FUSION PROTEIN CAPABLE OF SELF-ASSEMBLING INTO PROTEIN NANOPARTICLES AND APPLICATION THEREOF

Abstract

The present invention relates to a recombinant fusion protein and a polysaccharide conjugate vaccine. More specifically, the present invention relates to a fusion protein including a pentamer substrate protein and a peptide sequence capable of forming a trimer, an immunogenic composition including the fusion protein, the application of the fusion protein in immunology, a polynucleotide sequence encoding the fusion protein, an expression vector containing the polynucleotide sequence, and a host cell containing the expression vector.

Claims

1-27. (canceled)

28. A fusion protein, comprising: an AB5 toxin B subunit and a trimerization domain sequence.

29. The fusion protein according to claim 28, wherein the trimerization domain sequence comprises an amino acid sequence shown in SEQ ID No. 9.

30. The fusion protein according to claim 28, wherein the AB5 toxin B subunit is selected from the group consisting of cholera toxin B subunit (CTB), Shiga toxin B subunit (StxB), Escherichia coli heat-labile enterotoxin B subunit (LTB), and Bacillus subtilis toxin B subunit (SubB); preferably, the CTB has an amino acid sequence shown in SEQ ID No. 28; preferably, the StxB has an amino acid sequence shown in SEQ ID No. 3; preferably, the LTB has an amino acid sequence shown in SEQ ID No. 5; preferably, the SubB has an amino acid sequence shown in SEQ ID No. 7.

31. The fusion protein according to claim 28, wherein the fusion protein comprises, from N terminus to C terminus, the AB5 toxin B subunit and the trimerization domain sequence.

32. The fusion protein according to claim 31, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence consisting of amino acid residues at positions 20 to 172 of the sequence shown in SEQ ID NO. 21; (2) an amino acid sequence consisting of amino acid residues at positions 20 to 157 of the sequence shown in SEQ ID NO. 23; (3) an amino acid sequence consisting of amino acid residues at positions 20 to 172 of the sequence shown in SEQ ID NO. 25; and (4) an amino acid sequence consisting of amino acid residues at positions 20 to 209 of the sequence shown in SEQ ID NO. 27.

33. The fusion protein according to claim 28, further comprising: a glycosylation sequence containing an O-glycosylation site.

34. The fusion protein according to claim 33, wherein the glycosylation sequence is selected from a peptide fragment containing O-glycosylation site-of the pilin protein PilE of Neisseria meningitidis; preferably, the O-glycosylation site is the serine at position 63 of the PilE.

35. The fusion protein according to claim 34, wherein the glycosylation sequence comprises a peptide fragment consisting of amino acid residues at positions 45 to 73 of the PilE; preferably, the glycosylation sequence comprises a sequence shown in SEQ ID NO. 29.

36. The fusion protein according to claim 28, wherein the fusion protein, from N terminus to C terminus, comprises the AB5 toxin B subunit, the trimerization domain sequence, and the glycosylation sequence.

37. The fusion protein according to claim 36, wherein the fusion protein comprises an amino acid sequence selected from the group consisting of: (1) an amino acid sequence consisting of amino acid residues at positions 20 to 205 of the sequence shown in SEQ ID NO. 21; (2) an amino acid sequence consisting of amino acid residues at positions 20 to 190 of the sequence shown in SEQ ID NO. 23; (3) an amino acid sequence consisting of amino acid residues at positions 20 to 205 of the sequence shown in SEQ ID NO. 25; and (4) an amino acid sequence consisting of amino acid residues at positions 20 to 242 of the sequence shown in SEQ ID NO. 27.

38. The fusion protein according to claim 28, wherein any two adjacent domains are optionally linked by a peptide linker.

39. The fusion protein according to claim 28, further comprising a signal peptide and/or a protein tag.

40. Biomaterials as described in any of the following: (A). An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the fusion protein according to claim 28; (B). A vector, comprising an isolated nucleic acid molecule, comprising a nucleotide sequence encoding the fusion protein; (C). A host cell, comprising an isolated nucleic acid molecule, comprising a nucleotide sequence encoding the fusion protein.

41. A method for preparing a glycoprotein nanoparticle, comprising the following steps: (1) providing a bacterial host cell lacking O-antigen ligase; and (2) expressing the fusion protein according to claim 33 and the glycosyltransferase PglL in the bacterial host cell, thereby producing the target glycoprotein nanoparticle.

42. The method according to claim 41, wherein the bacterial host cell is a pathogenic bacterial cell; or wherein the method further comprises: (3) purifying the target glycoprotein nanoparticle; preferably, purifying the target glycoprotein nanoparticle by affinity chromatography and/or molecular sieve; or wherein step (2) comprises the following steps: (2a) introducing a first nucleotide sequence encoding the fusion protein 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; (2b) culturing the bacterial host cell; and (2c) recovering the target glycoprotein nanoparticle from a cell culture.

43. Biomaterials as described in any of the following: (I). A glycoprotein nanoparticle, comprising a protein-polysaccharide conjugate, wherein the conjugate is formed by coupling a bacterial O-polysaccharide and the fusion protein according to claim 33, and the bacterial O-polysaccharide is coupled to the fusion protein through the O-glycosylation site in the glycosylation sequence in the fusion protein. (II). An immunogenic composition or vaccine, comprising: (i) an immunologically effective dose of the glycoprotein nanoparticle, or (ii) an immunologically effective dose of the fusion protein or a mixture of a protein nanoparticle formed thereby and bacterial 0-polysaccharide; preferably, the immunogenic composition or vaccine does not comprise an adjuvant; preferably, the immunogenic composition or vaccine comprises a pharmaceutically acceptable carrier and/or excipient.

44. The Biomaterials according to claim 43, wherein the bacterial O-polysaccharide is selected from pathogenic bacteria O-polysaccharide; preferably, the bacterial O-polysaccharide is selected from Shigella O polysaccharide, Escherichia coli O157O polysaccharide, Shigella dysenteriae O polysaccharide, Salmonella paratyphi A O polysaccharide, and Vibrio cholerae O polysaccharide.

45. The Biomaterials according to claim 43, characterized by one or more of the following characteristics: (i) in the glycoprotein nanoparticle, the AB5 toxin B subunit of the fusion protein exists in a form of a pentamer, and the trimerization domain sequence of the fusion protein exists in a form of a trimer; and/or (ii) an average diameter of the glycoprotein nanoparticle is about 25-50 nm.

46. The Biomaterials according to claim 43, wherein the glycoprotein nanoparticle is prepared by the method according.

47. A method for inducing a specific immune response to bacterial O-polysaccharide in a subject or preventing and/or treating a bacterial infection in a subject, comprising administrating an immunologically effective dose of the glycoprotein nanoparticle according to claim 43 or the immunogenic composition or vaccine to a subject in need.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0118] FIGS. 1A-1C show detection results of a monomer size of CTBTri after purification and a size of a particle formed thereby. FIG. 1A: detection results of Coomassie brilliant blue staining analysis and western blot; FIG. 1B: detection result of dynamic light scattering; FIG. 1C: detection result of transmission electron microscopy.

[0119] FIGS. 2A-2C show detection results of a monomer size of StxBTri after expression and purification and a size of a particle formed thereby. FIG. 2A: detection results of Coomassie brilliant blue staining analysis and western blot; FIG. 2B: detection result of dynamic light scattering; FIG. 2C: detection result of transmission electron microscopy.

[0120] FIGS. 3A-3C show detection results of a monomer size of LTBTri after expression and purification and a size of a particle formed thereby. FIG. 3A: detection results of Coomassie brilliant blue staining analysis and western blot; FIG. 3B: detection result of dynamic light scattering; FIG. 3C: detection result of transmission electron microscopy.

[0121] FIGS. 4A-4C show detection results of a monomer size of SubBTri after expression and purification and a size of a particle formed thereby. FIG. 4A: detection results of Coomassie brilliant blue staining analysis and western blot; FIG. 4B: detection result of dynamic light scattering; FIG. 4C: detection result of transmission electron microscopy.

[0122] FIG. 5 shows detection results of expressions of four glycoproteins CTBTri4573-OPS, StxBTri4573-OPS, LTBTri4573-OPS, and SubBTri4573-OPS.

[0123] FIG. 6 shows Coomassie brilliant blue staining analysis of four glycoproteins CTBTri4573-OPS, StxBTri4573-OPS, LTBTri4573-OPS, and SubBTri4573-OPS after passing through a molecular sieve.

[0124] FIGS. 7A and 7B show particle sizes of four glycoproteins CTBTri4573-OPS, StxBTri4573-OPS, LTBTri4573-OPS, and SubBTri4573-OPS detected by dynamic light scattering and transmission electron microscopy analysis. FIG. 7A: dynamic light scattering; FIG. 7B: transmission electron microscopy.

[0125] FIG. 8 shows determination results of antibody titer of mice immunized with B5Tri4573-OPS (CTBTri4573-OPS, StxBTri4573-OPS, LTBTri573-OPS, SubBTri4573-OPS). *p<0.05, ***p<0.001, ****p<0.0001.

[0126] FIG. 9 shows challenge test results of mice immunized with B5Tri4573-OPS (CTBTri4573-OPS, StxBTri4573-OPS, LTBTri573-OPS, SubBTri4573-OPS).

[0127] FIG. 10 shows determination results of antibody titer of different IgG subtypes after mice are immunized with CTBTri4573-OPS. ****p<0.0001.

[0128] FIG. 11 shows a comparison of antibody titers of different IgG subtypes after mice are immunized with CTBTri4573-OPS with and without an adjuvant. *p<0.05, **p<0.01.

[0129] FIGS. 12A and 12B show tests of in vitro bactericidal activity of an antiserum induced by CTBTri4573-OPS.

[0130] FIGS. 13A and 13B show challenge test results of mice immunized with CTBTri4573-OPS.

[0131] Sequence Information

TABLE-US-00001 TABLE 1 Information of sequences involved in the present invention is provided in the table below. SEQ ID NO: Description 1 Amino acid sequence of CTB 2 Nucleotide sequence of CTB 3 Amino acid sequence of StxB 4 Nucleotide sequence of StxB 5 Amino acid sequence of LTB 6 Nucleotide sequence of LTB 7 Amino acid sequence of SubB 8 Nucleotide sequence of SubB 9 Amino acid sequence of Tri 10 Nucleotide sequence of Tri 11 Nucleotide sequence of tacCTBTri expression cassette 12 Amino acid sequence of CTBTri protein 13 Nucleotide sequence of tacStxBTri expression cassette 14 Amino acid sequence of StxBTri protein 15 Nucleotide sequence of tacLTBTri expression cassette 16 Amino acid sequence of LTBTri protein 17 Nucleotide sequence of tacSubBTri expression cassette 18 Amino acid sequence of SubBTri protein 19 Amino acid sequence of PglL 20 Nucleotide sequence of PglL expression cassette 21 Amino acid sequence of CTBTri4573 22 Nucleotide sequence of CTBTri4573 expression cassette 23 Amino acid sequence of StxBTri4573 24 Nucleotide sequence of StxBTri4573 expression cassette 25 Amino acid sequence of LTBTri457 26 Nucleotide sequence of LTBTri4573 expression cassette 27 Amino acid sequence of SubBTri4573 28 Nucleotide sequence of SubBTri4573 expression cassette 29 Glycosylation sequence (PilE S45-K73) 30 Linker 31 DsbA signal peptide 32 Primer SacI-UP 33 Primer XhoI-Down

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0132] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods. The materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0133] The non-toxic Shigella flexneri 301DWP lacking O-antigen ligase gene waal in the following embodiments was obtained by modifying 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). The public can reach it from the Institute of Bioengineering, Academy of Military Medicine, PLA Academy of Military Sciences. The biological material was only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.

[0134] pET28a(+) in the following embodiments was purchased from Novagen.

[0135] HRP-labeled Anti-His antibody in the following embodiments was an Abmart product, with a product number of M20020.

[0136] Rabbit-derived Freund's type II serum in the following embodiments was a product of Denka Seiken Co., Ltd., with a number of 210227.

[0137] HRP-labeled goat anti-rabbit in the following embodiments was a product of TransGen Biotech Co., LTD, with a number of HS-101-01.

[0138] HRP-labeled donkey anti-mouse IgG, goat anti-mouse IgG1, IgG2a, IgG2b, and IgG3 in the following embodiments were purchased from Abcam, with numbers of ab6820, ab97240, ab97245, ab97250, and ab97260, respectively.

[0139] Aluminum hydroxide adjuvant Rehydragel LV in the following embodiments was purchased from General Chemical Company, with a number of 203120070602.

[0140] Complement in the following embodiments was purchased from Pel-Freez LLC, with a number of 31061-3.

Embodiment 1. Construction and Expression of Nanoparticle Vector

[0141] 1) Construction of B5Tri Fusion Protein Expression Vector

[0142] In the present embodiment, a vector for expressing the fusion protein (B5Tri) including B5 protein and Tri sequence was constructed, where the B5 protein included cholera toxin B subunit (CTB), Shiga toxin B subunit (StxB), Escherichia coli heat-labile enterotoxin B subunit (LTB), and Bacillus subtilis toxin B subunit (SubB), and the Tri sequence was shown in SEQ ID NO. 9. The construction method was as follows:

[0143] CTBTri expression vector construction: According to the amino acid sequence of cholera toxin B subunit (X76390.1) published by GeneBack, a signal peptide (first 21 amino acids) was replaced with a DsbA signal peptide (SEQ ID NO. 31), and Tri sequence was fused at the C terminus of CTB to construct CTB fusion protein CTBTri. In the CTBTri expression cassette, the expression of CTBTri was initiated by the tac promoter. The expression cassette was named tacCTBTri, and had a nucleotide sequence shown in SEQ ID No. 11, wherein the nucleotides at positions 103 to 131 were the tac promoter sequence, the nucleotides at positions 178 to 729 were the coding sequence of CTBTri (CTBTri protein sequentially included DsbA signal peptide, CTB, Tri and His tag from N terminal to C terminal). Specifically from the 5′ end, the nucleotides at positions 178 to 234 were the signal peptide sequence, the nucleotides at positions 235 to 543 were the CTB coding sequence, and the nucleotides at positions 556 to 693 were the Tri sequence. The amino acid sequence of the CTBTri protein was shown in SEQ ID No. 12, in which from the N terminus, the nucleotides at positions 1 to 19 were the DsbA signal peptide sequence, the nucleotides at positions 20 to 122 were the CTB amino acid sequence, and the nucleotides at positions 123 to 126 were a flexible linker, and the nucleotides at positions 127 to 172 were the Tri sequence.

[0144] StxBTri expression vector construction: In the StxBTri expression cassette, the expression of StxBTri was initiated by the tac promoter. The expression cassette was named tacStxBTri, and had a nucleotide sequence shown in SEQ ID No. 13, wherein the nucleotides at positions 103 to 131 were the tac promoter sequence, and the nucleotides at positions 178 to 684 were the coding sequence of StxBTri (StxBTri protein sequentially included DsbA signal peptide, StxB, Tri, and His tag from N terminal to C terminal). Specifically from the 5′ end, the nucleotides at positions 178 to 234 were the signal peptide sequence, the nucleotides at positions 235 to 498 were the StxB coding sequence, and the nucleotides at positions 511 to 648 were the Tri sequence. The amino acid sequence of the StxBTri protein was shown in SEQ ID No. 14, in which from the N terminus, the nucleotides at positions 1 to 19 were the DsbA signal peptide sequence, the nucleotides at positions 20 to 107 were the StxB amino acid sequence, the nucleotides at positions 108 to 111 were a flexible linker, and the nucleotides at positions 112 to 157 were the Tri sequence.

[0145] LTBTri expression vector construction: In the LTBTri expression cassette, the expression of LTBTri was initiated by the tac promoter. The expression cassette was named tacLTBTri, and had a nucleotide sequence shown in SEQ ID No. 15, wherein the nucleotides at positions 103 to 131 were the tac promoter sequence, and the nucleotides at positions 178 to 729 were the coding sequence of LTBTri (LTBTri protein sequentially included DsbA signal peptide, LTB, Tri, and His tag from N terminal to C terminal). Specifically from the 5′ end, the nucleotides at positions 178 to 234 were the signal peptide sequence, the nucleotides at positions 235 to 543 were the LTB coding sequence, and the nucleotides at positions 556 to 693 were the Tri sequence. The amino acid sequence of the LTBTri protein was shown in SEQ ID No. 16, in which from the N terminus, the nucleotides at positions 1 to 19 were the DsbA signal peptide sequence, the nucleotides at positions 20 to 122 were the LTB amino acid sequence, the nucleotides at positions 123 to 126 were a flexible linker, and the nucleotides at positions 127 to 172 were the Tri sequence.

[0146] SubBTri expression vector construction: In the SubBTri expression cassette, the expression of SubBTri was initiated by the tac promoter. The expression cassette was named tacSubBTri, and had a nucleotide sequence shown in SEQ ID No. 17, wherein the nucleotides at positions 103 to 131 were the tac promoter sequence, and the nucleotides at positions 178 to 840 were the coding sequence of SubBTri (SubBTri protein sequentially included DsbA signal peptide, SubB, Tri, and His tag from N terminal to C terminal). Specifically from the 5′ end, the nucleotides at positions 178 to 234 were the signal peptide sequence, the nucleotides at positions 235 to 654 were the SubB coding sequence, and the nucleotides at positions 667 to 804 were the Tri sequence. The amino acid sequence of the SubBTri protein was shown in SEQ ID No. 18, in which from the N terminus, the nucleotides at positions 1 to 19 were the DsbA signal peptide sequence, the nucleotides at positions 20 to 159 were the SubB amino acid sequence, the nucleotides at positions 160 to 163 were a flexible linker, and the nucleotides at positions 164 to 209 were the Tri sequence.

[0147] The DNA molecules shown in SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, and SEQ ID No. 17 were respectively digested with XbaI and XhoI to obtain gene fragments. pET28a(+) was digested with XbaI and XhoI to obtain large fragments of the vector. The gene fragments and the large fragments of the vector were ligated to obtain recombinant vectors respectively containing the DNA molecules shown in SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, and SEQ ID No. 17, and the recombinant vectors were named pET28-tacB5Tri (B5 was CTB, StxB, LTB, and SubB, respectively).

[0148] 2) Construction and Expression of B5Tri Expression Strains

[0149] The pET28-tacB5Tri vector was transformed into BL21 competence, coated with LB solid medium containing kanamycin at a final concentration of 50 μg/mL, and subjected to a positive clone to obtain BL21/pET28-tacB5Tri. The BL21/pET28-tacB5Tri was selected and inoculated in LB medium containing kanamycin at a final concentration of 50 μg/mL for culture at 37° C. and 220 rpm for 10 h, and then transferred to 5 mL of LB medium containing kanamycin at a final concentration of 50 μg/mL for culture at 37° C. and 220 rpm until an OD600 reached about 0.6, followed by adding IPTG with a final concentration of 1 mM, reducing a temperature to 30° C., and inducing for 10 h.

[0150] 1 mL of each bacterial solution induced at 30° C. for 10 h was taken and centrifuged to collect bacterial cells. The bacterial cells were suspended with 1× reduction buffer (50 mM pH 6.8 Tris-HCl, 1.6% SDS, 0.02% bromophenol blue, 8% glycerol, 20 mM DTT), and underwent a boiling water bath for 10 min and a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After the electrophoresis, the proteins were transferred to poly (vinylidene fluoride) (PVDF) membrane at a constant voltage of 20 V for 1 h with Bio-Lab semi-dry transfer apparatus. The protein expression bands were detected using mouse-derived HRP-Anti-His tag monoclonal antibodies.

Embodiment 2. Purification and Detection of Nanoparticles

[0151] The BL21/pET28-tacB5Tri was inoculated in LB medium containing kanamycin at a final concentration of 50 μg/mL for culture at 37° C. and 220 rpm for 10 h, and then transferred to 5 L of LB medium for culture at 37° C. and 220 rpm until an OD600 reached about 0.6, followed by adding IPTG with a final concentration of 1 mM, reducing a temperature to 30° C., and inducing for 10 h.

[0152] Sample treatment: bacterial cells obtained above after an induction at 30° C. for 10 h were taken and added with 400 mL of a loading buffer (20 mM pH 7.5 Tris-HCl, 0.2 M NaCl, 10 mM imidazole), ultrasonically broken (ultrasound worked for 3 s and paused for 5 s, with cumulative ultrasonic time of 30 min), and centrifuged at 12000 g to collect a supernatant.

[0153] Sample purification: a column bed was first washed with 0.5 M NaOH aqueous solution for at least 3 bed volumes, and then deionized water was used to adjust pH to neutral. Subsequently, 0.5 M NiSO.sub.4 aqueous solution was used to equilibrate at least 3 bed volumes, and then B1 solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 500 mM imidazole) was used to equilibrate at least one bed volume, and finally Al solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 10 mM imidazole) was used to equilibrate at least 3 bed volumes, each of the above was performed at a flow rate of 4 mL/min. A sample was loaded from an A pipeline, washed with Al solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 10 mM imidazole) to remove unbound proteins until the ultraviolet absorption (280 nm) was close to 0 mAU, and finally eluted with 100% B1 (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 500 mM imidazole). 30 mL of eluate was collected to obtain a purified sample, and then the purified sample passed through a molecular sieve (superdex 200) with a mobile phase of 1×PBS, pH 7.4.

Four fusion proteins (CTBTri, StxBTri, LTBTri, SubBTri) were analyzed by Coomassie brilliant blue staining after passing through molecular sieves (FIGS. 1A-4A), and protein samples with purity greater than 90% can be obtained. Use His tag antibody to conduct Western blot verification, the detection results of the four fusion proteins are shown in FIG. 1A-4A. The sample size was detected by dynamic light scattering and transmission electron microscopy. The dynamic light scattering detection results of the four fusion proteins are shown in FIGS. 1B-4B, and the transmission electron microscopy detection results of the four fusion proteins are shown in FIG. 1C-4C. It can be seen that although the protein monomer is only about 20 kDa, it can form protein particles of about 10-20 nm in its natural state.

Embodiment 3. Preparation of Nanoscale Bacterial Polysaccharide Conjugate Vaccine by Biological Methods

3.1 Construction of B5Tri (B5 was CTB, StxB, LTB, and SubB, Respectively) Glycosylation Expression Vector

[0154] 3.1.1 Construction of Neisseria meningitidis glycosyltransferase PglL expression vector: the amino acid sequence of the Neisseria meningitidis glycosyltransferase PglL (GeneBank: JN200826.1) was shown in SEQ ID No. 19, and its coding sequence included the nucleotides at positions 180 to 1994 of SEQ ID No. 20. The nucleotides at positions 1 to 6 of SEQ ID No. 20 were the XbaI recognition site, and the nucleotides at positions 105 to 2240 of SEQ ID No. 20 were the sequence of the PglL expression cassette. In the PglL expression cassette, the expression of PglL was initiated by the tac promoter, and the expression cassette was named tacpglL. Specifically in SEQ ID No. 20, the nucleotides at positions 105 to 133 were the tac promoter sequence, the nucleotides at positions 180 to 1994 were the coding sequence of Neisseria meningitidis glycosyltransferase PglL, the nucleotides at positions 2475 to 2480 were the SacI recognition sequence, and the nucleotides at positions 2486 to 2491 were the XhoI recognition site.

[0155] The DNA molecule shown in SEQ ID No. 20 was digested with XbaI and XhoI to obtain gene fragments. pET28a(+) was digested with XbaI and XhoI to obtain large fragments of the vector. The gene fragments and the large fragments of the vector were ligated to obtain a recombinant vector containing the DNA molecule shown in SEQ ID No. 20, and the recombinant vector was named pET28-tacpglL.

[0156] 3.1.2 Fusion of B5Tri sequence and O-glycosylation site (B5Tri4573): PilE was the pilin of Neisseria meningitidis. In Neisseria meningitidis, PilE can be 0-glycosylated under the catalysis of glycosyltransferase PglL. The glycosylation site was the serine at position 63 of the PilE. In order to obtain the glycosylation sequence, 29 amino acids S45-K73, from the serine (S45) at position 45 of the N terminal of the PilE to the lysine (K73) at position 73, were intercepted and fused to the C terminus of B5Tri; at the same time, in order to avoid steric hindrance effect between B5 and Tri peptide fragment, the B5 and Tri peptide fragment were connected by 4 flexible amino acids.

[0157] The amino acid sequence of CTBTri4573 was shown in SEQ ID No. 21, and the gene sequence was shown in positions 178 to 816 of SEQ ID No. 22. In SEQ ID No. 22, the nucleotides at positions 1 to 6 were the XbaI recognition site, the nucleotides at positions 103 to 131 were the tac promoter sequence, the nucleotides at positions 178 to 792 were the coding sequence of CTBTri4573, the nucleotides at positions 793 to 798 were the XhoI recognition sequence, and the nucleotides at positions 799 to 816 were the His tag.

[0158] The amino acid sequence of StxBTri4573 was shown in SEQ ID No. 23, and the gene sequence was shown in positions 178 to 771 of SEQ ID No. 24. In SEQ ID No. 24, the nucleotides at positions 1 to 6 were the XbaI recognition site, the nucleotides at positions 103 to 131 were the tac promoter sequence, the nucleotides at positions 178 to 747 were the coding sequence of StxBTri4573, the nucleotides at positions 748 to 753 were the XhoI recognition sequence, and the nucleotides at positions 754 to 771 were the His tag.

[0159] The amino acid sequence of LTBTri4573 was shown in SEQ ID No. 25, and the gene sequence was shown in positions 178 to 816 of SEQ ID No. 26. In SEQ ID No. 26, the nucleotides at positions 1 to 6 were the XbaI recognition site, the nucleotides at positions 103 to 131 were the tac promoter sequence, the nucleotides at positions 178 to 792 were the coding sequence of LTBTri4573, the nucleotides at positions 793 to 798 were the XhoI recognition sequence, and the nucleotides at positions 799 to 816 were the His tag.

[0160] The amino acid sequence of SubBTri4573 was shown in SEQ ID No. 27, and the gene sequence was shown in positions 178 to 927 of SEQ ID No. 28. In SEQ ID No. 28, the nucleotides at positions 1 to 6 were the XbaI recognition site, the nucleotides at positions 103 to 131 were the tac promoter sequence, the nucleotides at positions 178 to 903 were the coding sequence of SubBTri4573, the nucleotides at positions 904 to 909 were the XhoI recognition sequence, and the nucleotides at positions 910 to 927 were the His tag.

[0161] The DNA molecules shown in SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, and SEQ ID No. 28 were respectively digested with XbaI and XhoI to obtain gene fragments. pET28a(+) was digested with XbaI and XhoI to obtain large fragments of the vector. The gene fragments and the large fragments of the vector were ligated to obtain recombinant vectors, and the recombinant vectors were named pET28-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively).

[0162] The specific method for constructing B5Tri4573 glycosylation expression vector was as follows:

[0163] Primers SacI-UP (SEQ ID NO. 32) and XhoI-Down (SEQ ID NO. 33) were respectively used to amplify pET28-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively) to obtain the corresponding amplification products, and the amplification products were digested with SacI and XhoI to obtain DNA fragments. pET28-tacpglL was digested with SacI and XhoI to obtain large fragments of the vector. The gene fragments were connected to the large fragments of the vector to obtain recombinant plasmids, named pET28-tacpglL-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively), in which the expression cassette (i.e., tacB5Tri4573) composed of the coding gene of B5Tri4573 was located downstream of the pglL expression cassette (tacpglL). The recombinant plasmids were sent for sequencing, and the results were all correct.

3.2 Preparation of 301DWP Competent Cells

[0164] The attenuated Shigella 301DWP where O-antigen ligase was knocked out was inoculated into LB liquid medium. Since the bacterium lacked O-antigen ligase, free O-antigen can be produced. After overnight culture at 30° C., 1 ml of culture solution was transferred to 100 mL of low-salt LB liquid medium for culture at 30° C. until an OD600 reached 0.6. Subsequently, bacterial cells were collected by centrifugation, washed four times with a 10 vol % sterile aqueous glycerol solution, and finally resuspended with 400 uL of the 10 vol % sterile aqueous glycerol solution to obtain 301DWP competent cells for transformation by electroporation, which were sub-packed for use.

3.3 Construction of Expression Strains

[0165] The plasmids pET28-tacpglL-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively) were transformed by electroporation into the 301DWP competent cells obtained in 3.2 above, coated with LB solid medium containing kanamycin at a final concentration of 50 μg/mL, and subjected to a positive clone to obtain 301DWP/pET28-tacpglL-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively).

3.4 Protein Expression and Detection

[0166] 301DWP/pET28-tacpglL-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively) were selected for monoclone, and inoculated in LB medium containing kanamycin at a final concentration of 50 μg/mL for culture at 37° C. until an OD600 reached approximately 0.6, followed by adding IPTG having a final concentration of 1 mM, reducing a temperature to 30° C., and inducing for 10 h.

[0167] Next day, 1 mL of each bacterial solution induced at 30° C. for 10 h above was taken and centrifuged to collect bacterial cells. The bacterial cells were suspended with 1× reduction buffer (50 mM pH 6.8 Tris-HCl, 1.6% SDS, 0.02% bromophenol blue, 8% glycerol, 20 mM DTT), and underwent a boiling water bath for 10 min and a 12% SDS-PAGE. After the electrophoresis, the proteins were transferred to PVDF membrane at a constant voltage of 20 V for 1 h with Bio-Lab semi-dry transfer apparatus, detected by using mouse-derived HRP-Anti-His tag monoclonal antibodies, the results were shown in FIG. 5. The whole bacterial sample was subjected to the SDS-PAGE and western blot (WB) detection in sequence. When PglL and B5Tri4573 were co-expressed, it can be seen from WB bands that upward migration of molecular weight occurred, because the O-antigen polysaccharide of the bacteria itself was transferred to the carrier protein under the catalysis of glycosyltransferase. At the same time, since the bacterial polysaccharide was a tandem repeat structure, a typical glycosylated ladder band was presented, indicating that four carrier proteins (CTBTri4573-OPS, StxBTri4573-OPS, LTBTri4573-OPS, SubBTri4573-OPS) were all glycosylated, and the bacterial polysaccharide was introduced, while the vector without PglL (B5Tri4573) in the control group only showed a carrier protein expression.

3.5 Protein Purification

[0168] The 301DWP/pET28-tacpglL-tacB5Tri4573 (B5 was CTB, StxB, LTB, and SubB, respectively) were inoculated in LB medium containing kanamycin at a final concentration of 50 μg/mL for culture at 37° C. and 220 rpm for 10 h, and then transferred to 5 L of LB medium for culture at 37° C. and 220 rpm until an OD600 reached about 0.6, followed by adding IPTG with a final concentration of 1 mM, reducing a temperature to 30° C., and inducing for 10 h.

[0169] Sample treatment: bacterial cells obtained above after an induction at 30° C. for 10 h were taken and added with 400 mL of a loading buffer (20 mM pH 7.5 Tris-HCl, 0.2 M NaCl, 10 mM imidazole), ultrasonically broken (ultrasound worked for 3 s and paused for 5 s, with cumulative ultrasonic time of 30 min), and centrifuged at 12000 g to collect a supernatant.

[0170] Sample purification: a column bed was first washed with 0.5 M NaOH aqueous solution for at least 3 bed volumes, and then deionized water was used to adjust pH to neutral. Subsequently, 0.5 M NiSO.sub.4 aqueous solution was used to equilibrate at least 3 bed volumes, and then B1 solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 500 mM imidazole) was used to equilibrate at least one bed volume, and finally Al solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 10 mM imidazole) was used to equilibrate at least 3 bed volumes, each of the above was performed at a flow rate of 4 mL/min. A sample was loaded from an A pipeline, washed with Al solution (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 10 mM imidazole) to remove unbound proteins until the ultraviolet absorption (280 nm) was close to 0 mAU, and finally eluted with 100% B1 (20 mM pH 7.5 Tris-HCl, 0.5 M NaCl, 500 mM imidazole). 30 mL of eluate was collected to obtain a purified sample, and then the purified sample passed through a molecular sieve (superdex 200) with a mobile phase of 1×PBS, pH 7.4.

[0171] Four glycoproteins (CTBTri4573-OPS, StxBTri4573-OPS, LTBTri4573-OPS, SubBTri4573-OPS) were subjected to a Coomassie brilliant blue staining analysis after passing through a molecular sieve. The results were shown in FIG. 6, and a glycoprotein sample with purity greater than 90% can be obtained. Dynamic light scattering was used to measure the particle size of the sample, and the results were shown in FIG. 7A. All the four glycoproteins can form 25-50 nm glycoprotein particles. The CTBTri4573-OPS was further observed by transmission electron microscope, and the resulting sizes were consistent with the results of dynamic light scattering (FIG. 7B).

Embodiment 4. Animal Immunity Evaluation

4.1 Evaluation of the Effect of B5Tri4573-OPS on Immunizing Mice

[0172] In the present embodiment, the immunological effect of B5Tri4573-OPS was evaluated. B5Tri4573-OPS referred to the glycosylated protein prepared in Embodiment 3 (B5 was CTB, StxB, LTB, and SubB, respectively). Forty 6-week-old female Balb/c mice were taken and randomly divided into 6 groups (PBS group, OPS group (polysaccharide alone), CTBTri4573-OPS group, StxBTri4573-OPS group, LTBTri573-OPS group, and SubBTri4573-OPS group). Each group of mice were injected subcutaneously with 2.5 μg of polysaccharide based on a polysaccharide content; immunized on the 1.sup.st, 15.sup.th, and 29.sup.th day, the tail bloods thereof were collected on the 10.sup.th day after the last immunization, and antibody titers of anti-Shigella flexneri type 2a O-antigen polysaccharide in the serum of each group of mice were measured by indirect enzyme-linked immunosorbent assay (ELISA). The enzyme-linked plate was coated with the extracted LPS of anti-Shigella flexneri type 2a 301 strain, with 10 μg of LPS per well, and other operating steps referred to the “Short Protocols in Molecular Biology”. The results were shown in FIG. 8. All the four glycoproteins can induce a generation of specific antibodies in mice, which effectively triggered the body's immune response, and showed antibody titers induced thereby significantly superior to those in the OPS group administered with polysaccharide alone. To further confirm the above effects, two weeks after the last immunization, the mice were injected intraperitoneally with Shigella flexneri 301 wild strains at a dose of 5 times the half lethal dose (4.35×10.sup.7 colony-forming units (CFU)), namely, 200 μl, followed by observing the death of the mice. The results were shown in FIG. 9. A protective effect was generated in the mice in each group of B5Tri4573-OPS at the dose of 5 times the half lethal dose, and the protection rate of CTBTri573-OPS and LTBTri573-OPS can reach 100%.

4.2 Further Evaluation of the Immunological Effect of CTBTri4573-OPS

4.2.1 Comparison of the Immunological Effects of CTBTri4573-OPS and CTB4573-OPS

[0173] Forty 6-week-old female Balb/c mice were taken and randomly divided into 4 groups (PBS group (Control), OPS group, CTB4573-OPS, and CTBTri4573-OPS group). Among them, CTB4573-OPS was a glycoprotein prepared with CTB (without Tri sequence) as a substrate protein, and its preparation method referred to Pan C et al., mBio. 2016 Apr. 26; 7(2):e00443-16. Each group of mice were injected subcutaneously with 2.5 μg of polysaccharide based on a polysaccharide content; immunized on the 1.sup.st, 15.sup.th, and 29.sup.th day, the tail bloods thereof were collected on the 10.sup.th day after the last immunization, and antibody titers of anti-Shigella flexneri type 2a O-antigen polysaccharide in the serum of each group of mice were measured by indirect ELISA. The enzyme-linked plate was coated with the extracted LPS of anti-Shigella flexneri type 2a 301 strain, with 10 μg of LPS per well, and other operating steps referred to the “Short Protocols in Molecular Biology”.

[0174] The results were shown in FIG. 10. Compared with the OPS group and the CTB4573-OPS group, the total IgG of nanoscale CTBTri4573-OPS was significantly increased, and the IgG1-dominated humoral immunity was mainly triggered. Meanwhile, the levels of IgG2a and IgG3 were also increased significantly, indicating that the cellular immunity was also activated.

[0175] Furthermore, the immunological effects of nanoscale CTBTri4573-OPS with and without an aluminum adjuvant (Adj) were compared. The immunization protocol was the same as that described above, and a dose of the aluminum adjuvant was 10% of the total volume. It was found that the titer of IgG in the nanoscale CTBTri4573-OPS without adjuvant was significantly higher than that with adjuvant group (FIG. 11).

4.2.2 Evaluation of Bactericidal Activity of Antibody Serum of CTBTri4573-OPS

[0176] Regarding whether the antibody in the serum after immunization had bactericidal activity, an in vitro bactericidal experiment was further carried out. The Shigella 301 wild strains were cultured in LB liquid medium at 37° C. in a shaker at 220 rpm until an OD600 reached 2.0, followed by diluting with normal saline to obtain a 100-200 CFU/10 μL dilution. 10 μL of the diluted bacterial solution and 10 μL of immunized mouse serum undergoing a doubling dilution (maintaining in a 56° C. water bath for 30 min in advance to inactivate complement components) were mixed and incubated at 37° C. for 1 h. After the incubation, 20 μL of complement was added to each tube for well mixing and incubation at 37° C. for 1 h. All the resultants were coated on an LB plate, and incubated overnight at 37° C. The total plate count was recorded next day, and the sterilization rate was calculated.

[0177] The results were shown in FIG. 12, the bactericidal activity of the antiserum induced by CTBTri4573-OPS was much higher than those induced by polysaccharide alone and CTB4573-OPS, indicating that the protective effect of the nanoscale polysaccharide conjugate vaccine was greatly improved, and a better effect was achieved without adding the adjuvant, thus avoiding the adverse effect caused by the adjuvant.

4.2.3 Evaluation of the Protection of CTBTri4573-OPS after Immunization

[0178] To further confirm the above effects, two weeks after the last immunization, the mice were injected intraperitoneally with Shigella flexneri 301 wild strains at a dose of 5 times the half lethal dose (4.35×10.sup.7 CFU), namely, 200 μl, followed by observing the death of the mice. The results were shown in FIG. 13. 100% protection was maintained in the nanoparticle group at the dose of 5 times the half lethal dose, reaching the optimal effect (FIG. 13A). Meanwhile, the protective effect in the group without adding an adjuvant was superior to that with an adjuvant (FIG. 13B).

[0179] Although the specific embodiments of the present invention have been described in details, those skilled in the art will understand that various modifications and variations can be made to the details according to all the disclosure published, and these changes are within the protection scope of the present invention. The whole of the present invention is defined by the appended claims and any equivalents thereof.