activated by free Ag and antigen-loaded PLGA microspheres.

[0029] Fig. 9 Shown is the in vivo distribution of fluorescently labeled bacterial antigens and antigens loaded into PLGA microspheres after subcutaneous injection.

[0030] Fig.10 Shows the distribution of different antigens in mouse organs 120 hours after injection of free antigen and microsphere vaccine.

[0031] Fig.11 It shows that the use of bone marrow cell NE can further reduce the hemolytic rate of MSV vaccine and improve its safety.

[0032] Fig.12 Figure 3 shows the immune evaluation of mice immunized with free Ag and MSV / NE, A is the curve of mouse body weight changes, B is the curve of serum IgM level changes over time, C is the curve of anti-α-toxin antibody level changes in serum over time, D is the curve of mouse lymph node changes after immunization, E is the activation efficiency of DC cells in mouse lymph nodes after immunization, F is the proportion of functional CD8+T cells in mouse spleen after immunization, G is the proportion of IFN-γ+CD4+ and IL-4+CD4+T cells in mouse spleen after immunization.

[0033] Fig.13 Shown are germinal centers of B cells in lymph node sections of mice after immunization with free Ag and MSV / NE.

[0034] Fig.14The weight-time curve and survival rate within 7 days after immunization of mice with free Ag and MSV / NE and challenge with MRSA bacteria one week later are shown. It can effectively resist MRSA bloodstream infection.

[0035] Fig.15 It shows the proportion of immune cells in the peripheral blood of mice after immunization with free Ag and MSV / NE and then challenged with MRSA bacteria.

[0036] Fig.16 The figure shows the number of bacteria colonizing in the peripheral blood of mice after immunization with free Ag and MSV / NE and then challenged with MRSA bacteria.

[0037] Fig.17 The figure shows the number of bacteria colonizing in the main organs of mice after immunization with free Ag and MSV / NE and then challenged with MRSA bacteria. DETAILED DESCRIPTION

[0038] The present invention is further described with reference to the accompanying drawings and implementation examples.

[0039] Example 1 Preparation and morphological characterization method of MRSA full antigen particles (Ag)

[0040] MRSA (Methicillin-resistant Staphylococcus aureus) was inoculated into LB liquid culture medium, cultured at 37°C until the logarithmic growth phase, and then centrifuged at 1000rpm for 5 minutes to collect the live bacterial precipitate. After the bacteria were repeatedly frozen and thawed by liquid nitrogen / room temperature, a bacterial cell protein lysate (purchased from Sangon Biotech (Shanghai) Co., Ltd., No. C500003-0010) was added in a volume 5 times the wet weight of the bacteria, and the bacterial cells were lysed on ice to obtain whole bacterial antigens. Subsequently, the bacterial lysate was diluted with pure water and subjected to probe ultrasonic disruption to obtain a whole bacterial suspension containing antigens with different physical and chemical properties; the hydrophilic and hydrophobic antigen components were rearranged by high-pressure co-extrusion to prepare whole bacterial antigen particles in the form of nanoparticles (the present invention uses Ag to refer to the whole bacterial antigen in the form of nanoparticles prepared in Example 1), and its morphology was characterized by transmission electron microscopy (TEM) ( Figure 1 The particle size of the whole bacterial antigen antigen particles is 20-200nm, and the appearance is a round and uniform sphere). In the embodiment, after repeated freezing and thawing of liquid nitrogen, the bacteria are lysed by a bacterial lysis solution, the purpose of which is to physically and chemically inactivate the bacteria; the bacterial lysis solution is diluted with pure water, and the bacteria are ultrasonically broken by a probe, and the antigen components with different physical and chemical properties are recombined, and the antigen can exist in the form of particles.

[0041] Example 2 Preparation method of a microsphere vaccine preparation (MSV)

[0042] This embodiment uses a water-in-oil-in-water (W / O / W) type emulsion as a carrier to prepare a microsphere vaccine MSV with Ag co-loaded inside and outside. Specifically as follows: First, 100 μL of 120 mg / mL Ag aqueous solution is used as the inner aqueous phase, and it is added to 1 mL of 60 mg / mL PLGA dichloromethane solution, and an oil-in-water type (W / O) colostrum is formed by probe ultrasound. Subsequently, the colostrum is quickly injected into 10 mL of an outer aqueous phase solution (2% polyvinyl alcohol PVA aqueous solution), and the mixed solution is sheared at a high speed of 1500 rpm to form a W / O / W emulsion. The emulsion is then injected into 100 mL of a solidifying solution (0.2% PVA aqueous solution), and after stirring overnight at room temperature to volatilize the organic solvent, the microsphere precipitate is collected by centrifugation at 10000 rpm for 10 minutes. Finally, the microsphere precipitate was resuspended in 1 mL PBS, and 20 μL 120 mg / mL Ag aqueous solution was added and stirred at low speed for 2 h, and then purified by centrifugation to obtain a microsphere vaccine preparation (MSV) with internal and external Ag. The MSV was resuspended in PBS, lyophilized with a lyophilization protectant, and stored at -20°C for later use. The antigen protein loading rate in the microspheres was determined to be 10% by using a BCA protein assay kit.

[0043] Example 3 Characterization of Microsphere Vaccine Formulation (MSV)

[0044] Firstly, the hydrodynamic size of MSV particles was measured by a particle size potentiometer to be 5 μm, and the surface potential (Zetapotential) was -42.1 mV. The distribution of Ag in MSV was observed by scanning electron microscopy (SEM). Figure 2 The scanning electron microscope images show the distribution of Ag in the microspheres, showing three states: one is distributed inside the microspheres, the second is adsorbed on the surface of the microspheres, and the third is mixed with the microsphere material to form a part of the sphere. Then, using purified Staphylococcus aureus protein A (SPA) as an internal reference, the distribution of free Ag on SDS-PAGE gel and antigen molecule protein in MSV was investigated by silver staining. Figure 3 The results show that after the antigen was loaded into the microspheres, the distribution of its protein bands did not change significantly compared with the free antigen. In order to evaluate the encapsulation efficiency and drug loading of Ag in PLGA, 20 mg of different batches of MSV lyophilized powder were dissolved in 1 mL of N-methylpyrrolidone (MMP) to fully release the antigen in the microspheres. Figure 4 It can be seen that the antigen in MSV first shows a sudden release phenomenon, and then can maintain a sustained release for more than 200 hours.

[0045] Example 4 In vitro biosafety assessment of MSV

[0046] Different cell lines (mouse macrophage-like cell line RAW264.7 and mouse bone marrow-derived dendritic cells DC2.4) were cultured at 5*104 100 cells / well were pre-seeded in a 96-well plate and cultured to the logarithmic phase. Then, PBS, low-dose free antigen (Ag-L, 1 μg / mL), medium-dose free antigen (Ag-M, 5 μg / mL), high-dose free antigen (Ag-H, 10 μg / mL), MS0 (blank microsphere group, 10 μg / mL) and microsphere vaccine MSV (containing Ag 5 μg / mL) were added. After 24 hours, the cell viability was detected using the CCK8 kit. Figure 5 It can be seen that the free antigens, because they contain bacterial toxins, have certain cytotoxicity to the immune cells RAW264.7 and DC2.4, and are dose-dependent. After being loaded with microspheres, the cytotoxicity of bacterial antigens to immune cells is significantly downregulated.

[0047] Furthermore, the blood of the rats was collected using an anticoagulant blood collection tube, and the red blood cells were separated by the natural sedimentation method, and the hemolysis phenomenon was observed. The specific method is as follows: 2 mL of orbital venous blood from the rat was taken, placed in a test tube with an anticoagulant, and gently mixed. The test tube was placed upright at room temperature or in a 37°C incubator for 30 to 60 minutes to allow the red blood cells to settle naturally. The obtained red blood cells were made into a 4% suspension with a 0.9% sodium chloride solution. Then, Triton-X-100 was used as a positive control, and Saline, Free Ag, blank microsphere group (MS0) and MSV group were set up. Different concentrations were set according to the Ag content. After mixing with the red blood cell suspension, it was added to a 96-well plate. After incubation at 60 rpm in a 37°C incubator for 1 hour, 100 μL of supernatant from each group was carefully pipetted into a new 96-well plate, and its OD540 nm was detected with an enzyme reader, and the hemolysis rate was calculated. Figure 6 The experimental results show that the lysis ability of free Ag on red blood cells is concentration-dependent, and the hemolysis rate of free Ag is significantly reduced compared with the same concentration of Ag loaded on microspheres.

[0048] Example 5 Study on Enhanced MSV Uptake by Antigen Presenting Cells

[0049] DC2.4 cells were cultured at 1x10 5 100 cells / well were inoculated in a 24-well plate, and MS0 and MSV groups were set up, that is, the corresponding DiD-labeled MS0 and MSV were added to the wells of the cell culture plate. After incubation for 6 hours, the cell uptake of particles was observed under a fluorescence microscope. Figure 7 The results show that under the same incubation conditions, the uptake of MSV by DC2.4 was significantly higher than that of blank microspheres.

[0050] Example 6: MSV promotes DC presentation of antigens

[0051] Differentiation-induced BMDC (mouse bone marrow derived dendritic cells) were inoculated in 24-well plates, and free Ag, MS0 and MSV groups were set up respectively, and the corresponding free Ag, MS0 and MSV were added to the wells of the cell culture plate. After incubation for 24 hours, the expression of CD86 and MHC-II positive cells in different treatment groups was analyzed by flow cytometry. Figure 8 The results showed that in the group with antigen presence, the expression levels of DC surface activation markers CD86 and MHC II were significantly upregulated.

[0052] Example 7 MSV in vivo biodistribution study

[0053] Proteins and peptides in Ag were labeled with Cy7-N-hydroxysuccinimide ester (Cy7-NHS), and PLGA was labeled with FITC to prepare MSV (FMSV) with dual fluorescence labeling. FMSV (2 mg) or free Cy7-Ag (100 μg) was injected subcutaneously into C57 mice. In vivo fluorescence imaging of mice was performed at different time points using an in vivo imaging system (IVIS). The results showed that the Cy7-Ag signal in the MSV group mice was the strongest within 120 hours, while the Cy7 fluorescence signal in the free Ag group was the weakest, indicating that MSV prolonged the retention time of Ag in mice and could achieve slow and sustained release of Ag. After 120 hours, the mice were euthanized, and the retention of the vaccine at the injection site was observed. The lymph nodes, heart, liver, spleen, lung, kidney and other tissues were removed for fluorescence imaging. Fig. 9 It can be seen that the fluorescence signal intensity of the antigen at the inoculation site of mice vaccinated with free Ag began to weaken after 72 hours, while the fluorescence intensity of the antigen at the inoculation site of mice vaccinated with microsphere vaccine still had strong antigen retention until 120 hours later. The tissues were then fixed with 4% paraformaldehyde and sliced ​​for immunofluorescence analysis. In vitro fluorescence imaging showed the distribution of Cy7-Ag in the important organs of mice after 120 hours. Fig.10 The results showed that the Ag in the MSV group was mainly concentrated in the lymph nodes, with very little retention in the heart, liver, spleen, lungs, and kidneys. This showed that MSV could prevent the spread of antigens to major organs in the body, reduce the damage of the vaccine to the body, and improve biosafety.

[0054] Example 8 Study on the immune response of MSV vaccine

[0055] In order to further reduce the impact of bacterial antigens adsorbed outside the MSV microsphere vaccine on mouse immune cells, we isolated mouse bone marrow cells (NE) during the immunization experiment, briefly incubated NE with MVS, and further reduced the toxicity and irritation of bacterial toxins to mice by adsorbing bacterial toxins. Fig.11It showed that after incubation with NE, the hemolysis rate of MSV on red blood cells was further reduced, indicating that the safety was further improved. Saline, free Ag and NE-attenuated MSV (MSV / NE) were used for immunization and immune evaluation in healthy mice. On the third day after immunization, mice treated with free Ag showed a trend of sudden weight loss. The possible reason was that high doses of Ag spread in the mice and bacterial toxins caused obvious harm to the mice. There was no significant difference in the weight change of mice in other groups within 21 days, which well demonstrated that MSV / NE can reduce the toxicity of inactivated bacterial antigens by adsorbing toxins and has good biosafety. The IgM antibody level in the peripheral blood serum of mice was monitored, and the results showed that the vaccine had good immunogenicity. Compared with other groups, the α-Toxin-IgG antibody level in the peripheral blood serum of mice in the MSV / NE group was the highest on the 14th day after immunization. After the immunization, the lymph nodes and spleen were separated, and the effect of the vaccine on the immune system was further evaluated by high-throughput flow cytometry. The results showed that the peripheral immune organ lymph nodes in the MSV / NE group were significantly enlarged. In the lymphoid tissues of mice treated with free Ag and MSV / NE, activated DC (CD11c + CD80 + CD86 + ) cell number significantly increased. Macrophages expressing MHC-II can not only phagocytize pathogens, but also present antigens and induce active CD4 + T cell immune response. Although there was little difference in T cells in the spleen of mice immunized by different methods, the killer T cells (IFN-γ + CD8 + T cells) was significantly increased, and Th2 cells (IL-4 + CD4 + The proportion of T cells) also increased significantly, indicating that MSV / NE effectively activated humoral and cellular immune responses ( Fig.12 In addition, immunofluorescence analysis of lymph node tissue sections of immunized mice revealed that the lymph nodes of mice treated with MSV / NE had obvious B cell germinal centers, which provided a prerequisite for the secretion of antibacterial specific antibodies ( Fig.13 ).

[0056] Example 9: MSV protects mice from MRSA sepsis infection

[0057] Three weeks after vaccination, all mice were intravenously injected with the MRSA strain homologous to the vaccine. During the 7-day observation period, the weight loss rate of the mice immunized with MSV was less than 5% and all survived, while the weight of the mice in the free Ag and Saline groups decreased significantly, with a weight loss rate of more than 15%. Correspondingly, the survival rates of these two groups of mice were only 20% and 40% ( Fig.14 In addition, the proportions of neutrophils (Neu), monocytes (Mon), and white blood cells (WBC) in the peripheral blood of mice in the Saline group and the free Ag group were higher, and the proportion of lymphocytes (Lym) was lower, while the proportions of various immune cells in the peripheral blood of mice in the MSV group were not significantly different from those in healthy mice, which suggests that there may be a large number of bacteria circulating in the blood of mice in the Saline group and the free Ag group ( Fig.15 To further verify this conclusion, the peripheral blood of mice was smeared on agarose medium, and a very obvious number of live bacterial colonies could be seen, among which the number of bacterial colonies in the peripheral blood of mice in the Saline group was the highest ( Fig.15 In addition to peripheral blood, the heart, liver, spleen, lungs and kidneys of mice immunized with MSV also showed the least bacterial load, and compared with the Saline and free Ag groups, the CD4 + T cells, CD8 + T cells, killer T cells (IFN-γ + CD8 + The proportion of T cells) and Th2 cells increased significantly, and the proportion of central memory T cells TCM in the lymph nodes also increased significantly. This shows that the vaccine induces a strong humoral and cellular immune response, which can effectively resist bacterial infection ( Fig.16 ).

[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a PLGA microsphere vaccine carrying a complete bacterial antigen, characterized in that: The steps include: 1) using bacterial cell protein lysis solution to lyse the target bacteria to obtain whole bacterial antigens, and preparing whole bacterial antigen particles in the form of nanoparticles by probe ultrasound and high-pressure co-extrusion; 2) dissolving PLGA in an organic solvent as an oil phase, suspending bacterial full antigen particles in water as an inner aqueous phase, and mixing the two to obtain colostrum; using a PVA solution as an outer aqueous phase, placing the colostrum in the outer aqueous phase, and obtaining a double emulsion after high-speed shearing; using the PVA aqueous solution as a solidifying liquid, adding the double emulsion to the solidifying liquid for solidification, and then volatilizing the organic solvent, washing, and centrifuging to enrich, to obtain PLGA microspheres with uniform particle size, namely the PLGA microsphere vaccine carrying bacterial full antigens.

2. The preparation method according to claim 1, characterized in that: The target bacteria include, but are not limited to, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa.

3. The preparation method according to claim 1, characterized in that: The step 1) is specifically as follows: The target bacteria are cultured to the logarithmic growth phase, and the live bacterial precipitates are collected by centrifugation; after the bacteria are repeatedly frozen and thawed in liquid nitrogen / room temperature, bacterial cell protein lysis buffer is added, and the bacterial cells are lysed on ice to obtain bacterial lysate; after the bacterial lysate is diluted with pure water, it is subjected to probe ultrasonic disruption, and then the hydrophilic and hydrophobic antigen components are rearranged by high-pressure co-extrusion to obtain nano-sized whole bacterial antigen particles.

4. The preparation method according to claim 3, characterized in that: In step 1), the particle size of the whole bacterial antigen particles is 20-200 nm.

5. The preparation method according to claim 1, characterized in that: Step 2) The organic solvent is dichloromethane; the colostrum is a water-in-oil emulsion; and the double emulsion is a W / O / W water-in-oil-in-water emulsion.

6. The preparation method according to claim 1, characterized in that: The mass percentage concentration of PVA in the external water phase is 2%, and the mass percentage concentration of PVA in the solidifying liquid is 0.2%.

7. The preparation method according to claim 1, characterized in that: The particle size of the PLGA microsphere vaccine is 1-10 μm, and the bacterial antigen loading on the PLGA microsphere vaccine is 4wt%-10wt%.

8. The PLGA microsphere vaccine loaded with complete bacterial antigens prepared by the method according to any one of claims 1 to 7.

9. The PLGA microsphere vaccine carrying complete bacterial antigens according to claim 8, characterized in that: The microspheres in the microsphere vaccine perform surface adsorption and internal loading of nanometer-scale antigen particles. The antigen particles adsorbed on the surface of the preparation can achieve a burst release, and the antigen particles loaded inside can achieve a long-acting release.

10. Use of the PLGA microsphere vaccine carrying complete bacterial antigens according to claim 8 in the preparation of vaccine products.

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