CN116478303A - Fusion proteins that can self-assemble into nanoparticles and their application in immunity - Google Patents

Abstract

The present invention relates to a fusion protein capable of self-assembling into nanoparticles, a glycoprotein nanoparticle formed from the fusion protein, an immunogenic composition containing them, and the application of the fusion protein, the glycoprotein nanoparticle and the immunogenic composition in immunization.

Description

Fusion proteins that can self-assemble into nanoparticles and their applications in immunity

Technical Field

The present application belongs to the field of biotechnology. Specifically, the present invention relates to fusion proteins that can self-assemble into nanoparticles, glycoprotein nanoparticles formed by the fusion proteins, immunogenic compositions containing them, and applications of the fusion proteins, glycoprotein nanoparticles and immunogenic compositions in immunization.

Background Art

Polysaccharide conjugate vaccines refer to covalently linking immunogenic polysaccharides to appropriate substrate proteins, which converts polysaccharide antigens from T-cell-independent antigens to T-cell-dependent antigens, allowing the body to produce a lasting protective effect. Polysaccharide conjugate vaccines can stimulate both T-cell-dependent and -independent immune responses at the same time, and are one of the vaccine forms with extremely high application potential. Although some polysaccharide conjugate vaccines have been reported, the field still needs polysaccharide conjugate vaccines with better immune effects and adaptability to more complex modular multi-antigen designs, intramolecular adjuvant designs, etc.

Summary of the invention

The inventors surprisingly found that the fusion expression of AB5 toxin B subunit and streptavidin subunit can self-assemble into nanoparticles. On this basis, a sequence containing a glycosylation site is further fused, so that a nano-scale polysaccharide conjugate vaccine can be prepared by co-expression with a glycosyltransferase, so that the body produces a long-lasting protective effect. The following invention is provided.

Fusion Protein

In one aspect, the present invention provides a fusion protein that can self-assemble into nanoparticles, wherein the fusion protein comprises an AB5 toxin B subunit capable of forming a pentameric structure (also referred to as B5 or B5 protein in this article), a streptavidin subunit capable of forming a tetrameric structure (also referred to as SA or SA sequence in this article), and a glycosylation sequence containing an O-glycosylation site.

AB5 toxin is a common form of microbial toxin in nature, mainly composed of an A subunit and a B subunit. Five B subunits form a ring pentamer, which is responsible for binding to the host cell surface and catalyzing the A subunit to destroy the host's cells.

AB5 toxins are known to those skilled in the art. In certain embodiments, the AB5 toxin B subunit is selected from cholera toxin B subunit (CTB), Shiga toxin B subunit (StxB), Escherichia coli heat-labile enterotoxin B subunit (LTB) or Bacillus subtilisin B subunit (SubB).

CTB is the B subunit of cholera toxin, which can specifically bind to ganglioside GM1 on the surface of most mammalian cells, promote the passage of antigenic proteins connected to it through the mucosal barrier, and enhance the presentation of antigens by antigen-presenting cells such as DCs, so CTB is a good carrier protein. In certain exemplary embodiments, the CTB has an amino acid sequence as shown in SEQ ID NO: 1.

StxB is the B subunit of Shiga toxin, which has strong immunomodulatory properties and can recognize exogenous antigens by MHC-I in antigen presenting cells, thereby mediating cellular immune responses. Therefore, StxB has great potential as a non-live, non-toxic vaccine carrier and has been shown to have anti-tumor protective immunity in mice. Its ability to induce cell-mediated immunity also indicates its potential use in the immunoprevention and treatment of infectious diseases. In certain exemplary embodiments, the StxB has an amino acid sequence as shown in SEQ ID NO: 13.

LTB is the B subunit of the heat-labile enterotoxin of Escherichia coli. Its structure and function are similar to those of CTB. Both can promote immune response through the GM1 receptor. In addition, LTB can also bind to some glycolipids or glycoproteins on the cell surface to promote antigen recognition and presentation. In certain exemplary embodiments, the LTB has the amino acid sequence shown in SEQ ID NO: 15

SubB is the B subunit of Bacillus subtilisin toxin, similar to cholera toxin and heat-labile enterotoxin, and SubB can also stimulate immune response, promote T cell activation and release of inflammatory factors. In certain exemplary embodiments, the SubB has the amino acid sequence shown in SEQ ID NO:17.

Streptavidin (SA) exists naturally as a tetramer of four identical subunits, i.e., it is a homotetramer, wherein each subunit contains a single binding site for biotin, a biotin derivative or analog, or a biotin mimetic. An exemplary sequence of a streptavidin subunit is shown in SEQ ID NO: 3, however, those skilled in the art understand that a streptavidin subunit may also include sequences present in homologs of other Streptomyces species. In certain exemplary embodiments, the streptavidin subunit has an amino acid sequence as shown in SEQ ID NO: 3 or has at least 90%, at least 95%, at least 98% or at least 99% identity compared thereto, and the sequence of the streptavidin subunit is a sequence present in a homolog of a Streptomyces species.

In certain embodiments, the fusion protein comprises adjacent AB5 toxin B subunits and streptavidin subunits, and the two subunits can be arranged in any order. In certain embodiments, the fusion protein comprises adjacent AB5 toxin B subunits and streptavidin subunits from N-terminus to C-terminus. A peptide linker is optionally included between the AB5 toxin B subunits and the streptavidin subunits.

The use of biological methods to prepare polysaccharide conjugate vaccines mainly utilizes the loose substrate specificity of glycosyltransferases, co-expresses them with bacterial surface polysaccharides and substrate proteins in bacteria (usually Escherichia coli), so that the polysaccharides are transferred to the substrate proteins under the catalysis of glycosyltransferases, and the glycoproteins can be obtained by one-step purification. Among them, the glycosyltransferase PglL of Neisseria meningitidis is widely used because it has loose substrate specificity and can recognize almost all polysaccharides.

In certain embodiments, the glycosylation sequence containing an O-glycosylation site can serve as a substrate protein for a glycosyltransferase (e.g., PglL) in an O-glycosylation modification system, and under the action of the glycosyltransferase, the bacterial O polysaccharide can be transferred to the glycosylation site contained in the glycosylation sequence.

In certain embodiments, the glycosylation sequence is selected from a peptide segment comprising an O-glycosylation site of the pilin PilE of Neisseria meningitidis.

In certain embodiments, the O-glycosylation site is the serine at position 63 from the N-terminus of PilE.

In certain embodiments, the peptide segment comprising the O-glycosylation site of the pilin PilE of Neisseria meningitidis comprises at least the serine at position 63 from the N-terminus of PilE.

In certain embodiments, the peptide segment containing the O-glycosylation site of the pilin protein PilE of Neisseria meningitidis is a peptide segment containing at least the amino acid residues from position 55 (such as glycine) to position 66 (such as valine) of PilE.

In certain embodiments, the peptide segment containing the O-glycosylation site of the pilin PilE of Neisseria meningitidis is a peptide segment containing at least the amino acid residues from position 45 (eg, serine) to position 73 (eg, lysine) of PilE.

In certain embodiments, the glycosylation sequence comprises a peptide segment consisting of amino acid residues from positions 45 to 73 of PilE.

In certain embodiments, the glycosylation sequence comprises the sequence shown in SEQ ID NO:9.

In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, an AB5 toxin B subunit, a streptavidin subunit, and a glycosylation sequence.

In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, a glycosylation sequence, an AB5 toxin B subunit, and a streptavidin subunit.

In certain embodiments, any two adjacent domains comprised by the fusion protein (eg, AB5 toxin B subunit and streptavidin subunit, streptavidin subunit and glycosylation sequence) are optionally connected via a peptide linker.

In certain embodiments, the peptide linker comprises one or more flexible amino acids.

In certain embodiments, the peptide linker is a flexible amino acid linker comprising glycine (G) and serine (S).

In certain embodiments, the peptide linker is a flexible amino acid linker comprising alanine (A) and lysine (K), and optionally glutamic acid (E).

In certain embodiments, the peptide linker consists of 1 to 20 amino acids (eg, 1 to 15 amino acids, 1 to 8 amino acids, 2 to 6 amino acids, or about 4).

In certain exemplary embodiments, the peptide linker has the sequence shown in SEQ ID NO:10 or 11.

In certain exemplary embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of:

(1) an amino acid sequence consisting of amino acid residues 20 to 326 of the sequence shown in SEQ ID NO: 5;

(2) an amino acid sequence consisting of amino acid residues 20 to 311 of the sequence shown in SEQ ID NO: 19;

(3) an amino acid sequence consisting of amino acid residues 20 to 326 of the sequence shown in SEQ ID NO: 20;

(4) An amino acid sequence consisting of amino acid residues 20 to 363 of the sequence shown in SEQ ID NO:21.

In certain embodiments, the fusion protein may further comprise a signal peptide, thereby facilitating secretory expression of the fusion protein of the present invention in a host cell. In certain embodiments, the fusion protein of the present invention comprises a signal peptide at its N-terminus.

The signal peptide originally possessed by the AB5 toxin B subunit can be completely fused to the fusion protein of the present invention. Alternatively, it can be removed and replaced with a signal peptide of a protein derived from a prokaryotic cell, such as a signal peptide of a secretory protein from Escherichia coli (e.g., an outer membrane protein or a periplasmic protein (such as DsbA)).

In certain exemplary embodiments, the signal peptide has the sequence shown in SEQ ID NO:12.

In certain exemplary embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of:

(1) an amino acid sequence consisting of amino acid residues 1 to 326 of the sequence shown in SEQ ID NO: 5;

(2) an amino acid sequence consisting of amino acid residues 1 to 311 of the sequence shown in SEQ ID NO: 19;

(3) an amino acid sequence consisting of amino acid residues 1 to 326 of the sequence shown in SEQ ID NO: 20;

(4) An amino acid sequence consisting of amino acid residues 1 to 363 of the sequence shown in SEQ ID NO: 21.

In certain embodiments, the fusion protein may further comprise a protein tag. Such protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, etc., and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection, or tracing). In certain exemplary embodiments, the fusion protein of the present invention comprises a His tag. In certain embodiments, the fusion protein of the present invention comprises a protein tag at its C-terminus.

In certain exemplary embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

Nanoparticles and their preparation

The fusion protein of the present invention can be self-assembled into nanoparticles after being expressed in cells. The protein nanoparticles have an average particle size of about 20-30 nm, and the protein nanoparticles contain pentamers of B5 protein and tetramers of SA subunits.

In another aspect, the present invention also relates to a protein nanoparticle obtained by expressing the fusion protein of the present invention in a host cell comprising a nucleotide sequence encoding the fusion protein.

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of the present invention.

In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is, for example, a plasmid, a cosmid, a phage, and the like.

On the other hand, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In certain embodiments, the host cell is a prokaryotic cell (e.g., a bacterial cell, such as an Escherichia coli cell).

On the other hand, a method for preparing the fusion protein described in the first aspect is provided, comprising culturing the host cell described above under conditions allowing protein expression, and recovering the fusion protein from the cultured host cell culture, wherein the fusion protein is in the form of nanoparticles.

In another aspect, a method for preparing protein nanoparticles is provided, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the nanoparticles from the culture of the cultivated host cell.

Glycoprotein Nanoparticles

When the fusion protein of the present invention comprises a glycosylation sequence, by co-expression with a glycosyltransferase in bacterial cells, the glycosyltransferase can transfer bacterial polysaccharides to the fusion protein of the present invention, thereby obtaining glycoprotein nanoparticles.

In another aspect, the present invention provides a method for preparing glycoprotein nanoparticles, comprising the steps of:

(1) Providing a bacterial host cell lacking an O-antigen ligase. By lacking endogenous O-antigen ligase, the O-antigen polysaccharide can be prevented from being catalyzed by the O-antigen ligase to transfer to the lipid A core polysaccharide to form lipopolysaccharide, thereby generating free O-antigen polysaccharide;

(2) expressing the fusion protein of the present invention and glycosyltransferase PglL in the bacterial host cells to produce target glycoprotein nanoparticles.

Neisseria meningitidis glycosyltransferase PglL is an O-oligosaccharide transferase well known to those skilled in the art, and its sequence can be found in various public databases, such as GeneBank: JN200826.1.

In some embodiments, the method further comprises: (3) purifying the target glycoprotein nanoparticles. In some embodiments, the target glycoprotein nanoparticles are purified by affinity chromatography and/or molecular sieves. In some embodiments, when the fusion protein comprises a protein tag, the method further comprises the step of detecting the target glycoprotein nanoparticles by using an antibody specific to the protein tag.

In certain embodiments, in step (1), an O-antigen ligase-deficient bacterial host cell is provided by knocking out an endogenous O-antigen ligase gene in the bacterial host cell. In this article, "knockout" in the context of a gene means that the host cell carrying the knockout does not produce a functional protein product of the gene. Knockout can be produced in a variety of ways by removing all or part of the coding sequence, introducing a frameshift mutation so that a functional protein (truncated or nonsense sequence) is not produced, removing or changing a regulatory component (e.g., a promoter) so that the gene is not transcribed, preventing translation by combining with mRNA, etc. Usually, the knockout is carried out at the genomic DNA level so that the offspring of the cell also permanently carry the knockout.

In certain embodiments, the O-antigen ligase gene is the waaL gene.

In certain embodiments, the bacterial host cell is a pathogenic bacterial cell. In certain embodiments, the bacterial host cell is a pathogenic Gram-negative bacteria (Gram-negative pathogen) cell. In certain embodiments, the bacterial host cell is selected from Shigella, Escherichia coli, Shigella dysenteriae, Salmonella paratyphi A, Vibrio cholerae.

In certain embodiments, the fusion protein of the present invention and PglL are co-expressed in the same bacterial host cell. Co-expression is optionally performed by expressing each protein on the same or different expression vectors. The bacterial host cell can be co-transfected with two expression vectors, the first vector encoding the fusion protein and the second vector encoding PglL. Alternatively, a single vector encoding and expressing the fusion protein and PglL can be used.

In certain embodiments, step (2) comprises the following steps:

(2a) introducing a first nucleotide sequence encoding the fusion protein of the present invention and a second nucleotide sequence encoding PglL into the bacterial host cell, wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different expression vectors (e.g., prokaryotic expression vectors);

(2b) culturing the bacterial host cell (e.g., under conditions that allow protein expression);

(2c) Recovering the target nanoglycoprotein particles from the cell culture (eg, cell lysate).

On the other hand, the present invention provides a glycoprotein nanoparticle comprising a conjugate of protein and polysaccharide, wherein the conjugate is formed by coupling bacterial O polysaccharide and the fusion protein of the present invention, and the bacterial O polysaccharide is coupled to the fusion protein via the O-glycosylation site in the glycosylation sequence in the fusion protein.

In certain embodiments, the bacterial O-polysaccharide is selected from the O-polysaccharide of a pathogenic bacteria.

In certain embodiments, the bacterial O antigen polysaccharide is selected from Shigella O polysaccharide, Escherichia coli O157 O polysaccharide, Shigella dysenteriae O polysaccharide, Salmonella paratyphi A O polysaccharide, and Vibrio cholerae O polysaccharide.

In certain embodiments, in the glycoprotein nanoparticle, the AB5 toxin B subunit in the fusion protein exists in the form of a pentamer, and the SA subunit in the fusion protein exists in the form of a tetramer.

In certain embodiments, the glycoprotein nanoparticles have an average particle size of about 25-50 nm.

In certain embodiments, the glycoprotein nanoparticles are obtained by the preparation method provided above.

Compound

The inventors surprisingly found that the nanoparticles formed by the fusion protein provided by the present invention can be connected to various bioactive molecules labeled with biotin through the streptavidin-biotin system, and can also change the size and geometry of the chassis particles, and enable different arrangements of antigen orientation combinations, and may support superior delivery and/or stimulation configurations.

In another aspect, the present invention provides a complex comprising: the fusion protein, protein nanoparticle or glycoprotein nanoparticle of the present invention, and another biologically active molecule.

In certain embodiments, the additional biologically active molecule is labeled with biotin.

In certain embodiments, the additional biologically active molecule is attached via a biotin-streptavidin interaction.

In certain embodiments, the additional biologically active molecule is a nucleic acid, a protein and/or a sugar.

In certain embodiments, the additional biologically active molecule is an immunogen.

In certain embodiments, the additional biologically active molecule is an adjuvant.

In certain embodiments, the additional biologically active molecule is a CpG adjuvant.

Immunotherapy Applications

On the other hand, the present invention provides an immunogenic composition or vaccine comprising: (i) an immunologically effective amount of the glycoprotein nanoparticles described in the present invention, or (ii) an immunologically effective amount of a mixture comprising the fusion protein of the present invention or protein nanoparticles formed by the fusion protein, and bacterial polysaccharides (such as bacterial O polysaccharide), or (iii) an immunologically effective amount of the complex described in the present invention.

In certain embodiments, the immunogenic composition or vaccine does not comprise an adjuvant.

In certain embodiments, the immunogenic composition or vaccine is for inducing an immune response in a subject and/or for preventing and/or treating an infection in a subject.

In certain embodiments, the immune response is an immune response (eg, an antibody response) to bacterial O polysaccharide.

In certain embodiments, the infection is a bacterial infection (eg, an infection caused by a bacterium expressing an O-polysaccharide).

In certain embodiments, the immunogenic composition or vaccine is used to induce an immune response (e.g., an antibody response) against bacterial O polysaccharide in a subject, and/or to prevent and/or treat bacterial infection (e.g., infection caused by bacteria expressing O polysaccharide).

In certain embodiments, the bacterium is a pathogenic bacterium. In certain embodiments, the bacterium is a gram-negative bacterium. In certain embodiments, the bacterium is a pathogenic gram-negative bacterium (gram-negative pathogenic bacteria). In certain embodiments, the bacterium is selected from Shigella, Escherichia coli, Shigella dysenteriae, Salmonella paratyphi A, Vibrio cholerae.

In certain embodiments, the bacterial O polysaccharide is selected from the O polysaccharide of pathogenic bacteria. In certain embodiments, the bacterial O antigen polysaccharide is selected from the O polysaccharide of Shigella, Escherichia coli O157 O polysaccharide, Shigella dysenteriae O polysaccharide, Salmonella paratyphi A O polysaccharide, Vibrio cholerae O polysaccharide.

In certain embodiments, the immunogenic composition or vaccine further comprises a pharmaceutically acceptable carrier and/or excipient.

The glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention can be in any form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, lyophilized powders), inhalants, sprays, etc. The technician will understand that the preferred dosage form depends on the intended mode of administration and therapeutic use. In certain embodiments, the glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention are injectable preparations, such as injections, sterile powders for injection, or concentrated solutions for injection.

The glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention can be administered by any suitable method known in the art, including but not limited to oral, rectal, parenteral or topical administration. In certain embodiments, the glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention are administered parenterally, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection and injection. The dosage form for parenteral administration can be an injection preparation, including an injection solution, a sterile powder for injection or a concentrated solution for injection. In addition to the active ingredient, the injection formulation can contain a pharmaceutically acceptable carrier such as sterile water, Ringer's solution and isotonic sodium chloride solution, and suitable additives such as antioxidants, buffers and antibacterial agents can also be added according to the properties of the drug.

In addition, other carrier materials and administration methods known in the pharmaceutical field may also be used. The glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention may be prepared by any known pharmaceutical process, such as effective formulations and administration methods.

In another aspect, the present invention provides the use of the glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention in the preparation of a product for inducing an immune response in a subject and/or for preventing and/or treating an infection in a subject.

In certain embodiments, the product is for inducing a specific immune response (eg, an antibody response) against bacterial O polysaccharide in a subject and/or for preventing and/or treating bacterial infection (eg, infection caused by bacteria expressing O polysaccharide).

In certain embodiments, the product is a vaccine.

In certain embodiments, the bacterium is a pathogenic bacterium. In certain embodiments, the bacterium is a gram-negative bacterium. In certain embodiments, the bacterium is a pathogenic gram-negative bacterium (gram-negative pathogenic bacteria). In certain embodiments, the bacterium is selected from Shigella, Escherichia coli, Shigella dysenteriae, Salmonella paratyphi A, Vibrio cholerae.

In certain embodiments, the bacterial O polysaccharide is selected from the O polysaccharide of pathogenic bacteria. In certain embodiments, the bacterial O antigen polysaccharide is selected from the O polysaccharide of Shigella, Escherichia coli O157 O polysaccharide, Shigella dysenteriae O polysaccharide, Salmonella paratyphi A O polysaccharide, Vibrio cholerae O polysaccharide.

On the other hand, the present invention also provides a method for inducing an immune response in a subject and/or for preventing and/or treating an infection in a subject, comprising: administering an immunologically effective amount of the glycoprotein nanoparticles, complexes or immunogenic compositions or vaccines of the present invention to a subject in need thereof.

In certain embodiments, the methods are used to induce a specific immune response (eg, an antibody response) against bacterial O polysaccharide in a subject and/or to prevent and/or treat bacterial infection (eg, infection caused by bacteria expressing O polysaccharide).

In certain embodiments, the bacterium is a pathogenic bacterium. In certain embodiments, the bacterium is a gram-negative bacterium. In certain embodiments, the bacterium is a pathogenic gram-negative bacterium (gram-negative pathogenic bacteria). In certain embodiments, the bacterium is selected from Shigella, Escherichia coli, Shigella dysenteriae, Salmonella paratyphi A, Vibrio cholerae.

In certain embodiments, the bacterial O polysaccharide is selected from the O polysaccharide of pathogenic bacteria. In certain embodiments, the bacterial O antigen polysaccharide is selected from the O polysaccharide of Shigella, Escherichia coli O157 O polysaccharide, Shigella dysenteriae O polysaccharide, Salmonella paratyphi A O polysaccharide, Vibrio cholerae O polysaccharide.

In certain embodiments, the subject is a mammal, such as a human.

Definition of terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the microbiology, immunology, biochemistry, nucleic acid chemistry and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "O polysaccharide" refers to the outermost part of lipopolysaccharide, which is composed of dozens of identical oligosaccharide units. O polysaccharide has antigenic properties, so it is also called O-antigen. O antigen is a bacterial antigen of Gram-negative bacteria-specific polysaccharide.

As used herein, the term "AB5 toxin B subunit" can be used interchangeably with "B5" or "B5 protein". The AB5 toxin produced by bacteria is a protein exotoxin produced by toxin-producing bacteria and secreted outside the bacterial cell. The AB5 toxin consists of one active subunit (A subunit) with toxicity and five binding subunits (B subunits) with cell binding activity. The AB5 toxin B subunit can self-assemble into a pentamer.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, preservatives, stabilizers, and the like. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

As used herein, the term "immunologically effective amount" is an amount sufficient to provide a protective immune response against bacterial infection (e.g., infection caused by Gram-negative pathogens) or to induce the body to produce a protective immune response against an immunogen (e.g., a bacterial polysaccharide antigen). In certain embodiments, the amount should be sufficient to significantly prevent the possibility or severity of bacterial infection (e.g., infection caused by Gram-negative pathogens). The immunologically effective amount of the glycoprotein nanoparticles, or immunogenic compositions or vaccines of the present invention may vary depending on the overall state of the patient's own immune system, the patient's general condition (e.g., age, weight, and sex), the mode of administration of the drug, and other treatments administered simultaneously.

Beneficial Effects

The inventors first discovered that by fusing the AB5 toxin B subunit with streptavidin tetramers and linking bacterial polysaccharides into them using a glycosylation system, nanoscale glycoproteins can be obtained. The nanoscale glycoproteins are significantly superior to single polysaccharide antigens in terms of induced antibody levels and protective effects. In addition, streptavidin tetramers can be coupled with biotin-labeled macromolecules (nucleic acids, proteins, sugars), which further expands the Nano-B5 platform to accommodate more complex modular multi-antigen designs and intramolecular adjuvant designs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of the monomer size and particle size detection of purified CTBSA4573. A: Coomassie brilliant blue staining analysis and western blot detection results; B: transmission electron microscopy; C: dynamic light scattering detection results.

Figure 2 shows the results of the detection of the monomer size and the size of the formed particles after LTBSA4573 purification. A: Coomassie brilliant blue staining analysis and western blot detection results; B: transmission electron microscopy; C: dynamic light scattering detection results.

Figure 3 shows the results of the detection of the monomer size and the size of the formed particles after purification of SubBSA4573. A: Coomassie brilliant blue staining analysis and western blot detection results; B: transmission electron microscopy; C: dynamic light scattering detection results.

Figure 4 shows the results of the detection of the monomer size and the size of the formed particles after purification of StxBSA4573. A: Coomassie brilliant blue staining analysis and western blot detection results; B: transmission electron microscopy; C: dynamic light scattering detection results.

FIG5 shows the expression detection results of glycoprotein CTBSA4573-OPS.

Figure 6 shows the results of the detection of the monomer size and the size of the formed particles after the expression and purification of the glycoprotein CTBSA4573-OPS. A: Coomassie brilliant blue staining analysis and western blot detection results; B: transmission electron microscopy; C: dynamic light scattering detection results.

Figure 7 shows the results of antibody titer determination after mice were immunized with CTBSA4573-OPS. ****p<0.0001.

Figure 8 shows the results of the determination of antibody titers of different IgG subtypes after mice were immunized with CTBTri4573-OPS. ****p<0.0001.

FIG. 9 shows the results of the challenge test after immunization of CTBSA4573-OPS mice.

FIG. 10 shows the ability of nanoparticles to bind biotinylated CpG adjuvant.

Sequence information

Table 1: Information on the sequences involved in the present invention is provided in the table below.

DETAILED DESCRIPTION

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

The quantitative tests in the following examples were all repeated three times, and the results were averaged.

The avirulent Shigella flexneri 301DWP lacking the O-antigen ligase gene waaI in the following examples is transformed from the wild strain of Shigella flexneri 2a 301 (S.flexneri 2a 301) (Jin Q et al. Nucleic Acids Res. 2002 Oct 15; 30(20): 4432-41), which can be obtained by the public from the Institute of Bioengineering, Academy of Military Medical Sciences, Chinese People's Liberation Army Academy of Military Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

pET28a(+) in the following examples was purchased from Novagen.

The Trans5a competent cells in the following examples were purchased from TransGen.

The HRP-labeled Anti-His antibody in the following examples is a product of Abmart, with the product number M20020.

The HRP-labeled goat anti-rabbit antibody in the following examples is a product of TransGen, with the serial number HS-101-01.

The rabbit-derived Flexner 2a serum in the following examples is a product of Denka Seiken Co., Ltd., with the serial number 041011.

The HRP-labeled donkey anti-mouse IgG, goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 in the following examples were purchased from Abcam, with the numbers ab6820, ab97240, ab97245, ab97250, and ab97260, respectively.

The Balb/c mice in the following examples were purchased from Beijing Weitonglihua Co., Ltd.

Example 1. Construction and expression of B5SA fusion protein expression vector

In this example, a vector expressing a fusion protein of B5 protein and SA sequence (B5SA) was constructed, wherein the B5 protein includes cholera toxin B subunit (CTB), Shiga toxin B subunit (StxB), Escherichia coli heat-labile enterotoxin (LTB), and Bacillus subtilis toxin B subunit (SubB), and the SA sequence is SEQ ID NO. 3. The construction method is as follows:

1.1 Construction of CTBSA4573 fusion protein expression vector

CTBSA4573 refers to a fusion protein formed by CTB, SA, and a glycosylation sequence (PilE S45-K73). A DNA molecule of a fully gene-synthesized fusion protein, the amino acid sequence of the fusion protein is shown in SEQ ID No.5, and the nucleotide sequence is shown in SEQ ID No.6, wherein nucleotides 103-131 of SEQ ID No.6 are the sequence of the tac promoter, nucleotides 178 to 234 are signal peptide sequences, nucleotides 241 to 549 are CTB coding sequences, nucleotides 550 to 585 are flexible linkers, nucleotides 586 to 1062 are SA coding sequences, and nucleotides 1075 to 1161 are amino acids 45 to 73 of the pili protein PilE of Neisseria meningitidis, and the serine at position 63 of the pili protein PilE of Neisseria meningitidis is a glycosylation site recognized by the glycosyltransferase PglL of Neisseria meningitidis. The DNA molecule was digested with SacI and XhoI to obtain the target gene fragment, and the pET28(a)+ vector was digested with SacI and XhoI to obtain the vector fragment. The gene fragment and the vector fragment were connected to obtain the recombinant vector pET28-tacSubBSA4573.

1.2 Construction of StxBSA4573 fusion protein expression vector

StxBSA4573 refers to a fusion protein formed by StxB, SA, and a glycosylation sequence (PilE S45-K73). A DNA molecule of a fully gene-synthesized fusion protein, the amino acid sequence of the fusion protein is shown in SEQ ID No.19, and the nucleotide sequence is shown in SEQ ID No.22, wherein the nucleotides 103-131 of SEQ ID No.22 are the sequence of the tac promoter, the nucleotides 178 to 234 are the signal peptide sequence, the nucleotides 241 to 504 are the StxB coding sequence, the nucleotides 505 to 540 are the flexible linker, the nucleotides 541 to 1017 are the SA coding sequence, the nucleotides 1030 to 1116 are the amino acids 45 to 73 of the pili protein PilE of Neisseria meningitidis, and the serine at position 63 of the pili protein PilE of Neisseria meningitidis is the glycosylation site recognized by the glycosyltransferase PglL of Neisseria meningitidis. The DNA molecule was digested with NcoI and XhoI to obtain the target gene fragment, and the pET28(a)+ vector was digested with NcoI and XhoI to obtain the vector fragment. The gene fragment and the vector fragment were connected to obtain the recombinant vector pET28-tacStxBSA4573.

1.3 Construction of LTBSA4573 fusion protein expression vector

LTBSA4573 refers to a fusion protein formed by LTB, SA, and a glycosylation sequence (PilE S45-K73). A DNA molecule of a fully gene-synthesized fusion protein, the amino acid sequence of the fusion protein is shown in SEQ ID No. 20, and the nucleotide sequence is shown in SEQ ID No. 24, wherein nucleotides 103-131 of SEQ ID No. 24 are the sequence of the tac promoter, nucleotides 178 to 234 are the signal peptide sequence, nucleotides 241 to 549 are the LTB coding sequence, nucleotides 550 to 585 are the flexible linker, nucleotides 586 to 1062 are the SA coding sequence, and nucleotides 1075 to 1161 are amino acids 45 to 73 of the pili protein PilE of Neisseria meningitidis, and the serine at position 63 of the pili protein PilE of Neisseria meningitidis is a glycosylation site recognized by the glycosyltransferase PglL of Neisseria meningitidis. The DNA molecule was digested with NcoI and XhoI to obtain the target gene fragment, and the pET28(a)+ vector was digested with NcoI and XhoI to obtain the vector fragment. The gene fragment and the vector fragment were connected to obtain the recombinant vector pET28-tacLTBSA4573.

1.4 Construction of SubBSA4573 fusion protein expression vector

SubBSA4573 refers to a fusion protein formed by SubB, SA, and a glycosylation sequence (PilE S45-K73). A DNA molecule of a fully gene-synthesized fusion protein, the amino acid sequence of the fusion protein is shown in SEQ ID No.21, and the nucleotide sequence is shown in SEQ ID No.23, wherein nucleotides 103-131 of SEQ ID No.23 are the sequence of the tac promoter, nucleotides 178 to 234 are signal peptide sequences, nucleotides 241 to 660 are SubB coding sequences, nucleotides 661 to 696 are flexible linkers, nucleotides 697 to 1173 are SA coding sequences, and nucleotides 1186 to 1272 are amino acids 45 to 73 of the pili protein PilE of Neisseria meningitidis, and the serine at position 63 of the pili protein PilE of Neisseria meningitidis is a glycosylation site recognized by the glycosyltransferase PglL of Neisseria meningitidis. The DNA molecule was digested with NcoI and XhoI to obtain the target gene fragment, and the pET28(a)+ vector was digested with NcoI and XhoI to obtain the vector fragment. The gene fragment and the vector fragment were connected to obtain the recombinant vector pET28-tacSubBSA4573.

1.5 Construction and expression of B5SA4573 expression strain

The pET28-tacB5SA4573 vector was transformed into the Trans5a competent cell and coated with LB solid culture medium containing kanamycin with a final concentration of 50 μg/mL. The positive clone, i.e. Trans5a/pET28-tacCTBSA4573, was inoculated in LB culture medium containing kanamycin with a final concentration of 50 μg/mL, cultured at 37°C, 220 rpm for 10 hours, then transferred to 5 mL LB culture medium containing kanamycin with a final concentration of 50 μg/mL and cultured at 37°C, 220 rpm until OD600 was about 0.6, IPTG with a final concentration of 1 mM was added, and the temperature was lowered to 30°C for induction for 10 hours.

Take 1 mL of each bacterial solution induced at 30°C for 10 h, centrifuge to obtain the bacteria, suspend with 1X reducing buffer (50 mM pH 6.8 Tris-HCl, 1.6% SDS, 0.02% bromophenol blue, 8% glycerol, 20 mM DTT), boil in boiling water for 10 min, and perform 12% SDS-PAGE electrophoresis. After electrophoresis, use Bio-Lab semi-dry transfer instrument to transfer the protein to PVDF membrane, transfer at 20 V constant voltage for 1 h, and detect the protein expression bands with mouse HRP-Anti-His tag monoclonal antibody.

Example 2. Nanoparticle purification and detection

Inoculate trans5a/pET28-tacB5SA4573 in 5 ml LB medium (final kanamycin concentration is 50 μg/mL), culture at 37°C for 12 h, transfer to 100 mL LB medium and culture for 7 h, transfer to 5 L LB medium, culture at 37°C until OD600 is about 0.6, add IPTG to a final concentration of 1 mM, culture at 30°C for 12 h, and centrifuge at 6000 r/min for 10 min to collect the bacteria.

Sample treatment: Resuspend the bacteria in 200 mL of loading buffer (20 mM pH 7.5 Tris-HCl, 0.2 M NaCl, 10 mM imidazole), break the bacteria by ultrasonication, and collect the supernatant by centrifugation at 8000 r/min.

Sample purification: First, balance at least 3 column volumes with deionized water, then balance at least 3 column volumes with A1 solution (20mM pH7.5 Tris-HCl, 0.5M NaCl, 10mM imidazole), load the sample from the A1 pipeline at a flow rate of 4mL/min, and then elute the impurities with A1 (20mM pH7.5 Tris-HCl, 0.5M NaCl, 10mM imidazole) until the UV value is close to 0mAU, then elute the target protein with B1 solution (20mM pH7.5 Tris-HCl, 0.5M NaCl, 500mM imidazole) to obtain a purified sample, and then pass it through a molecular sieve (superdex 200) with a mobile phase of 1× PBS, pH7.4.

Four fusion proteins (CTBSA4573, StxBSA4573, LTBSA4573, SubBSA4573) were sieved and then analyzed by Coomassie brilliant blue staining (Figures 1A-4A), and a protein sample with a purity greater than 90% was obtained. Western blot verification was performed using His tag antibody, and the detection results of the four fusion proteins were shown in Figures 1A-4A. The sample size was detected using dynamic light scattering and transmission electron microscopy, and the transmission electron microscopy detection results of the four fusion proteins were shown in Figures 1B-4B, and the dynamic light scattering detection results of the four fusion proteins were shown in Figures 1C-4C. It can be seen that although the protein monomer is only about 35kDa, it can form protein particles of about 20-30nm in the natural state.

Example 3. Preparation of Nano-scale Bacterial Polysaccharide Conjugate Vaccine by Biological Method

3.1 Construction of the expression vector for Neisseria meningitidis glycosyltransferase PglL

The DNA molecule shown in SEQ ID No. 8 was artificially synthesized. In SEQ ID No. 8, nucleotides 1 to 6 from the 5' end are XbaI recognition sites, nucleotides 105 to 2240 are sequences of the expression cassette of the glycosyltransferase PglL of Neisseria meningitidis, wherein nucleotides 105 to 133 are sequences of the tac promoter, nucleotides 180 to 1994 are coding sequences of the glycosyltransferase PglL of Neisseria meningitidis (encoding the protein shown in SEQ ID No. 7), nucleotides 2475 to 2480 are SacI recognition sequences, and nucleotides 2486 to 2491 are XhoI recognition sites.

The DNA molecule shown in SEQ ID No.8 was digested with XbaI and XhoI to obtain a gene fragment; the pET28a(+) vector was digested with XbaI and XhoI to obtain a large vector fragment; the gene fragment was connected to the vector fragment to obtain a recombinant vector pET28-tacpglL.

3.2 Construction of expression vector pET28-tacpgIL-CTBSA4573

pET28-tacCTBSA4573 was double-digested with SacI and XhoI to obtain the target gene fragment, pET28-tacpglL was double-digested with SacI and XhoI to obtain the vector fragment, and the two fragments were connected to obtain the recombinant vector pET28-tacpglL-tacCTBSA4573.

3.3 Construction of glycoengineered Shigella and detection of protein glycosylation

3.3.1 Preparation of 301DWP electroporation competent cells

The attenuated Shigella 301DWP with O-antigen ligase knocked out was streaked on a solid LB plate and cultured in a 37°C incubator overnight. The next day, a single clone was picked and inoculated into 5 mL LB liquid culture medium, cultured at 37°C for 12 h, transferred to 50 mL low-salt LB culture medium at a ratio of 1:100, cultured at 30°C until OD600 was approximately 0.6, ice-bathed for 30 min, centrifuged at 4°C, 8000 r/min for 15 min to collect the bacteria, washed three times with 10% sterile glycerol, and finally resuspended with 400 μL of 10% sterile glycerol in an ice bath, and 50 μL per tube was dispensed and stored at -80°C for later use.

3.3.2 Expression and glycosylation of fusion protein expression vector pET28tacpglL-tacCTBSA4573 in Shigella 301DWP

pET28tacpglL-tacCTBSA4573 of Example 1 was introduced into the competent cells of Shigella 301DWP obtained in the above steps, and the bacteria were named pET28tacpglL-tacCTBSA4573/301DWP. A positive single clone was picked and inoculated into 5mL LB (final concentration of kanamycin was 50μg/mL) liquid culture medium, and cultured at 37°C until OD was about 0.6, IPTG was added to make its final concentration 1mM, and after induction at 30°C for 12h, 1mL of bacterial solution was taken, and the bacteria were collected by centrifugation, and the bacteria were resuspended with 100μL dd _H2O , and then 100μL 2xSDS was added, and the samples were separated by 12% SDS-PAGE. After electrophoresis, the protein was transferred to a PVDF membrane, and the expression of glycoprotein was detected by mouse HRP-Anti-His tag monoclonal antibody. Western blot results were analyzed, as shown in Figure 5. The whole bacterial sample was subjected to SDS-PAGE and then WB detection. After the addition of the inducer (WB right lane), when PglL and CTBSA4573 were expressed at the same time, the O-antigen polysaccharide of the bacteria itself was transferred to the carrier protein under the catalysis of glycosyltransferase, so the WB band could be seen to migrate upward in molecular weight; at the same time, since the bacterial polysaccharide was a tandem repeat structure, it showed a typical glycosylation ladder band, indicating that the carrier protein was glycosylated and connected to the bacterial polysaccharide. When the inducer was not added (WB left lane), no obvious bands were seen.

3.3.3 Protein purification

301DWP/pET28-tacpglL-tacCTBSA4573 was inoculated into 5 mL LB (final concentration of kanamycin was 50 μg/mL) medium, cultured at 37°C for 12 h, transferred to 100 mL LB medium and cultured for 7 h, transferred to 5 L LB medium, and cultured at 37°C until OD600 was approximately 0.6, IPTG was added to a final concentration of 1 mM, and after culturing at 30°C for 12 h, the cells were centrifuged at 6000 r/min for 10 min to collect the bacteria.

Sample treatment: Resuspend the bacteria in 200 mL of loading buffer (20 mM pH 7.5 Tris-HCl, 0.2 M NaCl, 10 mM imidazole), break the bacteria by ultrasonication, and collect the supernatant by centrifugation at 8000 r/min.

Sample purification: First, balance at least 3 column volumes with deionized water, then balance at least 3 column volumes with A1 solution (20mM pH7.5 Tris-HCl, 0.5M NaCl, 10mM imidazole), load the sample from the A1 pipeline at a flow rate of 4mL/min, and then elute the impurities with A1 (20mM pH7.5 Tris-HCl, 0.5M NaCl, 10mM imidazole) until the UV value is close to 0mAU, then elute the target protein with B1 solution (20mM pH7.5 Tris-HCl, 0.5M NaCl, 500mM imidazole) to obtain a purified sample, and then pass it through a molecular sieve (superdex 200) with a mobile phase of 1×PBS, pH7.4.

Glycoprotein CTBSA4573-301OPS was analyzed by Coomassie Brilliant Blue staining after molecular sieve, and the results are shown in Figure 6A, and a glycoprotein sample with a purity greater than 90% can be obtained. The particle size of the sample was detected by transmission electron microscopy, and the results are shown in Figure 6B, and the glycoprotein can form glycoprotein particles of 25-50nm. CTBSA4573-OPS was further observed by dynamic light scattering, and the size results were consistent with the transmission electron microscopy results (Figure 6C).

Example 4. Animal Immunity Evaluation

4.1 Evaluation of the effect of CTBSA4573-OPS in immunizing mice

In this implementation case, the immune effect of CTBSA-OPS was evaluated. Thirty 6-week-old Balb/c mice were randomly divided into three groups (PBS, OPS, CTBSA-301OPS), 10 mice in each group, and 2.5 μg of polysaccharide was injected subcutaneously. Immunization was performed on days 0, 14, and 28, and blood was collected from the tail vein 10 days after the last immunization. The antibody titer against Shigella flexneri type 2a O polysaccharide antigen was determined based on ELISA.

The cells were coated with LPS against Shigella flexneri type 2a strain 301, 10 μg per well, and HRP-labeled donkey anti-mouse IgG was used as the secondary antibody (the antibody was an Abcam product, catalog number GR160830-14). The results are shown in Figure 7. Compared with OPS, the antibody titer of mice treated with nanoglycoprotein was significantly increased. For verification, we further measured the titer of IgG antibody subtypes (IgG1, IgG2a, IgG2b, IgG3). The results are shown in Figure 8. Compared with OPS, the four antibody subtypes of nanoglycoprotein were significantly increased, indicating that the glycoprotein significantly stimulated the immune response in mice.

4.2 Evaluation of protective efficacy of CTBSA4573-OPS after immunization

In order to determine the protective effect of the nano vaccine, 14 days after the last immunization, the mice were intraperitoneally injected with 100 μL of Shigella flexneri 301 wild strain at a dose of 3 times the half-lethal dose (2.61×10 ⁷ CFU), and the survival of the mice was observed within 7 days. The results are shown in Figure 9, which shows that the mice treated with nano glycoprotein can still produce a protective effect at 3 times the half-lethal dose, and the protection rate can reach 100%.

Example 5. Preparation of glycoprotein nanoparticle complex and verification of its activity

This implementation case evaluates the ability of nanoparticles as carriers to connect biotin-labeled macromolecules. First, CpG was labeled with biotin and Cy5.5 fluorescence (Biotin-CpG _Cy5.5 ), where the Cy5.5 fluorescence was labeled to facilitate observation of the results under a two-color infrared laser scanning imager. Then, 1 μg of Biotin-CpG _Cy5.5 was mixed with 2 μg, 4 μg, 6 μg and 8 μg of nanoparticles, respectively, and placed in a 37°C incubator for 1 hour. After the mixed product was separated by agarose gel, it was observed under a two-color infrared laser scanning imager. The results are shown in Figure 10. As the amount of nanoparticles input increased, the fluorescence signal of Biotin-CpG _Cy5.5 moved up and the fluorescence signal intensity gradually increased. When 8 μg of nanoparticles were added, the fluorescence signal of Biotin-CpG _Cy5.5 disappeared, and the fluorescence signal intensity of the coupling product CTB-SA-Biotin-CpG _Cy5.5 reached the maximum, indicating that the nanoparticles can couple the biotin-labeled nucleic acid adjuvant CpG. The above results show that the nanoparticles provided by the present invention can be connected to various biotin-labeled bioactive molecules through the streptavidin-biotin system, thereby being used as a carrier platform to support different delivery and/or stimulation configurations.

Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims (18)

CLAIMS 1. A fusion protein comprising: AB5 toxin B subunit, streptavidin subunit and a glycosylation sequence containing an O-glycosylation site. 2. The fusion protein according to claim 1, wherein the glycosylation sequence is selected from peptides comprising O-glycosylation sites of pili protein PilE of Neisseria meningitidis; Preferably, the O-glycosylation site is serine at position 63 of PilE; Preferably, the glycosylation sequence is selected from the peptide segment comprising the 55th to 66th amino acid residues of PilE; Preferably, the glycosylation sequence comprises a peptide segment consisting of the 45th to 73rd amino acid residues of PilE; Preferably, the glycosylation sequence comprises the sequence shown in SEQ ID NO:9. 3. The fusion protein according to claim 1 or 2, wherein the streptavidin subunit comprises the amino acid sequence shown in SEQ ID NO:3. 4. The fusion protein according to any one of claims 1-3, wherein the AB5 toxin B subunit is selected from cholera toxin B subunit (CTB), Shiga toxin B subunit (StxB), Escherichia coli heat-labile enterotoxin B subunit (LTB) or subtilis toxin B subunit (SubB); Preferably, the CTB has the amino acid sequence shown in SEQ ID NO:1; preferably, the StxB has the amino acid sequence shown in SEQ ID NO:13; preferably, the LTB has the amino acid sequence shown in SEQ ID NO:15; preferably, the SubB has the amino acid sequence shown in SEQ ID NO:17. 5. The fusion protein according to any one of claims 1-4, wherein said fusion protein comprises AB5 toxin B subunit, streptavidin subunit and glycosylation sequence from N-terminus to C-terminus; or comprises glycosylation sequence, AB5 toxin B subunit and streptavidin subunit from N-terminus to C-terminus. 6. The fusion protein of any one of claims 1-5, wherein any two adjacent domains are optionally connected by a peptide linker. 7. The fusion protein of any one of claims 1-6, comprising an amino acid sequence selected from the group consisting of: (1) an amino acid sequence consisting of the 20th to 326th amino acid residues of the sequence shown in SEQ ID NO:5; (2) an amino acid sequence consisting of the 20th to 311th amino acid residues of the sequence shown in SEQ ID NO:19; (3) an amino acid sequence consisting of the 20th to 326th amino acid residues of the sequence shown in SEQ ID NO:20; (4) An amino acid sequence consisting of the 20th to 363rd amino acid residues of the sequence shown in SEQ ID NO:21. 8. The fusion protein according to any one of claims 1-7, further comprising a signal peptide and/or a protein tag. 9. Protein nanoparticles, which are formed by self-assembly of the fusion protein according to any one of claims 1-8; Preferably, the average diameter of the protein nanoparticles is about 20-30 nm. 10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-8. 11. A vector comprising the isolated nucleic acid molecule of claim 10. 12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the vector of claim 11. 13. A method of preparing protein nanoparticles comprising culturing the host cell of claim 12 under conditions that permit expression of the protein, and recovering the nanoparticles from the cultured host cell culture. 14. A method for preparing glycoprotein nanoparticles, comprising the steps of: (1) providing a bacterial host cell lacking O-antigen ligase; (2) expressing the fusion protein and glycosyltransferase PglL according to any one of claims 1-8 in the bacterial host cell, thereby producing target glycoprotein nanoparticles; Preferably, said bacterial host cell is a pathogenic bacterial cell; Preferably, the method further includes: (3) purifying the target glycoprotein nanoparticles; preferably, purifying the target glycoprotein nanoparticles by affinity chromatography and/or molecular sieves; Preferably, step (2) comprises the following steps: (2a) introducing the first nucleotide sequence encoding the fusion protein of any one of claims 1-8 and the second nucleotide sequence encoding PglL into the bacterial host cell, wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different expression vectors; (2b) culturing said bacterial host cell; (2c) recovering the target nanoglycoprotein particles from the cell culture. 15. Glycoprotein nanoparticles, comprising a conjugate of protein and polysaccharide, the conjugate is formed by coupling bacterial O polysaccharide and the fusion protein according to any one of claims 1-8, and the bacterial O polysaccharide is coupled to the fusion protein through the O-glycosylation site in the glycosylation sequence in the fusion protein; Preferably, the bacterial O-polysaccharide is selected from the O-polysaccharide of pathogenic bacteria; Preferably, the bacterial O polysaccharide is selected from Shigella O polysaccharide, Escherichia coli O157O polysaccharide, Shigella O polysaccharide, Salmonella paratyphi A O polysaccharide, Vibrio cholerae O polysaccharide; Preferably, in the glycoprotein nanoparticles, the AB5 toxin B subunit in the fusion protein exists in the form of a pentamer, and the SA subunit in the fusion protein exists in the form of a tetramer; Preferably, said glycoprotein nanoparticles have an average diameter of about 30-50 nm; Preferably, the glycoprotein nanoparticles are prepared by the method of claim 14. 16. A complex comprising: the fusion protein of any one of claims 1-8, the protein nanoparticle of claim 9, or the glycoprotein nanoparticle of claim 15, and another biologically active molecule; Preferably, said additional bioactive molecule is labeled with biotin; Preferably, said additional biologically active molecule is attached via a biotin-streptavidin interaction; Preferably, said additional biologically active molecule is a nucleic acid, protein and/or sugar; Preferably, said additional biologically active molecule is an immunogen and/or an adjuvant. 17. An immunogenic composition or vaccine, comprising: (i) the glycoprotein nanoparticle of claim 15 of an immunologically effective amount, or (ii) the fusion protein of any one of claims 1-8 or a mixture of protein nanoparticles formed therefrom, and bacterial O polysaccharide, or (iii) the complex of claim 16 of an immunologically effective amount; Preferably, said immunogenic composition or vaccine does not comprise an adjuvant; Preferably, the immunogenic composition or vaccine comprises pharmaceutically acceptable carriers and/or excipients. 18. Use of the glycoprotein nanoparticle of claim 15, the complex of claim 16, or the immunogenic composition or vaccine of claim 17 for the preparation of a product for inducing an immune response in a subject and/or for preventing and/or treating infection in a subject; Preferably, the product is used to induce a specific immune response against bacterial O polysaccharides in a subject; preferably, the specific immune response comprises an antibody response; Preferably, the product is for the prevention and/or treatment of bacterial infections in a subject; Preferably, the product is a vaccine; Preferably, said specific immune response comprises an antibody response; Preferably, the subject is a human.