CN112773901A

CN112773901A - Bacterial outer membrane vesicle carrier and preparation method and application thereof

Info

Publication number: CN112773901A
Application number: CN202110129743.9A
Authority: CN
Inventors: 聂广军; 赵潇; 赵宇亮; 梁洁
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2021-05-11
Anticipated expiration: 2041-01-29
Also published as: CN112773901B

Abstract

The invention provides a bacterial outer membrane vesicle carrier and a preparation method and application thereof. The invention provides a vaccine universal carrier OMV-SpyC-SPAb which is constructed based on B structural domain SPAb of staphylococcus aureus protein A and can target dendritic cells, the B structural domain SPAb of the staphylococcus aureus protein A is displayed on the bacterial outer membrane vesicle and can be quickly combined with an antibody (such as Anti-DEC205 antibody) of the targeted dendritic cells, and an antigen is displayed on the surface of the bacterial outer membrane vesicle through Spycatcher/SpyTag molecular glue to prepare a novel OMV-OVA-DEC vaccine which can target the dendritic cells and obviously enhance the antigen intake, can promote the dendritic cells to cross-present the antigen and induce specific immune response, thereby achieving the aim of resisting tumors and having wide application prospect.

Description

Bacterial outer membrane vesicle carrier and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a bacterial outer membrane vesicle carrier and a preparation method and application thereof.

Background

The tumor vaccine has been used as one of the tumor immunotherapy for over one hundred years, and shows potential advantages and good application prospects in the tumor immunotherapy. However, single antigen therapy has poor immunogenicity, and the anti-tumor immune effect is not ideal. To solve this problem, antigen and adjuvant can be efficiently delivered using vaccine vectors, and antigen-specific adaptive immune responses can be enhanced by increasing the efficiency of uptake, processing and presentation of antigen by Dendritic Cells (DCs). At present, liposome, nanoparticles, nano-micelles, hydrogel, biological materials and the like based on biological materials are used as vaccine carriers, so that the delivery efficiency of antigens can be improved, the immunogenicity is enhanced, DC cells are targeted, the cross presentation is promoted, the relatively durable immunological memory can be generated, and the unique advantages of the vaccine carriers are fully exerted. Among them, the biologically derived carrier materials have potential immunogenicity compared to synthetic materials, and when pathogen-based carriers are used for vaccine delivery, the immunogenicity of the vaccine can be substantially improved due to their adjuvant efficacy. Therefore, the development of a nano vaccine vector capable of rapidly obtaining and having adjuvant effect is a problem to be solved in the field of tumor vaccines.

The bacterial Outer Membrane Vesicle (OMV) is a natural nanoparticle secreted by gram-negative bacteria, is rich in a large amount of biogenic substances, contains abundant Lipopolysaccharide (LPS) on the surface of the Outer membrane, can stimulate natural immunity and quickly induce DC cells to mature, and is an antigen display platform and a vaccine carrier with great potential. Host bacteria are genetically engineered to express and display heterologous antigens in fusion on the surface proteins of OMVs secreted by the bacteria. OMV is used as a vaccine carrier, not only can activate antigen-specific immune response, but also can effectively improve tumor immune microenvironment. In addition, the bacteria can be rapidly propagated in a fermentation mode, and OMVs can be rapidly obtained in a large batch, so that the large-scale production of the OMVs is feasible.

However, since OMVs have a rich pathogen-associated molecular pattern on their Surface (PAMPs), and LPS in particular, facilitates recognition and activation of Toll-like receptor 4(TLR4) in dendritic cells, OMVs can be rapidly taken up and processed by DC cells, and induce DC high maturation through the LPS-TLR4 signaling pathway during antigen uptake by DC cells. Although DC cell maturation is a prerequisite for successful antigen presentation, premature maturation of dendritic cells results in greatly reduced antigen uptake by OMVs. Through specific modification of OMV vectors, development of vaccines that can be rapidly recognized by DCs is an urgent task.

Therefore, it is urgently needed to develop a multifunctional modified tumor vaccine vector, modify an OMV vaccine vector by using a targeting antibody, and construct a novel tumor vaccine, so that the ability of DC cells to take in specific antigens in a targeted manner is increased, thereby effectively promoting the processing and presentation of the antigens and enhancing the specific immune response induced by the antigens.

Disclosure of Invention

The invention aims to provide a bacterial outer membrane vesicle carrier and a preparation method and application thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a bacterial outer membrane vesicle vector, which is a bacterial outer membrane vesicle carrying the B domain of staphylococcus aureus protein a and SpyCatcher protein.

The B structure domain of the staphylococcus aureus protein A and the bacterial outer membrane protein are fused and expressed in the bacterial outer membrane vesicle, and the SpyCatcher protein and the bacterial outer membrane protein are fused and expressed in the bacterial outer membrane vesicle. The bacteria are gram-negative bacteria, preferably Escherichia coli.

In the invention, the amino acid sequence of the B structural domain of the staphylococcus aureus protein A is shown as SEQ ID NO. 4, and the amino acid sequence of the SpyCatcher protein is shown as SEQ ID NO. 5.

The bacterial outer membrane protein can be ClyA, and the amino acid sequence of the bacterial outer membrane protein is shown as SEQ ID NO. 6.

The particle size of the bacterial outer membrane vesicle carrier is 20-40 nm (preferably about 27.9 nm).

In a second aspect, the present invention provides a method for preparing a bacterial outer membrane vesicle vector, comprising:

(1) artificially synthesizing a nucleic acid construct ClyA-SpyCatcher-ClyA-SPAb (SEQ ID NO:7), and constructing the nucleic acid construct into a prokaryotic expression vector to obtain a recombinant expression vector;

(2) introducing the recombinant expression vector into escherichia coli to obtain recombinant bacteria; adding an inducer into a culture solution of the recombinant bacteria for induction expression, centrifugally collecting the bacteria solution, concentrating and purifying the bacteria solution, ultracentrifuging, then discarding the supernatant, adding PBS into an ultracentrifuge tube for heavy suspension, then ultracentrifuging again, discarding the supernatant, and carrying out heavy suspension precipitation by using PBS to obtain the bacterial outer membrane vesicle vector (OMV-SpyC-SPAb).

Wherein, ClyA is the outer membrane protein of bacteria, SPAb is the B structural domain of staphylococcus aureus protein A.

Preferably, the prokaryotic expression vector is pETDuet-1.

Preferably, the E.coli is Rosetta (DE 3).

Preferably, the inducer is IPTG, and the optimal induction concentration is 0.1-1 mM (preferably 1 mM).

In the method, the conditions for centrifuging the bacterial liquid are as follows: centrifuging at 5000-8000 rpm for 5-10 min (preferably at 5000rpm for 10 min); and/or

Further, concentrating and purifying the bacterial liquid through a sterile filter membrane and an ultrafiltration tube, wherein the aperture of the filter membrane is 0.45 μm and 0.22 μm; the molecular weight cut-off of the ultrafiltration tube is 50KDa, the bacterial liquid passes through a 0.45 mu m filter membrane before ultrafiltration, and then passes through a 0.22 mu m filter membrane after ultrafiltration is finished.

In the method, the ultracentrifugation conditions are as follows: centrifuge at 150000g for 3 h.

In a third aspect, the invention provides the use of the bacterial outer membrane vesicle vector, or a bacterial outer membrane vesicle vector prepared according to the above method, as a vaccine vector or a drug delivery system.

In a fourth aspect, the present invention provides a dendritic cell-targeting nano-vaccine (OMV-SPA-DEC) prepared by mixing an antibody targeting dendritic cells, an antigen containing a SpyTag tag, and the bacterial outer membrane vesicle vector, or the bacterial outer membrane vesicle vector prepared according to the above method.

In the invention, the amino acid sequence of the SpyTag label is shown in SEQ ID NO. 3.

Preferably, the mass ratio of the dendritic cell-targeting antibody to the antigen containing the SpyTag tag to the bacterial outer membrane vesicle carrier is 1:1: 1-4: 4:1, preferably 1:1: 1.

The dendritic cell-targeting antibody can be an Anti-DEC205 antibody.

The antigen is tumor specific antigen, preferably model antigen OVA_257-264The amino acid sequence is SIINFEKL; the C end of the antigen is connected with a SpyTag label. The SpyTag tag can be linked to the antigen via an amide bond by solid phase synthesis.

In a fifth aspect, the invention provides application of the nano vaccine in preparation of tumor immunotherapy vaccines and drugs.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention provides a vaccine universal carrier OMV-SpyC-SPAb which is constructed based on a B structural domain SPAb of staphylococcus aureus protein A and can target dendritic cells, the B structural domain SPAb of the staphylococcus aureus protein A is displayed on the bacterial outer membrane vesicle and can be rapidly combined with an antibody (such as an Anti-DEC205 antibody) of the targeted dendritic cells, and an antigen is displayed on the surface of the bacterial outer membrane vesicle through a Spycatcher/SpyTag molecular glue, so that a novel OMV-OVA-DEC vaccine which can target the dendritic cells and obviously enhance the antigen intake is prepared, the cross presentation of the dendritic cells to the antigen can be promoted, and a specific immune response is induced, thereby achieving the aim of resisting tumors.

The invention uses the genetic engineering method to perform fusion expression on the SpyCatcher of the molecular glue and the B structural domain SPAb of the staphylococcus aureus protein A and the ClyA protein on the bacteria, and obtains the vaccine universal vector (OMV-SpyC-SPAb) capable of specifically adsorbing the antibody after ultracentrifugation and purification.

And thirdly, the vaccine universal carrier OMV-SpyC-SPAb can quickly combine different antigens and antibodies containing SpyTag by two-step reaction, namely molecular glue reaction and B domain SPAb specific binding antibody reaction of the staphylococcus aureus protein A.

According to the rapid reaction of the molecular glue and the characteristic that staphylococcus aureus protein A can rapidly adsorb an antibody, the nano vaccine OMV-SPA-DEC with the DC targeting function is prepared.

Aiming at the characteristic that the OMV can rapidly induce the DC cell to mature, the OMV vaccine has the function of targeting the DC cell by modifying the targeting antibody, realizes the high-efficiency uptake of the DC cell to the antigen, and enhances the cross presentation of the antigen.

The OMV vaccine with the DC cell targeting function provided by the invention can enhance the immune response efficiency of the vaccine and enhance the immune stimulation and anti-tumor effects.

Drawings

FIG. 1 is a representation of the OMV vaccine universal vector in a preferred embodiment of the invention; wherein A is the particle size characterization of the OMV vaccine universal carrier; b is the electron microscope characterization of OMV vaccine universal vector, and the scale is 50 nm.

FIG. 2 shows the successful construction of OMV universal vaccine vectors according to a preferred embodiment of the present invention; wherein A is a result of a protein immunoblot band successfully expressed by the B structural domain SPAb fusion of the staphylococcus aureus protein A, and B is a result of a protein immunoblot band successfully expressed by the SpyCatcher fusion.

FIG. 3 shows the results of testing whether OMV universal vaccine vectors and targeting antibodies can be linked to SpyTag in a preferred embodiment of the present invention; wherein A is a protein immunoblotting band result of a B structural domain SPAb specific adsorption antibody of staphylococcus aureus protein A; b is the result of Western blot band of specific reaction between Spycatcher and SpyTag.

FIG. 4 shows the results of testing the efficiency of uptake of antigen by DC, as demonstrated by OMV nano-vaccines in accordance with a preferred embodiment of the present invention.

FIG. 5 shows fluorescence imaging results of lymph node drainage to verify that OMV nano-vaccine can target lymph node in the preferred embodiment of the present invention; wherein A is the condition that whether Cy5.5 fluorescence is drained to a lymph node in a mouse body or not is detected by a small animal imaging system, and B is the fluorescence imaging condition that a nano vaccine is drained to the lymph node after the lymph node of the mouse is dissected; c is the statistical result of the relative fluorescence intensity of the vaccines of the experimental group and the control group at the lymph node part.

FIG. 6 shows the cross-presentation results of the nano-vaccine enhanced antigens in the preferred embodiment of the present invention; wherein A is the cross presentation result of the in vitro nano vaccine promoted antigen, and B is the cross presentation result of the in vivo nano vaccine promoted antigen.

FIG. 7 is a photograph of lung metastases of mice after administration of the nano-vaccine in a preferred embodiment of the present invention.

Detailed Description

The invention provides a universal vector for a bacterial outer membrane vesicle vaccine, and a preparation method and application thereof. The invention also provides a nano vaccine targeting dendritic cells, and preparation and application thereof. The universal carrier for the bacterial outer membrane vesicle vaccine displays the target antibody of the specific antigen on the bacterial outer membrane vesicle through the SpyCatcher protein of the molecular glue and the B structural domain SPAb of the staphylococcus aureus protein A expressed on the bacterial outer membrane vesicle, so that the antigen intake efficiency is efficiently improved, the cross presentation of the antigen is promoted, the specific immunoreaction of the antigen of an organism is activated, and the inhibition effect on tumors is enhanced.

The invention adopts the following technical scheme:

in a first aspect, the invention provides a bacterial outer membrane vesicle vaccine vector comprising SpyCatcher protein of molecular glue and the B domain spa of staphylococcus aureus protein a.

Furthermore, the nucleotide sequence of the encoding SpyCatcher protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding B structural domain SPAb of the staphylococcus aureus protein A is shown as SEQ ID NO. 2.

Further, the SpyCatcher protein of the molecular glue, the B domain spa of staphylococcus aureus protein a, and the bacterial outer membrane protein on the bacterial outer membrane vesicle are expressed in the bacterial outer membrane vesicle in a fusion manner.

Preferably, the bacterial outer membrane protein is ClyA.

The invention further provides an application of the bacterial outer membrane vesicle vaccine vector as an antigen delivery vector.

In a second aspect, the present invention provides a nano-vaccine comprising the bacterial outer membrane vesicle, the B domain spa of staphylococcus aureus protein a on the bacterial outer membrane vesicle displaying an antibody in a form specifically binding to the antibody, and the bacterial outer membrane vesicle linking an antigen by reaction of the surface SpyCatcher with SpyTag to form an irreversible covalent bond, the antigen containing a SpyTag tag.

The targeting antibody can link the antibody and the B domain of the yellow staphylococcus protein A together in a form of specific binding with the antibody, and the binding mode is stable and rapid.

SpyTag can link antigens to bacterial outer membrane vesicles through a reaction between Tag and Catcher, which forms isopeptide bonds.

Furthermore, the amino acid sequence of the SpyTag is shown in SEQ ID NO. 3.

Preferably, solid phase synthesis is used to link the SpyTag to the antigen via an amide bond.

Preferably, the antigen is a tumor specific antigen, preferably a model antigen OVA_257-264(SIINFEKL)。

Preferably, the antibody is an antibody targeting DC cells, preferably an Anti-DEC205 antibody.

Preferably, the particle size of the tumor vaccine is about 27.9nm, and the nano-particles are stable, have a lymph node enrichment effect, are beneficial to antigen presenting cell recognition, and induce specific immune response.

In a third aspect, the invention provides a preparation method of the vaccine vector and the nano-vaccine, which comprises the following steps: 1) after the encoding genes of the SpyCatcher protein and the B structural domain SPAb of the staphylococcus aureus protein A are recombined with the encoding gene of the ClyA protein in a gene recombination mode, carrying out fusion expression in gram-negative bacteria to obtain the outer membrane vesicle of the bacteria; 2) an antigen containing SpyTag and an antibody targeting DC were mixed with the bacterial outer membrane vesicles, respectively.

Furthermore, the mass ratio of the antigen containing the SpyTag, the antibody targeting the DC and the bacterial outer membrane vesicle vaccine vector is 1: 1.

Preferably, the gram-negative bacterium is escherichia coli.

As a preferred technical scheme, the invention provides a preparation method of a nano vaccine, which comprises the following steps:

(1) designing and fusing a plasmid (starting plasmid is pETDuet-1) containing B structural domain SPAb of SpyCatcher and staphylococcus aureus protein A by using a genetic engineering method to obtain a recombinant plasmid pETDuet-ClyA-SpyCatcher-ClyA-SPAb;

(2) introducing plasmid pETDuet-ClyA-SpyCatcher-ClyA-SPAb into Rosetta (DE3) competent escherichia coli, adding an inducer for induced expression, carrying out low-speed centrifugation to collect bacterial liquid, concentrating and purifying the bacterial liquid through an aseptic filter membrane and an ultrafiltration tube, removing supernatant after ultracentrifugation, adding PBS again into the ultrafiltration tube for resuspension, then carrying out ultracentrifugation, finally removing the supernatant, and carrying out resuspension precipitation by using PBS to obtain OMV-SpyC-SPAb;

(3) coupling antigen peptide into SpyTag by adopting a solid-phase synthesis method, adding SpyTag-antigen into OMV-SpyC-SPAb according to a certain protein proportion, and then adding a targeting antibody with a certain protein proportion to obtain the nano vaccine.

Further, the inducer is IPTG, and the optimal concentration for induction is 1 mM.

Further, the low-speed centrifugation condition is 5000rpm centrifugation for 10 min;

further, the pore size of the filter membrane is 0.45 μm and 0.22 μm;

furthermore, the molecular weight cut-off of the ultrafiltration tube is 50KDa, the bacterial culture medium is filtered through a 0.45 mu m filter membrane before ultrafiltration, and then is filtered through a 0.22 mu m filter membrane after ultrafiltration is finished;

further, the ultracentrifugation conditions were 150000g centrifugation for 3 h;

further, the SpyTag-antigen and the target antibody are added into the OMV-SpyC-SPAb according to a certain protein ratio, wherein the mass ratio of the SpyTag-antigen to the target antibody to the OMV-SpyC-SPAb is 1:1: 1.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Anti-DEC205(DEC) antibody was purchased from BioXcell, USA in the following examples.

Example 1 preparation of bacterial outer Membrane vesicle vectors

Through a genetic engineering method, SpyCatcher, the B structural domain SPAb of staphylococcus aureus protein A and the C end of ClyA protein are subjected to fusion expression, after a plasmid is constructed, the plasmid is transformed into escherichia coli Rosetta (DE3), and a target protein is expressed under the action of an IPTG inducer. Separating and purifying by ultrafiltration and superseparation to obtain universal vaccine carrier OMV-SpyC-SPAb. Characterizing the obtained OMV vaccine universal carrier by using a laser particle size analyzer and a transmission electron microscope, wherein A in the graph 1 is the particle size of the OMV vaccine universal carrier, and the particle size is 27.9 nm; b in figure 1 is the electron microscope topography of the OMV vaccine universal vector obtained by preparation. The construction method of the recombinant plasmid pETDuet-ClyA-Spycatcher-ClyA-SPAb comprises the following steps: a backbone sequence (artificially synthesized nucleic acid construct ClyA-SpyCatcher-ClyA-SPAb, SEQ ID NO:7) and IPTG-induced promoter T7-lac were inserted into pETDuet-1 plasmid to obtain recombinant plasmid.

The method for transforming the recombinant plasmid into the escherichia coli comprises the following steps: add 1. mu.l of the desired DNA into 100. mu.l of the competent cells, mix them gently, and leave them on ice for 30 min. The tube was heat-shocked at 42 ℃ for 90 s. The centrifuge tube was quickly placed on ice and allowed to cool for 2 min. 900. mu.l of LB medium without antibiotics was added to each tube and incubated at 37 ℃ for 1 hour on a shaker. 100. mu.l of the culture broth was directly applied to LB solid plates containing 75. mu.g/ml ampicillin. The plates were cultured in an inverted format at 37 ℃ for 14-16h.

The induction expression of the recombinant bacteria and the purification method of the outer membrane vesicles of the bacteria comprise the following steps: single colonies were picked into 10mL shake tubes containing 3mL LB medium. Then 75. mu.g/mL ampicillin was added to the shake tube. Culturing at 37 deg.C and 180rpm for 12 h. 100. mu.l of the resulting suspension was transferred to a 200mL Erlenmeyer flask containing 100mL of LB medium. Then, 75. mu.g/mL ampicillin was added to the Erlenmeyer flask. Culturing for 4-8 h at 37 ℃ and 180 rpm. To be OD₆₀₀The value reached 0.6 and 0.1mM IPTG was added. Culturing at 16 deg.C and 160rpm for 16h. Centrifuging the bacterial liquid at 8000rpm for 5min, concentrating and purifying the bacterial liquid through a sterile filter membrane and an ultrafiltration tube, wherein the pore diameter of the filter membrane is 0.45 μm and 0.22 μm, the cut-off molecular weight of the ultrafiltration tube is 50KDa, filtering the bacterial liquid through the filter membrane with the diameter of 0.45 μm before ultrafiltration, and filtering through the filter membrane with the diameter of 0.22 μm after ultrafiltration is finished. The conditions of the ultracentrifugation were: centrifuge at 150000g for 3 h.

In order to verify that the fusion expression of the SpyCatcher and the B structural domain SPAb of the staphylococcus aureus protein A is successful, the detection is further carried out by a protein immunoblotting method, a corresponding label antibody is incubated after membrane conversion, and a developing solution is added to detect the position of a strip after a second antibody is incubated. Fusion expressed protein was detected at a position of about 60kDa using tag 3-Cmyc (EQKLISEEDL) attached to the C-terminus of SpyCatcher, see A in FIG. 2; using the tag 3-flag (DYKD DDDK) attached to the C-terminus of the B domain SPAb of Staphylococcus aureus protein A, fusion expressed proteins were detected at the position of about 45kDa as shown in B in FIG. 2.

Example 2

The purpose of this example was to verify whether the B domain SPAb of Staphylococcus aureus protein A binds to the antibody and whether a reaction between Spycatcher and SpyTag occurred.

The B domain SPAb of Staphylococcus aureus protein A expressed on the OMV universal vaccine vector of example 1 can specifically adsorb antibody; the SpyCatcher can specifically react with SpyTag to form a stable isopeptide bond.

In order to detect the binding of SPAb and antibody, horseradish peroxidase HPR labeled Rat IgG antibody (Rat IgG-HPR) and universal vaccine carrier OMV-SpyC-SPAb are incubated, the detection is carried out by a protein immunoblotting method, a developing solution is added to detect the position of a strip, the SPAb successfully adsorbs Rat IgG-HPR, and a protein strip expressed by fusion of B structural domain SPAb of staphylococcus aureus protein A and ClyA protein is detected at a position of about 45KDa, which is shown as A in figure 3.

To detect the molecular glue protein SpyCatcher/SpyTag reaction, an HA polypeptide (YPYDVPDYA) was coupled to the SpyTag, and the HA tag was detected by western blotting, and the SpyCatcher and SpyTag proteins were linked and detected at a position of about 45kDa, as shown in fig. 3B.

Example 3

The purpose of this example was to demonstrate that dendritic cell-targeting antibodies can effectively increase DC uptake.

By mixing Anti-DEC205(DEC) antibody with OVA carrying pattern antigen_257-264The SpyTag (SpyTag-OVA) of (SIINFEKL) is connected with the vaccine universal carrier OMV-SpyC-SPAb of the embodiment 1 to obtain the OMV nano vaccine OMV-OVA-DEC.

Bone marrow derived cells of femur and tibia of C57BL/6 mouse were extracted, bone marrow derived cells were induced to differentiate into dendritic cells (BMDC cells) by GM-CSF and IL-4 co-stimulation, and DC cells were stimulated by adding OMV-OVA-DEC and other control groups, and their uptake was examined by flow cytometry. As shown in fig. 4, the addition of the targeting antibody DEC was effective in enhancing DC cell uptake.

Example 4

The purpose of this example was to verify that the targeting antibody can target the lymph nodes of mice.

The SpyTag-OVA polypeptide prepared in example 3 was further coupled with a fluorescent molecule, cy5.5, and mixed with Anti-DEC205(DEC) antibody or control IgG antibody, OMV-SpyC-spa, and then the nano vaccine, OMV-OVA-DEC and its control group, OMV-OVA-IgG, was subcutaneously injected into the root of the tail of a mouse (C57BL/6 black mouse, 20-25 g, 6-8 weeks old), and imaged using a small animal imaging system to observe the migration of DC cells to lymph nodes. A in FIG. 5 is the result of in vivo fluorescence imaging (12 h subcutaneous injection), and it can be seen that the nano vaccine group OMV-OVA-DEC has stronger lymph node drainage effect than the control group. B in FIG. 5 is the distribution of the nano-vaccine in the inguinal lymph node of the mouse (12 h subcutaneous injection), and the results also show that the OMV-OVA-DEC vaccine efficiently drains to the lymph node. C in FIG. 5 is the relative fluorescence intensity in lymph nodes of OMV-OVA-DEC vaccine and control group (100% for OMV-OVA-DEC group). The above results indicate that the DEC vaccine modified with antigen and targeting antibody can enhance the efficiency of DC uptake and enrichment in lymph nodes.

Example 5

The purpose of this example is to verify that the nano-vaccine promotes cross-presentation of antigens.

Using the nano vaccine OMV-OVA-DEC prepared in example 3, after incubation with BMDC cells for 12h in vitro, expression of MHC-I OVA in DC cells was detected by flow cytometry, and as shown in A in FIG. 6, OMV-OVA-DEC was effective in promoting cross-presentation of antigen compared to control group. In vivo, OMV-OVA-DEC and its control group were injected subcutaneously into the roots of the tail of mice, lymph nodes were removed 12h later, and MHC-I OVA expression in DC cells was examined by flow cytometry, as shown in B of FIG. 6, and OMV-OVA-DEC was also effective in promoting cross-presentation of antigen in vivo. The results show that the nano vaccine group OMV-OVA-DEC effectively promotes the cross presentation of the antigen compared with the control group, thereby causing stronger adaptive immune response.

Example 6

The purpose of this example is to verify the antitumor effect of the nano-vaccine.

The tumor-inhibiting effect of the nano vaccine platform was verified using the nano vaccine OMV-OVA-DEC prepared in example 3.

The animal model corresponding to FIG. 7 is a melanoma model, with 20 ten thousand B16-OVA tumor cells (cell bank of the Chinese academy) inoculated into the tail vein at day 0, and mice (C57BL/6 melanos, 20-25 g, 6-8 weeks old) treated at days 6 and 12 with an equivalent of 50 μ g of OMV vaccine injected per mouse. Efficacy analysis was performed at day 18. It can be seen that the nano vaccine has a significant inhibiting effect on lung metastasis of B16-OVA cells.

In conclusion, the dendritic cell-targeted antibody modified vaccine universal vector provided by the invention can effectively promote the ingestion and cross presentation of DC to antigen, thereby inducing immune response to achieve the purpose of inhibiting lung metastasis, and has better application prospect in tumor vaccines.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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<400> 7

ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag 60

gagatatacc atgactgaaa tcgttgcaga taaaacggta gaagtagtta aaaacgcaat 120

cgaaaccgca gatggagcat tagatcttta taataaatat ctcgatcagg tcatcccctg 180

gcagaccttt gatgaaacca taaaagagtt aagtcgcttt aaacaggagt attcacaggc 240

agcctccgtt ttagtcggcg atattaaaac cttacttatg gatagccagg ataagtattt 300

tgaagcaacc caaacagtgt atgaatggtg tggtgttgcg acgcaattgc tcgcagcgta 360

tattttgcta tttgatgagt acaatgagaa gaaagcatcc gcccagaaag acattctcat 420

taaggtactg gatgacggca tcacgaagct gaatgaagcg caaaaatccc tgctggtaag 480

ctcacaaagt ttcaacaacg cttccgggaa actgctggcg ttagatagcc agttaaccaa 540

tgatttttca gaaaaaagca gctatttcca gtcacaggta gataaaatca ggaaggaagc 600

atatgccggt gccgcagccg gtgtcgtcgc cggtccattt ggattaatca tttcctattc 660

tattgctgcg ggcgtagttg aaggaaaact gattccagaa ttgaagaaca agttaaaatc 720

tgtgcagaat ttctttacca ccctgtctaa cacggttaaa caagcgaata aagatatcga 780

tgccgccaaa ttgaaattaa ccaccgaaat agccgccatc ggtgagataa aaacggaaac 840

tgaaacaacc agattctacg ttgattatga tgatttaatg ctttctttgc taaaagaagc 900

ggccaaaaaa atgattaaca cctgtaatga gtatcagaaa agacacggta aaaagacact 960

ctttgaggta cctgaagtcg gaagttccat ggtaaccacc ttatcaggtt tatcaggtga 1020

gcaaggtccg tccggtgata tgacaactga agaagatagt gctacccata ttaaattctc 1080

aaaacgtgat gaggacggcc gtgagttagc tggtgcaact atggagttgc gtgattcatc 1140

tggtaaaact attagtacat ggatttcaga tggacatgtg aaggatttct acctgtatcc 1200

aggaaaatat acatttgtcg aaaccgcagc accagacggt tatgaggtag caactgctat 1260

tacctttaca gttaatgagc aaggtcaggt tactgtaaat ggcgaagcaa ctaaaggtga 1320

cgctcatact ggatccagtg gtagctaaga attcgagctc ggcgcgcctg caggtcgaca 1380

agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta cacggccgca 1440

taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccatct 1500

tagtatatta gttaagtata agaaggagat atacatatga ctgaaatcgt tgcagataaa 1560

acggtagaag tagttaaaaa cgcaatcgaa accgcagatg gagcattaga tctttataat 1620

aaatatctcg atcaggtcat cccctggcag acctttgatg aaaccataaa agagttaagt 1680

cgctttaaac aggagtattc acaggcagcc tccgttttag tcggcgatat taaaacctta 1740

cttatggata gccaggataa gtattttgaa gcaacccaaa cagtgtatga atggtgtggt 1800

gttgcgacgc aattgctcgc agcgtatatt ttgctatttg atgagtacaa tgagaagaaa 1860

gcatccgccc agaaagacat tctcattaag gtactggatg acggcatcac gaagctgaat 1920

gaagcgcaaa aatccctgct ggtaagctca caaagtttca acaacgcttc cgggaaactg 1980

ctggcgttag atagccagtt aaccaatgat ttttcagaaa aaagcagcta tttccagtca 2040

caggtagata aaatcaggaa ggaagcatat gccggtgccg cagccggtgt cgtcgccggt 2100

ccatttggat taatcatttc ctattctatt gctgcgggcg tagttgaagg aaaactgatt 2160

ccagaattga agaacaagtt aaaatctgtg cagaatttct ttaccaccct gtctaacacg 2220

gttaaacaag cgaataaaga tatcgatgcc gccaaattga aattaaccac cgaaatagcc 2280

gccatcggtg agataaaaac ggaaactgaa acaaccagat tctacgttga ttatgatgat 2340

ttaatgcttt ctttgctaaa agaagcggcc aaaaaaatga ttaacacctg taatgagtat 2400

cagaaaagac acggtaaaaa gacactcttt gaggtacctg aagtcggcgg tggaggaagt 2460

ggtggcggtg gatccggagg tggtgggtcc gcggataaca aattcaacaa agaacaacaa 2520

aatgctttct atgaaatctt acatttacct aacttaaacg aagaacaacg caatggtttc 2580

atccaaagcc taaaagatga cccaagccaa agcgctaacc ttttagcaga agctaaaaag 2640

ctaaatgatg ctcaagcacc aaaataagat atcggccggc cacgcgatcg ctgacgtcgg 2700

taccctcgag tctggtaaag aaaccgctgc tgcgaaattt gaacgccagc acatggactc 2760

gtctactagc gcagcttaat taacctaggc tgctgccacc gctgagcaat aactagcata 2820

accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc 2880

cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 2940

acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 3000

ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 3060

ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 3120

ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 3180

acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 3240

tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 3300

atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat ttctggcggc 3360

acgatggcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 3420

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 3480

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 3540

ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 3600

taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 3660

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 3720

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3780

ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 3840

aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 3900

gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 3960

cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 4020

actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 4080

caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 4140

gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 4200

ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 4260

caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4320

tactcatact cttccttttt caatcatgat tgaagcattt atcagggtta ttgtctcatg 4380

agcggataca tatttgaatg tatttagaaa aataaacaaa taggtcatga ccaaaatccc 4440

ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4500

ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4560

agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4620

cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 4680

caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 4740

tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 4800

ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 4860

ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 4920

gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 4980

gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 5040

tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 5100

cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 5160

gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 5220

ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 5280

gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc 5340

agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg 5400

actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 5460

gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 5520

agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt 5580

ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct 5640

ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct 5700

gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga 5760

tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg 5820

aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc 5880

agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc 5940

atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac 6000

tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc 6060

agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc 6120

aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcta gtcatgcccc 6180

gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg 6240

gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt 6300

cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 6360

tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg 6420

cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc 6480

aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta 6540

tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc ggtaatggcg 6600

cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc 6660

tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt 6720

tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga 6780

cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg 6840

accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg 6900

ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca 6960

gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg 7020

agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc 7080

accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc 7140

gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt 7200

tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc 7260

cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag 7320

acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat 7380

tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg 7440

tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag 7500

gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa 7560

cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc 7620

gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc 7680

acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatcgatct 7740

cgatcccgcg aaattaatac gactcactat a 7771

Claims

1. The bacterial outer membrane vesicle carrier is characterized in that the bacterial outer membrane vesicle carrier is a bacterial outer membrane vesicle carrying a B structural domain of staphylococcus aureus protein A and Spycatcher protein;

wherein, the amino acid sequence of the B structural domain of the staphylococcus aureus protein A is shown as SEQ ID NO. 4, and the amino acid sequence of the SpyCatcher protein is shown as SEQ ID NO. 5.

2. The bacterial outer membrane vesicle vector according to claim 1, wherein the B domain of staphylococcus aureus protein a is expressed in bacterial outer membrane vesicles fused to a bacterial outer membrane protein, and the SpyCatcher protein is expressed in bacterial outer membrane vesicles fused to a bacterial outer membrane protein;

preferably, the bacterial outer membrane protein is ClyA.

3. The bacterial outer membrane vesicle carrier according to claim 2, wherein the particle size of the bacterial outer membrane vesicle carrier is 20-40 nm.

4. A method for preparing a bacterial outer membrane vesicle vector, comprising:

(1) artificially synthesizing a nucleic acid construct ClyA-SpyCatcher-ClyA-SPAb, and constructing the nucleic acid construct into a prokaryotic expression vector to obtain a recombinant expression vector;

(2) introducing the recombinant expression vector into escherichia coli to obtain recombinant bacteria; adding an inducer into a culture solution of the recombinant bacteria for induced expression, centrifugally collecting the bacteria solution, concentrating and purifying the bacteria solution, discarding the supernatant after ultracentrifugation, adding PBS into an ultracentrifugation tube for heavy suspension, then carrying out ultracentrifugation again, discarding the supernatant, and carrying out heavy suspension precipitation by using PBS to obtain the recombinant bacteria;

wherein, ClyA is the outer membrane protein of bacteria, SPAb is the B structural domain of staphylococcus aureus protein A, the amino acid sequence is shown as SEQ ID NO. 4, and the amino acid sequence of Spycatcher protein is shown as SEQ ID NO. 5.

5. The method of claim 4, wherein the prokaryotic expression vector is pETDuet-1; and/or

The Escherichia coli is Rosetta (DE 3); and/or

The inducer is IPTG, and the induction concentration is 0.1-1 mM; and/or

The conditions of bacterial liquid centrifugation are as follows: centrifuging at 5000-8000 rpm for 5-10 min; and/or

Concentrating and purifying the bacterial liquid through a sterile filter membrane and an ultrafiltration tube, wherein the aperture of the filter membrane is 0.45 mu m and 0.22 mu m; the cut-off molecular weight of the ultrafiltration tube is 50KDa, the bacterial liquid passes through a 0.45 mu m filter membrane before ultrafiltration, and passes through a 0.22 mu m filter membrane after ultrafiltration is finished; and/or

The conditions of the ultracentrifugation were: centrifuge at 150000g for 3 h.

6. Use of a bacterial outer membrane vesicle vector according to any of claims 1-3, or prepared according to the method of claims 4 or 5, as a vaccine vector or a drug delivery system.

7. A dendritic cell-targeting nano-vaccine, wherein the nano-vaccine is prepared by mixing a dendritic cell-targeting antibody, an antigen containing a SpyTag tag, and the bacterial outer membrane vesicle vector according to any one of claims 1 to 3, or the bacterial outer membrane vesicle vector prepared by the method according to claim 4 or 5;

wherein, the amino acid sequence of the SpyTag label is shown as SEQ ID NO. 3.

8. The nano-vaccine according to claim 7, wherein the mass ratio of the dendritic cell-targeting antibody, the SpyTag-tag-containing antigen and the bacterial outer membrane vesicle carrier is 1:1: 1-4: 4: 1.

9. The nano-vaccine according to claim 7 or 8, characterized in that the dendritic cell targeting antibody is Anti-DEC205 antibody; and/or

The antigen is tumor specific antigen, preferably model antigen OVA_257-264The amino acid sequence is SIINFEKL; the C end of the antigen is connected with a SpyTag label.

10. Use of the nano-vaccine of any one of claims 7 to 9 in the preparation of a tumor immunotherapeutic vaccine and a medicament.