A pre-fusion F protein of respiratory syncytial virus and its application

Abstract

This invention discloses a pre-fusion F protein of respiratory syncytial virus (RSV) and its applications. The pre-fusion F protein is a recombinant pre-fusion F protein of RSV modified with amino acid mutations. The mutation modification method is selected from any of the following: (1) point mutation of amino acids based on the full-length sequence of wild-type pre-fusion F protein; (2) first deleting the transmembrane / intracellular region of the full-length sequence of wild-type pre-fusion F protein, and then attaching the fibritin / Throm / 6his / Stretaq sequence to its C-terminus, and then mutating K at amino acid position 75 to C and S at amino acid position 215 to C. This invention utilizes protein genetic engineering techniques to rationally optimize the amino acid sequence of the pre-fusion F protein of RSV. Compared with the wild-type F protein, the pre-fusion F recombinant protein modified with amino acid mutations has improved stability and immunogenicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a respiratory syncytial virus (RSV) pre-fusion F protein and its applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the major pathogens causing non-bacterial respiratory diseases and is also the most important viral pathogen causing lower respiratory tract infections (LRTIs) in infants, the elderly, and immunocompromised adults.

[0003] Given the serious challenges posed by RSV, RSV vaccination is a more economical and effective way to prevent RSV infection, and the WHO has listed RSV vaccines as one of the world's top priority vaccines for development. Therefore, developing a vaccine to prevent RSV infection is of great significance for improving the health of infants and the elderly and contributing to the Healthy China strategy.

[0004] Respiratory syncytial virus (RSV) is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the order Cytovirales, family Pneumoviridae, and genus Orthopneumovirus. Its genome consists of single-stranded negative-sense RNA molecules encoding 11 proteins (including 9 structural proteins (3 glycoproteins and 6 internal proteins) and 2 non-structural proteins). The structural proteins include three transmembrane surface glycoproteins: attachment protein G, fusion protein F, and small hydrophobic SH protein. Two RSV subtypes exist: A and B, differing primarily in the G glycoprotein, while the F glycoprotein sequence is more conserved between the two subtypes.

[0005] The respiratory syncytial virus (RSV) fusion protein (F protein) is a class I transmembrane protein composed of 574 amino acid residues. Initially, it is generated in the host cell as the precursor F0. F0 protein undergoes glycosylation at the Golgi apparatus and is subsequently hydrolyzed by intracellular furin protease, releasing a 27-amino acid polypeptide, pep27. At the N and C-terminal cleavage sites of this polypeptide, two subunits, F1 and F2, are generated, respectively. The F2 subunit consists of a signal peptide SP and a heptapeptide repeat sequence HRC. The F1 subunit consists of the fusion peptide FP, heptapeptide repeat regions HRA and HRB, Domain I and Domain II transmembrane regions TM, and a cytoplasmic domain CP. F1 and F2 are linked by disulfide bonds, forming a heterodimer. The three heterodimers assemble into a mature F protein trimer. The RSV F protein trimer is unstable, exhibiting two conformations: prefusion and postfusion. The F protein on the viral envelope initially exists in a metastable pre-F conformation. Upon viral adsorption to the cell membrane, the pre-F protein undergoes a conformational change triggered by factors such as cell receptors, temperature, and ion concentration, generating a highly stable post-F trimer. This process releases energy and mediates the fusion of the viral envelope with the cell membrane. Due to the high conformational instability of pre-F, the protein obtained through in vitro isolation and purification is usually post-F. However, the metastable pre-F conformation is essential for viral-mediated membrane fusion and is also an important antigen for inducing an immune response in humans.

[0006] Pre-fusion F protein (i.e., pre-F protein), as one of the main antigens of respiratory syncytial virus (RSV), possesses good immunogenicity. However, the natural RSV pre-fusion F protein is easily destroyed under conditions such as high temperature, high pressure, and extreme pH, resulting in poor protein structural stability and difficulty in ensuring its antigenicity. Therefore, further research is needed on the structure and function of RSV pre-fusion F protein. However, designing and obtaining highly stable and effective RSV pre-fusion F recombinant proteins remains a challenge, and currently, there are no relevant RSV recombinant protein vaccine products on the market in China. Therefore, an immunogen derived from the RSV F protein is needed, possessing improved properties compared to the corresponding natural RSV F protein, such as enhanced immunogenicity or improved stability of the RSV pre-fusion F protein, to lay the foundation for the development of RSV recombinant protein vaccines. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a pre-fusion F protein of respiratory syncytial virus (RSV) and its applications. By rationally optimizing the amino acid sequence of the pre-fusion F protein, a recombinant pre-fusion F protein with enhanced stability is obtained. This recombinant protein immunomodulator further enhances the immunogenicity and stability of the pre-fusion F protein, effectively preventing diseases caused by RSV infection and strengthening the protective effect.

[0008] This invention is achieved through the following technical solution:

[0009] A respiratory syncytial virus (RSV) pre-fusion F protein, wherein the pre-fusion F protein is a recombinant RSV pre-fusion F protein modified by amino acid mutation, wherein the mutation modification is selected from any of the following methods:

[0010] (1) Based on the full-length sequence of the wild-type pre-fusion F protein with an amino acid sequence as shown in SEQ ID NO.1, point mutations were performed to change K at position 75 to C and S at position 215 to C.

[0011] (2) First, delete the transmembrane / intracellular region in the full-length sequence of the wild-type pre-fusion F protein as shown in SEQ ID NO.1, and connect the fibritin / Throm / 6his / Stretaq sequence to its C-terminus to obtain the mutant as shown in SEQ ID NO.2. Then, perform point mutations on the amino acid sequence SEQ ID NO.2, mutating K at position 75 to C and S at position 215 to C.

[0012] Preferably, the amino acid sequence of the pre-fusion recombinant F protein obtained by method (2) is shown in SEQ ID NO.3.

[0013] The method for preparing the above-mentioned respiratory syncytial virus pre-fusion F protein includes the following steps:

[0014] The nucleic acid molecule encoding the pre-fusion F protein of the above-mentioned respiratory syncytial virus was introduced into CHO cells to obtain recombinant cells; the recombinant cells were cultured to obtain the desired product.

[0015] The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0016] A biomaterial, said biomaterial being at least one of the following (i)-(iv):

[0017] (i) A nucleic acid molecule encoding the pre-fusion F protein of the above-mentioned respiratory syncytial virus, the nucleotide sequence of which is shown in SEQ ID NO.4;

[0018] (ii) a recombinant expression vector containing the nucleic acid molecules described in (i);

[0019] (iii) Recombinant microorganisms containing nucleic acid molecules as described in (i) or recombinant microorganisms containing recombinant expression vectors as described in (ii);

[0020] (iv) A recombinant cell line containing the nucleic acid molecule described in (i) or a recombinant cell line containing the recombinant expression vector described in (ii).

[0021] The above-mentioned respiratory syncytial virus pre-fusion F protein, or the respiratory syncytial virus pre-fusion F protein prepared by the above-mentioned preparation method, or the above-mentioned biological materials are used in the preparation of respiratory syncytial virus antibodies.

[0022] The above-mentioned respiratory syncytial virus pre-fusion F protein, or the respiratory syncytial virus pre-fusion F protein prepared by the above-mentioned preparation method, or the above-mentioned biological material, are used in the preparation of drugs for the prevention and / or treatment of respiratory syncytial virus infection.

[0023] The above-mentioned respiratory syncytial virus pre-fusion F protein, or the respiratory syncytial virus pre-fusion F protein prepared by the above-mentioned preparation method, or the above-mentioned biological material in the preparation of diagnostic reagents for respiratory syncytial virus.

[0024] The above-mentioned respiratory syncytial virus pre-fusion F protein, or the respiratory syncytial virus pre-fusion F protein prepared by the above-mentioned preparation method, or the above-mentioned biological materials, are used in the preparation of nucleic acid vaccines, protein vaccines, virus-like particle vaccines, or vector vaccines of respiratory syncytial virus.

[0025] A respiratory syncytial virus (RSV) vaccine comprising the RSV pre-fusion F protein described above, or the RSV pre-fusion F protein prepared by the above preparation method, or the above-described biological material.

[0026] Preferably, it also includes pharmaceutically acceptable adjuvants and / or excipients.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) This invention optimizes the amino acid sequence of RSV protein through genetic engineering. The RSV pre-fusion F recombinant protein modified by amino acid mutation has higher stability than the corresponding wild-type RSV pre-fusion F protein. The vaccine designed based on this recombinant protein mainly modifies the amino acids of RSV pre-fusion F protein by mutation, which can make the protein maintain its structural stability and antigen cluster function under different environments, including high temperature, acidity and high osmotic pressure. Even after undergoing chemical reaction, it can still retain its good antigenicity.

[0029] (2) This invention enhances the stability of the pre-fusion F protein structure by designing amino acid mutations of the RSV-specific antigen, thus solving the problem of poor stability of the wild-type protein antigen. The stability is measured by the binding of the mutant to antibody AM22. Experiments have shown that the modified pre-fusion F protein prepared in this invention exhibits significantly better stability than the unmodified pre-fusion F protein at different temperatures, pH levels, and osmotic pressures. Furthermore, the modified protein still shows high antigen-binding activity after treatment with high temperature, acidity, and high osmotic pressure.

[0030] (3) This invention designs amino acid mutations in the RSV pre-fusion F protein, which, while ensuring protein structural stability, can effectively induce neutralizing antibodies in the body, thereby providing effective immune protection. Experiments have shown that, compared with the unmodified RSV pre-fusion F protein, the recombinant RSV pre-fusion F protein vaccine prepared in this invention, modified with amino acid mutations, can produce serum with higher protective titers and better immunization effects after immunizing mice. Attached Figure Description

[0031] Figure 1 Temperature stability of the F protein before RSV fusion in Example 2 was tested;

[0032] Figure 2 pH stability of the modified and unmodified RSV F protein before fusion in Example 3 was measured.

[0033] Figure 3 The osmotic stability of the F protein before RSV fusion in Example 4 was measured.

[0034] Figure 4 The results of detecting protein antibody titers in mouse immune serum of formulations one and two in Example 5;

[0035] Figure 5 The results show the detection of protein antibody titers in the mouse immune serum of formulations three and four in Example 5. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0037] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0038] Unless otherwise specified, the reagents, equipment, etc. used in the following examples are all commercially available.

[0039] Example 1: Preparation of RSV recombinant protein

[0040] 1. Protein Construction

[0041] The term "wild-type" as used in this invention refers to a product that exists in nature and has not undergone any artificial modification or processing. Those skilled in the art will understand that wild-type RSV F protein can have multiple sequences, which may have minor differences but have essentially the same biological activity. The wild-type full-length F protein mentioned in this invention refers to the amino acid sequence provided in GenBank, as shown in SEQ ID NO.1 (Fusion glycoprotein F0OS = Human respiratory syncytialvirus A(strain A2) OX = 11259 GN = F PE = 1 SV = 1).

[0042] (1) Mutation modification of amino acids

[0043] The present invention provides an RSV pre-fusion F protein with enhanced stability obtained through amino acid mutation modification, wherein the amino acid mutation modification methods include the following two:

[0044] Method I: Amino acid point mutations were performed on the full-length sequence of the wild-type pre-F protein: K at position 75 was mutated to C, and S at position 215 was mutated to C.

[0045] Method II: The transmembrane / intracellular region of the wild-type pre-F protein full-length sequence was deleted, and the fibritin / Throm / 6his / Stretaq sequence was ligated to its C-terminus, resulting in the mutant sequence shown in SEQ ID NO.2. Further amino acid point mutations were then performed based on the SEQ ID NO.2 sequence. Specifically, the K at position 75 of the wild-type pre-fusion F protein amino acid sequence was mutated to C, and the S at position 215 was mutated to C, resulting in the full-length mutant F protein, whose amino acid sequence is shown in SEQ ID NO.3.

[0046] In this embodiment, method II is used to obtain the RSV fusion-pre-fusion recombinant protein.

[0047] (2) Synthesis of the target gene

[0048] The corresponding coding sequence was determined based on the designed amino acid sequence SEQ ID NO.3, and the restriction endonuclease XbaI sequence was added to the 5' end of the gene segment, and the restriction endonuclease EcoRI sequence was added to the 3' end. Based on the codon preference of the host cell, the designed nucleotide sequence (SEQ ID NO.4) was chemically synthesized.

[0049] (3) Plasmid amplification and target gene extraction

[0050] The pUC19 plasmid vector was digested with EcoRI and XbaI restriction enzymes, ligated to the synthesized gene, and introduced into the host DH5α for amplification. Single clones were screened using LB (Amp+) agar solid medium. Single clones containing the target gene were inoculated into LB (Amp+) liquid medium and amplified at 37°C and 200 rpm. The plasmid pUC19-preF was extracted using the Sigma-Aldrich GenElute™ HP plasmid medium-scale preparation kit. The target gene fragment was recovered from the plasmid extracted by digestion with EcoRI and XbaI restriction enzymes using the TaKaRaMiniBestAgarose GelExtraction Kit.

[0051] (4) Construction of eukaryotic expression vectors

[0052] The mammalian cell expression plasmid pGN-M, containing the CMV promoter and dihydrofolate reductase (DHFR) gene, was digested with EcoRI and XbaI restriction enzymes. The vector DNA fragment was recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0. The vector DNA fragment and the target gene fragment were ligated at sticky ends and introduced into the DH5α amplification host. Single clones containing the eukaryotic expression plasmid pGN-M_preF were screened and amplified. The amplified plasmid was extracted using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

[0053] 2. Protein expression and clone screening

[0054] Using ATCC (CHO) cells as the host cell, the cells were cultured in DMEM (Sigma-Aldrich) medium containing 10% newborn calf serum after resuscitation, and passaged every 3 days. After two passages, the cells were observed to be growing well. Then, the CHO cells were cultured at a rate of 0.75 × 10⁻⁶ cells / year. 6 Three 9.6cm cells / wells 2The cells were placed in wells containing Iscove's optimized DMEM medium (Sigma-Aldrich) and 10% fetal bovine serum (IMEM+FBS) (Gibco). Cells were incubated in a humidity-saturated incubator at 37°C and 5% CO2. Each well contained 4 μg of pcDNA vector. The DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two wells. Lipofectamine 2000 was added separately to a third well as a negative control. After 48 h, the medium was removed, and the cells were centrifuged at 200 × g for 5 min. The supernatant was stored at -20°C. Add IMDM+FBS culture medium and 10 μg / mL Blastidin-HCl (Invitrogen) to one well of transfected cells. Wash the other well with PBS, then lyse the cells with a mixture of 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% (v / v) Triton X-100 containing complete, and EDA-free protease inhibitor (Roche Diagnostics). Centrifuge at 16000×g for 10 min at 4°C, and store the lysate at -20°C. Use Western blot to detect the presence of recombinant proteins in the supernatant and lysate. After culturing in selective medium for 5 days, wash the cells with trypsin (Invitrogen) and then seed them onto 9 cm Petri dishes for serial dilutions to isolate monoclonal cells. Over subsequent days 7-11, select 42 single clones and transfer them to wells of a 96-well plate. Use Western blot to analyze the culture supernatant and screen for high-expression proteins. The clone that secretes the highest amount of RSV protein is selected for the next round of screening, and the cells are finally expanded. Thirty new clones are then selected and preserved.

[0055] The selected clones were amplified into three T175 flasks (NETS). After trypsin digestion and washing with PBS, the clones were resuspended in 250 mL rotational flasks containing 100 mL of ProCHO4 (Lonza), 1×ProHT, 4 mM L-glutamine, and 2% FBS (Lonza). The flasks were incubated at 37°C in a humidified incubator with 5% CO2 and a stirring speed of 90 rpm, with the lid slightly ajar to ensure air diffusion. Samples were taken daily, stained with trypan blue (Sigma-Aldrich), and cells were counted. Cells were passaged every 3-5 days until the viable cell concentration exceeded 0.3 × 10⁻⁶. 6 After the plateau phase, when the cell count exceeds 90% and the viable cell count is above 90%, and the cells have adapted and are growing well, the BFS (Bio-free suspension) is gradually removed. At this point, the cells are considered fully adapted for serum-free suspension growth.

[0056] 3. Production of RSV pre-fusion F protein in a bioreactor

[0057] A 1.5L perfusion culture was configured in a 3L bioreactor, equipped with a rotary filter (10μm). Culture parameters were set as follows: temperature controlled at 37℃ using a heating blanket; pH adjusted to 6.9 using CO2 or 0.3M sodium hydroxide; stirring speed at 200–300 rpm; dissolved oxygen (dO2) adjusted to 40% of saturated air using a N2 and O2 mixture at a maximum flow rate of 200 mL / min. The perfusion rate was 0.3–0.8 V dilutions / day, and cell counting was performed daily by sampling the culture medium. Trypan blue staining was used, and glucose and lactate concentrations in the supernatant were detected offline.

[0058] The collected cell-free culture medium was centrifuged at 8000×g for 30 min at 4°C, filtered through a 0.45 μm membrane, and then concentrated by ultrafiltration using a 10 kDa membrane. The sample was washed with buffer and concentrated to 0.5 L. 0.5 L of PBS was added, and the concentration was further reduced to 0.5 L. This process was repeated five times.

[0059] 4. Protein purification

[0060] The sample solution was loaded onto a Q-Sepharose fast flow (GE Bioscience) column and washed with 20 mM Tris-HCl pH 7.5. Then, the column was washed again with 20 mM Tris-HCl pH 7.5 containing 200 mM sodium chloride to further remove adsorbed protein impurities. RSV protein was eluted with a solution containing 300 mM sodium chloride. Ammonium sulfate was added to the combined buffer to a concentration of 800 mM, and the solution was loaded onto a Butyl-Sepharose (GE Bioscience) column. The column was washed with phosphate-buffered saline (PBS, 6 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.15 M sodium chloride, pH 6.8) containing 800 mM ammonium sulfate, followed by washing with PBS containing 400 mM ammonium sulfate. Finally, the pre-fusion F protein of RSV was eluted with purified water. Finally, the sample was loaded onto a Sephacryl S-400HR (GE Bioscience), the column was washed with PBS, the protein peak was collected, a co-solvent was added, and the sample was freeze-dried in a vacuum freeze dryer and stored at -70°C for later use.

[0061] In this invention, both the unmodified and modified RSV proteins were prepared using the methods described above. The unmodified protein refers to the RSV pre-fusion F protein (SEQ ID NO.2) that has not undergone amino acid mutation modification, while the modified protein refers to the RSV pre-fusion F protein (SEQ ID NO.3) that has undergone amino acid mutation modification.

[0062] Example 2: Temperature stability test of F protein before RSV fusion

[0063] 1. Experimental Procedure

[0064] (1) Dilute the test protein (the unmodified protein and the modified protein prepared in Example 1) to 20 μg / mL with 1×PBS pH=7.4 buffer and place it in a 1.5 mL centrifuge tube with a total volume of 1 mL.

[0065] (2) Incubate at different temperatures according to Table 1 below.

[0066] Table 1 Temperature stability test

[0067] Serial Number Temperature (°C) Time (min) Quantity (pieces) Concentration (μg / mL, diluted with PBS) 1 4 60 1 20 2 50 60 1 20 3 70 60 1 20 4 90 60 1 20 5 -80 (control) - 1 20

[0068] (3) After incubation, each sample was temporarily stored at 4℃.

[0069] (4) Perform ELISA test

[0070] Dilute the samples stored at 4℃ to 1μg / mL with 1×PBS pH=7.4, add 100μL / well to a microplate (NUNC442404), and coat overnight at 4℃. Shake the plate dry, add 150μL / well of 1% BSA-PBS, and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add 100μL / well of the detection antibody AM22 (Antibody System), and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add 100μL / well of AP-labeled goat anti-human secondary antibody at a 1:2000 dilution, and incubate at 37℃ for 1 hour. Wash the plate three times according to the programmed procedure, add 100μL / well of pNPP substrate solution, set the microplate reader to 405nm, and read the value.

[0071] 2. Experimental Results

[0072] Test results as follows Figure 1 As shown, there are significant differences in binding activity. The antibody detection OD values ​​of the modified RSV pre-fusion F protein at 4℃, 50℃, and 70℃ are significantly higher than those of the unmodified protein. This indicates that the modified RSV pre-fusion F protein prepared in this invention can still maintain high antigen-binding activity after treatment at different temperatures. That is, compared with the unmodified protein, the temperature stability of the RSV modified protein prepared in this invention is significantly enhanced.

[0073] Example 3: pH stability test of F protein before RSV fusion

[0074] 1. Experimental steps (1) Solution preparation

[0075] ① 25mM acetate buffer, pH=3.5. Preparation method (based on 100mL volume): Accurately weigh 98.953mg sodium acetate into a 200mL beaker; accurately weigh 1.429g acetic acid into a 5mL centrifuge tube; add approximately 80mL of purified water to the sodium acetate and dissolve it completely, then add the acetic acid; adjust the pH of the solution to 3.5; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0076] ② 25mM acetate buffer, pH=5.0. Preparation method (based on 100mL volume): Accurately weigh 1.381g sodium acetate into a 200mL beaker; accurately weigh 490.3mg acetic acid into a 5mL centrifuge tube; add approximately 80mL of purified water to the sodium acetate and dissolve it completely, then add the acetic acid; adjust the pH of the solution to 5.0; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0077] ③ 25mM Tris-HCl buffer, pH=8.0. Preparation method (based on a 100mL volume): Add 80mL of purified water to a 200mL beaker; accurately measure 302.85mg Tris and add it; add 1mL of 1N hydrochloric acid (theoretical value 1.42mL), then add a small amount to adjust the pH of the solution to 8.0; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0078] ④ 25mM Tris-HCl buffer, pH=10.0. Preparation method (based on a 100mL volume): Add 80mL of purified water to a 200mL beaker; accurately measure and add 302.85mg Tris; add 20μL of 1N hydrochloric acid (theoretical value 33μL), then add a small amount to adjust the pH of the solution to 10.0; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0079] ⑤ 25mM PBS buffer control, pH=7.5. Preparation method (based on 100mL volume): Add 80mL of purified water to a 200mL beaker; accurately measure 142mg Na2HPO4, 27mg KH2PO4, 800mg NaCl, and 20mg KCl and add; add a small amount of HCl to adjust the pH of the solution to 7.5; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0080] (2) Dilute the test proteins (unmodified protein and modified protein prepared in Example 1) to 20 μg / mL with buffers of different pH values ​​and place them in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0081] (3) The samples were incubated at different pH values ​​according to Table 2 below.

[0082] Table 2 pH stability test

[0083] Serial Number Solution preparation number pH Time (min) Temperature (°C) Quantity (pieces) Final concentration (μg / mL) 1 ① 3.5 60 23-25°C (room temperature) 1 20 2 ② 5.0 60 23-25°C (room temperature) 1 20 3 ③ 8 60 23-25°C (room temperature) 1 20 4 ④ 10 60 23-25°C (room temperature) 1 20 5 ⑤ 7.5 60 23-25°C (room temperature) 1 20

[0084] (4) After incubation, neutralize the pH of each sample to 7.5 with acid or alkali (measured with pH test paper) and store at 4°C.

[0085] (5) Perform ELISA testing

[0086] Dilute the samples stored at 4℃ to 1μg / mL with 1×PBS pH=7.4, add 100μL / well to a microplate (NUNC442404), and coat overnight at 4℃. Shake the plate dry, add 150μL / well of 1% BSA-PBS, and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add 100μL / well of the detection antibody AM22 (Antibody System), and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add 100μL / well of AP-labeled goat anti-human secondary antibody at a 1:2000 dilution, and incubate at 37℃ for 1 hour. Wash the plate three times according to the programmed procedure, add 100μL / well of pNPP substrate solution, set the microplate reader to 405nm, and read the value.

[0087] 2. Experimental Results

[0088] Test results as follows Figure 2 As shown, the binding activity of modified and unmodified RSV pre-fusion F protein with antibody AM22 differed significantly after different pH treatments. Specifically, the OD values ​​of the modified RSV pre-fusion F protein detected by AM22 antibody at pH values ​​of 3.5, 5.0, 7.5, 8.0, and 10 were significantly higher than those of the unmodified RSV pre-fusion F protein. This indicates that the RSV modified protein prepared in this invention can still maintain high antigen-binding activity after different pH treatments, that is, compared with the unmodified protein, the pH stability of the RSV modified protein prepared in this invention is significantly enhanced.

[0089] Example 4: Osmotic stability test of F protein before RSV fusion

[0090] 1. Experimental steps (1) Solution preparation

[0091] ① 10mM Tris-HCl buffer, pH=7.5. Preparation method (based on a 25mL volume): Add 10mL of 25mM Tris-HCl buffer (pH=8.0) to a 50mL centrifuge tube; add purified water to a volume of 25mL; add 6mL of 1N hydrochloric acid (theoretical value 6.45mL), then add a small amount to adjust the pH of the solution to 7.5; bring the volume to 25mL, store at room temperature, shelf life 3 months.

[0092] ② 80mM Tris-HCl buffer, pH=7.5. Preparation method (based on a 100mL volume): Add 80mL of purified water to a 200mL beaker; accurately measure and add 969.12mg Tris; add 6mL of 1N hydrochloric acid (theoretical value 6.45mL), then add a small amount to adjust the pH of the solution to 7.5; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0093] ③ 1.5M MgCl2 solution. Preparation method (based on a 100mL volume): Accurately measure 20mL of 3M MgCl2 solution and add it to a 50mL centrifuge tube; add purified water to 20mL and mix thoroughly; store at room temperature, shelf life is 3 months.

[0094] ④ 3M MgCl2 solution. Preparation method (based on a 100mL volume): Accurately measure 60.99g of magnesium chloride hexahydrate into a 200mL beaker; add purified water to about 80mL, stir to dissolve completely; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0095] ⑤ 150mM Tris-HCl buffer, pH=7.5. Preparation method (based on a 100mL volume): Add 80mL of purified water to a 200mL beaker; accurately measure and add 1.815mg Tris; add 6mL of 1N hydrochloric acid (theoretical value 6.45mL), then add a small amount to adjust the pH of the solution to 7.5; bring the volume to 100mL, store at room temperature, shelf life 3 months.

[0096] ⑥ 4M NaCl solution. Preparation method (based on a volume of 125mL): Accurately weigh 29.22g of sodium chloride and add it to a 200mL beaker; add 100mL of purified water and stir thoroughly to dissolve; bring the volume up to 125mL, store at room temperature, shelf life 3 months.

[0097] (2) Dilute the test protein (the unmodified protein and the modified protein prepared in Example 1) to 20 μg / mL with buffers of different osmotic pressure values, and place them in a 1.5 mL centrifuge tube with a total volume of 1 mL.

[0098] (3) The samples were incubated with different osmotic pressure values ​​according to Table 3 below.

[0099] Table 3 Osmotic Stability Testing

[0100]

[0101]

[0102] (4) The 10mM and 80mM groups were diluted with Tris buffer, and the 1500mM and 3000mM groups were diluted with MgCl2 buffer respectively.

[0103] (5) After incubation, the 10mM and 80mM groups were adjusted to 150mM with 4M NaCl buffer, and the 1500mM and 3000mM groups were diluted 10 times and 20 times with purified water, respectively, and stored at 4℃.

[0104] (6) Perform ELISA testing

[0105] Dilute the samples stored at 4℃ to 1μg / mL with 1×PBS pH=7.4, add 100μL / well to a microplate (NUNC442404), and coat overnight at 4℃. Shake the plate dry, add 150μL / well of 1% BSA-PBS, and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add 100μL / well of the detection antibody AM22 (Antibody System), and incubate at 37℃ for 2 hours. Wash the plate three times according to the programmed procedure, add 100μL / well of AP-labeled goat anti-human secondary antibody at a 1:2000 dilution, and incubate at 37℃ for 1 hour. Wash the plate three times according to the programmed procedure, add 100μL / well of pNPP substrate solution, set the microplate reader to 405nm, and read the value.

[0106] 2. Experimental Results

[0107] Test results as follows Figure 3 As shown, the binding activity of unmodified and modified RSV pre-fusion F protein to antibody AM22 differed significantly after incubation at different osmotic pressures. The OD values ​​of the modified RSV pre-fusion F protein at antibody detection at 10mM, 80mM, 150mM, 1500mM, and 3000mM were significantly higher than those of the unmodified RSV pre-fusion F protein. This indicates that the RSV modified protein prepared in this invention can still maintain high antigen-binding activity after incubation at different osmotic pressures, meaning that the stability of the RSV modified protein prepared in this invention is significantly enhanced under different osmotic pressures compared to the unmodified protein.

[0108] The experimental results of the above embodiments show that, compared with the unmodified RSV pre-fusion F protein, the RSV pre-fusion F protein prepared by the present invention with amino acid mutation modification still maintains its structural stability and antigen cluster function under different environments, including high temperature, acidity and high osmotic pressure. Even after undergoing chemical reactions, it can still retain its good antigenicity.

[0109] Example 5: Application of RSV pre-fusion F protein immunomodulator

[0110] 1. Preparation of Immunotherapy Agent 1 and Agent 2

[0111] Using the RSV unmodified protein and modified protein obtained in Example 1, samples of 5 μg each were added to phosphate buffer (pH 5.8), sterilized by filtration through a 0.22 μm membrane, stirred at 4°C for 1 h, aseptically dispensed into 0.5 mL / bottle, and stored at 4°C for use in immunization, thus obtaining formulation one (unmodified protein) and formulation two (modified protein).

[0112] 2. Preparation of Immunotherapy Agent III and Agent IV

[0113] Using the RSV unmodified protein and modified protein obtained in Example 1, samples of 5 μg each were added to phosphate buffer (pH 5.8) and sterilized by filtration through a 0.22 μm membrane. Sterile aluminum phosphate gel (Benetag) was added to achieve a final aluminum ion concentration of 0.125 mg. The mixture was stirred at 4°C for 1 h, aseptically dispensed into 0.5 mL / bottle, and stored at 4°C for use in immunization, thus obtaining formulation three (unmodified protein) and formulation four (modified protein).

[0114] 3. Immunization of mice with RSV pre-fusion F protein preparation and blood collection

[0115] Female BALB / c mice aged 4-6 weeks were randomly divided into four groups of 10 mice each. Each group was treated with one of the four immunizing agents prepared above: Agent 1, Agent 2, Agent 3, and Agent 4. Subcutaneous immunization was performed every two weeks, with 0.1 mL administered each time, for a total of two immunizations. Blood was collected 35 days after immunization. The blood was then left at room temperature for 4 hours, centrifuged at 10,000 rpm at room temperature, and the supernatant serum was collected and stored at -70°C for later analysis.

[0116] 4. Detection of RSV pre-fusion F protein antibody titer in mouse immune serum by ELISA.

[0117] Prepare a purified RSV pre-fusion F protein stock solution of 1 mg / mL (1×PBS solution) and store at 4°C. Dilute the protein stock solution to 4 μg / mL coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash four times with wash buffer, add 150 μL of blocking buffer, incubate at 37°C for 2 h, wash three times with 300 μL of wash buffer per well, and it can be stored at 4°C for one week.

[0118] Dilute the corresponding test serum obtained from mouse vaccination in step 3 to prepare working sample serum. Add 100 μL to each well in the first row of ELISA plates, starting with the first row and performing serial 2-fold dilutions downwards. Incubate at 37°C for 2 hours. Wash each well three times with 300 μL of wash buffer. Add 100 μL of AP-labeled anti-mouse secondary antibody at a 1:2000 dilution and incubate at 37°C for 1 hour. Wash the plate three times according to the programmed sequence. Add 100 μL of pNPP substrate solution per well. Set the microplate reader to 405 nm and take the reading.

[0119] 5. Experimental Results

[0120] like Figure 4 As shown, in the absence of adjuvants in the immunizing agents, there were significant differences in the antibody titers of proteins in the serum of mice immunized with different agents. The antibody titer of mice immunized with the pre-fusion F recombinant protein preparation modified with amino acid mutations (Preparation 2) was significantly higher than that of the unmodified pre-fusion F protein preparation (Preparation 1). Furthermore, even after different dilutions, the antibody titer of the pre-modified RSV recombinant protein preparation (Preparation 2) remained significantly higher than that of the unmodified RSV protein preparation (Preparation 1) at all concentrations. Moreover, the antibody titers of both pre- and post-modification RSV proteins showed a decreasing trend as the serum dilution factor gradually increased.

[0121] like Figure 5 As shown, when adjuvants were added to all immunizing agents, there were significant differences in the antibody titers of proteins in the serum of mice immunized with different agents. The antibody titer of mice immunized with the pre-fusion F recombinant protein preparation after amino acid mutation modification (Preparation 4) was significantly higher than that of the unmodified pre-fusion F protein preparation after amino acid mutation modification (Preparation 3). Furthermore, after different dilutions of the serum of immunized mice, at all concentrations, the antibody titer of the pre-modified RSV recombinant protein preparation (Preparation 4) was still significantly higher than that of the unmodified RSV protein preparation (Preparation 3). Moreover, as the serum dilution factor gradually increased, the antibody titers of RSV proteins before and after modification showed a decreasing trend.

[0122] The experimental results of this embodiment show that, compared with the unmodified RSV pre-fusion F protein, the RSV pre-fusion F recombinant protein preparation modified by amino acid mutation can obtain serum with higher protective titer, indicating that the RSV recombinant protein preparation modified by amino acid mutation prepared in this invention has better immune effect.

[0123] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A respiratory syncytial virus (RSV) pre-fusion F protein, characterized in that, The amino acid sequence of the pre-fusion F protein is shown in SEQ ID NO.

3.

2. The method for preparing the pre-fusion F protein of respiratory syncytial virus as described in claim 1, characterized in that, Includes the following steps: A nucleic acid molecule encoding the pre-fusion F protein of the respiratory syncytial virus as described in claim 1 is introduced into CHO cells to obtain recombinant cells; the recombinant cells are then cultured to obtain the desired product. The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

3. A biomaterial, characterized in that, The biomaterial is at least one of the following (i)-(iv): (i) A nucleic acid molecule encoding the pre-fusion F protein of respiratory syncytial virus as described in claim 1, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4; (ii) A recombinant expression vector containing the nucleic acid molecule described in (i); (iii) Recombinant microorganisms containing nucleic acid molecules as described in (i) or recombinant microorganisms containing recombinant expression vectors as described in (ii); (iv) A recombinant cell line containing a nucleic acid molecule as described in (i) or a recombinant cell line containing a recombinant expression vector as described in (ii).

4. The use of the pre-fusion F protein of respiratory syncytial virus as described in claim 1, or the pre-fusion F protein of respiratory syncytial virus prepared by the preparation method described in claim 2, or the use of the biomaterials described in claim 3 in the preparation of anti-respiratory syncytial virus serum.

5. The use of the pre-fusion F protein of respiratory syncytial virus as described in claim 1, or the pre-fusion F protein of respiratory syncytial virus prepared by the preparation method described in claim 2, or the use of the biomaterial as described in claim 3 in the preparation of a medicament for preventing respiratory syncytial virus infection.

6. The respiratory syncytial virus pre-fusion F protein as described in claim 1, or the respiratory syncytial virus pre-fusion F protein prepared by the preparation method as described in claim 2, or the application of the biomaterial as described in claim 3 in the preparation of diagnostic reagents for respiratory syncytial virus.

7. The use of the pre-fusion F protein of respiratory syncytial virus as described in claim 1, or the pre-fusion F protein of respiratory syncytial virus prepared by the preparation method described in claim 2, or the use of the biomaterials as described in claim 3 in the preparation of nucleic acid vaccines, protein vaccines, virus-like particle vaccines, or vector vaccines of respiratory syncytial virus.

8. A respiratory syncytial virus vaccine, characterized in that, Includes the pre-fusion F protein of respiratory syncytial virus as described in claim 1, or the pre-fusion F protein of respiratory syncytial virus prepared by the preparation method described in claim 2, or the biomaterial as described in claim 3.

9. A respiratory syncytial virus vaccine according to claim 8, characterized in that, It also includes pharmaceutically acceptable adjuvants and / or excipients.

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