CN112662695A - Construction method and application of Bacterial Biofilm Vesicle (BBV) as vaccine vector - Google Patents

Abstract

The invention provides a construction method and application of a Bacterial Biofilm Vesicle (BBV) as a vaccine carrier, wherein an ultra-high pressure is used for driving an engineered bacterial biofilm to complete high-efficiency self-assembly for the first time, and the ability of ClyA to assemble a pore on a bacterial outer membrane is used for carrying RBD to construct a burred bacterial vesicle (RBD-BBV) in a prokaryotic system, and the RBD-BBV efficiently exposes the correctly folded RBD on the surface of the vesicle and shows the burred vesicle structure. The RBD-BBV can be efficiently taken up by DC cells and stimulates the maturation of the DC cells, and meanwhile, the bacterial biomembrane mediates the lysosome escape of the RBD. After RBD-BBV immunization of mice, SARS-COV-2 specific neutralizing antibodies and SARS-COV-2 specific CD4+ T and CD8+ T cell responses are induced in the mice. RBD-BBV also enhances the local stability of the antigen in vivo and induces the production of memory T cells. The RBD-BBV vaccine form has the unique characteristics that other vaccine forms cannot be realized, and can provide a new idea for the development of SARS-CoV-2 vaccine.

Description

Construction method and application of Bacterial Biofilm Vesicle (BBV) as vaccine vector

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a Bacterial Biofilm Vesicle (BBV) as a vaccine vector.

Background

SARS-CoV-2 is a new type of coronavirus, whose surface has obvious spike structure, and RBD on spike protein can combine with ACE2 of receptor cell, thereby mediating virus invasion. While some drugs have been found to treat COVID-19 in experimental animal models, the development of SARS-CoV-2 vaccines has held more promise than therapeutic drugs. At present, SARS-CoV-2 vaccines such as inactivated vaccines, mRNA vaccines, subunit vaccines, virus vector vaccines and the like have already entered clinical experiments. However, the current vaccine forms have various limiting problems of high production cost, low yield, difficult transportation and the like.

RBD is considered an effective vaccine target and vaccines directed against RBD have been shown to induce anti-viral neutralizing antibodies after expression in eukaryotic expression systems. Starting from the structure of SARS-CoV-2, the trimer structure of RBD has important function, and it has been proved that the vaccine form of RBD polymeric structure can raise the neutralization effect induced by vaccine. The prokaryotic cell expression system can realize high-efficiency expression of foreign protein and greatly improve the vaccine yield at lower cost, but the prokaryotic expression system is difficult to realize correct folding and assembly of target protein, so the development of the prokaryotic expression system in the field of viral vaccines is severely limited, and if the prokaryotic expression system can realize correct exposure and assembly of RBD, the prokaryotic expression system is favorable for developing SARS-CoV-2 cheap vaccines.

Normally, the ability of correctly folding foreign proteins in escherichia coli is poor, the foreign proteins often exist in the form of inclusion bodies which are folded incorrectly, the folding ability of the foreign proteins can be improved by displaying the foreign proteins through bacterial outer membrane vesicles, and the immune response ability is improved by means of the characteristic that OMVs are efficiently taken up by DC cells. Although there have been many studies demonstrating that OMVs can be effective vaccine vectors, including bacterial, viral, and tumor vaccines, unfortunately, OMVs have a very limited ability to present foreign proteins and are very low in yield because they are difficult to artificially control by natural secretion from bacteria. More seriously, OMV is used as a communication medium secreted by bacteria, is rich in a plurality of intracellular proteins and nucleic acid components, brings about the harm of protein toxicity and drug-resistant gene transfer, and the application of OMV as a vaccine carrier is greatly limited by the problems of yield, gene engineering capability, safety and the like.

Techniques for self-assembly of cell membranes have been widely reported in mammalian cells, including erythrocyte membranes, neutrophil membranes, macrophage membranes, and the like. However, the bacterial biofilm contains more outer membrane proteins, polysaccharides, peptidoglycans and the like, which brings difficulty to the membrane assembly technology, the current assembly technology of the bacterial biofilm still stays on the basis of using OMVs released by bacteria, for example, using the OMVs to fuse with tumor cells, using the OMVs including gold particles, using the OMVs to carry medicines and the like, but the yield and modification efficiency of the OMVs are low, which seriously limits the development of the bacterial membrane carrier technology. If the bacterial cells can be directly utilized to realize the assembly of the biological membrane, the vesicle yield and the loading efficiency of exogenous modified protein can be greatly improved.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to overcome the defects of OMV (OMV), provides a construction method and application of a Bacterial Biofilm Vesicle (BBV) as a vaccine vector, directly utilizes bacterial cells to realize biofilm assembly, and improves vesicle yield and loading efficiency of exogenous modified protein.

The purpose of the invention is realized by the following technical scheme:

a method for constructing a Bacterial Biofilm Vesicle (BBV) as a vaccine vector comprises the following steps:

step 1, connecting an exogenous gene to the 3' end of a gene for encoding the outer membrane anchoring protein by using a genetic engineering technology, cloning one or more expression units into escherichia coli plasmids to obtain recombinant plasmids, converting the recombinant plasmids into escherichia coli, culturing in a culture medium, and adding an inducer to induce the expression of the recombinant protein when the OD600 of a bacterial liquid reaches 0.4-0.6;

step 2, when the OD600 reaches 1.0, centrifuging the culture medium, removing supernatant, washing the bacterial sediment, and then resuspending to obtain bacterial suspension;

step 3, high-pressure treatment: carrying out high-pressure homogenization treatment on the obtained bacterial suspension, centrifuging the obtained suspension, harvesting supernatant, ultracentrifuging the supernatant, removing supernatant, taking bacterial precipitate, and carrying out heavy suspension to obtain a genetic engineering modified vesicle suspension;

step 4, purifying the genetic engineering modified vesicle suspension by utilizing column chromatography purification or density gradient centrifugation to obtain purified genetic engineering modified BBV;

the density gradient centrifugation uses centrifugation media including iodixanol, cesium chloride and sucrose.

Further, the anchoring protein in the step 1 is a protein with a membrane targeting effect or a traction protein with a non-membrane targeting effect. The protein with membrane targeting effect includes but is not limited to ClyA protein, OmpA, OmpW and Hbp, the traction protein with non-membrane targeting effect includes but is not limited to TAT, Flag, Trx and His, and the foreign gene in step 1 includes genes encoding various viral antigens, bacterial antigens, self-antigens and tumor antigens.

Further, step 2 is to centrifuge before C₁₀H₁₄N₂Na₂O₈(EDTA-2 Na) was slowly added to the medium, incubated under shaking, and the bacterial biofilm was softened and centrifuged.

Further, the pressure range of the high-pressure homogenization treatment in the step 3 is 200 bar-2000 bar.

Another aspect of the invention:

the vaccine is prepared by taking the BBV modified by the genetic engineering obtained by the construction method as an active ingredient.

Further, the application modes of the vaccine comprise subcutaneous injection, intramuscular injection, inhalation and oral administration.

Another aspect of the invention:

the use of the vaccine as a carrier for other vaccines, including peptide vaccines, subunit vaccines, DNA vaccines, RNA vaccines;

the vaccine is used as a drug delivery carrier, and the drugs comprise antitumor drugs, antibacterial drugs and tracer drugs;

the immune stimulation comprises innate immunity activation, tumor immune regulation and vaccine adjuvant.

Compared with the prior art, the invention has the beneficial effects that:

1. ClyA is utilized to simulate the structure of S2 protein support S1 of SARS-CoV-2, and the construction method of the invention is used to efficiently present RBD on self-assembled Bacterial Biofilm Vesicles (BBV), and due to the high efficiency of ClyA-RBD, spike-like nano-structure is presented on the vesicles, so that the RBD-BBV is closer to natural SARS-CoV-2, and the innovation is unprecedented;

2. the invention realizes gene modification on the nano scale of the bacterial biomembrane for the first time by using a high-pressure driving technology, and compared with the gene modification technology of OMV, the BBV technology of the invention improves the vesicle yield of 107 times of gene modification;

3. the Bacterial Biofilm Vesicles (BBV) have the same nanostructure and PAMPs components on the surface as OMVs, so that the uptake and maturation of DC cells are facilitated;

4. the full-length RBD protein displayed on the Bacterial Biofilm Vesicle (BBV) is improved by at least 28.16 times compared with the presentation level of OMV, multiple antibody protection can be provided aiming at the presentation technology of RBD whole protein, and the problem caused by virus mutation is avoided;

5. the BBV modified by genetic engineering obtained by the construction method has triple functions of inducing neutralizing antibodies, cellular immunity and immunological memory;

6. the BBV modified by genetic engineering obtained by the construction method helps RBD to realize lysosome escape by virtue of the biocompatibility characteristic of a bacterial biofilm, so that the cellular immune response level is improved

7. Compared with OMV, the BBV modified by genetic engineering releases intracellular ineffective protein and bacterial nucleic acid, and improves safety;

8. the production of the BBV modified by the genetic engineering is an artificially controllable process, the quality control performance of the vesicle is improved, and the preparation process can be completed in a 'continuous flow' manner, thereby being beneficial to the future large-scale preparation process.

Drawings

The invention is further illustrated by the following examples in conjunction with the accompanying drawings:

FIG. 1 is a diagram of the design and action of RBD-loaded bacterial spike-like biofilm vesicles (RBD-BBV) according to example 1 of the present invention;

FIG. 2 is a graph showing the analysis of natural E.coli BL21 whole cell thallus (WT-WC), RBD-expressing whole cell thallus (RBD-WC), RBD-loaded BBV (RBD-BBV), and RBD-loaded OMV (RBD-OMV) by silver staining after SDS-PAGE;

FIG. 3 is a graph showing the results of Image analysis of the load content of RBD protein in total protein by Image Lab software;

FIG. 4 is a graph comparing the yields of RBD-modified OMVs and BBVs;

FIG. 5 is SDS-PAGE silver staining analysis of samples of natural BBV and RBD-BBV without genetic engineering modification, and protein expression levels of whole cells before and after IPTG induction;

FIG. 6 shows the presence of RBD protein analyzed by Western blot assay using the S1 antibody against SARS-COV-2;

FIG. 7 is a diagram of a bioinformatic structural mimic of the ClyA-RBD fusion protein and its assembly on the bacterial outer membrane;

FIG. 8 is a TEM image of RBD-BBV;

FIG. 9 is a schematic diagram of the affinity analysis of RBD-BBV with ACE 2;

FIG. 10 is a graph of the immunofluorescent staining of BMDC cells using confocal hyperintension analysis, with BBV or RBD-BBV 0.05mg/ml added to the cells for 4h to observe the efficiency of BMDC uptake;

FIG. 11 is a graph of the statistics of the ratio of CD11c + CD80+ and CD11c + CD86 +;

FIG. 12 is an ELISA assay for cytokine levels in supernatants of BMDC stimulated with 0.05mg/ml BBV or RBD-BBV for 24 h;

FIG. 13 is a schematic diagram of the immunization schedule and blood sampling time for RBD-BBV;

FIG. 14 is a graph of the IgG content of anti-SARS-CoV-2 spike protein after 50-fold dilution of serum by ELISA;

FIG. 15 is a graph showing experimental results of binding of S1-Fc protein to ACE2 by RBD-BBV-induced antibody;

FIG. 16 is a graph showing the results of blocking the invasion of SARS-CoV-2 live virus into Vero cells after 64-fold dilution of antiserum;

FIG. 17 is a graph of the results of SARS-CoV-2EC50 titer calculated from the results of neutralization experiments with antibodies;

FIG. 18 is a graph showing the results of pathological examination of major organs 7 days after immunization with RBD-BBV at 0.5. mu.g and 5. mu.g doses three times.

Detailed Description

Example 1

In this embodiment, a Bacterial Biofilm Vesicle (BBV) is used as a vaccine vector to construct a novel coronary disease vaccine, and the specific method comprises:

the DNA sequence of the encoded recombinant protein containing SARS-CoV-2RBD structural domain Asn331-Val524 (YP _009724390) and ClyA protein (AAL55667) is directly synthesized, the RBD gene is connected to the 3' end of ClyA gene by using the genetic engineering technology and cloned into pThioHisA plasmid, and then positive plasmid is confirmed by restriction endonuclease analysis and sequencing. Transforming the recombinant plasmid into E.coli BL21, culturing in LB culture medium, adding 1mmol/L isopropyl beta-D-1-thiogalactopyranoside (IPTG, Solarbio) when bacterial liquid OD600 reaches 0.4-0.6, and inducing the expression of the recombinant protein ClyA-RBD overnight at 30 ℃; adding 2mM EDTA & 2Na the next day, and continuing culturing for 2 h; the culture was centrifuged at 10000rpm at 4 ℃ for 30min to collect the cells. E.coli BL21 overexpressing recombinant protein Clya-RBD and wild type cells were resuspended in HBSS buffer (Servicobio), bacteria were driven through the nip by a high pressure homogenizer (APV-2000, SPX, Germany) at conditions of 1200pa, 3 times, 4 ℃), the passed sample was centrifuged at 6000g at 4 ℃ for 30min, the cells were removed, and the supernatant was collected; ultracentrifuging the supernatant at 100000g at 4 deg.C for 30 min; resuspending the pellet with HBSS using iodixanol OptiPrep^TM(STEMCELL) medium was subjected to gradient centrifugation to purify RBD-BBV, each gradient layer from bottom to top being OptiPrep^TMHBSS concentration 45%, 35%, 30%, 25%, 20%, 15%, 10% and RBD-BBV, centrifuging at 40000rpm for 3-4h, and collecting sample layer as purified RBD-BBV.

As shown in FIG. 1, the action mechanism of RBD-BBV is:

after expressing the ClyA-RBD fusion protein monomer in escherichia coli, the efficient assembly of the ClyA-RBD on the bacterial outer membrane is realized by virtue of the outer membrane targeting assembly characteristic of ClyA. The bacteria are driven by ultrahigh pressure and pass through cracks to form a membrane component carrying ClyA-RBD polymers, and the bacterial membranes are self-assembled with high efficiency to form RBD-loaded bacterial spike-like biomembrane vesicles (RBD-BBV). RBD-BBV as vaccine can mediate high-efficiency uptake of DC cells and realize lysosome escape, thereby inducing organism to generate specific neutralizing antibody and T cell response against SARS-COV-2.

Comparative example 1

The specific method for constructing the novel coronary disease vaccine by using the Outer Membrane Vesicles (OMVs) as the vaccine carrier comprises the following steps:

according to the method for inducing expression described in example 1, the culture supernatant after inducing expression was filtered through 0.45 μm membrane (Millipore), the filtrate was concentrated by ultrafiltration using 500kDa column (Millipore), the concentrate was ultracentrifuged at 200000g and 4 ℃ for 2h, then the vesicle precipitate was resuspended in Phosphate Buffered Saline (PBS) and washed twice with PBS, and filtered again through 0.45 μm membrane, and the filtrate was purified according to the density gradient centrifugation method described in example 1 to obtain purified RBD-OMV sample.

Example 2 RBD-BBV Structure and Loading

This example compares the gene modification techniques of BBV and OMV in example 1 and comparative example 1, respectively, and samples were stained with a rapid silver staining kit (Beyotime) and analyzed by Image Lab for RBD loading in RBD-BBV (before iodixanol density gradient centrifugation purification), RBD-OMV; BBV production was analyzed using Bradford kit (Sangon Biotech); the results shown in FIG. 2 demonstrate that the RBD loading on the BBV is significantly higher than that of OMV, the position of RBD protein is marked by the arrow in the figure, and that BBV is about 28.16 times higher than OMV in RBD-loading (FIG. 3), and that RBD-BBV is about 107 times higher in RBD-OMV production (FIG. 4). More importantly, the nucleic acid component of the bacteria is not detected in the RBD-BBV, which greatly improves the safety of the vaccine.

In conclusion, the BBV genetic engineering modification technology developed by the invention breaks through the technical bottleneck of OMV engineering modification, so that the efficient presentation of RBD on BBV is realized in the invention.

The assembled RBD-BBV was separated by iodixanol density gradient centrifugation of the RBD-BBV described in example 1, and the SDS-PAGE electrophoretic analysis shown in FIG. 5 demonstrated that the RBD was efficiently loaded on the BBV, and further the western blot analysis using the S1 antibody specifically recognizing SARS-COV-2 demonstrated that the protein presented on the BBV by the genetic engineering was indeed an RBD (FIG. 6). In this example, bioinformatics analysis of amino acid structure of ClyA-RBD fusion protein demonstrated that there is no protein interaction between ClyA and RBD under this design, they can fold independently into correct protein structure and expose on bacterial outer membrane (fig. 7), and we also observed the assembled spurt-like structure of ClyA-RBD on vesicle surface under transmission electron microscope (fig. 8). In addition, this example utilizes the specific binding characteristics of natural RBD and ACE2 to design an affinity experiment of RBD-BBV and ACE2 (FIG. 9), and we demonstrate that RBD-BBV has almost the same ACE2 binding ability with S1-Fc protein expressed by eukaryotic cells. Since Proteinase K (PK) can remove proteins on the surface of the vesicle, most of proteins disappear after the RBD-BBV is treated by PK, and the protein composition of the RBD-BBV is mainly composed of RBD and membrane proteins, no redundant intracellular proteins exist, and the RBD is completely exposed on the surface of the BBV, and the complete exposure of the RBD on the surface is confirmed by using an S1 antibody which specifically recognizes SARS-COV-2. DLS particle size analysis proves that RBD-BBV has a complete structure as unmodified BBV, and the particle size does not change significantly.

In conclusion, the RBD-BBV constructed by the invention efficiently exposes the correctly folded RBD on the surface of the vesicle and displays a spike-like vesicle structure.

Example 3 evaluation of RBD-BBV as vaccine

1. RBD-BBV promotes DC cell uptake and maturation, and mediates lysosome escape of RBD

The efficient uptake of antigen by DC cells is the key for the success of vaccines, experiments show that both RBD-BBV and BBV can be efficiently taken by DC cells (FIG. 10), and we also see that both RBD-BBV and BBV can effectively stimulate the maturation of DC cells, as shown in FIG. 11, which is specifically represented by the high expression of CD11c + CD80+ and CD11c + CD86+ on DC cells. As shown in FIG. 12, both RBD-BBV and BBV can stimulate the secretion of IL-6 and IL-1. beta. which are DC cell inflammatory factors. In conclusion, we observed that RBD-BBV and BBV have the same characteristics in terms of DC cell uptake and stimulatory maturation, suggesting that these effects are derived from the nanostructure of BBV, and the stimulation and targeting of PAMPs on BBV to DC cells, thus representing that BBV is crucial as a vaccine vector for RBD.

In addition, the RBD-BBV has a high-efficiency lysosome escape phenomenon, the lysosome escape effect is generated by synchronizing the RBD escape and the BBV escape, and the lysosome escape phenomenon can be mediated by the RBD-BBV or BBV empty vectors, so that the RBD loaded on the RBD-BBV can realize the high-efficiency lysosome escape together with the BBV. Therefore, we believe that the RBD protein loaded on RBD-BBV accomplishes lysosomal escape by virtue of the BBV's own properties, which are crucial for inducing T cell responses.

2. RBD-BBV can induce anti-SARS-CoV-2 specific neutralizing antibody

As shown in FIG. 13, in this example, mice immunized with RBD-BBV were evaluated for their stimulated B cell response, and mice immunized with RBD-BBV at 5. mu.g and 0.5. mu.g three times induced IgG responses specific for SARS-CoV-2 prod, wherein the 5. mu.g dose of RBD-BBV produced IgG antibody responses after two immunizations (FIG. 14), and both IgG1 and IgG2a responses were significantly induced, with consistent results observed in IgM detection. The antiserum was diluted in duplicate to analyze the antibody titer of antibody induced by RBD-BBV binding to SARS-CoV-2 spurs protein, and the results showed that the antibody titers induced after immunization at 5. mu.g and 0.5. mu.g doses were 1600 and 300, respectively. As shown in fig. 15, this example examined the ability of RBD-BBV-induced antibodies to block the binding of S1 to ACE2, and it was observed that 5 μ g of RBD-BBV-induced antibodies could block the binding of S1 to ACE 2. More importantly, we investigated the ability of RBD-BBV-induced antibodies to block live SARS-CoV-2 from invading Vero cells, and the results shown in fig. 16 demonstrate that RBD-BBV-induced antiserum can inhibit SARS-CoV-2 from invading Vero cells, as the antibodies prevent the Vero cells from disease. As shown in FIG. 17, it was calculated that 5. mu.g and 0.5. mu.g of RBD-BBV produced antibodies after three immunizations blocked SARS-CoV-2 invading cells, and that 5. mu.g and 0.5. mu.g of RBD-BBV had EC50 titers of 102.4 and 64, respectively. As described above, RBD-BBV is capable of inducing potent neutralizing antibodies specific for anti-SARS-CoV-2.

3. RBD-BBV can induce SARS-CoV-2 specific cellular immune response

The role of the cellular immune response in combating SARS-CoV-2 appears to be increasingly important, and therefore this example evaluates the ability of RBD-BBV to induce SARS-CoV-2 specific cellular immunity in the spleen and lung of mice. It was found that the cellular immune response of SARS-CoV-2 spurs-specific CD4+ T and CD8+ T was induced in the spleen of mice after RBD-BBV immunization. RBD-BBV can not only induce systemic T cell response, but also induce SARS-CoV-2 spur specific CD4+ T and CD8+ T cellular immune response in lung. Furthermore, we also evaluated the ability of RBD-BBV to induce Th1 cells of CD4+ IFN γ + and CTL cells of CD8+ IFN γ +, and we found that RBD-BBV induced SARS-CoV-2 specific Th1 responses in spleen and lung, and similarly RBD-BBV induced SARS-CoV-2 specific CTL responses in spleen and lung. We also detected that splenocytes from mice immunized with RBD-BBV secreted significantly functional molecules GzmB and IFN-gamma, and IL-2 which promoted T cell proliferation, after stimulation by the S1-Fc protein of SARS-CoV-2. We examined the spots of functional cells secreting IFN-. gamma.induced by RBD-BBV using Elispot assay, and demonstrated that mice immunized with RBD-BBV were able to generate SARS-CoV-2 spike-specific cellular immune responses both systemically (spleen) and locally (lung). As described above, RBD-BBV is capable of inducing a latent SARS-CoV-2 specific cellular immune response in the system and lung to combat SARS-CoV-2.

4. RBD-BBV enhances local stability of antigen in vivo, and can induce immune T cell production

The in vivo local stability of the vaccine is beneficial to improving the strength and duration of antigen immune response, and we observed that the in vivo stability time of the RBD-BBV can be improved by at least 3 times compared with that of the RBD protein by 5ug of the RBD-BBV and the RBD protein.

The ability of the vaccine to induce T cell immune memory reflects its potential level of persistence of protection, which may be very important for the SARS-CoV-2 vaccine. This example evaluates SARS-CoV-2 specific memory T cell levels both systemically (spleen) and locally (lung) using flow cytometry. We observed the post-immune potency of RBD-BBVCentral memory T cells (T) inducing CD4 and CD8 in spleen_CM) However, statistically significant CD4 and CD8 central memory T cells were not observed in the lungs. We also observed that RBD-BBV induced CD4 and CD8 effector memory T cells in the spleen (T cells)_EM) Furthermore, RBD-BBV induces CD4 and CD8 effector memory T cells (T cells) in lung tissue_EM) Lung T induced by this vaccine_EMMemory cells may be important to fight lung infection against SARS-CoV-2. In conclusion, we demonstrated that RBD-BBV enhances the local stability of antigen and induces T cell immunological memory in vivo.

EXAMPLE 4 biocompatibility of RBD-BBV

This example initially evaluated the safety of RBD-BBV in vivo, and we did not observe significant fluctuations in animal body temperature nor did we find significant weight loss in the immunization protocol of 5 μ g RBD-BBV vaccine. As shown in FIG. 18, no significant tissue lesions were seen in the major organs after completion of the entire immunization program with RBD-BBV at 0.5 μ g or 5 μ g doses. We examined the inflammatory factor profile in the serum 2h and 24h after immunization of mice with 5. mu.g and 0.5. mu.g RBD-BBV, respectively, and we did not observe significant increases in IFN-. gamma.levels caused by RBD-BBV in the serum. IL-6 levels in serum were elevated at 2h of both 5. mu.g and 0.5. mu.g RBD-BBV injection, but returned to normal 24h later. TNF- α was elevated only 2h after injection of 5 μ g RBD-BBV, but returned to normal 24h, with no significant increase in TNF- α levels seen at the 0.5 μ g dose. The PAMPs carried by RBD-BBV inevitably cause weak inflammatory response, but the inflammatory response disappears after 24 h.

Therefore, we believe that RBD-BBV has good biocompatibility.

Claims (10)

1. A method for constructing a Bacterial Biofilm Vesicle (BBV) as a vaccine vector, which is characterized by comprising the following steps:

step 1, connecting an exogenous gene to the 3' end of a gene encoding an outer membrane anchoring protein or a non-anchoring protein by using a genetic engineering technology, cloning one or more expression units into escherichia coli plasmids to obtain recombinant plasmids, transforming the recombinant plasmids into escherichia coli, culturing in a culture medium, and adding an inducer to induce the expression of the recombinant protein when the OD600 of a bacterial liquid reaches 0.4-0.6;

step 2, when the OD600 reaches 1.0, centrifuging the culture medium, removing supernatant, washing the bacterial sediment, and then resuspending to obtain bacterial suspension;

step 3, high-pressure treatment: carrying out high-pressure homogenization treatment on the obtained bacterial suspension, centrifuging the obtained suspension, harvesting supernatant, ultracentrifuging the supernatant, removing supernatant, taking bacterial precipitate, and carrying out heavy suspension to obtain a genetic engineering modified vesicle suspension;

and 4, purifying the genetic engineering modified vesicle suspension by utilizing column chromatography purification or density gradient centrifugation to obtain the purified genetic engineering modified BBV.

2. The method for constructing a Bacterial Biofilm Vesicle (BBV) as a vaccine vector according to claim 1, wherein the anchor protein in step 1 is a protein with membrane targeting effect or a traction protein with non-membrane targeting effect.

3. The method of claim 1, wherein the foreign genes in step 1 include genes encoding viral antigens, bacterial antigens, autoantigens, and tumor antigens.

4. The method for constructing Bacterial Biofilm Vesicles (BBV) as vaccine vectors according to claim 1, wherein step 2 comprises centrifuging C₁₀H₁₄N₂Na₂O₈(EDTA-2 Na) was slowly added to the medium, incubated under shaking, and the bacterial biofilm was softened and centrifuged.

5. The method for constructing a Bacterial Biofilm Vesicle (BBV) as a vaccine vector according to claim 1, wherein the pressure of the high-pressure homogenization treatment in step 3 is in a range of 200bar to 2000 bar.

6. A vaccine prepared from the genetically modified BBV obtained by the construction method according to any one of claims 1 to 5 as an active ingredient.

7. The vaccine of claim 6, wherein the vaccine is administered by subcutaneous injection, intramuscular injection, inhalation, or oral administration.

8. Use of the vaccine of claim 6 as a carrier for other vaccines including peptide vaccines, subunit vaccines, DNA vaccines, RNA vaccines.

9. Use of the vaccine of claim 6 as a drug delivery vehicle, wherein the drug comprises an anti-tumor drug, an antibacterial drug, or a tracer drug.

10. Use of the vaccine of claim 6 as an immunostimulant, including innate immunity activation, tumor immunomodulation, vaccine adjuvants.

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