Design and preparation of recombinant e protein of dengue virus and its use as vaccine antigen

Abstract

This invention discloses a method for designing and preparing a recombinant dengue virus E protein and its application as a vaccine antigen, belonging to the field of biopharmaceuticals. This invention identifies the design and preparation methods for a novel dengue virus E protein, A6, and discloses its nucleotide and amino acid sequences, determining its biological characteristics and immunogenicity. In this invention, A6, as a vaccine antigen, can produce effective immune protection against DENV1-4, showing potential for preventing dengue virus infection. This invention not only provides a highly promising candidate vaccine for broad-spectrum dengue, but also demonstrates a general paradigm for computational protein design in solving the metastable state problem of complex viral antigens, possessing significant biological and medical implications.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to a method for designing and preparing a recombinant E protein of dengue virus and its application as a vaccine antigen. Background Technology

[0002] Dengue virus (DENV) is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family and the Flavivirus genus. It contains three structural proteins and seven non-structural proteins. The envelope glycoprotein E is the most important and most fully exposed protein on the surface of mature viral particles. Based on the different E proteins, the virus is classified into four serotypes (DENV I-IV). The E protein is involved in multiple functions, including host cell attachment, viral infection membrane fusion, and immune evasion, and is also a major target of neutralizing antibodies. DENV is mainly transmitted by mosquitoes such as Aedes albopictus and Aedes aegypti. Due to factors such as intensified global warming, accelerated urbanization, and increased international travel and trade, mosquito habitats are constantly expanding, and the threat of dengue fever (DF) caused by dengue virus is becoming increasingly severe, posing a significant challenge to global public health.

[0003] Faced with the increasingly serious threat of dengue virus outbreaks, vaccines are one of the most economical and effective preventative measures. Currently, two live attenuated dengue vaccines have been approved for use in some countries, and several others are in clinical trials. Marketed vaccines have not been widely adopted due to efficacy and safety concerns, thus necessitating the development of safe and effective dengue vaccines. Various types of vaccines are under development, including live attenuated vaccines, inactivated virus vaccines, subunit vaccines, viral vector vaccines, and nucleic acid vaccines. However, due to the significant ADE effect of dengue virus, vaccines need to provide durable and effective protection against all four serotypes, posing a significant challenge to vaccine development and explaining why no ideal vaccine has been approved to date. Early live attenuated vaccines (LAV), such as CYD-TDV and TAK-003, often cause viral interference and immune response imbalances due to differences in in vivo replication kinetics, posing potential safety risks. To mitigate the risks of live viruses, research has shifted its focus to recombinant subunit vaccines and novel delivery systems.

[0004] Subunit vaccines are vaccines containing specific antigenic components (proteins or peptides) of the target pathogen. They are characterized by rapid production, low cost, and wide applicability, and are widely used in the development of pathogen vaccines. The development of dengue subunit vaccines also faces the challenge of mitigating the effects of adverse drug reactions (ADE). To address this complex immunological challenge, dengue vaccine development has shifted from traditional empiricism to structure-oriented rational design, but it has not yet fully overcome the inherent biophysical barriers of the E protein. However, the core obstacle of this approach lies in the thermodynamic metastability of the E protein: to drive membrane fusion, the natural E protein exhibits dynamic "respiratory" characteristics under physiological conditions, leading to dimer dissociation and exposure of the fusion loop epitope (FLE), which induces ADE. Despite the emergence of numerous sophisticated engineering strategies—such as using glycosylation masking to physically block bad epitopes, constructing consensus sequences using COBRA technology to enhance broad applicability, and even utilizing ferritin nanoparticles and mRNA platforms to improve antigen delivery efficiency—these strategies are often based on an "immunology-first" logic, neglecting the underlying "physics foundation." While consensus sequences are theoretically broad, they often suffer from decreased folding stability due to disruption of co-evolutionary networks. Nanoparticles and mRNA, on the other hand, follow a "garbage-in, garbage-out" principle; if the antigen payload itself has poor thermal stability and is prone to aggregation, even the most advanced LNP delivery systems cannot save the antigen from inducing antibodies in the correct conformation. Therefore, a new paradigm for antigen design is urgently needed that can "lock in" protective conformations from a thermodynamic perspective while adapting to the demands of high-volume industrial production. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a novel dengue virus vaccine. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides a recombinant protein, which is any of the following proteins: A1) The amino acid sequence of the protein is SEQ ID NO:1; A2) The amino acid sequence is the protein of positions 1-397 of SEQ ID NO:1; A3) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A4) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1), A2) or A3).

[0007] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of the present invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the protein isolated in the present invention, as long as they encode and possess the aforementioned protein function, are derived from and equivalent to the nucleotide sequence of the present invention.

[0008] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting Gapexistencecost, Perresiduegapcost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0009] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0010] In some specific embodiments of the present invention, the tag is a histidine tag.

[0011] The present invention also provides a nucleic acid molecule that encodes the aforementioned recombinant protein.

[0012] The present invention also provides an expression cassette containing the aforementioned nucleic acid molecules.

[0013] The present invention also provides a recombinant vector containing the aforementioned nucleic acid molecule or the aforementioned expression cassette.

[0014] The present invention also provides recombinant cells containing the aforementioned nucleic acid molecules, or containing the aforementioned expression cassettes, or containing the aforementioned recombinant vectors.

[0015] In some specific embodiments of the present invention, the recipient cells for obtaining the recombinant cells are HEK293-F cells.

[0016] In some specific embodiments of the present invention, the recombinant cells are obtained by transfecting HEK293-F cells with the aforementioned recombinant vector.

[0017] The expression cassette described herein refers to a DNA molecule capable of expressing the proteins described above in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all the regulatory sequences necessary for the expression of any of the aforementioned proteins. The regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0018] The present invention also provides any of the following applications of the aforementioned recombinant protein: C1) Prepare products for the prevention and / or treatment of diseases caused by dengue virus infection; C2) Preparation of agents for inducing an immune response to dengue virus antigens; C3) Prepare vaccines to prevent diseases caused by dengue virus infection; C4) Preparation of drugs against dengue virus.

[0019] This invention also provides any of the following applications of the aforementioned nucleic acid molecules, expression cassettes, recombinant vectors, or recombinant microorganisms: C1) Prepare products for the prevention and / or treatment of diseases caused by dengue virus infection; C2) Preparation of agents for inducing an immune response to dengue virus antigens; C3) Prepare vaccines to prevent diseases caused by dengue virus infection; C4) Preparation of drugs against dengue virus.

[0020] In some embodiments of the present invention, the anti-dengue virus drug is a reagent that neutralizes dengue virus.

[0021] The present invention also provides a vaccine for the prevention or control of dengue virus, said vaccine comprising the aforementioned recombinant protein.

[0022] In practical applications, the vaccine of this invention can be administered directly to patients or animals as a drug, or mixed with a suitable carrier or excipient and then administered to patients or animals to prevent or treat diseases caused by dengue virus. The carrier materials used here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Various dosage forms can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To formulate unit-dose dosage forms for injection, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injection formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; via cavities, such as rectal and vaginal; via the respiratory tract, such as nasal; and via mucosal administration. Injection is the preferred route of administration.

[0023] The present invention also provides a method for producing the aforementioned recombinant protein, comprising expressing the coding gene of the recombinant protein in a mammal, including introducing the coding gene of the recombinant protein into a recipient cell to obtain a recombinant cell expressing the recombinant protein, culturing the recombinant cell, and expressing the recombinant protein.

[0024] In some specific embodiments of the present invention, the recipient cells for obtaining the recombinant cells are HEK293-F cells.

[0025] In some specific embodiments of the present invention, the recombinant cells are obtained by transfecting HEK293-F cells with the aforementioned recombinant vector.

[0026] This invention also provides a novel dengue virus E protein design method, comprising the following steps: S1: Obtain the original dataset, which includes data of four domains obtained by dividing the extracellular domain of the dengue virus E protein using the Unidoc algorithm. The dengue virus includes four serotypes: dengue virus type I, dengue virus type II, dengue virus type III, and dengue virus type IV. The four domains are named Part-1, Part-2, Part-3, and Part-4, respectively. Each Part-1, Part-2, Part-3, and Part-4 of the E protein is a non-contiguous but spatially independent domain. S2: Obtain the assembly dataset. Assemble candidate molecules according to the rule that Part-1, Part-2, Part-3, and Part-4 are derived from different serotypes, and obtain the assembly dataset, which includes 24 backbone sequences. S3: Obtain the optimized dataset. Perform sequence optimization on the candidate molecules in the assembly dataset according to the PROSS algorithm (Protein Repair One-Stop Shop) to obtain stable variants with different mutation loads, and obtain the optimized dataset, which includes 86 candidate molecules. S4: Obtain the high-energy state sequence dataset. The REF2015 all-atom energy function in the Rosetta molecular modeling suite was used to evaluate the energy of all 86 candidates. Candidate molecules with energy cutoff values ​​within a certain range were screened to obtain the high-energy state sequence dataset, which includes 36 candidate sequences. S5: Obtain a high-scoring dataset. Perform structural orthogonal validation based on AlphaFold 3 deep learning, and remove sequences with pTM < 0.8 to obtain a high-scoring dataset containing 30 candidate sequences. S6: Obtain the final candidate molecules. Use the MMseqs2 algorithm to perform sequence spatial clustering analysis on the candidate sequences in the high-scoring dataset, and screen sequences with consistency of more than 95% (Clusters). From each color-marked cluster, select the sequence with the largest bubble (energy closest to 0) and pTM>0.8 as the representative, resulting in 8 final candidate molecules, named A1–A8.

[0027] The advantages of this invention are that the novel dengue virus E protein provided by this invention can be expressed and purified efficiently, and the purified component is single, stable, non-toxic, and free of impurities, making it suitable for large-scale preparation. As a vaccine antigen, it can produce effective immune protection against dengue virus DENV1-4 and can be used to effectively prevent dengue virus infection or prevention.

[0028] This invention also demonstrates a general paradigm for computational protein design in solving the metastable state problem of complex viral antigens, which has important biological and medical significance. Attached Figure Description

[0029] Figure 1 The sequences and secondary structures of the dengue virus E protein of four serotypes were compared.

[0030] Figure 2 A schematic diagram of UniDoc segmentation and mosaic assembly.

[0031] Figure 3 For computer-aided immunogen maturation and screening: A: Rosetta energy screening; B: Molecular PTM value assessment; C: Molecular cluster analysis.

[0032] Figure 4 Expression, purification, and SDS-PAGE detection of A1-A8.

[0033] Figure 5 Western blot identification for A1-A8.

[0034] Figure 6 The thermal stability of A6 molecules was tested.

[0035] Figure 7 For the detection of specific antibodies in mouse serum.

[0036] Figure 8 To detect the titer of neutralizing antibodies in mouse serum. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0039] In the quantitative experiments in the following examples, two replicate experiments were set up, and the results were averaged.

[0040] Unless otherwise specified, the experimental methods described in the following examples are conventional methods. For detailed steps, please refer to: *Molecular Cloning: A Laboratory Manual* (Sambrook, J., Russell, David W., *Molecular Cloning: A Laboratory Manual*, 3rd edition). rd (edition, 2001, NY, Cold Spring Harbor) and the Pharmacopoeia of the People's Republic of China (edited by the National Pharmacopoeia Commission, Vol. 3, 2020, Chemical Industry Press).

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The BHK-21 cells, dengue virus type I (DENVI), dengue virus type II (DENV II), dengue virus type III (DENV III), and dengue virus type IV (DENV IV) involved in the following examples are disclosed in the non-patent literature “Guo J, Lu J, Du P, et al. Fluorescence Reduction Neutralization Test: A Novel, Rapid, and Efficient Method for Characterizing the Neutralizing Activity of Antibodies Against Dengue Virus. Curr Issues Mol Biol. 2025;47(3):140. Published 2025 Feb 21.doi:10.3390 / cimb47030140”, and are publicly available from the Academy of Military Medical Sciences of the Chinese People's Liberation Army. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0042] Example 1: Design of a novel E protein for dengue virus First, based on structural homology, a systematic deconstruction and recombination design of the envelope glycoprotein E of four dengue virus serotypes was performed. Multiple sequence alignment (MSA) and secondary structure fingerprinting were then used. Figure 1 The inventors confirmed that, despite significant sequence differences (approximately 60-70% homology), the E protein of four dengue virus serotypes still strictly maintains a highly conserved β-sheet topology and disulfide bond network. This characteristic of "sequence variability and structure conservation" provides a solid biophysical basis for modular recombination.

[0043] To accurately define interchangeable structural modules, the inventors utilized the latest domain resolution algorithm, UniDoc, to perform energy-directed segmentation of the E protein extracellular domain sequence. Based on residue contact density and local globularity, UniDoc divided the original three regions of the E protein extracellular domain into four discontinuous but spatially independent domains (Part-1, Part-2, Part-3, and Part-4). The segmentation boundaries (e.g., Part-1: aa 1-68; Part-2: aa 69-115, etc.) cleverly avoided core secondary structural elements and conserved N-glycosylation sites (N67, N153), thereby minimizing damage to the protein's hydrophobic core and post-translational modifications caused by scaffold breakage. This ensures that each independent module maintains its native local conformation after recombination. Figure 2 ).

[0044] Based on the module boundaries defined above, the inventors established a "order-preserving mosaic assembly" strategy. To maximize epitope diversity within a single molecule and avoid the immunogenicity of a single serotype, the inventors introduced a strict combinatorial constraint: in the constructed chimera, the four domain positions (P1-P2-P3-P4) must originate from different serotypes. Under this constraint, the combinatorial space converges to 24 possible permutations (…). Figure 2 This design not only ensures that all chimeric molecules theoretically cover the antigenic characteristics of all four serotypes, but also, through forced cross-serotype splicing, obtains highly compatible backbones that can tolerate evolutionary divergence, laying a diverse foundation for subsequent stability screening.

[0045] Although the 24 mosaic frameworks constructed above achieve modular compatibility in terms of topology, side chain stacking defects or high-energy steric hindrance inevitably exist at non-natural splicing interfaces. This physical mismatch can easily cause proteins to get trapped in high-energy traps or aggregate along the folding path. In order to transform these rough chimeras into foldable entities, the inventors have developed a multi-stage computational screening pipeline designed to simulate and accelerate the "stability maturation" of proteins within a computer.

[0046] First, the inventors introduced the PROSS (Protein Repair One-Stop Shop) algorithm to optimize the original 24 mosaic backbone sequences. PROSS, based on phylogenetic analysis and all-atom energy calculations, can identify and repair evolutionary setbacks in natural proteins. The inventors ran the design under default parameters, but to ensure vaccine efficacy, they imposed strict structural constraints: known broad-spectrum neutralizing epitope residues and key N-glycosylation sites were designated as non-mutagenic regions. This strategy ensured that any improvement in stability did not come at the expense of immunogenicity. Through this step, the inventors generated a series of stable variants with different mutational loads, expanding the candidate library from 24 to 86 (including the original sequence and variants).

[0047] Subsequently, to rigorously control the quality from a thermodynamic perspective, the inventors used the REF2015 all-atom energy function in the Rosetta molecular modeling suite to perform energy assessments on all 86 candidates. For example... Figure 3 As shown in Figure A, the inventors used four natural DENV E proteins (Origin_denv1-4) as energy benchmarks (red area in the figure), with their total Rosetta scores ranging from -550 to -650 REU. In contrast, the computationally optimized designed molecules (blue area in the outer circle of the figure) exhibit a significant downward trend in energy. Notably, the length of the blue band in the pie chart represents the extent of negative energy scores; a longer band indicates higher stability in the computational prediction. Most designed molecules (such as Design_Des24_1) achieved energy scores exceeding -650 and even reaching -662 REU, occupying deeper minimum positions in the energy landscape than natural viruses. This theoretical energy "surpassing" of natural molecules by artificial molecules provides a solid physicochemical basis for subsequent high expression. Based on this, the inventors set a strict energy cutoff value, eliminating sequences that failed to outperform the natural benchmark, and significantly narrowing the candidate range from 86 to 36 high-energy state sequences.

[0048] Next, to eliminate potential biases introduced by a single algorithm, the inventors introduced AlphaFold 3, a deep learning-based algorithm, for structural orthogonality verification. This step aims to confirm whether these low-energy sequences can truly fold into the expected three-dimensional conformation, specifically checking for predicted alignment errors (PAEs) in the manually assembled splice seams. The inventors performed single-unit structure prediction on all sequences filtered by Rosetta and used the prediction template modeling score (pTM score) as the core indicator of structural confidence. (See figure...) Figure 3 (B) The inventors visually compared the pTM values ​​of the designed molecules (cyan region) with those of the natural dengue virus E protein (pink region). The length of the bands in the annular plot directly corresponds to the pTM value. The results showed that the vast majority of the designed molecules exhibited extremely high structural confidence, with pTM values ​​generally exceeding 0.8, which is at the same level as or even higher than the folding confidence of the natural viral protein. This result indicates that despite the inventors' extensive sequence mutations and module recombinations, these molecules maintained a high degree of folding certainty in the AI ​​prediction model. Based on this, the inventors discarded sequences with pTM < 0.8 as potential folding failures, ultimately retaining 30 candidate sequences with "structure-energy dual superiority".

[0049] Ultimately, to select the most representative molecules from these 30 high-scoring sequences for wet experiments and avoid testing highly homologous redundant sequences, the inventors employed the MMseqs2 algorithm for sequence spatial clustering analysis. At a sequence consistency threshold of 95%, the inventors divided the candidate library into 8 independent sequence clusters. The clustering results are presented in bubble chart format. Figure 3 (C) In this visualization, each bubble represents a specific candidate sequence, and the size of the bubble is negatively correlated with the normalized Rosetta energy—that is, the larger the bubble, the lower the energy (the more stable). This visualization strategy clearly reveals the "energy winner" within different clusters. The inventors adopted a "best-in-class" strategy: from each color-coded cluster, the sequence with the largest bubble (lowest energy) and excellent pTM performance was selected as the representative, resulting in 8 final candidate molecules (named A1–A8).

[0050] Example 2: Construction of a novel recombinant expression plasmid for the E protein and its expression and identification in mammalian cells. (1) Construction and identification of novel recombinant expression plasmids for E protein The designed molecular genes (A1-A8) were optimized for human codons and synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. The synthesized gene sequences were then utilized... Hind III and BamH I Two restriction enzyme sites were cloned into the pcDNA3.1(+) (Invitrogen, V79020) vector using conventional molecular biology techniques. After the recombinant plasmid was verified to be correct by sequencing, it became the eukaryotic expression plasmid of the recombinant A1-A8 protein.

[0051] The pcDNA3.1(+)-A6 recombinant protein expression plasmid is formed by replacing the pcDNA3.1(+) DNA fragment with the nucleotide sequence SEQ ID NO:2. Hind III and BamH I A recombinant expression vector was obtained by obtaining a small fragment between two restriction endonuclease recognition sites while maintaining the other nucleotide sequence of pcDNA3.1(+), which expresses the protein whose amino acid sequence is SEQ ID NO:1.

[0052] (2) Expression and identification in mammalian cells The recombinant protein expression plasmids of the constructed A1-A8 proteins were transfected into FreeStyle using the FectoPRO DNA Transfection Reagent (Polyplus, 116-001). TM HEK293-F cells (Invitrogen, R79007). Details are as follows: FreeStyle cells were prepared one day before transfection. TM HEK293-F cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cell pellet was resuspended in FreeStyle 293 medium (Gibco, 12338-018). The cell density was adjusted to 1.0 × 10⁶ cells / min. 6 / mL, aliquoted into 30 mL cell suspensions per bottle, and cultured in a shaker at 125 rpm, 5% CO2, and 37°C. The next day, prepare the transfection complex: take 24 μg of the plasmid to be transfected, dilute it in 3 mL of Opti-MEM medium, gently mix, add 24 μL of FectoPRO transfection reagent, mix well, and incubate at room temperature for 15 min. Then add the mixture to the FreeStyle medium prepared the previous day. TM In HEK293-F cells, gently mix and return to the cell shaker; monitor cell viability starting 48 h after transfection. When cell viability drops to 80-85%, centrifuge at 8,000 rpm for 10 min and collect the supernatant for purification.

[0053] The cell expression supernatant was filtered through a 0.45 μm filter membrane to remove impurities. HisTrap TM Install the 1 mL pre-packed HP column into the AKTA system and continue rinsing with deionized water for 10 min. Then, continue rinsing with PB loading buffer (pH 7.0) after filtration until the baseline stabilizes. Load the filtered cell supernatant at 1 mL / min. After the loading is complete, continue rinsing with PB loading buffer (pH 7.0) until all baselines tend to stabilize. Stop rinsing and switch to tube B for gradient rinsing with PB elution buffer (pH 7.0) (10%, 20%, 50%, 100%) to elute proteins. Start collecting when UV280 reaches 50 and stop collecting when it drops to 50. After the baseline stabilizes, rinse the system with PB loading buffer (pH 7.0).

[0054] Use Hitrap TM Desalting chromatography column will use HisTrap TM Affinity chromatography purified samples underwent desalting treatment using 1×PBS buffer (pH 7.4) as the mobile phase. First, the entire system was washed with 1×PBS buffer until the baseline stabilized, then washed for another 3-5 column volumes. Sample loading was then performed (the loading volume should not exceed one-fifth of the total column volume). The UV280 value was observed to ensure it was close to zero. If not, manual zeroing was required, followed by buffer replacement at a flow rate of 2.5 mL / min. Collection began when UV280 reached 50 and ended when it dropped back to 50. Replacement was terminated if the salt peak overlapped with the protein peak. The obtained elution peak represents the purified protein sample. A portion of the sample was analyzed by SDS-PAGE for concentration determination, while the remainder was aliquoted and stored at -80°C.

[0055] Testing revealed that the expression level of A6 in the purified sample was significantly higher than that of other molecules, and the A6 molecule had high purity with no impurities. The molecular weight of the sample was approximately 60 kDa, and the band size was consistent with the expected molecular weight. Figure 4 (Reducing electrophoresis). The purified protein solution was filtered through a filter membrane for sterilization, and the concentration of A6 protein (500 ng / μL) was determined using a NanoDrop UV spectrophotometer (Thermo Scientific).

[0056] Example 3: Verification of the biological characteristics of molecules First, the obtained protein was subjected to SDS-PAGE. The initial voltage was 80 V, which was adjusted to 120 V after the sample was broken up. After SDS-PAGE electrophoresis, the gel was cut to an appropriate size. Based on the gel size, a suitable PVDF membrane (activated with methanol and then soaked in protein transfer buffer for 10 min) and filter paper (soaked in transfer buffer for 10 min) were cut. Following the order of lower filter paper, PVDF membrane, SDS-PAGE gel, and upper filter paper, the membranes were carefully placed on a semi-dry transfer apparatus, removing as many air bubbles as possible. Transfer was performed using a semi-dry transfer method, with the voltage and current adjusted to 25 V and 1.3 A, respectively, for 10 min. After transferring the protein onto a PVDF membrane, the membrane was removed, the protein side was labeled, and the membrane was blocked with 5% skim milk powder at room temperature for 2 h. After blocking, the concentration of the universal flavivirus detection antibody 4G2 (Abcam, catalog number AB41349) was adjusted to 1 ng / μL using blocking buffer, and the membrane was incubated at 4°C for 2 h. Afterward, the membrane was washed three times with TBST (containing 0.1% Tween-20) for 10 min each time. Finally, goat anti-mouse IgG / horseradish enzyme labeled (Zhongshan Jinqiao, ZB-2305) was added and incubated at room temperature for 45 min. After secondary antibody incubation, the membrane was washed three times with TBST for 10 min each time. Finally, Western blot developing solutions A and B were prepared in a 1:1 volume ratio. The membrane was laid flat on a plate, and the freshly prepared developing solution was added to wet it. The membrane was then exposed and developed using a gel imaging system. Results are as follows: Figure 5 As shown in the (non-reducing electrophoresis) results, the lanes for proteins A4, A6, A7, and A8 show clear and specific bands, indicating that the above molecules are successfully expressed and can specifically bind to the detection antibody 4G2. However, the band for A6 is more prominent. Based on the SDS-PAGE results, A6, which has a better expression level, was selected as the candidate molecule.

[0057] The amino acid sequence of A6 is as follows (SEQ ID NO:1): MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASLSNITTDSRCPTQGEAVLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKGGSHHHHHH.

[0058] Amino acid sequence of dengue virus type I E protein: MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQA.

[0059] Amino acid sequence of dengue virus type II E protein: MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQA.

[0060] Amino acid sequence of type III dengue virus E protein: MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAVLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNALKINWYKKGSSIGKMFEATERGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWVMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGAVVQA.

[0061] The amino acid sequence of dengue virus type IV E protein is: MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVT FKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNI ELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQA.

[0062] The amino acid sequence was optimized using codons commonly used in mammalian cells. The optimized nucleotide sequence encoding A6 is as follows (SEQ ID NO:2):

[0063] Example 4: Detection of the thermal stability of a novel dengue virus E protein Thermal stability is one of the core indicators for evaluating protein drugs, and it is closely related to drug storage, formulation, and manufacturing process. The Tm value, which refers to the temperature at which a protein begins to dissolve, is a key indicator of antibody thermal stability; a higher Tm value indicates stronger heat resistance. To further determine the thermal stability of the novel E protein molecule A6, the inventors used the Uncle protein stability screening platform to detect the Tm and Tagg values ​​(protein aggregation temperature) of A6. First, the protein to be tested was placed in a Uni tube with two replicates. Then, the intrinsic fluorescence of the protein sample was excited at 266 nm, and spectral data was collected. The protein dissolution temperature (Tm value) was determined by the changes in spectral data. Simultaneously, the spectral changes at 473 nm were detected, and the sample aggregation process was recorded to determine the initial aggregation temperature (Tagg value). Finally, the spectral data were analyzed to plot the antibody aggregation and denaturation curve. The results are as follows: Figure 6 As shown, A6 exhibits typical characteristics of a denatured polymer as the temperature increases. Differentiating the aggregation denaturation curves of the two experiments reveals that A6 has a Tm1 value of 70.70℃, which is higher than 65℃, indicating good thermal stability. The Tagg value is 52.30℃, which is higher than 55℃, indicating no abnormality in aggregation temperature (Table 1).

[0064] Table 1. Tm & Tagg Analysis for A6

[0065] Example 5: Preparation and Immunization of a Novel Dengue Virus E Protein Subunit Vaccine in Mice Recombinant protein A6 was dissolved in PBS (pH 7.4), followed by the addition of aluminum hydroxide adjuvant (Alhydrogel). TM 2.0% (purchased from Brenntag Biosector) was used to make the final concentrations of recombinant protein A6 100, 20 and 4 μg / mL, and the final concentration of aluminum hydroxide adjuvant 1 mg / mL. Mice were immunized after incubation at 4°C overnight.

[0066] The mice were immunized as follows: Balb / c mice (6-8 weeks old, female, SPF grade, purchased from Spiford (Beijing) Biotechnology Co., Ltd.) were randomly divided into four groups of 10 mice each. The experimental groups were immunized with the dengue virus novel E protein subunit vaccine prepared above, with doses of 10, 2 and 0.4 μg per mouse, respectively. The control group was injected with an equal volume of aluminum hydroxide adjuvant with a final concentration of 1 mg / mL. The immunization interval was two weeks. Starting from the second immunization, blood was collected from the retro-orbital venous plexus two weeks after each immunization.

[0067] Example 6: Detection of specific antibody levels in mouse serum First, the dengue virus E protein of the four serotypes (Yiqiao Shenzhou, 40533-V08B2, 40532-V08H1, 40471-V08B, 40531-V08H) was diluted to 2 μg / ml using carbonate coating buffer (pH 7.4). Then, 100 μL / well was added to each well of a 96-well ELISA plate (Corning, 9018), with blank control wells not coated with antigen. The plate was incubated overnight at 4°C. The next day, 200 μL of 2% (w / w) skim milk powder in PBS blocking buffer was added to each well, and the plate was incubated at 37°C for 2 h. The plate was then washed 6 times with PBST (PBS containing 0.1% Tween-20). Mouse serum samples (serum from mice obtained in Example 5) were then diluted with PBS blocking buffer containing 2% skim milk powder at an initial dilution of 1:1000, followed by serial dilutions of 2-fold. The diluted samples (100 μL / well) were added to the washed ELISA plate and incubated at 37°C for 1.5 h. After washing 6 times with PBST, 100 μL of horseradish enzyme-labeled goat anti-mouse IgG diluted 1:4000 was added to each well, and the plate was incubated at 37°C for 1 h, followed by 6 washes with PBST. Then, 50 μL of OPD substrate chromogenic solution (50 μL / well) was added to the cleaned ELISA plate, and the plate was incubated at room temperature in the dark for 5 min. After chromogenic development, the reaction was terminated with 2 M sulfuric acid (50 μL / well), and the absorbance was read at 492 nm using a microplate reader. Blank wells were considered negative controls (N), and immunized groups with an OD 492 value (P) greater than 0.3 and a P / N ≥ 2 were considered positive. Antibody levels in each group are expressed as mean ± standard deviation.

[0068] The results are as follows Figure 7 As shown, after two immunizations, all immunization doses could elicit a specific immune response in mice, but the effects of each dose were not significantly different. After three immunizations, the 10 μg / mouse dose (DE-A6-10 μg) significantly increased the specific antibody titer in mouse serum, showing a more obvious dose-dependent relationship compared to the 0.4 μg / mouse (DE-A6-0.4 μg) and 2 μg / mouse (DE-A6-2 μg) dose groups. After four immunizations, the specific antibody titers against dengue virus E protein of all four serotypes in the dose group were 2 × 10⁻⁶. 5 The above demonstrates that A6 successfully stimulated humoral immunity in mice, exhibited good immunogenicity, and suggests the potential for broad-spectrum protective efficacy.

[0069] Example 7: Detection of neutralizing antibody titer in mouse serum BHK-21 cells in good growth condition were digested with trypsin, and the cell density was adjusted to 4 × 10⁻⁶. 5Approximately 2 mL / well was seeded into 6-well cell culture plates and incubated overnight (37°C, 5% CO2). The next day, when the cell density reached over 80%, the 6-well plates were removed, the supernatant was discarded, and 1 mL of fresh DMEM medium was added to each well, gently mixed. The serum from the mice to be tested (serum obtained in Example 5) was then serially diluted 2-fold with DMEM medium, with an initial dilution ratio of 1:10. Viruses (dengue virus type I (DENVI), dengue virus type II (DENV II), dengue virus type III (DENV III), and dengue virus type IV (DENV IV)) stored at -80°C were then retrieved, and the titers of each virus were adjusted to 1 pfu / μL with DMEM medium. 100 pfu of virus solution was mixed with the diluted serum and incubated at 4°C for 1 h to obtain the incubated mixed solutions of each virus. The incubated mixture was then used to infect BHK-21 cells. The cells were incubated statically for 1 h (37℃, 5% CO2), with the 6-well plate gently shaken every 15 min to ensure even contact between the mixture and the cells. After infection, the supernatant was discarded, and the cells were washed twice with sterile PBS buffer. Semi-solid viral culture medium (2% low-melting-point agarose solution mixed 1:1 with filtered sterilized 2×DMEM medium) was quickly added at 2 mL / well. After the medium solidified, the cells were incubated statically for 4–6 days (37℃, 5% CO2) until obvious cytopathic effects appeared (obvious plaques could be observed under a microscope). Finally, 4% paraformaldehyde was added to each well at 1 mL / well, and the cells were fixed at 4℃ for 30 min. The supernatant of solid culture medium was then removed with water, and 1 mL of 0.1% crystal violet solution was added to each well. The cells were stained at room temperature for 30 min, the number of empty plaques was counted, and the serum neutralizing titer was calculated.

[0070] The results are as follows Figure 8 As shown, A6+ adjuvant induced neutralizing antibodies against all four dengue virus serotypes, with neutralizing titers all above 300. In conclusion, although A6 is a completely artificially designed molecule, the neutralization assay results demonstrate that it perfectly retains the natural broad-spectrum neutralizing epitopes, inducing an effective and balanced cross-neutralizing antibody response in mouse models.

[0071] The amino acid sequence of A1: MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTATRCPTQGEPYLPEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFK.

[0072] Amino acid sequence of A2: MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFK.

[0073] Amino acid sequence of A3: MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKLTNITTATRCPTQGEPYLNEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFK.

[0074] The amino acid sequence of A4: MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASLTNITTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKEMLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNALKINWYK.

[0075] The amino acid sequence of A5: MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTDSRCPTQGEATLPEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFK.

[0076] Amino acid sequence of A7: MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNALKINWYK.

[0077] Amino acid sequence of A8: MRCVGIGNRDFVEGLSGATWVDVVLEHGSCCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTATRCPTQGEPYLKEEQDQQYICRRDVVD RGWGNGCGLFGKGGVVTCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMIL LTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFK.

[0078] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A recombinant protein, characterized in that, The recombinant protein is any of the following proteins: A1) The amino acid sequence of the protein is SEQ ID NO:1; A2) The amino acid sequence is the protein of positions 1-397 of SEQ ID NO:1; A3) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A4) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1), A2) or A3).

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant protein of claim 1.

3. An expression box, characterized in that, The expression cassette contains the nucleic acid molecule as described in claim 2.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2 or the expression cassette as described in claim 3.

5. Recombinant cells, characterized in that, The recombinant cells contain the nucleic acid molecules of claim 2, the expression cassette of claim 3, or the recombinant vector of claim 4.

6. Any of the following applications of the recombinant protein according to claim 1: C1) Prepare products for the prevention and / or treatment of diseases caused by dengue virus infection; C2) Preparation of agents for inducing an immune response to dengue virus antigens; C3) Prepare vaccines to prevent diseases caused by dengue virus infection; C4) Prepare drugs to combat dengue virus.

7. Any of the following applications of the nucleic acid molecule of claim 2, the expression cassette of claim 3, the recombinant vector of claim 4, or the recombinant cell of claim 5: C1) Prepare products for the prevention and / or treatment of diseases caused by dengue virus infection; C2) Preparation of agents for inducing an immune response to dengue virus antigens; C3) Prepare vaccines to prevent diseases caused by dengue virus infection; C4) Prepare drugs to combat dengue virus.

8. A vaccine for the prevention or treatment of diseases caused by dengue virus, said vaccine comprising the recombinant protein of claim 1.

9. A method for preparing the recombinant protein of claim 1, comprising expressing the coding gene of the recombinant protein in a mammal, comprising introducing the coding gene of the recombinant protein into a recipient cell to obtain a recombinant cell expressing the recombinant protein, culturing the recombinant cell, and expressing the recombinant protein.

10. A novel dengue virus E protein design method, comprising the following steps: S1: Obtain the original dataset, where, The original dataset includes data from four domains obtained by dividing the extracellular domain of the dengue virus E protein using the Unidoc algorithm. The dengue virus includes four serotypes: dengue virus type I, dengue virus type II, dengue virus type III, and dengue virus type IV. The four domains are named Part-1, Part-2, Part-3, and Part-4, respectively. Each Part-1, Part-2, Part-3, and Part-4 of the E protein is a non-contiguous but spatially independent domain. S2: Obtain the assembly dataset. Assemble candidate molecules according to the rule that Part-1, Part-2, Part-3, and Part-4 are derived from different serotypes, and obtain the assembly dataset, which includes 24 backbone sequences. S3: Obtain the optimized dataset. Perform sequence optimization on the candidate molecules in the assembly dataset according to the PROSS algorithm to obtain stable variants with different mutation loads, and obtain the optimized dataset, which includes 86 candidate molecules. S4: Obtain the high-energy state sequence dataset. The REF2015 all-atom energy function in the Rosetta molecular modeling suite was used to evaluate the energy of all 86 candidates. Candidate molecules with energy cutoff values ​​within a certain range were screened to obtain the high-energy state sequence dataset, which includes 36 candidate sequences. S5: Obtain a high-scoring dataset. Perform structural orthogonal validation based on AlphaFold 3 deep learning, and remove sequences with pTM < 0.8 to obtain a high-scoring dataset containing 30 candidate sequences. S6: Obtain the final candidate molecules. Use the MMseqs2 algorithm to perform sequence spatial clustering analysis on the candidate sequences in the high-scoring dataset. Select sequences with consistency of more than 95%. From each color-marked cluster, select the sequence with the largest bubble (energy closest to 0) and pTM>0.8 as the representative, resulting in 8 final candidate molecules, named A1–A8.

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