A recombinant fusion protein derived from the stem region of dengue virus E protein, preparation method and application thereof

Abstract

The application discloses a recombinant fusion protein derived from a stem region of dengue virus E protein, a preparation method and application thereof. The structure of the recombinant fusion protein is Loop-H2-H1-Loop-H2. The recombinant fusion protein can be expressed in Expi293F cells, has high expression amount, and has simple preparation method and easiness in purification. The recombinant fusion protein H212-Fc provided by the application has high-efficiency and broad-spectrum anti-4-type dengue virus activity at a cell level, and has anti-zika virus activity, and has no antibody-enhanced virus infection effect at a cell and animal level. In-vivo experiments prove that the fusion protein has effects of inhibiting dengue virus replication, protecting pathological damage and reducing mortality. The fusion protein provided by the application can be used as a detection raw material for a membrane fusion process of dengue virus and zika virus, and has wide application prospects in the field of dengue virus and zika virus protein drug development.

Description

Technical Field

[0001] This invention relates to recombinant fusion proteins, and particularly to a recombinant fusion protein derived from the stem region of dengue virus E protein, its preparation method, and its applications. Background Technology

[0002] dengue virus ( dengue virus Dengue fever (DENV) belongs to the genus Flaviviridae in the family Flaviviridae. It is a single-stranded positive-sense RNA virus with a genome length of approximately 10.7 kb. In tropical and subtropical regions, it is mainly transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes, causing varying degrees of dengue fever. Dengue fever is the most widespread, most prevalent, and most harmful mosquito-borne viral disease globally. Approximately 400 million people worldwide are infected with DENV each year. According to incomplete statistics from the WHO, in 2024, more than 14.43 million cases of dengue fever were reported globally, including 11,201 deaths. Currently, there are no effective drugs against dengue virus; existing treatments are mainly symptomatic.

[0003] Dengue virus is a typical type II enveloped virus, and its surface membrane protein (E) plays a major role in its entry into cells. The E protein consists of five domains: EDI, EDII, EDIII, the stem (S), and the transmembrane (TM). On the surface of mature viral particles, the E protein is a dimer. The EDI, EDII, and EDIII domains are rich in β-sheets, forming antiparallel dimer structures on the membrane surface. The stem (S) is the membrane-adjacent region, composed of two conserved α-helix-rich domains, H1 and H2, and a loop peptide linking them. The transmembrane (TM) anchors the E protein to the viral lipid bilayer membrane.

[0004] Dengue virus entry into cells can be divided into two stages: adsorption and membrane fusion. (1) Adsorption: Dengue virus binds specifically or non-specifically to receptors on the cell membrane surface through the E protein E1, EDII, and EDIII, especially the EDIII domain. Subsequently, the virus is endocytosed into the endosome. (2) Membrane fusion: In the acidic pH (<6.2) environment of the endosome, the conformation of the viral E protein undergoes a series of changes: the dimer conformation changes to the trimer, the EDIII domain folds and binds to the E1 domain, and the fusion loop of the EDII domain inserts into the endosome membrane, thus forming an open E protein trimer conformation, called the fusion intermidiate. Subsequently, the H1-Loop-H2 in the stem region binds to the surface groove of the fusion intermidiate, similar to a zipper, polymerizing the open E protein trimer to form a closed E protein trimer, called the post-fusion structure. The closure of the stem region, resembling a zipper, brings the endosomal membrane and viral membrane closer together, promoting their fusion to form a fusion pore, allowing viral genetic material to be released into the cytoplasm. Currently, research on the mechanism of the dengue virus membrane fusion stage and drug development are limited. Dengue virus (DENV) has four serotypes, and repeated infection with different serotypes easily induces antibody-dependent enhancement (ADE): non-neutralizing antibodies induced by previous infection can mediate viral entry into cells and massive replication, significantly increasing the risk of severe illness. The biggest bottleneck in dengue vaccine development is currently the difficulty in balancing broad-spectrum neutralization with avoiding ADE.

[0005] In earlier studies, researchers synthesized peptides of different lengths derived from the dengue virus stem region, specifically H1 and H2. Activity assays revealed that the H1-derived peptides lacked antiviral activity, but several H2-derived peptides could bind to the dengue virus E protein trimer at a concentration of μM, exhibiting broad-spectrum activity against four dengue virus serotypes. Among these, DV-2 (419-447 aa) showed the highest antiviral activity, with an IC50 value of [missing information]. 90 The peptide reaches 250 nM and even exhibits anti-Zika virus activity. However, the peptide derived from the H2 domain of the dengue virus stem region has low water solubility and is easily degraded. Designing drugs that mimic the native conformation of the H1 and H2 domains before fusion to inhibit E protein trimer polymerization and block the fusion process between the viral membrane and the cell membrane is one of the breakthroughs in the design of antiflavin drugs (such as dengue and Zika viruses). Simultaneously, designing drugs that enhance water solubility and stability while avoiding ADE (anti-adsorption-desorption) is one of the current challenges in antiflavin drug design. Summary of the Invention

[0006] To address the above technical problems, the present invention aims to provide a recombinant fusion protein derived from the stem region of dengue virus E protein and its applications.

[0007] The technical solution of the present invention is as follows: A recombinant fusion protein derived from the stem region sequence of dengue virus E protein, named H212, has a protein structure of Loop-H2-H1-Loop-H2. The H212 protein is composed of amino acid sequences from the stem region sequence of dengue virus E protein, specifically the H2, Loop, and H1 sequences linked together. Specifically, the H1 amino acid sequence is as shown in SEQ ID NO: 1, and one or more amino acids are substituted, deleted, and / or added to obtain amino acids with the same function. Similarly, the H2 amino acid sequence is as shown in SEQ ID NO: 2, and one or more amino acids are substituted, deleted, and / or added to obtain amino acids with the same function. Finally, the Loop amino acid sequence is as shown in SEQ ID NO: 3, and one or more amino acids are substituted, deleted, and / or added to obtain amino acids with the same function.

[0008] Preferably, the recombinant fusion protein further includes Fc, with the protein structure Loop-H2-H1-Loop-H2-linker-Fc, that is, the H212 protein of claim 1 is coupled with the Fc fragment of human IgG2 to form the H212-Fc fusion protein, and the amino acid sequence of the linker is shown in SEQ ID NO: 4.

[0009] Furthermore, the amino acid sequence of the recombinant fusion protein H212 is shown in SEQ ID NO: 5, and the nucleotide coding sequence encoding the protein is shown in SEQ ID NO: 7.

[0010] Furthermore, the amino acid sequence of the recombinant fusion protein H212-Fc is shown in SEQ ID NO: 6, and the nucleotide coding sequence encoding the protein is shown in SEQ ID NO: 8.

[0011] Furthermore, the recombinant protein H212-Fc can specifically bind to the H2 stem region of the dengue virus E protein, inhibiting the fusion of dengue virus with the host cell membrane, and does not induce antibody-dependent enhancement.

[0012] The present invention also protects the gene encoding the recombinant fusion protein.

[0013] The present invention also protects a recombinant expression vector for expressing the recombinant fusion protein, the recombinant expression vector containing the gene encoding the recombinant fusion protein.

[0014] The present invention also protects a method for preparing a recombinant fusion protein, comprising the following steps: (1) constructing a recombinant expression vector encoding a nucleotide sequence of the recombinant fusion protein as claimed in any one of claims 1-5; (2) transfecting the recombinant expression vector into host cells Expi293F cells to induce expression; (3) purifying the recombinant protein by affinity chromatography and size exclusion chromatography to obtain a recombinant protein with a purity ≥95%.

[0015] The present invention also protects the recombinant fusion protein, the gene encoding the recombinant fusion protein, the recombinant expression vector of the recombinant fusion protein, and the use of the recombinant fusion protein prepared by the method in the preparation of dengue virus and Zika virus antigen detection reagents, anti-dengue virus vaccines or specific antibodies, and preventive and / or therapeutic drugs or reagents.

[0016] Furthermore, the dengue virus mentioned includes DENV-1, DENV-2, DENV-3, and DENV-4.

[0017] Because it is derived from the dengue virus E protein stem region sequence H1, Loop, and H2, the present invention provides a recombinant fusion protein H212-Fc derived from the dengue virus E protein stem region sequence, which can mimic the function of the dengue virus E protein stem region H2 sequence and act on the membrane fusion stage of dengue virus and Zika virus. It is a novel protein drug for inhibiting these two types of viruses and is significantly different from peptides derived from H2.

[0018] Based on this, the inventors designed a stem region H2-H1-H2 fusion protein, coupled it with a human Fc fragment, to form the recombinant fusion protein H212-Fc. This protein can be efficiently expressed in eukaryotic cells Expi293F, is easy to purify, and exhibits broad-spectrum anti-dengue virus and Zika virus activity both in vitro and in vivo. Moreover, compared to the dengue virus stem region H2-derived peptide, the recombinant fusion protein H212-Fc exhibits longer stability in vitro and better antiviral activity in mice.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The recombinant fusion protein H212-Fc provided by this invention can be efficiently expressed in eukaryotic cells Expi293F and is easy to purify.

[0020] 2. The recombinant fusion protein H212-Fc provided by this invention exhibits better stability compared to peptide H2. It demonstrates longer-lasting antiviral activity at the cellular level and better antiviral efficacy at the animal level.

[0021] 3. The recombinant fusion protein H212-Fc provided by the present invention can form and maintain a stable dimer structure, mimicking the conformation of the dengue virus membrane fusion intermediate state, and can be used as a detection raw material for detecting the dengue virus membrane fusion process.

[0022] 4. The recombinant fusion protein H212-Fc provided by this invention exhibits broad-spectrum dengue and Zika virus activity. Furthermore, it does not demonstrate antibody-dependent enhancement (ADE) at either the cellular or animal levels. It shows great promise for the development of protein drugs for the prevention or treatment of dengue and Zika viruses.

[0023] The H212-Fc recombinant protein of this invention has a completely different mechanism of action compared with the neutralizing antibodies targeting E protein DIII / prM in the prior art: the molecule of this invention targets the recessive epitope of the H2 stem region of the E protein, binds only after the virus enters the endosome (37°C conformation), inhibits the membrane fusion stage, and has been confirmed by mouse experiments to not induce ADE at all, while achieving broad-spectrum inhibition of dengue virus type IV and Zika virus. Attached Figure Description

[0024] Figure 1 These are schematic diagrams of the recombinant fusion protein H212-Fc structure and its expression and purification. Figure 1 A is a schematic diagram of the structure of H212-Fc. H212-Fc is composed of the Loop-H2-H1-Loop-H2 from the dengue virus stem region and the Fc of human IgG2 linked together by GGSGG. Figure 1 B is the SDS-PAGE electrophoresis image of H212-Fc; Figure 1 C is a Naive-PAGE electrophoresis image of H212-Fc; Figure 1 D is the Western-Blotting plot of H212-Fc; Figure 2 The image shows the construction, physicochemical properties, purification, and identification results of the H212-Fc fusion protein. Figure 2 A represents the predicted amino acid sequence and secondary structure of the fusion protein; Figure 2 B represents the predicted results of the protein's physicochemical properties; Figure 2 C is the affinity chromatography pattern of Protein A; Figure 2 D is the SDS-PAGE identification result of the purified product; Figure 3 This is a graph showing the toxicity of the recombinant fusion protein H212-Fc to Vero cells. Compared with control cells, H212-Fc protein did not show detectable toxicity to Vero cells, and its CC... 50 Value greater than 5 μM; Figure 4 This is a graph illustrating the broad-spectrum anti-dengue virus activity of the recombinant fusion protein H212-Fc at the cellular level. Figure 4 A shows that immunofluorescence assays revealed that H212-Fc can inhibit the replication of dengue virus type II (DENV-2); Figure 4 B indicates that Western blotting experiments have shown that H212-Fc can inhibit the replication of dengue virus type II. Figure 4 C indicates that plaque assays showed H212-Fc can inhibit dengue virus type II replication; Figure 4 D indicates that flow cytometry experiments showed that H212-Fc can inhibit the replication of dengue virus types I, II, III, and IV; Figure 5 This is a graph showing the anti-Zika virus activity of the recombinant fusion protein H212-Fc at the cellular level. Immunofluorescence experiments showed that H212-Fc can inhibit the replication of dengue virus type II (DENV-2). Figure 6 This is a diagram illustrating the long-lasting antiviral activity of the recombinant fusion protein H212-Fc at the cellular level. Figure 6 A indicates that, compared to the dengue virus H2 peptide, H212-Fc is less prone to degradation in Vero cells; Figure 6 B indicates that, compared to the dengue virus H2 peptide, H212-Fc still exhibits highly efficient antiviral activity after co-incubation with Vero cells for 96 h. Figure 7 This study investigated the in vitro ADE effect of the recombinant fusion protein H212-Fc. Compared with the positive control 4G2 antibody, the H212-Fc protein did not induce the ADE effect in THP-1 cells. Figure 8 This study investigated the ADE effect of the recombinant fusion protein H212-Fc in AG129 mice. Figure 8 A is the flowchart for ADE modeling in AG129 mice infected with dengue virus. Compared to the positive control 4G2 antibody, Figure 8 B, the H212-Fc protein, does not reduce the body weight of AG129 mice; Figure 8 C indicates that H212-Fc protein does not enhance the death of AG129 mice. Figure 8 D indicates that H212-Fc protein does not enhance the pathological damage in the brain tissue of AG129 mice; Figure 8 E indicates that the H212-Fc protein does not enhance viral replication in the liver tissue of AG129 mice; Figure 9 The protective effect of a single dose of recombinant fusion protein H212-Fc against DENV-2 infection in AG129 mice is shown in the figure. Figure 9 A is a flowchart of a single drug administration; Figure 9 B represents a reduction in viral load in the blood; Figure 9C reduces viral load in tissues; Figure 10 This is a graph showing the protective effect of repeated administration of the recombinant fusion protein H212-Fc against DENV-2 infection in AG129 mice. Figure 10 A is a flowchart of multiple dosing procedures; Figure 10 B represents a reduction in viral load in the blood; Figure 10 C reduces viral load in tissues; Figure 11 Here are electrophoresis images and antiviral activity images of another recombinant fusion protein, H121-Fc, after purification. Figure 11 A is the electrophoresis image of H121-Fc purification; Figure 11 B represents the anti-DENV-2 activity of H121-Fc at the cellular level; Figure 12 Drug administration experiments to different cells revealed that the recombinant integrin H212-Fc acts on the membrane fusion phase of viral infection. Figure 12 A. Flowchart and results of a timed dosing experiment; Figure 12 B shows the experimental process and results of directly killing the virus. Figure 12 C shows the adsorption experiment flow and results. Figure 12 D is a diagram showing the experimental process and results of fusion blocking; Figure 13 The interaction between the recombinant integrin H212-Fc and viral H2 inhibits membrane fusion; Figure 13 A shows the colocalization of H212-Fc and viral E protein on the endosomal membrane; Figure 13 Figure B shows the results of the Co-IP experiment, which revealed that the virus particles bound to H212-Fc at 37℃. Figure 13 Figure C shows the results of the liposome fusion blocking experiment, which revealed that H212-Fc inhibits dengue virus membrane fusion. Figure 13 D is the result of the ELISA experiment, which showed that H212-Fc has a high affinity for H2; Figure 13 E is the result of the ELISA experiment. It was found that the high affinity between H2 and H2 may be due to the high affinity between H212-Fc and H2. Figure 13 F is the molecular docking result diagram, which supports the high affinity between H212-Fc and H2. Detailed Implementation

[0025] The technical solution of the present invention will be further explained below with reference to experiments.

[0026] I. Materials and Reagents: 1. Main materials Viruses: Clinical isolates of dengue virus (DENV) types I, II, III, and IV (GenBank numbers: JQ317743.1; KY882458; KR296743.1; KY672960.1, respectively); clinical isolate of Zika virus (GenBank number: MH055376.1). Plasmid amplification strain: E. coli DH5α competent cells; protein expression strain: E. coli BL21(DE3) competent cells (Beijing Novozymes). Vero cells, C6 / 36 cells, THP-1 cells, and Expi293F cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). AG129 mice: derived from the National Institutes for Food and Drug Control, China

[0027] 2. Main reagents LB medium, 2×YT medium (Sigma); DMEM medium, FBS (Gibco); Linear PEI transfection reagent (Yeasen) Ampicillin (Sigma-Aldrich, St Louis, USA) Endotoxin-free plasmid large-scale extraction kit (Tiangen, Beijing) SMM 293-ENH Expression MediuM Enhancer (SionBiological, Beijing) SMS 293-SUPI Expression MediuM Supplement (SionBiological, Beijing) TaKaRa BCA Protein Assay Kit (Takara Bio, Beijing) Trizol reagent (Invitrogen, CA, USA) Protein A 4FF (GE) H2 control peptide: AWDFGSLGGVFTSIGKALHQVFGAIYGAA, synthesized from Shanghai Jierui Biotechnology. Anti-flavin 4G2 antibody (Genetex) Anti-DENV-1 / 3 / 4 Envelope protein E / EDIII domain Antibody, Rabbit MAb (Yi Qiao Shen Zhou) Ultra High Sensitivity ECL Kit (Guangzhou Levobio)

[0028] This invention protects a recombinant fusion protein derived from the stem region sequence of the dengue virus E protein. The recombinant fusion protein is named H212, and its protein structure is Loop-H2-H1-Loop-H2. The H212 protein is composed of amino acid sequences from the stem region sequence of the dengue virus E protein, specifically the sequences of H2, Loop, and H1. Specifically, the amino acid sequence of H1 is as shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 is obtained by substituting, deleting, and / or adding one or more amino acids to achieve the same function; the amino acid sequence of H2 is as shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 2 is obtained by substituting, deleting, and / or adding one or more amino acids to achieve the same function; and the amino acid sequence of Loop is as shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3 is obtained by substituting, deleting, and / or adding one or more amino acids to achieve the same function.

[0029] The amino acid sequence of the recombinant fusion protein H212 is shown in SEQ ID NO: 5, and the nucleotide coding sequence encoding the protein is shown in SEQ ID NO: 7.

[0030] The recombinant fusion protein further includes Fc, with the protein structure Loop-H2-H1-Loop-H2-linker-Fc, which means that the H212 described in claim 1 is coupled with the Fc fragment of human IgG2 to form the H212-Fc fusion protein, and the amino acid sequence of the linker is shown in SEQ ID NO: 4.

[0031] The amino acid sequence of the recombinant fusion protein H212-Fc is shown in SEQ ID NO: 6, and the nucleotide coding sequence encoding the protein is shown in SEQ ID NO: 8.

[0032] Example 1: Construction and preparation method of recombinant fusion protein eukaryotic expression vector pCAGGS-H212-Fc

[0033] 1.1. Construction of the eukaryotic expression vector pCAGGS-H212-Fc for the recombinant fusion protein: Based on the amino acid sequence corresponding to the H212-Fc fusion protein (SEQ ID NO: 6), its encoding nucleotide sequence (SEQ ID NO: 7) was artificially designed and optimized. Sequence optimization and human codon preference optimization of the full-length gene were performed, and gene synthesis was commissioned to Beijing Haichuang Biotechnology Co., Ltd. The optimized target gene fragment was directionally inserted into the PCAGGS vector carrying the tPA signal peptide to construct the eukaryotic recombinant expression vector pCAGGS-H212-Fc capable of expressing the secretory H212-Fc fusion protein. The results are shown in Figures 1 and 2.

[0034] The constructed pCAGGS-H212-Fc expression vector was transformed into E. coli DH5α competent cells and placed in LB liquid medium containing 50 μg / mL ampicillin. The cells were cultured at 37 °C with shaking for 12 h. After amplification, a large number of plasmids were extracted for later use.

[0035] 1.2 Expression of recombinant fusion protein H212-Fc (1) Culture of Expi-293F cells: One hour before transfection, the density of Expi-293F cells was adjusted to 2.5-3×10⁻⁶. 6 Transfer cells / mL to the desired volume of a cell shake flask and place on a shaker for further adaptation culture for 30 minutes.

[0036] (2) Plasmid transfection: Taking a 200 mL system as an example, the amount of target plasmid used is 200 μg.

[0037] ① Dilute the plasmid: Take 200 μg of plasmid and add it to 10 mL of serum-free 293 medium (generally 5% of the total volume required for transfection), and mix gently (avoid air bubbles).

[0038] ② Dilute PEI: Take 1200 μL of 1 mg / mL PEI, add it to 10 mL of serum-free 293 medium, and mix gently.

[0039] ③ Mixing the complex: Slowly add the diluted PEI to the diluted plasmid (do not add in reverse), mix gently immediately, and let stand at room temperature for 15 minutes to form the PEI-DNA complex.

[0040] Slowly add 20 mL of PEI-DNA complex to 180 mL of 293F cell suspension with adjusted density, gently shake the flask to mix, and return it to a shaker at 37°C, 8% CO2, and 120 rpm for incubation.

[0041] 24 hours after transfection, add the enhancer according to the SMM 293-ENH Expression Medium Enhancer instructions. 48 hours after adding the enhancer, add the SMS 293-SUPI Expression Medium Supplement according to the instructions. (Note that the SMS 293-SUPI Expression Medium Supplement instructions specify adding it every 48 hours after transfection, but since the enhancer contains this component, repeated additions are unnecessary. Therefore, the supplement only needs to be added 1-2 times during the 7 days of culture after transfection. The main component of this supplement is sugar; adding too much can lead to excess nutrients and inhibit expression.)

[0042] 1.3 Purification of recombinant fusion protein H212-Fc (1) Concentration of supernatant: Centrifuge the collected culture medium supernatant sample at 8000 rpm for 5 min, keep the supernatant and discard the cell pellet, and further concentrate the collected supernatant to 20 mL using a 30 kDa ultrafiltration tube.

[0043] (2) Desalting and purification: 5 mL of the sample was passed through a 15 mL Sephadex G-25 desalting column for column treatment, and the bound protein was separated using elution buffer. Then, a Protein A 4FF affinity chromatography column was connected to the chromatography system, and the Protein A 4FF affinity chromatography column was equilibrated using equilibration buffer at a flow rate of 1 mL / min. The column volume was washed with 5-10 column volumes. 100 mL of the desalted sample was purified per 1 mL Protein A 4FF affinity chromatography column. A pre-calculated volume of neutralization buffer (e.g., 500 μL of 1M Tris-HCl pH 8.0 per tube, corresponding to 1 mL of elution buffer collected later) was added to each collection tube in advance to prevent protein denaturation due to the acidic environment after elution. The target protein was eluted with elution buffer at a low flow rate of 1 mL / min, and the A280 value was observed in real time. When the A280 value begins to rise (>0.02AU), focus on collecting the fraction corresponding to the elution peak until the A280 value falls back to the baseline and then stop collecting; immediately mix the liquid in the tube after the collection is complete and place it on ice.

[0044] The collected eluents were combined and concentrated to a volume of 1-1.5 mL by centrifugation at 3000×g for 40 min at 4 °C using an ultrafiltration tube with a molecular weight cutoff of 30 kDa. During the concentration process, the buffer was replaced 2-3 times with equilibration buffer (PBS pH 7.2-7.4) to remove residual elution buffer components and avoid affecting subsequent experiments.

[0045] Example 2 Electrophoretic analysis and identification of purified recombinant fusion protein H212-Fc 2.1. Protein content determination: The purified protein was quantified using a BCA kit.

[0046] 2.2. SDS-PAGE: 50 μg of H212-Fc protein was separated by electrophoresis using a 10% SDS-PAGE gel. After electrophoresis, Coomassie Brilliant Blue staining and destaining were performed. Analysis of the results showed that the H212-Fc monomer protein was approximately 32 kDa. Figure 1 B).

[0047] 2.3 Native-PAGE: 50 μg of H212-Fc protein was separated by electrophoresis using a 10% Native-PAGE gel. After electrophoresis, Coomassie Brilliant Blue staining and destaining were performed. Analysis of the results showed that the H212-Fc protein was approximately 65 kDa, indicating that it has a dimer structure. Figure 1 C).

[0048] 2.4 Western Blotting to Verify H212-Fc Protein Expression: 10 μg of H212-Fc protein was separated by electrophoresis using a 10% Native-PAGE gel. After electrophoresis, the protein was transferred to a PVDF membrane and developed using anti-human IgG-Fc antibody (HRP).

[0049] The results showed that the H212-Fc monomeric protein size was 32 kDa. Figure 1 D). SDS-PAGE analysis of the purified product showed that the target band was located at approximately 35 kDa, which is consistent with the theoretical molecular weight, indicating that high-purity H212-Fc fusion protein was obtained. Figure 2 D).

[0050] Example 3: Cellular toxicity study of recombinant fusion protein H212-Fc The cytotoxicity of recombinant H212-Fc protein to Vero cells was evaluated using the CCK-8 assay. The specific method was as follows: (1) Cell seeding: Select Vero cells in good growth condition and seed 100 μL of cell suspension (i.e., 1×10⁻⁶ cells per well) into each well. 4 Cells (per well) were seeded in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours until 80% cell adhesion was achieved before use.

[0051] (2) Adding the drug: Dilute H212-Fc protein serially with culture medium, and add 100 μL / well to Vero cells. Incubate at 37°C and 5% CO2 for 24 hours.

[0052] CCK-8 assay: Following the instructions, add 10 μL of CCK-8 reagent to each well and incubate for 4 hours. After incubation, measure the absorbance (OD value) at 450 nm using a multi-mode microplate reader (reference wavelength 450 nm). Based on the experimental results, plot the dose-response curve using GraphPad Prism 10 software. Calculate the drug's CCK-8 concentration (CC) using the Reed & Muench method. 50 value.

[0053] No cytotoxicity of H212-Fc protein to Vero cells was detected; compared with the control, its CC... 50 Value greater than 5 μM ( Figure 3 ).

[0054] Example 4: Study on the anti-dengue virus activity of recombinant fusion protein H212-Fc at the cellular level

[0055] 4.1 Immunofluorescence assays showed that H212-Fc could inhibit the replication of dengue virus type II (DENV-2). (1) Cell infection and drug treatment: Cell spreaders of the corresponding size were added to 24-well plates for plating. Vero cells were seeded in 24-well plates (3.5 × 10⁻⁶ cells / well). 5 DENV-2 virus (MOI=0.05) and serially diluted H212-Fc protein were added to the culture medium (cells / well); after incubation at 37 ℃ for 4 h, the cells were washed three times with PBS. 200 μL of fresh culture medium was added, and the cells were incubated for another 24 h.

[0056] (3) Fixation: Discard the culture medium, slowly add 0.6 mL of pre-cooled 4% paraformaldehyde along the side wall, fix at -20℃ for 20 min, add 0.1% Triton X-100 after the fixation, and permeate the cells at room temperature for 30 min.

[0057] (3) Washing: After washing 3 times with PBS, add 400 μL of 1% BSA blocking solution to each well, block at room temperature for 1 h, and wash 3 times with PBST;

[0058] (4) Antibody color development: The cells were incubated with anti-flavin 4G2 mouse monoclonal antibody (1 μg / mL) at room temperature for 2 h, followed by washing 3 times. Anti-mouse Fc-FITC antibody (1:5000) was added as secondary antibody. The cells were incubated at room temperature in the dark for 1 h, followed by washing 3 times with PBST. After the cell slides were dried in the dark, 5 fields of view were randomly selected for photographing using an immunofluorescence microscope.

[0059] The results showed that, compared with the positive control, H212-Fc protein at a concentration of 0.0448 μM reduced viral E protein expression by more than half, indicating that it inhibits the EC50 of DENV-2 replication. 50 Below 0.0448 μM ( Figure 4 A).

[0060] 4.2 Western blotting experiments showed that H212-Fc can inhibit the replication of dengue virus type II. (1) Cell drug administration and infection treatment: 3.5 × 10⁻⁶ cells were added to a 24-well plate. 5 Vero cells were cultured in wells. Type II dengue virus (DENV-2, MOI=0.05) and serially diluted H212-Fc protein, along with control H2 peptide, were added to the culture medium. After incubation at 37°C for 4 h, the cells were washed three times with PBS. 200 μL of fresh culture medium was added, and the cells were cultured for another 24 h.

[0061] (2) Protein sample collection: Collect sample cells, positive control cells, and negative control cells, every 10 7 Add 200 μL of cell lysis buffer containing PMSF and aprotinin to each cell pellet, incubate on ice for 30 min to allow the cells to fully lyse, and then centrifuge at 12000 g for 5 min to collect the supernatant protein.

[0062] (3) Electrophoresis and transfer: Take 20 μL of supernatant protein, pass it through 10% SDS-PAGE electrophoresis, and then transfer it to a PVDF membrane using a wet transfer apparatus at a constant current of 300 mA for 45 min.

[0063] (4) Antibody color development: Pan Dengue virus (DENV) E / Envelope Protein (Domain III) Antibody at a dilution of 1:5000 was used as the primary antibody, and goat anti-rabbit IgG was used as the secondary antibody. The target band was detected by ECL Kit.

[0064] The results showed that, compared with the positive control, H212-Fc protein at a concentration of 0.224 μM reduced viral E protein expression by more than half, indicating that it inhibits the EC2 expression of DENV-2 replication. 50 Below 0.224 μM ( Figure 4 B).

[0065] 4.3 Plaque assays showed that H212-Fc can inhibit the replication of dengue virus type II. (1) Cell drug administration and infection treatment: 3.5 × 10⁻⁶ cells were added to a 24-well plate. 5Vero cells per well. Type II dengue virus (DENV-2, MOI=0.05) and serially diluted H212-Fc protein were added to the culture medium; after incubation at 37°C for 4 h, the cells were washed three times with PBS.

[0066] (2) Fixation: Take out 4% low melting point agarose kept at 40℃ and mix it with 4% FBS serum DMEM preheated to 37℃ at a volume ratio of 1:1 (final agarose concentration of 1% to reduce toxicity to cells). Gently mix to avoid generating bubbles. Slowly add 2 mL of the above 2% agarose mixture to each well (slowly flow down the well wall to avoid dispersing the cell monolayer). Place at room temperature for 15-20 minutes. After the agarose has completely solidified, put the 24-well plate back into the 37℃, 5% CO2 incubator and incubate for 7 days.

[0067] (3) Staining: After incubation, take out the 6-well plate and slowly add 2 mL of 0.1% crystal violet staining solution along the well wall at room temperature to ensure that the entire agarose surface is covered. Stain for 30 minutes. Discard the staining solution and slowly rinse the 6-well plate with tap water (avoid washing away the agarose and plaques) until the background color fades and the plaques are clearly visible in purple. Use an ELISA speckle analyzer to take pictures and record the number of plaques in 3 replicates for each concentration group and calculate the average value.

[0068] The results showed that, compared with the positive control, H212-Fc protein at a concentration of 0.224 μM reduced viral plaque formation by more than half, indicating that it inhibits the EC50 of DENV-2 replication. 50 Below 0.224 μM ( Figure 4 C).

[0069] 4.4 Flow cytometry experiments showed that H212-Fc can inhibit the replication of dengue virus types I, II, III, and IV. (1) Cell drug administration and infection treatment: 3.5 × 10⁻⁶ cells were added to a 24-well plate. 5 Vero cells were cultured at 100 cells / well. Types I, II, III, and IV dengue virus (DENV-1, DENV-2, DENV-3, DENV-4, MOI=0.05) were added to the culture medium along with serially diluted H212-Fc protein and control H2 peptide, respectively. After incubation at 37°C for 4 h, the cells were washed three times with PBS. 200 μL of fresh culture medium was added, and the cells were cultured for another 24 h.

[0070] (2) Fixation and permeabilization: Aspirate the culture medium, wash with PBS and digest the cells with trypsin, then transfer to a 1.5 mL centrifuge tube, add PBS and centrifuge at 1000 g for 5 minutes, discard the supernatant and keep the cell pellet; add 300 μL of fixation and permeabilization solution, resuspend the cells and incubate at 4℃ for 1 h;

[0071] (3) Primary antibody: After washing, add 10 μg / mL of 4G2 monoclonal antibody solution, incubate overnight, wash again, and resuspend the cells in 2 μg / mL of Alexa Fluor 488 goat anti-mouse IgG (H+L). Detect DENV-2 E+ cells using flow cytometry. Calculate the drug inhibition rate.

[0072] The results showed that the H212-Fc protein possesses broad-spectrum antiviral activity against four types of dengue virus, and its antiviral activity is similar to that of the H2 peptide. It inhibits DENV-1 replication via ECMO. 50 The EC50 value was 0.76 μM; its EC50 value inhibited DENV-2 replication. 50 The concentration was 0.21 μM; its EC50 inhibitory activity against DENV-3 replication was 0.21 μM. 50 The concentration was 0.60 μM; its EC50 inhibitory activity against DENV-4 replication was 0.60 μM. 50 0.03 μM ( Figure 4 D).

[0073] Example 5: Study on the anti-Zika virus activity of recombinant fusion protein H212-Fc at the cellular level (1) Cell infection and drug treatment: Cell spreaders of the corresponding size were added to 24-well plates for plating. Vero cells were seeded in 24-well plates (3.5 × 10⁻⁶ cells / well). 5 Zika virus (MOI=0.05) and serially diluted H212-Fc protein or control peptide H2 were added to the culture medium (samples / well). After incubation at 37°C for 4 h, the samples were washed three times with PBS. 200 μL of fresh culture medium was added, and the samples were incubated for another 24 h before the supernatant was collected.

[0074] Viral RNA extraction and viral load detection: RNA was extracted from the supernatant using the Trizol method; the viral load in the supernatant was quantified using the ABScript III One Step RT-qPCR Probe Kit with UDG V5. Specifically, the ZIKA-Forward Primer was 5'-CCGCTGCCCAACACAAG-3' (SEQ ID NO: 9); the ZIKA-Reverse Primer was 5'-CCACTAACGTTCTTTTGCAGAC AT-3' (SEQ ID NO: 10); and the ZIKA-Probe was 5'-FAM-AGCCTACCTTGACAAGCAGTCAGACA-TAMRA-3' (SEQ ID NO: 11).

[0075] (2) Calculate the sample EC using GraphPad Prism 10.0.1 software. 50 value.

[0076] The results showed that the H212-Fc protein also exhibited good anti-Zika virus activity, similar to that of the H2 peptide. Its EC2 inhibitory activity against Zika virus replication... 50 0.05 μM ( Figure 5 ).

[0077] Example 6: Study on the long-lasting antiviral activity of recombinant fusion protein H212-Fc at the cellular level To compare the long-term antiviral efficacy of the recombinant fusion protein H212-Fc, the H2 peptide (amino acid sequence as follows: AWDFGSLGGVFTSIGKALHQVFGAIYGAA (sequence shown in SEQ ID NO: 12) was used as a control to study the long-term antiviral efficacy of H212-Fc.

[0078] (1) Drug treatment: Vero cells were seeded into 24-well plates (3.5 × 10⁻⁶ cells per well) one day in advance. 5 (cells / well), observe cell growth the next day, and carry out subsequent experiments when the cells reach 80%-90% confluence; remove the original culture medium, add fresh culture medium containing different concentrations of H212-Fc protein and H2 peptide, incubate at 37 ℃ and 5% CO2, and collect the supernatant at different time points to detect the content of H212-Fc protein and H2 peptide by ELISA.

[0079] (2) Cell infection: Add the same drug concentration to another parallel Vero cell culture plate. After incubation, add DENV-2 virus (MOI=0.05) for 4 h. After the incubation, wash the cells 3 times with PBS, add fresh DMEM complete medium and continue to culture at 37 ℃ and 5% CO2 for 24 h. Collect the supernatant of the medium.

[0080] (3) Viral load detection: Viral RNA extraction and viral load detection were performed using the same method as in Example 5.

[0081] The results showed that, compared to the H2 peptide, the H212-Fc protein was less prone to degradation. Figure 6 A). After co-incubation with Vero cells for 96 hours, the antiviral activity of the H2 peptide was reduced by more than half. Figure 6 B), but the antiviral activity of the H212-Fc protein remained unchanged. Figure 6 C). This indicates that the antiviral activity of H212-Fc is long-lasting.

[0082] Example 7: In vitro ADE effect study of recombinant fusion protein H212-Fc Because the H212-Fc protein links to the Fc tag of IgG antibodies, and this tag may induce an enhanced effect of infection (ADE) in dengue and Zika virus infections, the potential ADE effect of the H212-Fc protein was investigated at the cellular level, using the 4G2 antibody, which can induce ADE, as a control.

[0083] (1) Cell drug administration and infection: THP-1 cells were seeded in 24-well plates (3.5 × 10⁻⁶ cells per well) one day in advance. 5 Cells were counted per well. Cell growth was observed the following day. When cells reached 70% confluence, H212-Fc protein and 4G2 monoclonal antibody solution were serially diluted 5-fold, starting with 25 μg. Dengue virus (MOI=0.05) and serially diluted H212-Fc protein or control peptide H2 were added to the culture medium. After incubation at 37°C for 4 h, the cells were washed three times with PBS. 200 μL of fresh culture medium was added, and the cells were incubated for another 24 h before collecting the supernatant.

[0084] (2) Viral load detection: Viral RNA was extracted using the same method as in Example 5, and the viral RNA content in the supernatant was detected by quantitative PCR. The DENV-Forward Primer was 5'-AGGCTCTCCACCAAGTTTTCGG-3' (SEQ ID NO: 13); the DENV-Reverse Primer was 5'-TTCCTATCCATGTGATAATGACTCCTA-3' (SEQ ID NO: 14); and the DENV-Probe was 5'-FAM-CCATGAGACCCCACTGAAGGCAGC-TAMRA-3' (SEQ ID NO: 15).

[0085] The results showed that, compared with the positive control 4G2 antibody, the H212-Fc protein did not induce the ADE effect in THP-1 cells. Figure 7 ).

[0086] Example 8: In vivo ADE effect study of recombinant fusion protein H212-Fc At the animal level, the potential ADE effect of H212-Fc protein was detected using the 4G2 antibody, which can induce ADE, as a control.

[0087] (1) Mouse separation: Select 7-week-old all-female AG129 mice, record the original weight of the mice before use, and separate the mice into cages of 4 mice per cage.

[0088] (2) Mouse administration: Mice were given the drug via intraperitoneal injection at a dose of 10 mg / kg body weight. The mice were divided into the following groups: positive control group (administered PBS during administration), treatment group (administered H212-Fc protein during administration), ADE effect control group (administered 4G2 monoclonal antibody), and blank control group (no treatment).

[0089] (3) Mouse challenge: 2 h after administration, except for the blank control group which was not treated, AG129 mice were given DENV-2 mouse strain at 500 FFU / mouse via tail vein.

[0090] (4) Mouse weight recording and survival rate detection: The mice were weighed and their condition was observed every day. When the weight of the mice in the positive control group decreased by 20%, the mice were euthanized and their brains, livers and other organs were removed.

[0091] (5) H&E staining of mouse tissue sections: Tissue sections and H&E staining were performed using conventional methods and scanned using the Shengqiang Technology slide scanning system.

[0092] (6) Immunohistochemical assessment of viral gene expression level in mouse tissues: Tissue sections were prepared according to conventional methods, using 4G2 antibody as the primary antibody and anti-mouse IgG as the secondary antibody, and scanned using the Shengqiang Technology slide scanning system.

[0093] The results showed that in AG129 mice, a 30% neutralizing concentration of 4G2 antibody mixed with the virus ( Figure 8 A), after infection, it was found that compared with viral infection alone, there was a significant decrease in weight ( Figure 8 B), the mortality rate increased ( Figure 8 C). Simultaneously, significant pathological changes were observed in the liver tissue. Figure 8 D), the viral E protein increases ( Figure 8 E), indicating enhanced viral replication. Compared to the positive control 4G2 antibody, in AG129 mice, the H212-Fc protein did not reduce mouse weight or increase mortality, nor did it enhance pathological damage to brain tissue; nor did it enhance viral replication in liver tissue. This indicates that the positive control antibody 4G2 antibody has a significant ADE effect, while the H212-Fc protein does not induce an ADE effect in vivo. Figure 8 ).

[0094] Example 9: In vivo antiviral activity study of recombinant fusion protein H212-Fc After the recombinant fusion protein H212-Fc was found to have no ADE effect in vitro and in vivo, the anti-DENV-2 activity of H212-Fc in AG129 mice was evaluated using the H2 peptide as a control.

[0095] 9.1 A single dose of the recombinant fusion protein H212-Fc significantly reduced viral load in various organs and tissues. (1) Mouse separation: Select 7-week-old all-female AG129 mice, record the original weight of the mice before use, and separate the mice into cages of 6 mice per cage.

[0096] (2) Mouse administration: Mice were given the drug via intraperitoneal injection at a dose of 10 mg / kg body weight. The mice were divided into the following groups: positive control group (administered PBS during administration), treatment group (administered H212-Fc protein during administration), and Fc control group (administered H212-Fc protein during administration).

[0097] (3) Mouse challenge: 2 h after administration, AG129 mice were given DENV-2 mouse-adapted strain via tail vein at a dose of 500 FFU / mouse.

[0098] (4) Collection of mouse blood and organs: Blood from the orbital area of ​​mice was collected 3 and 7 days after infection. The mice were euthanized after infection and organs such as lungs, liver, and kidneys were collected.

[0099] (5) Method for detecting viral load in blood and tissues: After homogenization of tissues, RNA was extracted from the tissues using Trizol and its content was determined. The viral RNA content in the supernatant was detected using quantitative PCR, similar to the method in Example 7. The inhibitory effect of a single dose of H212-Fc protein on viral load in blood and tissues was calculated based on the viral RNA content.

[0100] The results showed that in AG129 mice, a single dose ( Figure 9 Compared to the H2 control peptide, the H212-Fc protein exhibited better antiviral activity in AG129 mice. At 3 days post-infection, the viral load in the blood decreased by 0.5 log values; at 7 days post-infection, it decreased by 1 log value. Figure 9 B). Furthermore, dissection of mice 7 days after infection revealed a decrease in viral load in the lungs, liver, and kidneys of 0.8, 0.3, and 1.2 log values, respectively. Figure 9 C).

[0101] 9.2 Repeated administration of the H212-Fc recombinant fusion protein significantly reduced viral load and pathological damage in various organs and tissues. (1) Mouse separation: Select 7-week-old all-female AG129 mice, record the original weight of the mice before use, and separate the mice into cages of 6 mice per cage.

[0102] (2) Mouse administration: Mice were given the drug via intraperitoneal injection at a dose of 10 mg / kg body weight, 2 hours before infection and 1, 3 and 5 days after infection. The mice were divided into the following groups: positive group (administered PBS during administration), treatment group (administered H212-Fc protein during administration), H2 peptide control group (administered H2 peptide during administration), and blank group (no treatment).

[0103] (3) Mouse challenge: 2 h after administration, AG129 mice were given DENV-2 mouse-adapted strain via tail vein at a dose of 500 FFU / mouse.

[0104] (4) The methods for recording mouse weight and detecting survival rate, H&E staining of mouse tissue pathological tissue sections and immunohistochemical staining of mouse tissue are the same as in Example 8.

[0105] The results showed that in AG129 mice, repeated administration ( Figure 10 Compared to the H2 control peptide, the H212-Fc protein exhibited better antiviral activity in AG129 mice. The H212-Fc protein caused less weight loss in mice and provided better protection against mortality. Figure 10 B). It does not enhance pathological damage to brain tissue, nor does it enhance viral replication in liver tissue. Figure 10 C).

[0106] Example 10: Expression, purification, and antiviral activity study of recombinant fusion protein H121-Fc Simultaneously, the inventors also constructed and prepared the recombinant fusion protein H121-Fc to compare its antiviral activity. Details are as follows:

[0107] 10.1 Expression and purification of recombinant fusion protein H121-Fc In addition, based on the amino acid sequences of dengue virus H1, Loop, and H2, Loop-H1-Loop-H2-H1 were tandemly linked to form the H121 protein, which was then coupled to Fc to form the H121-Fc recombinant protein. The nucleotide coding sequence corresponding to the amino acid sequence of dengue virus type II H121-Fc was optimized using gene and codon analysis (Beijing Haichuang Biotechnology Co., Ltd.) and ligated into the pCAGSS plasmid containing the tPA signal peptide. Its expression and purification methods were the same as in Examples 1 and 2.

[0108] The results showed that after purification, the molecular weight of H121-Fc was similar to that of H212-Fc, around 32 kDa. Figure 11 A).

[0109] 10.2 Study on the cellular-level antiviral activity of recombinant fusion protein H121-Fc The anti-DENV-2 activity of the recombinant fusion protein H121-Fc was detected by quantitative PCR. Plaque assays showed that its activity was low. Figure 11 B), quantitative experiments revealed that its activity decreased by approximately 100 times compared to H212-Fc ( Figure 11 C).

[0110] (1) Cell drug administration and infection treatment: 3.5 × 10⁻⁶ cells were added to a 24-well plate. 5 Vero cells per well. Type II dengue virus (DENV-2, MOI=0.05) and serially diluted H212-Fc protein were added to the culture medium; after incubation at 37 °C for 4 h, the cells were washed three times with PBS.

[0111] (2) Viral load detection: Viral RNA extraction and viral load detection were performed using the same method as in Example 5.

[0112] The results showed that, compared to H212-Fc, the anti-DENV-2 value was EC 50 At a concentration of 0.21 μM, the antiviral activity of H121-Fc decreased by approximately 100-fold, and its inhibitory activity against DENV-2 EC was reduced. 50 The value is 3.9 μM ( Figure 11 C).

[0113] The above results indicate that the recombinant dengue virus protein H212-Fc possesses high activity and broad-spectrum antiviral activity against both dengue and Zika viruses, and its Fc fragment does not induce ADE (anti-depression) effects in vitro or in vivo. Therefore, it has broad application prospects in the field of drug development. However, the recombinant dengue virus protein H121-Fc exhibits weaker antiviral activity, which also demonstrates the specificity of the H212-Fc protein.

[0114] Example 11: Study on the mechanism of action of recombinant fusion protein H212-Fc in inhibiting dengue virus type II (DENV-2) infection.

[0115] 11.1 Recombinant fusion protein H212-Fc acts on the viral membrane fusion stage (1) Time-slot dosing experiment Through time-slot dosing experiments ( Figure 12 A) At different stages of viral infection of cells, recombinant dengue virus protein H212-Fc was added, and the antibody was detected at which stage of viral infection it acted upon.

[0116] ① The drug and cells were mixed and pre-incubated for 1 h, then washed to remove the drug adsorbed on the cell surface. Next, the cells were infected with a virus, and viral replication was assessed after 24 h. The results showed only a slight decrease in viral replication, indicating that the drug did not directly interact with the cells (pre-treatment).

[0117] ② The drug and virus were mixed and added to cells for infection. After 4 hours, uninfected virus and residual drug were washed away. After 24 hours, a sharp decrease in viral replication was observed, indicating that the drug acted in the early stage of viral infection (During treatment).

[0118] ③ The drug and virus were mixed and incubated for 0.5 h, then added to cells for infection. After 4 h, uninfected virus and residual drug were washed away. After 24 h, viral replication was found to have further decreased. This suggests that the drug acts on the early stage of viral infection or on the viral particles (direct treatment).

[0119] ④ Four hours after viral infection, cells were washed and then the drug was added. After 24 hours, no change in viral replication was observed, indicating that the drug did not act on the late stage of viral infection (post-treatment).

[0120] (2) Direct virus killing experiment High doses of the drug and virus were mixed and incubated at 37°C for 1 hour. The mixture was then divided into two portions: one undiluted and the other diluted 50-fold (to a concentration at which the drug does not inhibit replication). Both were then used to infect cells simultaneously. The results showed a sharp decrease in drug activity after dilution. This suggests that at high drug concentrations, the virus was not inactivated; that is, H212-Fc or H2 does not directly act on the viral particles and does not destroy the viral structure, thus not affecting its infectivity. Figure 12 B).

[0121] (3) Adsorption experiment The drug and virus were simultaneously added to pre-chilled (4°C) cells. After 1 hour of virus adsorption, the cells were washed three times with pre-chilled (4°C) PBS, then added to fresh culture medium and incubated at 37°C. The results showed that the drug did not inhibit viral adsorption to the cell surface. Figure 12 C).

[0122] (4) Fusion blocking experiment After the virus was added to pre-chilled (4°C) cells and adsorbed for 1 hour, the cells were washed three times with pre-chilled (4°C) PBS. Then, fresh culture medium containing the drug was added, and the cells were incubated at 37°C for 2 hours before the drug was washed off. The results showed that the drug inhibited viral replication, suggesting that the drug acts on the membrane fusion stage. Figure 12 D).

[0123] The above experiments show that: Experiments with different dosing sequences indicate that H212-Fc only exerts its antiviral effect when in direct contact with the virus (co-treatment, direct treatment), with no significant effect after pretreatment or post-infection treatment, suggesting that it directly targets viral particles or acts on the early stages of viral entry into cells; concentration gradient experiments show that the inhibitory effect of H212-Fc on DENV-2 is dose-dependent, and its activity is significantly higher than that of free H2 peptide, confirming that Fc fusion can enhance the antiviral activity of the peptide; adsorption and fusion blocking experiments show that H212-Fc does not affect the viral adsorption process to host cells, but rather exerts its anti-infection effect by inhibiting the viral-cell membrane fusion step, thereby blocking viral entry into cells.

[0124] (5) Study on the mechanism by which recombinant dengue virus protein H212-Fc inhibits membrane fusion During viral infection, dengue virus E protein and H212-Fc were observed to be enriched and co-localized on the endosome membrane surface under confocal microscopy using different fluorescent staining methods, suggesting that H212-Fc may play a role in the endosome. Figure 13 A).

[0125] Co-IP experiments revealed that dengue virus particles can bind to H212-Fc at 37°C. However, at 4°C, dengue virus particles cannot bind to H212-Fc. Figure 13 B). The results suggest that dengue virus has different conformations at 37℃ and 4℃. At 37℃, some latent epitopes of dengue virus may be exposed, which is consistent with previous reports on some structures (Proc Natl Acad Sci US A. 2013;110(17):6795-9; J Virol. 2013;87(13):7585-92) and antibody latent epitopes (Nat Struct Mol Biol. 2008;15(3):312-7).

[0126] The inventors also established a liposome fusion blocking assay for the specific detection of dengue virus membrane fusion. Trypsin was encapsulated in liposomes; after dengue virus fused with the liposome membrane, the trypsin in the liposomes could degrade the dengue virus nucleocapsid protein without affecting the dengue virus E protein. The results showed that H212-Fc could specifically inhibit the fusion of dengue virus with the liposome membrane. Figure 13 C) suggests that the target site of H212-Fc is in the membrane fusion stage.

[0127] In the study of the site of action, it was found that H212-Fc can specifically bind to H2 ( Figure 13(D) Furthermore, it was discovered for the first time that H2 has a high affinity for H2, H2 has a weak affinity for H1, and H1 also has a weak affinity for H1. Figure 13 E). Therefore, it is suggested that H212-Fc may bind to the latent epitope of viral H2 particles via its surface H2 peptide, co-entering the endosome with the viral particle and thus inhibiting membrane fusion. Further structural simulations and molecular docking experiments support a strong interaction between H212-Fc and H2. Figure 13 F).

[0128] In summary, the recombinant fusion protein H212-Fc of this invention is expected to be used for the preparation of novel dengue virus and Zika virus detection reagent raw materials, drugs, and drug optimization design phantoms.

[0129] For the purposes of ease of understanding by those skilled in the art, the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention fall within the scope of protection claimed by the present invention. For example, deleting or modifying the amino acids of the H1, Loop, or H2 peptides derived from the dengue virus stem region in the above embodiments, changing the host cell to yeast cells, Escherichia coli, or mammalian cells, etc., thereby selecting the appropriate backbone vector according to the host cell, and expressing dengue virus stem region-derived H212 in a similar manner to the above experimental examples, and using H212 as a preventive or therapeutic drug, antigen protein, or detection agent, etc., all fall within the scope of protection claimed by the present invention.

Claims

1. A recombinant fusion protein derived from the stem region sequence of dengue virus E protein, characterized in that: The recombinant fusion protein is named H212. The protein structure of H212 is Loop-H2-H1-Loop-H2, which is composed of amino acid sequences of H2, Loop, and H1 derived from the stem region sequence of dengue virus E protein. Specifically, the amino acid sequence of H1 is as shown in SEQ ID NO: 1 or is obtained by substituting, deleting, and / or adding one or more amino acids to achieve the same function; the amino acid sequence of H2 is as shown in SEQ ID NO: 2 or is obtained by substituting, deleting, and / or adding one or more amino acids to achieve the same function; and the amino acid sequence of Loop is as shown in SEQ ID NO: 3 or is obtained by substituting, deleting, and / or adding one or more amino acids to achieve the same function.

2. The recombinant fusion protein as described in claim 1, characterized in that: The recombinant fusion protein further includes Fc, with the protein structure Loop-H2-H1-Loop-H2-linker-Fc, which means that the H212 protein of claim 1 is coupled with the Fc fragment of human IgG2 to form the H212-Fc fusion protein, and the amino acid sequence of the linker is shown in SEQ ID NO:

4.

3. The recombinant fusion protein as described in claim 1, characterized in that: The amino acid sequence of the recombinant fusion protein H212 is shown in SEQ ID NO: 5, and the nucleotide coding sequence encoding the protein is shown in SEQ ID NO:

7.

4. The recombinant fusion protein as described in claim 2, characterized in that: The amino acid sequence of the recombinant fusion protein H212-Fc is shown in SEQ ID NO: 6, and the nucleotide coding sequence encoding the protein is shown in SEQ ID NO:

8.

5. The recombinant fusion protein as described in claim 1, characterized in that: The recombinant protein H212-Fc can specifically bind to the H2 stem region of the dengue virus E protein, inhibiting the fusion of dengue virus with the host cell membrane, and does not induce antibody-dependent enhancement.

6. A gene encoding the recombinant fusion protein as described in claim 1.

7. A recombinant expression vector for expressing the recombinant fusion protein as described in claim 1, characterized in that: The recombinant expression vector contains the gene encoding the recombinant fusion protein as described in claim 6.

8. A method for preparing a recombinant fusion protein, comprising the following steps: (1) Construct a recombinant expression vector encoding the nucleotide sequence of the recombinant fusion protein as described in any one of claims 1-5; (2) Transfect the recombinant expression vector into host cells Expi293F cells to induce expression; (3) Purify the recombinant protein by affinity chromatography and size exclusion chromatography to obtain a purity ≥95%.

9. The use of the recombinant fusion protein according to any one of claims 1-5, the gene encoding the recombinant fusion protein according to claim 6, the recombinant expression vector of the recombinant fusion protein according to claim 7, and the recombinant fusion protein prepared by the method according to claim 8 in the preparation of dengue virus and Zika virus antigen detection reagents, anti-dengue virus vaccines or specific antibodies, and preventive and / or therapeutic drugs or reagents.

10. The application as described in claim 9, characterized in that: The dengue viruses mentioned include DENV-1, DENV-2, DENV-3, and DENV-4.

Images