A bivalent subunit vaccine for Candida albicans, its preparation method and application

Abstract

This invention discloses a bivalent subunit vaccine against Candida albicans, its preparation method, and its application, relating to the field of biomedical technology. The bivalent subunit vaccine comprises recombinant Als3 protein and recombinant Cht3 protein as immunogens. The recombinant Als3 protein is obtained based on the sequence shown in SEQ ID NO:1, through site-directed mutagenesis of the N-glycosylation site and weak epitopes LDGTSASPG and IVIVATTRT. The recombinant Cht3 protein is obtained based on the sequence shown in SEQ ID NO:2, through site-directed mutagenesis of the weak epitopes TTSVTSSET and YTANGILQC to enhance immunogenicity. This invention overcomes the bottleneck of prokaryotic expression of Cht3 antigen through a combination strategy of antigen extraction, expression strain optimization, mild induction, and multiple site-directed mutagenesis, obtaining a high-purity, highly immunogenic antigen. Furthermore, the bivalent combination of Als3 and Cht3 produces a synergistic immune effect, significantly improving the protective effect against Candida albicans infection. Simultaneously, the process is simple and easy for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a bivalent subunit vaccine for Candida albicans, its preparation method, and its application. Background Technology

[0002] Candida albicans is a common opportunistic pathogenic fungus in clinical practice and an important pathogen of hospital-acquired infections. Invasive infections caused by Candida albicans have a mortality rate as high as 40%-50%, posing a particularly high risk to immunocompromised individuals (such as cancer patients, organ transplant recipients, and HIV / AIDS patients). Currently, antifungal drugs are the primary treatment for Candida albicans infections in clinical practice. However, with the emergence of drug-resistant strains and the continuous expansion of the immunocompromised population, the efficacy of existing antifungal drugs is gradually being limited. Therefore, the development of safe and effective preventative vaccines has become an urgent need to address Candida albicans infections.

[0003] In Candida albicans vaccine target research, Als3 and Cht3 are two key candidate antigens. Als3, as an adhesin and infiltrator, participates in the host adhesion and tissue invasion processes of Candida albicans. Its monoantigen vaccine (such as NDV-3A) has shown some protective effect in clinical trials, but the protective effect is limited, and a single antigen strategy is insufficient to address the complex pathogenic mechanisms of Candida albicans. Cht3 is a chitinase with strong immunogenicity, but its recombinant expression faces serious technical bottlenecks: expression is difficult in the E. coli prokaryotic expression system, specifically manifested as low expression levels, protein truncation, and easy formation of inclusion bodies; in the eukaryotic expression system, there are problems such as abnormal molecular weight, pigment contamination, and complex glycosylation modifications, making it difficult to prepare high-purity Cht3 antigen on a large scale, severely restricting its vaccine development and application.

[0004] Multivalent vaccine strategies can theoretically overcome the insufficient protective efficacy of single-antigen vaccines. Als3 and Cht3 play complementary roles in the pathogenesis of Candida albicans, and their combined application is expected to produce synergistic protective effects. However, a mature Als3-Cht3 bivalent vaccine technology platform has not yet been established, and there is no combined solution to address the Cht3 expression challenge, making it impossible to obtain high-quality antigens that meet vaccine standards. The core problems include: ① the protective efficacy of single-antigen vaccines cannot meet clinical needs; ② the expression and purification technology of Cht3 antigen is immature, making it impossible to obtain high-quality antigens that meet vaccine standards; ③ the lack of a vaccine preparation process that effectively integrates Als3 and Cht3 antigens while maintaining their immunogenicity. Therefore, developing a bivalent subunit vaccine for Candida albicans that can overcome the expression bottlenecks of Cht3 and Als3 antigens and achieve efficient integration of two antigens has significant clinical value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a bivalent subunit Candida albicans vaccine that can overcome the bottleneck of prokaryotic expression of Cht3 and Als3, achieve the required purity, have strong immunogenicity, and provide excellent protection.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A bivalent subunit vaccine for Candida albicans includes a recombinant Als3 protein and a recombinant Cht3 protein as immunogens; the recombinant Als3 protein is obtained by site-directed mutagenesis of the N-glycosylation site and the weak epitopes LDGTSASPG and IVIVATTRT based on the sequence shown in SEQ ID NO:1; the recombinant Cht3 protein is obtained by site-directed mutagenesis of the weak epitopes TTSVTSSET and YTANGILQC based on the sequence shown in SEQ ID NO:2 to enhance immunogenicity.

[0008] Furthermore, the mutation of the recombinant Cht3 protein is as follows: the two weak epitopes TTSVTSSET and YTANGILQC are mutated to TTSFTSSET and YTAFGILQC;

[0009] The mutation of the recombinant Als3 protein includes: [mutating the Nth...] 403 The asparagine N site was mutated to glutamine Q, and the weak epitopes LDGTSASPG and IVIVATTRT were mutated to LNGTSASPG and IVIVATTRY.

[0010] Furthermore, SEQ ID NO:1 represents amino acids 17 to 432 from the N-terminus of Als3, and the amino acid sequence of the recombinant Als3 protein is SEQ ID NO:3; SEQ ID NO:2 represents amino acids 23 to 567 from the N-terminus of Cht3, and the amino acid sequence of the recombinant Cht3 protein is SEQ ID NO:4.

[0011] Furthermore, the vaccine also includes an immune adjuvant, and the immune adjuvant is aluminum hydroxide adjuvant.

[0012] On the other hand, the present invention also provides a method for producing the above-mentioned Candida albicans bivalent subunit vaccine, comprising the following steps:

[0013] S1, construct a recombinant expression vector containing the mutant Als3 gene and the mutant Cht3 gene;

[0014] S2, the recombinant expression vector was transformed into prokaryotic expression strains and induced to express the recombinant protein to obtain bacterial cells containing the recombinant protein;

[0015] S3, the bacterial cells are disrupted, the supernatant is collected, and the fusion tag is removed by affinity chromatography;

[0016] S4 was further purified by ion exchange chromatography or molecular sieve chromatography to obtain high-purity recombinant Als3 antigen and recombinant Cht3 antigen.

[0017] S5, the recombinant Als3 antigen and recombinant Cht3 antigen are mixed with adjuvant to prepare a bivalent subunit vaccine.

[0018] Specifically, in step S2, the host strain expressing the recombinant Als3 protein is E. coli BL21(DE3) pLysS; the host strain expressing the recombinant Cht3 protein is E. coli Rosetta 2(DE3) which can supplement rare codons.

[0019] Specifically, the induction conditions in step S2 are: when the bacterial culture OD 600 When the concentration reaches 0.4-0.8, add IPTG to a final concentration of 0.1-0.5 mM and induce at low temperature for 12-20 hours at 15-20℃.

[0020] Specifically, in step S3, the affinity chromatography is GST affinity chromatography, and PP enzyme is added to the chromatography column for overnight enzymatic digestion to remove the GST tag; in step S4, the recombinant Als3 protein is purified by anion exchange chromatography combined with elution buffer of 50-100 mM NaCl gradient; the recombinant Cht3 protein is purified by anion exchange chromatography and molecular sieve chromatography in sequence, and the chromatography buffer contains 10% glycerol and 0.05% Tween-20.

[0021] In another aspect, the present invention also provides the use of the above-mentioned Candida albicans bivalent subunit vaccine in the preparation of medicaments for the prevention or treatment of Candida infectious diseases.

[0022] Specifically, the vaccine provides immune protection against Candida albicans by inducing the body to produce specific antibodies against Als3 and Cht3.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) This invention extracts amino acids 17 to 432 from the N-terminus of Als3 protein and amino acids 23 to 567 from the N-terminus of Cht3 protein as antigen fragments, removes the signal peptide sequence, and retains the mature protein domain with excellent immunogenicity. This avoids the problem that the signal peptide causes the protein to fail to fold correctly, form inclusion bodies or mislocalize in the prokaryotic expression system, and lays the molecular foundation for subsequent efficient soluble expression.

[0025] (2) This invention uses site-directed mutagenesis to target potential N-glycosylation sites (N-glycosylation sites) in the Als3 antigen. 403 The asparagine (N) of the protein is mutated to glutamine (Q), which eliminates the problem of protein conformational heterogeneity caused by the inability of glycosylation in the prokaryotic system. This ensures the production of conformationally homogeneous and stable antigenic proteins in the prokaryotic system, and significantly improves the solubility, stability and immunogenicity of the protein.

[0026] (3) Based on the principles of immunology and structural biology, this invention performs multiple site-directed mutations on the Als3 and Cht3 antigens: for the weak epitopes TTSVTSSET and YTANGILQC of Cht3, valine V is mutated to phenylalanine F and asparagine N is mutated to phenylalanine F, maximizing the filling of the P4 hydrophobic cavity of the MHC-II molecule with aromatic hydrophobic residues, thereby reducing the binding free energy; for the Als3 antigen, the D→N mutation in the weak epitope sequences LDGTSASPG and IVIVATTRT eliminates the charge repulsion between the peptide and the MHC-II molecule, and the T→Y mutation introduces a strong hydrophobic anchor to enhance the binding ability of the peptide to the antigen-presenting molecule. The above modifications enhance the binding force of weak epitopes from a weak binding state to a strong immunogenic epitope, broaden the CD4⁺ T cell immune epitope spectrum, and significantly enhance the level of immune response.

[0027] (4) In response to the technical problem of codon bias in prokaryotic systems, this invention selects E.coli Rosetta 2 (DE3) as the expression strain, which can supplement rare codons, and solves the problems of expression failure, protein truncation and molecular weight abnormality caused by codon bias, and realizes the complete expression of recombinant Cht3 protein.

[0028] (5) The present invention uses mild induction conditions of low temperature and low concentration of IPTG to promote correct protein folding, increase the proportion of soluble expression, effectively reduce inclusion body formation, and break through the traditional bottleneck of low expression level and easy aggregation of Cht3 protein in Escherichia coli.

[0029] (6) The present invention uses GST affinity chromatography combined with on-column enzyme digestion technology to directly remove the GST tag on the affinity chromatography column, which reduces purification steps and protein loss, significantly improves purification efficiency, and avoids the risk of protein degradation and activity loss in the traditional multi-step purification process.

[0030] (7) This invention designs optimized buffer systems and elution methods for Als3 and Cht3 proteins based on their different characteristics. A staged gradient elution is used for Als3, obtaining the target protein with a purity of over 90% in 50 mM and 100 mM NaCl elution solutions. A linear gradient elution is used for Cht3, with fine separation performed in a buffer system supplemented with glycerol and Tween-20, ultimately obtaining the Cht3 target protein with a purity of up to 95%. These optimizations achieve the goal of specifically separating high-purity antigens from complex lysis buffers.

[0031] (8) This invention uses a bivalent combination of Als3 and Cht3, utilizing the complementary effects of the two antigens in the pathogenic mechanism of Candida albicans to produce a significant synergistic immune effect. Animal experiments show that the bivalent vaccine can stimulate a stronger humoral immune response; challenge protection experiments have confirmed that the synergistic combination of the bivalent vaccine achieves a significant protective effect against Candida albicans infection, which is far superior to single antigen vaccines.

[0032] (9) The prokaryotic expression and purification process system established by this invention is mature and stable, does not require a complex eukaryotic expression system, has low production cost, short cycle, and is easy to scale up, laying a solid foundation for the industrial development of Candida albicans vaccine, and has good application prospects and market value. Attached Figure Description

[0033] Figure 1 The image shows a three-dimensional structural model of the Als3 protein; the left image is before the mutation, and the right image is after the mutation.

[0034] Figure 2 The image shows the three-dimensional structure of the Cht3 protein; the left image is before mutation, and the right image is after mutation.

[0035] Figure 3 Image showing double enzyme digestion verification of recombinant plasmid pGEX-6P-2-Als3;

[0036] Among them, lane M is the DNA molecular weight standard; lane 1 is the plasmid digested with BamHI and XhoI; lane 2 is the plasmid DNA.

[0037] Figure 4 Image showing double digestion verification of recombinant plasmid pGEX-6P-2-Cht3;

[0038] Among them, lane M is the DNA molecular weight standard; lane 1 is the plasmid digested with BamHI and XhoI; lane 2 is the plasmid DNA.

[0039] Figure 5 This is an SDS-PAGE image of Als3 recombinant protein digestion.

[0040] The sequence is as follows: Lane M: Protein molecular weight standard; Lane 1: Complete lysate; Lane 2: Supernatant; Lane 3: Lysate precipitate; Lane 4: Breakthrough buffer; Lane 5: Washing; Lane 6: Before enzyme digestion; Lane 7: Collection of packing material after enzyme digestion; Lane 8: Breakthrough buffer after enzyme digestion; Lane 9: Filtration of breakthrough buffer after enzyme digestion; Lane 10: Collection of packing material after breakthrough; Lane 11: Elution 1; Lane 12: Elution 2; Lane 13: Elution 3.

[0041] Figure 6 SDS-PAGE image of Als3 recombinant protein after ion exchange chromatography purification;

[0042] The following are the elution buffers: Lane M: Protein molecular weight standard; Lane 1: Before dialysis; Lane 2: After dialysis; Lane 3: After dialysis filtration; Lane 4: Breakthrough buffer; Lane 5: Washing buffer; Lane 6: 50 mM NaCl elution buffer; Lane 7: 100 mM NaCl elution buffer; Lane 8: 150 mM NaCl elution buffer; Lane 9: 200 mM NaCl elution buffer; Lane 10: 300 mM NaCl elution buffer; Lane 11: 400 mM NaCl elution buffer; Lane 12: 500 mM NaCl elution buffer.

[0043] Figure 7 This is an SDS-PAGE image of the Cht3 recombinant protease digestion.

[0044] The following are the components of the buffer: Lane M: Protein molecular weight standard; Lane 1: Complete lysate; Lane 2: Lysate precipitate; Lane 3: Lysate supernatant; Lane 4: Breakthrough buffer; Lane 5: Wash 1; Lane 6: Wash 2; Lane 7: Wash 3; Lane 8: Before enzyme digestion; Lane 9: Breakthrough buffer after enzyme digestion; Lane 10: Packing material collected after breakthrough; Lane 11: Elution 1; Lane 12: Elution 2; Lane 13: Elution 3; Lane 14: Elution 4.

[0045] Figure 8 This is an SDS-PAGE image of the Cht3 recombinant protein after ion exchange chromatography purification.

[0046] The following are the elution buffers: Lane M: Protein molecular weight standard; Lane 1: Before loading; Lane 2: Breakthrough buffer; Lane 3: Rebalancing buffer; Lane 4: 150mM NaCl washing buffer 1; Lane 5: 150mM NaCl washing buffer 2; Lane 6: Elution buffer 1; Lane 7: Elution buffer 2; Lane 8: Elution buffer 3; Lane 9: Elution buffer 4; Lane 10: Elution buffer 5; Lane 11: Elution buffer 6; Lane 12: Elution buffer 7; Lane 13: NaOH elution buffer.

[0047] Figure 9 This is an SDS-PAGE image of the Cht3 recombinant protein after molecular sieve chromatography purification.

[0048] The following are the elution buffers: Lane M: Protein molecular weight standard; Lane 1: Elution buffer 1; Lane 2: Elution buffer 2; Lane 3: Elution buffer 3; Lane 4: Elution buffer 4; Lane 5: Elution buffer 5; Lane 6: Elution buffer 6; Lane 7: Elution buffer 7; Lane 8: Elution buffer 8; Lane 9: Elution buffer 9; Lane 10: Elution buffer 10; Lane 11: Elution buffer 11; Lane 12: Elution buffer 12; Lane 13: Elution buffer 13.

[0049] Figure 10 The figure shows the titer of Als3-specific antibodies induced by the bivalent vaccine.

[0050] Figure 11 The figure shows the titer of Cht3-specific antibodies induced by the bivalent vaccine.

[0051] Figure 12 The graph shows the survival rate of mice challenged with the bivalent vaccine. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0053] This invention provides a bivalent subunit vaccine for Candida albicans, the preparation method of which is as follows:

[0054] Step 1: Cloning and constructing of the Als3 gene and the mutant Cht3 gene

[0055] (1) Sequence Design: The amino acid sequences of Als3 and Cht3 were compared among different strains of Candida albicans and found to be highly conserved. Based on the amino acid sequence of Als3, amino acids 17 to 432 of its N-terminal region (Als3-N) were extracted. This sequence has excellent immunogenicity, as shown in SEQ ID NO:1. To facilitate subsequent expression in prokaryotic systems, the Als3 sequence was rationally designed and molecularly modified as follows, as detailed in Tables 1 and 2. Figure 1 As shown ( Figure 1 The left side shows the area before the renovation. Figure 1 (The right image shows the modified version). The modified Als3 protein sequence is SEQ ID NO:3:

[0056] ① Glycosylation site mutation: This involves removing potential N-glycosylation sites (N...) 403 The asparagine (Asn, N) of the protein was mutated to glutamine (Gln, Q) to eliminate conformational heterogeneity caused by "post-translational modification loss", thereby improving the protein's solubility, stability and immunogenicity.

[0057] ② Weak Epitope Enhancement Mutations (D→N, T→Y): Site-directed mutations are performed on the two weak epitopes LDGTSASPG and IVIVATTRT of Als3. The corresponding sequences after mutation are LNGTSASPG and IVIVATTRY. The D→N mutation eliminates the charge repulsion between the peptide and the MHC-II molecule, as if removing the "roadblock" to binding. The T→Y mutation introduces a strong hydrophobic anchor to enhance the binding ability of the peptide to the antigen-presenting molecule, locking the peptide more firmly in the presenting molecule. The two work together to transform the weak epitopes into strong epitopes. Moreover, the amino acids D and N have similar structures, and amino acids T and Y share a hydroxyl group, without destroying the protein's natural three-dimensional structure and surface properties. This ensures that the mutated antigen can still be expressed efficiently, remain soluble, and be correctly presented to the immune system, thus laying an ideal foundation for stimulating a strong protective antibody response.

[0058] Table 1. Before the mutation of SEQ ID NO:1

[0059]

[0060] Table 2 SEQ ID NO:1 after mutation

[0061]

[0062] Based on the amino acid sequence of Cht3, amino acids 23 to 567 of its N-terminal region (Cht3-N) were extracted, as shown in SEQ ID NO:2. To facilitate subsequent expression in prokaryotic systems, the Cht3 sequence was rationally modified as follows, as detailed in Tables 3 and 4. Figure 2 As shown ( Figure 2 The left side shows the area before the renovation. Figure 2 (The right image shows the modified version). The sequence of the mutated Cht3 protein is SEQ ID NO:4.

[0063] Site-directed mutagenesis was performed on two weak epitopes, TTSVTSSET and YTANGILQC, in Cht3, resulting in TTSFTSSET and YTAFGILQC, respectively. Valine (V) and asparagine (Asn, N) were mutated to phenylalanine (Phe, F), replacing unsuitable aliphatic hydrophobic and polar hydrophilic residues with aromatic hydrophobic residues. The rigid benzene ring of phenylalanine maximizes the filling of the P4 hydrophobic cavity through hydrophobic interactions and the π-electron cloud effect, reducing the binding free energy and enhancing the epitope binding affinity from a weak state to a strong immunogenic epitope, thus broadening CD4 binding. + T-cell immune epitope profile enhances immune response levels.

[0064] Table 3 SEQ ID NO:2 before mutation

[0065]

[0066] Table 4 SEQ ID NO:2 after mutation

[0067]

[0068] The aforementioned multiple site-directed mutagenesis aims to enhance the affinity of peptides for MHC-II molecules, achieve the conversion of weak epitopes to strong epitopes, and at the same time rely on amino acid structural similarity to ensure the correct folding and soluble expression of recombinant proteins.

[0069] (2) Based on the extracted Als3 and mutated Cht3 gene sequences, the complete genes were synthesized. The synthesized genes were cloned into the expression vector pGEX-6P-2 using BamHI and XhoI restriction sites to construct recombinant plasmids pGEX-6P-2-Als3 and pGEX-6P-2-Cht3. The ligation products were transformed into TOP10 cloned strains, and sequencing confirmed that the target gene sequences were completely correct and without amino acid mutations.

[0070] (3) Recombinant plasmids were extracted from the correctly sequenced cloned strains and verified by double enzyme digestion. The results were consistent with expectations (e.g., Figure 3 , Figure 4 (As shown). The verified recombinant plasmid pGEX-6P-2-Als3 was transformed into the expression strain E. coli BL21(DE3) pLysS, resulting in the recombinant E. coli pGEX-6P-2 / Als3 / E. coli BL21(DE3) pLysS that expresses Als3. The recombinant plasmid pGEX-6P-2-Cht3 was transformed into the expression strain E. coli Rosetta 2(DE3) that can supplement rare codons, resolving expression failure, protein truncation, and molecular weight abnormalities caused by codon bias, to obtain the recombinant E. coli pGEX-6P-2 / cht3 / E. coli Rosetta 2(DE3) that expresses cht3.

[0071] Step 2: Induction of expression of Als3 and Cht3 antigens

[0072] (1) Activation of strains: Als3 and Cht3 expression strains pGEX-6P-2 / Als3 / E.coliBL21(DE3) pLysS and pGEX-6P-2 / cht3 / E.coli Rosetta 2(DE3) stored at -80℃ were inoculated and activated at a ratio of 1:1000, i.e., 20 μL of glycerol bacterial solution was added to 20 mL of LB medium containing Amp resistance and activated overnight at 37℃ and 220 rpm.

[0073] (2) Secondary activation induction: The bacteria were inoculated at a ratio of 1:100 for secondary activation. That is, 20 mL of overnight bacterial culture was added to 2 L of LB medium containing Amp resistance and cultured at 37℃ and 180 rpm until OD. 600 Approximately 0.6. Then, IPTG was added to a final concentration of 0.2 mM, and the mixture was placed on a shaker at 16°C and 150 rpm for overnight induction. The mild induction conditions of low temperature and low concentration of IPTG can promote correct protein folding, increase the proportion of soluble expression, and reduce inclusion body formation.

[0074] (3) Induction, expression, collection, and preliminary processing of recombinant proteins: After induction, bacterial cells were collected by centrifugation at 4°C and 6000 rpm for 15 minutes. The bacterial cells were resuspended in an appropriate amount of binding buffer and then lysed using a high-pressure homogenizer at 700-800 bar. After homogenization, the cells were centrifuged at 4°C and 12000 rpm for 50 minutes. The supernatant (soluble components) and precipitate (insoluble components such as inclusion bodies) were collected separately and stored for subsequent analysis.

[0075] Step 3: Affinity chromatography purification and tag removal

[0076] Sample loading: Filter the lysed supernatant collected in step 2 through a 0.45 μm filter membrane to remove impurities that may clog the chromatography column, and then load it into a GST affinity chromatography gravity column that has been pre-equilibrated with 5 column volumes of binding buffer.

[0077] Incubation: Place the chromatography column on a rotating shaker at 4°C and incubate for 2 hours to allow the GST fusion protein to fully bind with the packing material. After binding is complete, collect the permeate and take a sample.

[0078] Washing: Wash the chromatography column with 10 column volumes of binding buffer to remove non-specifically bound contaminating proteins, collect the wash buffer and take it;

[0079] Buffer replacement: Rinse the chromatography column with 10 column volumes of enzyme digestion buffer to ensure that the column environment is completely replaced from binding buffer to enzyme digestion buffer;

[0080] On-column digestion: Add 10 mL of digestion buffer to the chromatography column, mix gently, and take a sample (this is the sample before digestion). Add PP enzyme, mix well, and perform overnight digestion at 4°C to remove the GST tag, reduce purification steps and protein loss, and improve purification efficiency;

[0081] Product collection: After enzyme digestion, collect the permeate containing the target protein and take a sample. Then, elute 3 times with elution buffer (1-3 column volumes each time) and take samples separately.

[0082] SDS-PAGE Validation: All samples taken above (including whole lysate, lysate supernatant, lysate precipitate, breakthrough buffer, washing buffer, samples before and after enzyme digestion, and elution fractions) were analyzed by SDS-PAGE electrophoresis (conditions: 160V, 400mA, 60min). The results, observed under a gel imaging system, showed that the molecular weight of the enzyme-digested Als3 protein was approximately 45 kDa. Figure 5 The molecular weight of Cht3 protein is approximately 58 kDa. Figure 7 (The value is consistent with the expected size.)

[0083] Step 4: Ion exchange chromatography purification of Als3 protein

[0084] The Als3 digestion buffer collected in step 3 was dialyzed overnight at 4°C in dialysate (20 mM Tris-HCl, 20 mM NaCl, 2 mM TCEP, 0.1% Tween-80, pH 8.0). After dialyzing, the sample was clarified by high-speed centrifugation and membrane filtration. Fine purification was performed using an anion exchange chromatography column. The column bed was thoroughly equilibrated with equilibration buffer (components same as dialysate) to ensure compatibility between the packing material and the sample environment. After loading, a stepwise gradient elution was used, eluting with elution buffers (20 mM Tris-HCl, 2 mM TCEP, 0.1% Tween-80, pH 8.0) containing different concentrations of NaCl (50 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM) to progressively separate the target protein. Each elution volume was controlled at 3-5 column volumes to accurately capture protein components with different adsorption strengths. SDS-PAGE results showed that the Als3 target protein with a purity of over 90% was obtained in elution buffers of 50 mM and 100 mM NaCl (e.g., Figure 7 (As shown).

[0085] Step 5: Ion exchange chromatography purification of Cht3 protein

[0086] The Cht3 digestion buffer collected in step 3 was dialyzed overnight at 4°C in dialysate (20 mM Tris-HCl, 50 mM NaCl, 2 mM TCEP, 10% glycerol, 0.05% Tween-20, pH 8.5). After dialyzing, the sample was centrifuged at 12000 r / min at 4°C and filtered through a 0.45 μm filter to clarify.

[0087] Take a Capto™ Q anion exchange chromatography column (5 mL column volume, 1.6 cm × 2.5 cm column size), and wash the column with equilibration buffer (same as dialysate) at a flow rate of 1 mL / min until the UV 280 nm absorbance and conductivity baseline stabilize (approximately 5 column volumes, 25 mL).

[0088] Load the clarified sample at a flow rate of 0.5 mL / min and collect the flow-through. After loading, wash the column with equilibration buffer at a flow rate of 1 mL / min until the UV 280 nm absorbance and conductivity baseline stabilizes (approximately 5 column volumes, 25 mL), and collect the wash buffer; then wash with 20 mM Tris-HCl, 150 mM NaCl, 2 mM TCEP, 10% glycerol, 0.05% Tween-20, pH 8.5 until the UV 280 nm absorbance and conductivity baseline stabilizes, and collect the impurity wash buffer.

[0089] Linear gradient elution: Buffer A was prepared using elution buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM TCEP, 10% glycerol, 0.05% Tween-20, pH 8.5), and elution buffer B was prepared using 20 mM Tris-HCl, 550 mM NaCl, 2 mM TCEP, 10% glycerol, 0.05% Tween-20, pH 8.5). Elution was linearly increased from 0% B to 100% B in 15 column volumes (75 mL) at a flow rate of 1 mL / min, with elution peaks collected in fractions of 0.5 mL / tube. The addition of glycerol stabilized the protein structure, and low concentrations of Tween-20 reduced non-specific protein adsorption. Results were confirmed by SDS-PAGE (e.g., ...). Figure 8 (as shown)

[0090] After elution, rinse with 1 M NaCl for 2-3 column volumes, then rinse with pure water for 3-5 column volumes, followed by rinsing with 0.5-1 M NaOH for 2-3 column volumes and soaking for 30-60 minutes. Then rinse with pure water until the pH is neutral and the conductivity is <1 mS / cm. Finally, rinse with 20% ethanol and soak. Store at 4°C for later use.

[0091] In this invention, the breakthrough in soluble expression is the result of the synergistic effect of molecular design (truncation, mutation), expression system (strain), and process conditions (low temperature induction); efficient purification relies on the optimization of multi-step chromatography strategy. In addition, buffer systems and elution methods designed for the characteristics of Cht3 and Als3 proteins can achieve the goal of specifically separating high-purity antigens from complex lysates.

[0092] Step 6: Molecular sieve chromatography purification of Cht3 protein

[0093] The eluent 7 obtained from ion exchange chromatography was further purified by molecular sieve chromatography. The column was equilibrated with 5 column volumes of filtered degassed PBS buffer until the baseline stabilized. The eluent 7 sample was thoroughly dialyzed or diluted with the same PBS buffer, and after high-speed centrifugation and filtration clarification, it was loaded into the column at a volume not exceeding 5% of the column volume. After loading, isocratic elution was immediately performed with PBS buffer, and A280 UV absorption was monitored throughout the process. Elution peaks were collected in fractions of 1 mL / tube. After separation, the column was immediately washed with 2-3 column volumes of PBS, and the column was stored in 20% ethanol. SDS-PAGE analysis showed that the target Cht3 protein (e.g., [missing information]) was obtained with a purity of up to 95%. Figure 9 (as shown)

[0094] Step 7: Evaluation of antibody titer after immunization with Als3-Cht3 bivalent subunit vaccine

[0095] This step aims to assess the immunogenicity of the bivalent vaccine, as detailed below:

[0096] (1) Animal immunization: Six- to eight-week-old SPF-grade female BALB / c mice were randomly divided into four groups (PBS control group, ALS3 single antigen group, Cht3 single antigen group, and ALS3+Cht3 combined group), with five mice in each group. The immunogen was the high-purity recombinant protein prepared in steps 4 and 5 (ALS3 purity >90%, Cht3 purity >95%, both concentrations 1 mg / mL), and the adjuvant was aluminum hydroxide adjuvant (Alhydrogel). ® The immunization dose was 50 μg protein per mouse (2 mg / mL for the combined group). Subcutaneous injections were administered on day 0 (primary immunization), day 14 (booster immunization), and day 28 (second booster immunization) (immunization volume: 100 μL per mouse, containing 50 μg protein + an equal volume of adjuvant, fully emulsified before injection). Blood was collected from the orbital rim 14 days after the second booster (day 42), allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and stored at -80℃ for later use.

[0097] (2) Antibody titer detection (indirect ELISA method):

[0098] Coating antigen: Dilute Als3 and Cht3 proteins to 2 μg / mL with coating buffer and add 100 μL to each well. Coat overnight at 4°C. Discard the coating solution and wash 3 times with PBST for 3 min each time.

[0099] Blocking: Add 200 μL of blocking solution to each well, incubate at 37°C for 2 h, and wash 3 times with PBST;

[0100] Add test serum: Serially dilute the mouse serum of each group with blocking buffer (starting from 1:100, serially dilute 2 times to 1:102400), add 100 μL of diluted serum to each well, incubate at 37°C for 1 h, and wash 5 times with PBST;

[0101] Add secondary antibody: Dilute HRP-labeled goat anti-mouse IgG secondary antibody at a ratio of 1:5000 with blocking buffer, add 100 μL to each well, incubate at 37°C for 1 h, and wash 5 times with PBST;

[0102] Color development and detection: Add 100 μL of TMB substrate solution to each well, incubate at room temperature in the dark for 15 min, and then stop the reaction by adding 50 μL of 2M H2SO4. Measure the OD value of each well at 450 nm using a microplate reader.

[0103] Through the above process, the intensity of the antibody response of immunized mice to the antigen protein can be systematically evaluated, and the results show (e.g.) Figure 10 , Figure 11 As shown in the figure, the antibody titers against both Als3 and Cht3 antigens in the serum of mice in the Als3+Cht3 combined immunization group were significantly higher than those in the corresponding single antigen immunization group, indicating that the bivalent vaccine can elicit a stronger humoral immune response.

[0104] Step 8: Protection experiment of bivalent subunit vaccine against challenge in mice

[0105] This step aims to evaluate the protective efficacy of the bivalent vaccine, as detailed below:

[0106] (1) Animal grouping and immunization: Fifty 6-8 week old female BALB / c mice were randomly divided into 5 groups (n=10):

[0107] Group 1 (bivalent high dose): Take Als3 and Cht3 protein stock solutions (1 mg / mL), dilute with PBS to a final concentration of 50 μg / mL for each protein, and vortex emulsify with aluminum hydroxide adjuvant at a volume ratio of 1:1 to prepare a vaccine containing 10 μg of Als3 and 10 μg of Cht3 per 0.2 mL. Each mouse is immunized with Als3 (10 μg) + Cht3 (10 μg).

[0108] Group 2 (bivalent low dose): Prepare the protein working solution as described above, so that the final concentrations of Als3 and Cht3 are both 25 μg / mL. After emulsification, each 0.2 mL of the vaccine contains 5 μg of Als3 and 5 μg of Cht3. Each mouse is immunized with Als3 (5 μg) + Cht3 (5 μg).

[0109] Group 3 (ALS3 single antigen): Prepare a protein working solution with a final concentration of 50 μg / mL of Als3. After emulsification, each 0.2 mL contains 10 μg of Als3. Each mouse is immunized with Als3 (10 μg).

[0110] Group 4 (Cht3 single antigen): Prepare a protein working solution with a final Cht3 concentration of 50 μg / mL. After emulsification, each 0.2 mL contains 10 μg of Cht3. Each mouse is immunized with Cht3 (10 μg).

[0111] Group 5 (Adjuvant control): PBS + adjuvant.

[0112] (2) Immunization and challenge: Mice in each group were immunized intramuscularly on days 0, 14, and 28, with 0.2 mL per mouse. Blood was collected from the tail vein on day 35 to separate serum, which was stored at -80℃. On day 42, Candida albicans SC5314 bacterial suspension (0.2 mL / mouse, containing 5 × 10⁻⁶) was injected via the tail vein. 6 Mice were challenged with CFU (carbohydrate, volatile organic compound). The survival status of mice was observed and recorded daily for 14 days after challenge, and the survival rate was analyzed using the Kaplan-Meier method.

[0113] The results of the challenge experiment showed (e.g.) Figure 12 As shown in the figure, the 14-day survival rate of mice in the high-dose divalent group was as high as 90%, significantly higher than that of the Als3 single antigen group (50%), the Cht3 single antigen group (40%), and the adjuvant control group (20%). The protection rate of the low-dose divalent group was also better than that of the single antigen group. This result fully demonstrates that the Als3 and Cht3 divalent combination provided by this invention can produce a synergistic protective effect and significantly improve resistance to Candida albicans infection.

[0114] In summary, this invention has successfully developed a bivalent subunit vaccine for Candida albicans with good soluble expression, high purity, and strong immunogenicity. Animal experiments have verified its excellent protective effect, providing a new and effective means for the prevention and treatment of Candida albicans infection.

[0115] The sequences involved in this invention are as follows:

[0116] SEQ ID NO:1 (before the Als3 amino acid sequence mutation)

[0117] AKTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTSASPGDTFTLNMPCVFKFTTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTPSIKALGTVTLPLAFNVGGTGSSVDLEDSKCFTAGTNTVTFNDGGKKISINVDFERSNVDPKGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLNDWNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAGGYWQRAPFTLRWTGYRNSDAGSNGIVIVATTRTVTDSTTAVTTLPFDPNRDKTKTIEILKPIPTTTITTSYVGVTTSYSTKTAPIGETATVIVDIPYHTTTTVTSKWTGTITSTTTHTNPTDSIDTVIVQVP

[0118] SEQ ID NO:2 (amino acid sequence of Cht3 before mutation)

[0119] SNVAVYWGQNSGGSQQRLSYYCDSDAVDIVILSFMHQFPSPIQLNFANACEGTYTANGILQCQTIAEDIKYCQNKGKTILLSLGGAAGSYGFSDDATAKQFAHTLWDLFGNSKNLATNDRPFYDAVLDGFDFDIENNWSTGYPALATELRTLFQKDTSKNYYLGAAPQCPYPDASVGPLLKQSEIDFVFIQFYNNYCNLGSSSFNWDTWLNYAETDSPNKNIKLFVGVPASSRAAGSGYNDPSAVSQYLTSDILNSKYFGGISMWDVSAGWSNTNSNGNFVENMKAIVKKASPGEETTSSSTTTTTTTTTSTTISSSSSSSKTSKTSTTSTTSSSISSTTSSTTSSTSSSSTSSSTSSTTSSSTTSSQISTTSTAPTSSTSLSSSTISTSASTSDTTSVTSSETTPVVTPSSLSSAITIPGDSTTTGISKSSSTKPATSTTSALSSSTTTVATIPDDKEIINTPTDTETTSKPPAIITESDATTITQNLTPSTTTKNVKTTSTNIVTEWVWAPTTLRTLTTTYQILTTRTHIETVFAEPSTVVIYN

[0120] SEQ ID NO:3 (After mutation of the Als3 amino acid sequence):

[0121] AKTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLNGTSASPGDTFTLNMPCVFKFTTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTPSIKALGTVTLPLAFNVGGTGSSVDLEDSKCFTAGTNTVTFNDGGKKISINVDFERSNVDPKGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLNDWNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAGGYWQRAPFTLRWTGYRNSDAGSNGIVIVATTRYVTDSTTAVTTLPFDPNRDKTKTIEILKPIPTTTITTSYVGVTTSYSTKTAPIGETATVIVDIPYHTTTTVTSKWTGTITSTTTHTQPTDSIDTVIVQVP

[0122] SEQ ID NO:4 (after mutation of the Cht3 amino acid sequence):

[0123] SNVAVYWGQNSGGSQQRLSYYCDSDAVDIVILSFMHQFPSPIQLNFANACEGTYTAFGILQCQTIAEDIKYCQNKGKTILLSLGGAAGSYGFSDDATAKQFAHTLWDLFGNSKNLATNDRPFYDAVLDGFDFDIEN NWSTGYPALATELRTLFQKDTSKNYYLGAAPQCPYPDASVGPLLKQSEIDFVFIQFYNNYCNLGSSSFNWDTWLNYAETDSPNKNIKLFVGVPASSRAAGSGYNDPSAVSQYLTSDILNSKYFGGISMWDVSAGWS NTNSNGNFVENMKAIVKKASPGEETTSSSSTTTTTTTTSTTISSSSSSSKTSKTSTTSTTSSSISSTTSSTTSSTSSSSTSSSTSSTTSSSTTSSQISTTSTAPTSSTSLSSSTISTSASTSDTTSFTSSETTPVVT PSSLSSAITIPGDSTTTGISKSSSTKPATSTTSALSSSTTTVATIPDDKEIINTPTDTETTSKPPAIITESDATTITQNLTPSTTTKNVKTTSTNIVTEWVWAPTTLRTLTTTYQILTTRTHIETVFAEPSTVVIYN

[0124] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A bivalent subunit vaccine for Candida albicans, characterized in that, The invention includes recombinant Als3 protein and recombinant Cht3 protein as immunogens; the recombinant Als3 protein is obtained by site-directed mutagenesis of the N-glycosylation site and weak epitopes LDGTSASPG and IVIVATTRT based on the sequence shown in SEQ ID NO:1; the recombinant Cht3 protein is obtained by site-directed mutagenesis of the weak epitopes TTSVTSSET and YTANGILQC based on the sequence shown in SEQ ID NO:2 to enhance immunogenicity.

2. The Candida albicans bivalent subunit vaccine according to claim 1, characterized in that, The mutation of the recombinant Cht3 protein is as follows: the two weak epitopes TTSVTSSET and YTANGILQC are mutated to TTSFTSSET and YTAFGILQC respectively; The mutation of the recombinant Als3 protein includes: [mutating the Nth...] 403 The asparagine N site was mutated to glutamine Q, and the weak epitopes LDGTSASPG and IVIVATTRT were mutated to LNGTSASPG and IVIVATTRY.

3. The Candida albicans bivalent subunit vaccine according to claim 1, characterized in that, The SEQ ID NO:1 represents amino acids 17 to 432 from the N-terminus of Als3, and the amino acid sequence of the recombinant Als3 protein is SEQ ID NO:3; The SEQ ID NO:2 represents amino acids 23 to 567 from the N-terminus of Cht3, and the amino acid sequence of the recombinant Cht3 protein is SEQ ID NO:

4.

4. The Candida albicans bivalent subunit vaccine according to claim 1, characterized in that, The vaccine also includes an immune adjuvant, which is aluminum hydroxide adjuvant.

5. A method for preparing a bivalent subunit vaccine of Candida albicans as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1, construct a recombinant expression vector containing the mutant Als3 gene and the mutant Cht3 gene; S2, the recombinant expression vector was transformed into prokaryotic expression strains and induced to express the protein to obtain bacterial cells containing the recombinant protein; S3, the bacterial cells are broken down, the supernatant is collected, and the fusion tag is removed by affinity chromatography; S4 was further purified by ion exchange chromatography or molecular sieve chromatography to obtain high-purity recombinant Als3 antigen and recombinant Cht3 antigen. S5, the recombinant Als3 antigen and recombinant Cht3 antigen are mixed with adjuvant to prepare a bivalent subunit vaccine.

6. The method according to claim 5, characterized in that, In step S2, the host strain expressing the recombinant Als3 protein is E. coli BL21(DE3) pLysS; the host strain expressing the recombinant Cht3 protein is E. coli Rosetta 2(DE3) which can supplement rare codons.

7. The method according to claim 5, characterized in that, The induction conditions in step S2 are: when the bacterial culture OD 600 When the concentration reaches 0.4-0.8, add IPTG to a final concentration of 0.1-0.5 mM and induce at low temperature for 12-20 hours at 15-20℃.

8. The method according to claim 5, characterized in that, In step S3, the affinity chromatography is GST affinity chromatography, and PP enzyme is added to the chromatography column for overnight enzymatic digestion to remove the GST tag; in step S4, the recombinant Als3 protein is purified by anion exchange chromatography combined with elution buffer of 50-100 mM NaCl gradient; the recombinant Cht3 protein is purified by anion exchange chromatography and molecular sieve chromatography in sequence, and the chromatography buffer contains 10% glycerol and 0.05% Tween-20.

9. The use of a bivalent subunit vaccine of Candida albicans as described in any one of claims 1 to 4 in the preparation of a medicament for the prevention or treatment of Candida infectious diseases.

10. The application according to claim 9, characterized in that, The vaccine provides immune protection against Candida albicans by inducing the body to produce specific antibodies against Als3 and Cht3.

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