Ferritin nanoparticle vaccine for preventing giardia disease and preparation method thereof

Abstract

The invention provides a ferritin nanoparticle vaccine for preventing giardiasis and a preparation method thereof, and ferritin nanoparticles are used as a vaccine carrier to construct a subunit vaccine for preventing giardiasis. According to the vaccine, the stability and immunogenicity of the antigen are remarkably improved; and by utilizing the characteristics of the nanotechnology, the targeted delivery of the vaccine can be realized, so that the vaccine can act on immune cells more accurately, and the immune response level of the system is effectively improved. The vaccine has the characteristics of low cost, high safety and capability of inducing good protective immunity, and effectively controls giardia infection and propagation thereof.

Description

Technical Field

[0001] This invention provides a ferritin nanoparticle vaccine for the prevention of Giardiasis and its preparation method. This vaccine can be used to prevent Giardiasis and belongs to the field of biotechnology. Background Technology

[0002] Giardiasis is a common protozoan disease caused by the duodenal flagellate Giardia duodenalis, which parasitizes the epithelial cells of the small intestine (especially the duodenum) in humans and various animals. It is one of the most important zoonotic parasitic diseases worldwide. Currently, the prevention and control of giardiasis mainly rely on drug treatment, such as metronidazole, tinidazole, and albendazole. However, drug-resistant strains and adverse drug reactions are common problems.

[0003] The life cycle of Giardia lamblia comprises two main stages: the trophozoite stage (active growth and reproduction) and the cyst stage (environmental resistance morphology). The adhesion and damage of small intestinal epithelial cells by the trophozoite via its ventral sucker is the primary cause of intestinal pathology. Giardia lamblia adhesion and pathogenic mechanisms involve various secretory factors. The trophozoite relies on a series of adhesion-related proteins and immunomodulatory molecules released from its secretome, employing a unique sucker-mediated attachment mechanism. Examples include variant surface protein (VSP), cysteine-rich enterotoxin proteins, and α-giardin. Among the Giardia lamblia antigens currently under study, variant surface protein (VSP) has been extensively explored. Notably, the Giardia lamblia macrophage migration inhibitory factor (MIF) homolog, as a key immunomodulatory molecule, plays a crucial role in mediating host immune evasion and promoting parasite colonization, and is considered a promising novel candidate vaccine antigen. Summary of the Invention

[0004] This invention discloses a ferritin nanoparticle vaccine for the prevention of Giardiasis and its preparation method, which is a ferritin nanoparticle vaccine capable of displaying Giardia MIF protein.

[0005] The present invention discloses a ferritin nanoparticle SC-FE, wherein the N-terminus of the ferritin is fused with a SpyCatcher short peptide of 136 amino acids, the sequence of which is shown in SEQ ID NO.1.

[0006] The present invention discloses a recombinant Giardia lamblia protein ST-MIF, wherein the N-terminus of MIF is fused with a 13-amino acid SpyTag tag peptide, the sequence of which is shown in SEQ ID NO.2.

[0007] The present invention discloses a self-assembled nanoparticle vaccine of Giardia lamblia, comprising the aforementioned ferritin nanoparticles SC-FE and the aforementioned recombinant Giardia lamblia protein ST-MIF; these two parts are covalently assembled into SC-FE / ST-MIF (i.e., SF-MIF) nanoparticles in a buffer environment (50mM Tirs; 150mM NaCl; pH=8.0), which can display the MIF protein on the surface of the ferritin nanoparticles.

[0008] The present invention discloses a method for preparing a ferritin nanoparticle vaccine for the prevention of giardiasis, comprising the following steps: 1) The SC sequence was tandemly linked to the N-terminus of the FE sequence via a flexible linker (GGGGS)4 and constructed into a prokaryotic expression vector. The expression was induced and purified using an E. coli expression system. The expression vector was pET28a and the competent cells were BL21. 2) The ST sequence was tandemly linked to the N-terminus of the Giardia antigen MIF sequence using a flexible linker (GGGGS)4 and constructed into a prokaryotic expression vector. The expression was induced and purified using an E. coli expression system. The expression vector was pET28a and the competent cells were BL21. 3) The purified ST-MIF and SC-FE were assembled and purified at a molar ratio of 1:4 in a buffer solution (50 mM Tirs; 150 mM NaCl; pH=8.0) at 4 °C, and the size and morphology of the prepared SF-MIF nanoparticles were characterized. 4) The expression vectors provided by ST-MIF and SC-FE are pET28a-ST-MIF and pET28a-SC-FE, and the recombinant strain is BL21 cell; 5) Dissolve 100 μg of SF-MIF and 300 μg of aluminum hydroxide adjuvant in 1 ml of sterile PBS to prepare a nanoparticle vaccine of MIF protein, and store it at -20℃.

[0009] Ferritin nanoparticles, with their regular near-spherical structure (12 nm outer diameter, 8 nm inner cavity) formed by the self-assembly of 24 subunits, have become an ideal vaccine delivery platform. This invention overcomes the stability issues that may exist in traditional antigen display methods by introducing SpyTag / SpyCatcher (ST / SC) linkage technology. ST / SC, as a protein covalent linking tool, relies on the fact that SpyTag and SpyCatcher can form a highly specific, affinity, and excellent temperature / pH-tolerant covalent bond within minutes. In this delivery system, SpyCatcher is modified at the N-terminus of ferritin, and SpyTag is modified at the N-terminus of the antigen. Utilizing the rapid and robust covalent binding properties of ST / SC, the antigen is stably and uniformly presented on the surface of the ferritin nanoparticles, significantly improving the efficacy and reliability of this vaccine delivery system.

[0010] The positive effects of this invention are as follows:

[0011] A novel subunit vaccine for Giardiasis has been successfully developed using ferritin nanoparticles as a vaccine carrier. This vaccine system, through the precise delivery capabilities of nanotechnology, targets immune cells, significantly enhancing the immunogenicity and stability of the vaccine. Compared to conventional vaccines, this nanovaccine offers the following advantages: 1) it induces a stronger specific immune response; 2) it has lower production costs; 3) it demonstrates good safety; and 4) it exhibits excellent protective efficacy in animal models. This nanocarrier-based vaccine strategy provides a new technological approach for the prevention and control of Giardiasis. Attached Figure Description

[0012] Figure 1 This is a diagram of the SC-FE gene amplification in this invention; Figure 2 This is a diagram of ST-MIF gene amplification according to the present invention; Figure 3 This is a diagram showing the expression and purification of the SC-FE protein in this invention (M: protein marker; 1: unpurified supernatant from recombinant protein pET-28a-His-SC-FE induction; 2: unpurified precipitate of recombinant protein pET-28a-His-SC-FE; 3: washing buffer of recombinant protein pET-28a-His-SC-FE induction supernatant; 4-5: elution buffer of recombinant protein pET-28a-His-SC-FE induction supernatant; 6-8: ultrafiltration concentration of recombinant protein pET-28a-His-SC-FE). Figure 4This is a diagram showing the expression and purification of the ST-MIF protein in this invention (M: protein marker; 1: flow-through buffer of recombinant protein pET-28a-His-ST-GdMIF induced supernatant; 2-3: washing buffer of recombinant protein pET-28a-His-ST-GdMIF induced supernatant; 4-7: elution buffer of recombinant protein pET-28a-His-ST-GdMIF induced supernatant; 8-9: ultrafiltration concentration of recombinant protein pET-28a-His-SC-FE). Figure 5 The diagram shows the combined SC-FE and ST-MIF components of this invention (1: SC-FE; 2: SF-MIF 3:2 molar ratio combination; 3: SF-MIF 2:3 molar ratio combination; 4: SF-MIF 1:4 molar ratio combination; 5: ST-MIF). Figure 6 This is a diagram showing the electrophoresis results of SF-MIF in non-denaturing and non-reducing Native-PAGE. Figure 7 These are transmission electron microscopy (TEM) images of the SC-FE and SF-MIF nanoparticles of the present invention (1 is a TEM image of the SC-FE nanoparticles, and 2 is a TEM image of the SF-MIF nanoparticles). Figure 8 The image shows the H&E staining of the duodenum in this invention (from left to right: PBS group; SC-FE immunization group; ST-MIF immunization group; SF-MIF immunization group; non-immune challenge group). Figure 9 This is a graph showing the specific IgG levels of this invention. Figure 10 This is a cytokine level diagram of the present invention (from left to right: IL-4; IL-6; IFN-γ). Detailed Implementation

[0013] To facilitate a better understanding of the present invention by those skilled in the art, the following detailed description and explanation, in conjunction with the accompanying drawings and embodiments, will provide a comprehensive overview of the invention. It should be noted that the embodiments described herein are merely a part of all embodiments of the present invention and should not be construed as limiting the invention to these specific embodiments.

[0014] Example 1

[0015] 1. Construction of pET28a-SC-FE Based on the published FE and SC sequences, codon optimization and gene synthesis were performed by Sangon Biotech (Shanghai) Co., Ltd. The synthesized FN and SC sequences were then cloned into the pET28a prokaryotic expression vector via fusion PCR, with the SC sequence tandemly linked to the N-terminus of the FN sequence in a flexible (GGGGS)4 configuration. BamHI and EcoRI were selected as restriction enzyme sites, and the cloned gene was transformed into E. coli DH5α competent cells.

[0016] Single colonies were selected and expanded for culture. Positive clones were identified by PCR and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Plasmids were extracted from the correctly sequenced bacterial cultures and named pET28a-SC-FE.

[0017] The results showed that the amplified SC-FE gene sequence was as shown in SEQ ID NO.1 (e.g.) Figure 1 ).

[0018] 2. Construction of pET28a-ST-MIF Using the DNA of the Giardia GS strain preserved in our laboratory as a template, the MIF gene was amplified using fusion PCR. ST was tandemly linked to the N-terminus of ROP38 via a flexible (GGGGS)4 restriction enzyme. BamHI and EcoRI were selected as restriction sites, and the gene was cloned into the pET28a prokaryotic expression vector, which was then transformed into *E. coli* DH5α competent cells. Single colonies were picked and cultured, and positive clones were identified by colony PCR. The colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Plasmids were extracted from correctly sequenced colonies and named pET28a-ST-MIF.

[0019] The results showed that the amplified ST-MIF gene sequence was as shown in SEQ ID NO.2 (as shown in the image). Figure 2 ).

[0020] 3. Expression of recombinant proteins Expression and purification of SC-FE 100 μg of the correctly sequenced pET28a-SC-FE plasmid was transformed into BL21 competent cells. The cells were incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and then incubated on ice for 5 minutes. 1 mL of antibiotic-free medium was added, and the cells were incubated at 37°C and 150 rpm for 1 hour. 100 μL of the incubated plasmid was then spread onto solid LB agar plates containing kanamycin and incubated overnight at 37°C and 150 rpm. The next day, single colonies were picked and incubated on liquid LB agar containing kanamycin for 12 hours at 37°C and 150 rpm to obtain a strain stably expressing SC-FE protein. The 10 mL of overnight activated SC-FE-expressing bacterial culture was expanded to 500 mL of medium at a 2% ratio. When the OD reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and induction continued at 37°C for 6-8 hours. The cells were collected by centrifugation at 12000 rpm for 10 minutes, sonicated, and the supernatant was purified using a nickel column. After the purity of the collected protein was verified by SDS-PAGE, it was concentrated using a 100kDa ultrafiltration tube, replaced with a buffer suitable for SC-FE, and the protein concentration was measured using a BCA kit. The protein was then aliquoted and stored at -80℃.

[0021] The results showed that SC-FE was expressed in supernatant form, and high-purity SC-FE protein could be obtained by elution with 500 mM imidazole. The monomeric SC-FE had a size of 35 kDa, and the protein concentration after buffer replacement was 1.5 μg / μL, which can be used for subsequent experiments (such as...). Figure 3 (As shown).

[0022] 4. Expression and purification of ST-MIF The pET28a-ST-MIF plasmid was transformed into BL21 competent cells using the same method as pET28a-SC-FE, followed by induced expression and purification. After verifying the purity of the purified ST-MIF using SDS-PAGE, it was concentrated using a 10kDa ultrafiltration tube, replaced with a buffer suitable for ST-MIF, and the protein concentration was measured using a BCA kit. The purified plasmid was then aliquoted and stored at -80°C.

[0023] The results showed that ST-MIF was expressed in the supernatant, and high-purity SC-FE protein could be obtained by elution with 500 mM imidazole. The ST-MIF size was 14 kDa, and the protein concentration after buffer replacement was 2 μg / μL, which was used for subsequent experiments (such as...). Figure 4 (As shown).

[0024] 5. Combination and assembly of ST-MIF and SC-FE ST-MIF and SC-FE were bonded overnight at 4°C in molar ratios of 4:1, 3:2, 2:3, and 1:4. Through this bonding process, SC-FE could automatically assemble into nanoparticles, displaying ST-MIF on the surface of the SC-FE nanoparticles to form SF-MIF nanoparticles. The bonding efficiency of different molar ratios was then verified by SDS-PAGE, and a suitable molar ratio was selected for large-scale bonding. Figure 5 As shown.

[0025] The size, morphology, and structure of the prepared SC-FE and SF-MIF nanoparticles were observed using Native-PAGE and transmission electron microscopy, such as... Figure 6 As shown.

[0026] The results showed that SF-MIF can assemble into uniform spherical nanoparticles with a size of about 12 nm (e.g., ...). Figure 7 (As shown).

[0027] The positive effects of the present invention are further illustrated by the following experimental examples:

[0028] The experimental design consisted of five groups: a blank control group, a non-immunized challenge group, an SC-FE immunization group, an ST-MIF protein immunization group, and an SF-MIF nanoparticle vaccine immunization group, with six long-clawed gerbils in each group. The immunization schedule included two immunizations on day 1 and day 14, with blood collection and weighing performed after each immunization. Except for the blank control group and the non-immunized challenge group, which received 1 ml of sterile PBS, the other groups received 1 ml of PBS solution containing 150 μg of the corresponding antigen (SC-FE, ST-MIF, or SF-MIF) and 300 μg of aluminum hydroxide adjuvant. Fourteen days after the second immunization, all groups except the blank control group received 2 × 10⁻⁶ oz. of oral PBS. 7 Giardia trophozoites were used in a challenge experiment.

[0029] Nine days after the worm challenge, duodenal tissue samples from the same location in each group were fixed and stained with H&E. The results showed that the blank control group exhibited normal duodenal histological characteristics, with intact duodenal villi, orderly cell arrangement, and clear and intact structure, without any abnormal lesions. In the non-immunized worm challenge group and the SC-FE protein immunization group, the duodenal tissue of the experimental gerbils was significantly damaged, with disordered cell arrangement and severe destruction of normal cell structure; Giardia trophozoites were clearly observable. The duodenal villi in the ST-MIF protein immunization group and the SF-MIF protein immunization group also showed some degree of damage, with Giardia trophozoites almost invisible, and the degree of damage to the duodenal epithelial cells was less severe. This indicates that after immunization with recombinant protein MIF, the experimental gerbils possessed a certain degree of resistance to Giardia infection (e.g., Figure 8 (As shown).

[0030] The purified ST-MIF protein was diluted to 1.5 μg / mL using ELISA coating buffer and coated onto an ELISA plate. Serum samples collected before the first immunization, 2 weeks after the first immunization, and 2 weeks after the second immunization were used as the primary antibody, and HRP-GAM was used as the secondary antibody. Absorbance was measured at 450 nm. Serological tests showed that the specific IgG level in the nanovaccine immunization group was significantly higher than that in the ST-MIF protein immunization group (e.g., ...). Figure 9 (As shown).

[0031] Serum cytokine levels in each group were measured using a cytokine assay kit before the first immunization, 2 weeks after the first immunization, and 2 weeks after the second immunization. Results showed that the levels of IL-4, IL-6, and IFN-γ in the ST-MIF protein immunization group and the nanovaccine immunization group were significantly higher than those in the control group, and the nanovaccine group exhibited a superior immune response (e.g., ...). Figure 10 (As shown).

Claims

1. A ferritin nanoparticle SC-FN, characterized in that: Ferritin contains a short peptide of 114 amino acids fused to its N-terminus, the sequence of which is shown in SEQ ID NO.

1.

2. A recombinant Giardia lamblia protein ST-MIF, characterized in that: The N-terminus of the MIF is fused with a 13-amino acid SpyTag-tagged short peptide, the sequence of which is shown in SEQ ID NO.

2.

3. A self-assembled nanoparticle vaccine of Giardia lamblia, characterized in that: The ferritin nanoparticles SC-FE as described in claim 1 and the Giardia recombinant protein ST-MIF as described in claim 2 are covalently assembled into SF-MIF nanoparticles in a buffer environment (50 mM Tirs; 150 mM NaCl; pH=8.0), thereby displaying the MIF protein on the surface of the ferritin nanoparticles.

4. A method for preparing a ferritin nanoparticle vaccine for the prevention of giardiasis as described in claim 3, comprising the following steps: 1) The SC sequence was tandemly linked to the N-terminus of the FN sequence via a flexible linker (GGGGS)4 and constructed into a prokaryotic expression vector. The expression was induced and purified using an E. coli expression system. The expression vector was pET28a and the competent cells were BL21. 2) The ST sequence was tandemly linked to the N-terminus of the Giardia antigen MIF sequence using a flexible linker (GGGGS)4 and constructed into a prokaryotic expression vector. The expression was induced and purified using an E. coli expression system. The expression vector was pET28a and the competent cells were BL21. 3) The purified ST-MIF and SC-FE were mixed at a molar ratio of 1:4 in a buffer solution (50 mM Tirs; 150 mM NaCl; pH=8.0) at 4 °C. SC-FE could automatically assemble into nanoparticles and ST-MIF was displayed on the surface of the SC-FE nanoparticles to form SF-MIF nanoparticles. The size and morphology of the prepared SF-MIF nanoparticles were characterized. 4) The expression vectors provided by ST-MIF and SC-FE are pET28a-ST-MIF and pET28a-SC-FE, and the recombinant strain is BL21 cell; 5) Dissolve 150 μg of SF-MIF and 300 μg of aluminum hydroxide adjuvant in 1 ml of sterile PBS to prepare a nanoparticle vaccine of MIF recombinant protein, and store it at -20℃.

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