Respiratory syncytial virus pre-fusion F protein and application thereof

Abstract

The invention discloses a respiratory syncytial virus pre-fusion F protein and application thereof, the pre-fusion F protein is a respiratory syncytial virus pre-fusion F recombinant protein modified by amino acid mutation, and the mutation modification mode is selected from any one of the following modes: (1) performing amino acid point mutation on the basis of a wild type pre-fusion F protein full-length sequence; and (2) deleting a transmembrane region / intracellular region in the full-length sequence of the wild-type F protein before fusion, connecting a fibritin / Throm / 6his / Stretaq sequence to the tail end C of the wild-type F protein before fusion, mutating K at the 75th site of amino acid into C, and mutating S at the 215th site of amino acid into C. The amino acid sequence of the pre-fusion F protein of the respiratory syncytial virus is reasonably optimized and designed by utilizing a protein gene engineering means; compared with a wild type F protein, the pre-fusion F recombinant protein modified by amino acid mutation has the advantage that the stability and the immunogenicity are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a respiratory syncytial virus pre-fusion F protein and an application thereof. Background Art

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

[0003] Faced with the severe challenges brought by RSV, RSV vaccination is a more economical and effective way to prevent RSV infection, and WHO has also listed RSV vaccine as one of the world's top priority vaccines. Therefore, developing a vaccine that can be used to prevent RSV infection is of great significance for improving the health of infants and the elderly and helping to achieve the development strategy of Healthy China.

[0004] RSV is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the order Monovirales, family Pneumoviridae, genus Orthopneumovirus. Its genome consists of a single-stranded, negative-sense RNA molecule encoding 11 proteins, including 9 structural proteins (3 glycoproteins and 6 internal proteins) and 2 nonstructural proteins. The structural proteins include 3 transmembrane surface glycoproteins: attachment protein G, fusion protein F, and small hydrophobic SH protein. There are two RSV subtypes: A and B, which differ mainly in the G glycoprotein, while the sequence of the F glycoprotein is more conserved between the two subtypes.

[0005] The respiratory syncytial virus fusion protein (F protein) belongs to the class I transmembrane protein and is composed of 574 amino acid residues. It is initially generated as the precursor F0 of the F protein in the host cell. The F0 protein is glycosylated on the Golgi apparatus and then hydrolyzed by intracellular furin protease to release the 27-amino acid polypeptide pep27. The N- and C-terminal cleavage sites of the peptide segment produce two subunits, F1 and F2, respectively. The F2 subunit consists of the signal peptide SP and the heptad repeat sequence HRC. The F1 subunit consists of the fusion peptide FP, the heptad repeat regions HRA and HRB, the domain I and domain II transmembrane regions TM, and the cytoplasmic domain CP. F1 and F2 are connected by disulfide bonds to form a heterodimer. Three heterodimers assemble into a mature F protein trimer. The respiratory syncytial virus F protein trimer is unstable and has two conformations: prefusion F protein and postfusion F protein. The F protein on the surface of the viral envelope is initially in a metastable pre-F conformation. When the virus is adsorbed to the cell membrane surface, the pre-F protein changes its conformation under the triggering of factors such as cell receptors, temperature, and ion concentration, generating a highly stable post-F trimer. This process releases energy and can mediate the fusion of the viral envelope and the cell membrane. Since the pre-F conformation is highly unstable, the protein isolated and purified in vitro is usually post-F. However, the metastable pre-F conformation is necessary for the virus-mediated membrane fusion process and is also an important antigen that induces an immune response in the human body.

[0006] As one of the main antigens of respiratory syncytial virus, the pre-fusion F protein (i.e., pre-F protein) has good immunogenicity, but because the natural respiratory syncytial virus pre-fusion F protein is easily destroyed under conditions such as high temperature, high pressure, and extreme pH, the protein structure stability is poor, and it is difficult to ensure its good antigenicity, so it is necessary to further study the structure and function of the pre-fusion F protein of respiratory syncytial virus. However, there are still many challenges in how to design a recombinant F protein before the fusion of respiratory syncytial virus with high stability and good immune effect, and there are currently no related respiratory syncytial virus recombinant protein vaccine products listed in China. Therefore, it is necessary to derive an immunogen from the F protein of respiratory syncytial virus, which has improved characteristics compared with the corresponding natural F protein of respiratory syncytial virus, such as the immunogenicity or improved stability of the F protein before the fusion of respiratory syncytial virus, to lay the foundation for the development of RSV recombinant protein vaccine. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a respiratory syncytial virus prefusion F protein and its application, and obtains a respiratory syncytial virus prefusion F recombinant protein with enhanced stability by rationally optimizing the amino acid sequence of the respiratory syncytial virus prefusion F protein. The recombinant protein immune preparation further enhances the immunogenicity and stability of the prefusion F protein, can effectively prevent diseases caused by respiratory syncytial virus infection, and enhances the protective effect.

[0008] The present invention is achieved through the following technical solutions:

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

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

[0011] (2) First, the transmembrane region / intracellular region in the full-length sequence of the wild-type pre-fusion F protein as shown in SEQ ID NO.1 was deleted, and the fibritin / Throm / 6his / Stretaq sequence was connected to its C-terminus to obtain a mutant with an amino acid sequence as shown in SEQ ID NO.2. Then, amino acid point mutations were performed on the basis of the amino acid sequence SEQ ID NO.2, such that K at position 75 was mutated to C, and S at position 215 was mutated to C.

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

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

[0014] Introducing the nucleic acid molecule encoding the respiratory syncytial virus pre-fusion F protein into CHO cells to obtain recombinant cells; culturing the recombinant cells to obtain;

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

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

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

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

[0019] (iii) a recombinant microorganism containing the nucleic acid molecule described in (i) or a recombinant microorganism containing the recombinant expression vector 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 use of the above-mentioned biological material in the preparation of respiratory syncytial virus antibodies.

[0022] The use of 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 drugs for preventing and / or treating respiratory syncytial virus infection.

[0023] The use of 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 respiratory syncytial virus diagnostic reagents.

[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 material is used in the preparation of nucleic acid vaccine, protein vaccine, virus-like particle vaccine or vector vaccine of respiratory syncytial virus.

[0025] A respiratory syncytial virus vaccine comprises 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.

[0026] Preferably, pharmaceutically acceptable adjuvants and / or auxiliary materials are also included.

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

[0028] (1) The present invention rationally optimizes and designs the amino acid sequence of RSV protein by genetic engineering means. The RSV prefusion F recombinant protein modified by amino acid mutation of the present invention has higher stability than the corresponding wild-type RSV prefusion F protein. The vaccine designed based on the recombinant protein mainly modifies the amino acids of RSV prefusion F protein by mutation, so that the protein can maintain its structural stability and antigen cluster function under different environments, including high temperature, acidity and high osmotic pressure, and can still preserve its good antigenicity even after undergoing chemical reactions.

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

[0030] (3) The present invention designs amino acid mutations in RSV prefusion F protein, which can effectively induce antibodies with neutralizing activity in the body while ensuring the stability of the protein structure, thereby giving the body effective immune protection. Experiments have shown that compared with unmodified RSV prefusion F protein, the RSV prefusion F recombinant protein vaccine modified by amino acid mutation prepared by the present invention can obtain serum with higher protective titer after immunizing mice, and the immune effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The temperature stability test of the modified and unmodified RSV pre-fusion F proteins in Example 2;

[0032] Figure 2 pH stability test of modified and unmodified RSV prefusion F proteins in Example 3;

[0033] Figure 3 The osmotic pressure stability test of the modified and unmodified RSV prefusion F proteins in Example 4;

[0034] Figure 4 The results of the protein antibody titer test in the mouse immune serum of Preparation 1 and Preparation 2 in Example 5;

[0035] Figure 5 These are the results of the protein antibody titer test in the mouse immune sera of Preparations 3 and 4 in Example 5. DETAILED DESCRIPTION

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

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

[0038] Unless otherwise specified, the reagents, equipment, etc. used in the following examples can be obtained from commercial sources.

[0039] Example 1 Preparation of RSV recombinant protein

[0040] 1. Protein construction

[0041] The term "wild-type" used in the present invention means that there is in nature, without any artificial modification or processed product. It is understood by those skilled in the art that wild RSV F protein can be a variety of sequences, and there may be subtle differences in these sequences, but the biological activity is basically the same. The wild-type full-length F protein mentioned in the present invention is provided with reference to the amino acid sequence of GenBank, as shown in SEQ ID NO.1 (Fusion glycoprotein FOOS=Human respiratory syncytialvirus A (strain A2) OX=11259GN=F PE=1SV=1).

[0042] (1) Amino acid mutation modification

[0043] The RSV pre-fusion F protein with enhanced stability obtained by amino acid mutation modification provided by the present invention includes the following two amino acid mutation modification methods:

[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 region / intracellular region in the wild-type pre-F protein full-length sequence is deleted, and the fibritin / Throm / 6his / Stretaq sequence is connected to its C-terminus to obtain a mutant sequence as shown in SEQ ID NO.2, and amino acid point mutations are performed on the basis of the SEQ ID NO.2 sequence. The specific mutation method is as follows: K at position 75 of the wild-type pre-fusion F protein amino acid sequence is mutated to C, and S at position 215 is mutated to C to obtain a full-length mutant of the F protein, whose amino acid sequence is shown in SEQ ID NO.3.

[0046] In this example, method II was used to obtain RSV pre-fusion F recombinant protein.

[0047] (2) Synthesis of 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 in this segment, and the restriction endonuclease EcoRI sequence was added to the 3' end. According to 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 double-digested with EcoRI and XbaI restriction enzymes and then connected with the synthesized gene, introduced into the amplification host DH5α, and single clones were screened using LB (Amp+) agar solid culture medium; single clones containing the target gene were inoculated in LB (Amp+) liquid culture medium, cultured and amplified at 37°C, 200 rpm, and the plasmid pUC19-preF was extracted using the Sigma-Aldrich GenEluteTMHP plasmid midi preparation kit; the extracted plasmid was double-digested with EcoRI and XbaI restriction enzymes, and the target gene fragment was recovered using the TaKaRaMiniBestAgarose GelExtraction Kit.

[0051] (4) Construction of eukaryotic expression vector

[0052] The mammalian cell expression plasmid pGN-M, which contains a CMV promoter and a dihydrofolate reductase (DHFR) gene, was double-digested with EcoRI and XbaI restriction enzymes, and the vector DNA fragment was recovered using TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0; the vector DNA fragment and the target gene fragment were connected by sticky end and introduced into the DH5α amplification host, and a single clone containing the eukaryotic expression plasmid pGN-M_preF was screened; the plasmid was inoculated in LB (Amp+) for amplification culture, and the amplified plasmid was extracted using the endotoxin-free plasmid midi purification kit TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

[0053] 2. Protein expression and cloning screening

[0054] CHO cells (ATCC) were used as host cells. After cell recovery, they were cultured in DMEM medium (Sigma-Aldrich) containing 10% newborn calf serum and passaged once every 3 days. After passage 2, the cells were observed to grow well, and then CHO cells were cultured at 0.75×10 6 Three 9.6cm cells / well 2The cells were incubated in a humidity-saturated incubator at 5% CO2 and 37°C. 4 μg of pcDNA vector was added to each well. DNA was mixed with Lipofectamine 2000 (Sigma-Aldrich) and added to two of the wells. Lipofectamine 2000 alone was added to the third well as a negative control. After 48 h, the medium was removed, the cells were centrifuged at 200 × g for 5 min, and the supernatant was stored at -20°C. Add IMDM+FBS culture medium and 10μg / mL Blasticidin-HCl (Invitrogen) to one well of transfected cells, wash the other well of transfected cells with PBS, and then lyse the cells with 50mM Tris-HCl, pH=8, 150mM NaCl, 1% (v / v) Triton X-100containing complete, EDA-free protease inhibitor cocktail (Roche Diagnostics). Centrifuge at 16000×g for 10min at 4℃, and store the lysate at -20℃. Use Western blot to detect whether there is recombinant protein in the supernatant and lysate. After 5 days of culture in selective medium, wash the cells with trypsin (Invitrogen) and then inoculate them on a 9cm Petri dish for serial dilution to isolate monoclonal cells. In the next 7-11 days, select 42 single clones and transfer them to the wells of a 96-well plate. Use Western blot to detect the culture supernatant and screen for highly expressed proteins. The clones secreting the highest amount of RSV protein were subjected to the next round of screening, the cells were finally expanded, and 30 new clones were screened and preserved.

[0055] The selected clones were expanded into three T175 flasks (NETS). Trypsin was added for digestion, washed with PBS, and resuspended in 100 mL of ProCHO4 (Lonza) in a 250 mL spinner bottle with 1×ProHT, 4 mM L-glutamine, and 2% FBS (Lonza). In a humidified incubator at 37°C, 5% CO2, the stirring speed was 90 rpm, and the lid was slightly opened to ensure air diffusion for culture. Samples were taken daily, stained with trypan blue (Sigma-Aldrich), the cells were counted, and passaged every 3-5 days. When the viable cell concentration was higher than 0.3×10 6 cells / mL, after the plateau when the number of viable cells exceeds 90%. When the cells adapt and grow well, BFS is gradually removed, and the cells are considered to be completely suitable for serum-free suspension growth.

[0056] 3. Production of RSV prefusion F protein in bioreactor

[0057] A 1.5L perfusion culture was configured in a 3L bioreactor with a rotary filter (10μm) separator. The culture parameters were set as follows: the temperature was controlled at 37°C by a heating blanket, the pH was adjusted to 6.9 by CO2 or 0.3M sodium hydroxide, the stirring speed was 200-300rpm, and the dissolved oxygen (dO2) was adjusted to 40% of saturated air with a maximum flow rate of 200mL / min of N2 and O2 mixed gas. The perfusion rate was 0.3-0.8V dilution / day, and samples were taken from the culture medium every day for cell counting. Trypan blue staining was used, and the concentrations of glucose and lactate in the supernatant were detected offline.

[0058] The collected cell-free culture fluid was centrifuged at 8000×g for 30 min at 4°C, filtered with a 0.45μm membrane, and then ultrafiltered and concentrated with a 10kDa membrane package. Ultrafiltered and diafiltered with buffer, the sample solution was concentrated to 0.5L, and after adding 0.5L PBS, it was concentrated to 0.5L. The above steps were repeated 5 times.

[0059] 4. Protein Purification

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

[0061] The RSV unmodified protein and modified protein in the present invention are prepared respectively by the above method. The unmodified protein in the present invention refers to the RSV pre-fusion F protein (SEQ ID NO.2) that has not been modified by amino acid mutation, and the modified protein refers to the RSV pre-fusion F protein (SEQ ID NO.3) that has been modified by amino acid mutation.

[0062] Example 2 Temperature stability test of RSV pre-fusion F protein

[0063] 1. Experimental steps

[0064] (1) The test protein (the unmodified protein and the modified protein prepared in Reference Example 1, respectively) was diluted to 20 μg / mL with 1×PBS pH=7.4 buffer and placed 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(℃) Time (min) Quantity (pcs) Concentration (μg / mL, diluted in 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°C.

[0069] (4) Perform ELISA test

[0070] The samples stored at 4°C were diluted to 1 μg / mL with 1×PBS pH=7.4, and 100 μL / well was added to the ELISA plate (NUNC442404), and coated at 4°C overnight. The ELISA plate was dried, and 1% BSA-PBS was added at 150 μL / well, and incubated at 37°C for 2 hours. The plate washer was washed 3 times according to the program, and the detection antibody AM22 (Antibody System) was added according to the design, 100 μL / well, and incubated at 37°C for 2 hours. The plate washer was washed 3 times according to the program, and AP-labeled goat anti-human secondary antibody was added at a dilution of 1:2000, 100 μL / well, and incubated at 37°C for 1 hour. The plate washer was washed 3 times according to the program, and pNPP substrate solution was added, 100 μL / well, and the ELISA reader was set to a wavelength of 405nm and read.

[0071] 2. Experimental results

[0072] Test results such as 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°C, 50°C, and 70°C are significantly higher than those of the unmodified protein, indicating that the modified RSV pre-fusion F protein prepared by the present invention can still maintain a 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 by the present invention is significantly enhanced.

[0073] Example 3 pH stability test of RSV pre-fusion F protein

[0074] 1. Experimental steps (1) Preparation of solution

[0075] ①25mM acetate buffer, pH=3.5. Preparation method (calculated based on 100mL volume) is as follows: accurately weigh 98.953mg sodium acetate and add it to a 200mL beaker; accurately weigh 1.429g acetic acid and add it to a 5mL centrifuge tube; add about 80mL purified water to the sodium acetate to fully dissolve it, then add acetic acid; adjust the solution pH to 3.5; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0076] ②25mM acetate buffer, pH=5.0. Preparation method (calculated based on the preparation of 100mL volume) is as follows: accurately weigh 1.381g sodium acetate and add it to a 200mL beaker; accurately weigh 490.3mg acetic acid and add it to a 5mL centrifuge tube; add about 80mL purified water to the sodium acetate to fully dissolve it, and then add acetic acid; adjust the solution pH to 5.0; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0077] ③25mM Tris-HCl buffer, pH=8.0. Preparation method (calculated based on 100mL volume) is as follows: add 80mL purified water to a 200mL beaker; accurately measure 302.85mg Tris and add it; add 1mL 1N hydrochloric acid (theoretical value 1.42mL), then add a small amount to adjust the solution pH to 8.0; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0078] ④25mM Tris-HCl buffer, pH=10.0. The preparation method (calculated based on the preparation of 100mL capacity) is as follows: add 80mL of purified water to a 200mL beaker; accurately measure 302.85mg of Tris and add it; take 20μL of 1N hydrochloric acid and add it (theoretical value 33μL), then add a small amount to adjust the solution pH to 10.0; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0079] ⑤25mM PBS buffer control, pH = 7.5. Preparation method (calculated based on the preparation of 100mL volume) is as follows: add 80mL 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 value of the solution to 7.5; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0080] (2) The test proteins (the unmodified protein and the modified protein prepared in Reference Example 1, respectively) were diluted to 20 μg / mL using buffers of different pH values ​​and placed in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0081] (3) Incubate the samples at different pH values ​​according to Table 2 below.

[0082] Table 2 pH stability test

[0083] Serial number Liquid No. pH Time (min) Temperature(℃) Quantity (pcs) Final concentration (μg / mL) 1 ① 3.5 60 23~25(Room temperature) 1 20 2 ② 5.0 60 23~25(Room temperature) 1 20 3 ③ 8 60 23~25(Room temperature) 1 20 4 ④ 10 60 23~25(Room temperature) 1 20 5 ⑤ 7.5 60 23~25(Room temperature) 1 20

[0084] (4) After incubation, the pH value of each sample was neutralized to 7.5 with acid or alkali (measured by pH test paper) and temporarily stored at 4°C.

[0085] (5) Perform ELISA test

[0086] The samples stored at 4°C were diluted to 1 μg / mL with 1×PBS pH=7.4, and 100 μL / well was added to the ELISA plate (NUNC442404), and coated at 4°C overnight. The ELISA plate was dried, and 1% BSA-PBS was added at 150 μL / well, and incubated at 37°C for 2 hours. The plate washer was washed 3 times according to the program, and the detection antibody AM22 (Antibody System) was added according to the design, 100 μL / well, and incubated at 37°C for 2 hours. The plate washer was washed 3 times according to the program, and AP-labeled goat anti-human secondary antibody was added at a dilution of 1:2000, 100 μL / well, and incubated at 37°C for 1 hour. The plate washer was washed 3 times according to the program, and pNPP substrate solution was added, 100 μL / well, and the ELISA reader was set to a wavelength of 405nm and read.

[0087] 2. Experimental results

[0088] Test results such as Figure 2 As shown, the modified and unmodified RSV prefusion F proteins have significant differences in binding activity with antibody AM22 after treatment at different pH values, wherein the OD values ​​of the modified RSV prefusion F protein in AM22 antibody detection at pH values ​​of 3.5, 5.0, 7.5, 8.0 and 10 are significantly higher than those of the unmodified RSV prefusion F protein, indicating that the RSV modified protein prepared by the present invention can still maintain a high antigen binding activity after treatment at different pH values, that is, compared with the unmodified protein, the pH stability of the RSV modified protein prepared by the present invention is significantly enhanced.

[0089] Example 4 Osmotic Stability Test of RSV Pre-fusion F Protein

[0090] 1. Experimental steps (1) Preparation of solution

[0091] ①10mM Tris-HCl buffer, pH=7.5. The preparation method (calculated based on the preparation of 25mL capacity) is as follows: Take 10mL of 25mM Tris-HCl buffer (pH=8.0) and add it to a 50mL centrifuge tube; add purified water to a volume of 25mL; take 6mL of 1N hydrochloric acid and add it (theoretical value 6.45mL), then add a small amount to adjust the pH value of the solution to 7.5; make up to 25mL and store at room temperature. The shelf life is 3 months.

[0092] ②80mM Tris-HCl buffer, pH=7.5. Preparation method (calculated based on 100mL volume) is as follows: add 80mL purified water to a 200mL beaker; accurately measure 969.12mg Tris and add it; take 6mL 1N hydrochloric acid and add it (theoretical value 6.45mL), then add a small amount to adjust the solution pH to 7.5; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0093] ③1.5M MgCl2 solution. The preparation method (calculated based on the preparation of 100mL volume) is as follows: accurately measure 20mL of 3MM MgCl2 solution and add it to a 50mL centrifuge tube; add purified water to 20mL and mix thoroughly; store at room temperature, with a shelf life of 3 months.

[0094] ④3M MgCl2 solution. The preparation method (calculated based on the preparation of 100mL volume) is as follows: accurately weigh 60.99g of magnesium chloride hexahydrate and add it to a 200mL beaker; add purified water to about 80mL, stir to fully dissolve; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0095] ⑤150mM Tris-HCl buffer, pH=7.5. Preparation method (calculated based on 100mL volume) is as follows: add 80mL purified water to a 200mL beaker; accurately measure 1.815mg Tris and add it; add 6mL 1N hydrochloric acid (theoretical value 6.45mL), then add a small amount to adjust the solution pH to 7.5; make up to 100mL and store at room temperature. The shelf life is 3 months.

[0096] ⑥4M NaCl solution. Preparation method (calculated based on 125mL volume) is as follows: accurately weigh 29.22g sodium chloride and add it to a 200mL beaker; add 100mL purified water and stir thoroughly to dissolve; make up to 125mL and store at room temperature. The shelf life is 3 months.

[0097] (2) The test proteins (the unmodified protein and the modified protein prepared in Reference Example 1, respectively) were diluted to 20 μg / mL using buffers with different osmotic pressure values ​​and placed in 1.5 mL centrifuge tubes with a total volume of 1 mL.

[0098] (3) Incubate the samples at different osmotic pressures according to Table 3 below.

[0099] Table 3 Osmotic pressure stability test

[0100]

[0101]

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

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

[0104] (6) Perform ELISA test

[0105] The samples stored at 4°C were diluted to 1 μg / mL with 1×PBS pH=7.4, and 100 μL / well was added to the ELISA plate (NUNC442404), and coated at 4°C overnight. The ELISA plate was dried, and 1% BSA-PBS was added at 150 μL / well, and incubated at 37°C for 2 hours. The plate washer was washed 3 times according to the program, and the detection antibody AM22 (Antibody System) was added according to the design, 100 μL / well, and incubated at 37°C for 2 hours. The plate washer was washed 3 times according to the program, and AP-labeled goat anti-human secondary antibody was added at a dilution of 1:2000, 100 μL / well, and incubated at 37°C for 1 hour. The plate washer was washed 3 times according to the program, and pNPP substrate solution was added, 100 μL / well, and the ELISA reader was set to a wavelength of 405nm and read.

[0106] 2. Experimental results

[0107] Test results such as Figure 3 As shown, the binding activity of unmodified and modified RSV prefusion F proteins with antibody AM22 after incubation at different osmotic pressure values ​​was significantly different, and the antibody detection OD values ​​of the modified RSV prefusion F protein at 10mM, 80mM, 150mM, 1500mM and 3000mM were significantly higher than those of the unmodified RSV prefusion F protein, indicating that the RSV modified protein prepared by the present invention can still maintain a high antigen binding activity after incubation at different osmotic pressure values, that is, compared with the unmodified protein, the stability of the RSV modified protein prepared by the present invention at different osmotic pressures is significantly enhanced.

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

[0109] Example 5 Application of RSV pre-fusion F protein immune preparation

[0110] 1. Preparation of immune preparations 1 and 2

[0111] The RSV unmodified protein and modified protein obtained in Example 1 were sampled at a content of 5 μg, phosphate buffer pH = 5.8 buffer was added, sterilized and filtered with a 0.22 μm membrane, stirred at 4°C for 1 h, aseptically packaged in 0.5 mL / bottle, and stored at 4°C until used for immunization, to obtain Preparation 1 (unmodified protein) and Preparation 2 (modified protein).

[0112] 2. Preparation of immune preparations 3 and 4

[0113] The RSV unmodified protein and modified protein obtained in Example 1 were sampled at a content of 5 μg, phosphate buffer pH = 5.8 buffer was added, and sterilized and filtered with a 0.22 μm membrane; sterile aluminum phosphate gel (Benetag) was added, and the final aluminum ion amount was 0.125 mg. The mixture was stirred at 4°C for 1 h, aseptically divided into 0.5 mL / bottle, and stored at 4°C for immunization, thereby obtaining Preparation 3 (unmodified protein) and Preparation 4 (modified protein).

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

[0115] Female BALB / c mice aged 4-6 weeks were randomly divided into 4 groups with 10 mice in each group. The immune preparations used in each group were Preparation 1, Preparation 2, Preparation 3 and Preparation 4 prepared above, respectively. Subcutaneous immunization was performed once every two weeks, 0.1 mL each time, for a total of two immunizations. Blood was collected 35 days after immunization, and the blood was then placed at room temperature for 4 hours, centrifuged at 10,000 rpm at room temperature, and the supernatant serum was aspirated and stored at -70°C for detection.

[0116] 4. ELISA method to detect RSV prefusion F protein antibody titer in mouse immune serum

[0117] Prepare a purified RSV prefusion F protein stock solution of 1 mg / mL (1×PBS solution) and store in a 4°C refrigerator. Dilute the protein stock solution to 4 μg / mL in coating buffer, add 100 μL of coating solution to each well to coat the ELISA plate, and incubate overnight at room temperature. Wash 4 times with plate washing buffer, add 150 μL of blocking buffer, incubate at 37°C for 2 hours, wash 3 times with 300 μL of plate washing buffer per well, and store at 4°C for one week.

[0118] Dilute the corresponding test serum obtained by mouse vaccination in step 3 into working sample serum, dilute it appropriately, add it to the first row of wells of the ELISA plate, 100 μL per well, and perform 2-fold serial dilutions from the first row downwards, and incubate at 37°C for 2 hours. Wash each well with 300 μL of plate washing buffer, wash 3 times, add AP-labeled anti-mouse secondary antibody at a dilution of 1:2000, 100 μL / well, and incubate at 37°C for 1 hour. Wash the plate 3 times according to the program of the plate washer, add pNPP substrate solution, 100 μL / well, set the wavelength of the microplate reader to 405nm, and read the results.

[0119] 5. Experimental results

[0120] like Figure 4 As shown in the figure, when no adjuvant was added to the immune preparations, there were obvious differences in the protein antibody titers in the sera of mice immunized with different preparations. The antibody titer of mice immunized with the RSV pre-fusion F recombinant protein preparation modified by amino acid mutation (Preparation 2) was significantly higher than that of the RSV pre-fusion F protein preparation not modified by amino acid mutation (Preparation 1); and the sera of the immunized mice were diluted at different multiples. At each concentration, the antibody titer of the RSV recombinant protein preparation after amino acid modification (Preparation 2) was still significantly higher than that of the RSV protein preparation before modification (Preparation 1), and as the serum dilution multiple gradually increased, the antibody titers of RSV proteins before and after modification showed a downward trend.

[0121] like Figure 5 As shown in the figure, when adjuvants were added to the immune preparations, there were obvious differences in the protein antibody titers in the sera of mice immunized with different preparations. The antibody titer of mice immunized with the RSV pre-fusion F recombinant protein preparation modified by amino acid mutation (Preparation 4) was significantly higher than that of the RSV pre-fusion F protein preparation not modified by amino acid mutation (Preparation 3); and the sera of the immunized mice were diluted at different multiples. At each concentration, the antibody titer of the RSV recombinant protein preparation after amino acid modification (Preparation 4) was still significantly higher than that of the RSV protein preparation before modification (Preparation 3), and as the serum dilution multiple gradually increased, the antibody titers of RSV proteins before and after modification showed a downward trend.

[0122] The experimental results of this example show that compared with the RSV pre-fusion F protein before modification, 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 by the present invention has better immune effect.

[0123] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. The scope of protection of the present invention shall be subject to the scope required by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

Claims

1. A respiratory syncytial virus prefusion F protein, characterized in that The pre-fusion F protein is a respiratory syncytial virus pre-fusion F recombinant protein modified by amino acid mutation, and the mutation modification method is selected from any one of the following methods: (1) performing amino acid point mutations on the full-length sequence of the wild-type pre-fusion F protein as shown in SEQ ID NO.1, mutating K at position 75 to C and S at position 215 to C; (2) First, the transmembrane region / intracellular region in the full-length sequence of the wild-type pre-fusion F protein as shown in SEQ ID NO.1 was deleted, and the fibritin / Throm / 6his / Stretaq sequence was connected to its C-terminus to obtain a mutant with an amino acid sequence as shown in SEQ ID NO.

2. Then, amino acid point mutations were performed on the basis of the amino acid sequence SEQ ID NO.2, such that K at position 75 was mutated to C, and S at position 215 was mutated to C.

2. A respiratory syncytial virus prefusion F protein according to claim 1, characterized in that: The amino acid sequence of the pre-fusion F recombinant protein obtained by the method (2) is shown in SEQ ID NO.

3.

3. A method for preparing a respiratory syncytial virus pre-fusion F protein as claimed in claim 1 or 2, characterized in that: The following steps are involved: Introducing a nucleic acid molecule encoding the respiratory syncytial virus pre-fusion F protein as claimed in claim 1 or 2 into a CHO cell to obtain a recombinant cell; culturing the recombinant cell to obtain; The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

4. A biomaterial, characterized in that: The biological material is at least one of the following (i)-(iv): (i) a nucleic acid molecule encoding the respiratory syncytial virus prefusion F protein according to claim 1 or 2, 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) a recombinant microorganism containing the nucleic acid molecule described in (i) or a recombinant microorganism containing the recombinant expression vector described in (ii); (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).

5. Use of the respiratory syncytial virus prefusion F protein as claimed in claim 1 or 2, or the respiratory syncytial virus prefusion F protein prepared by the preparation method as claimed in claim 3, or the biomaterial as claimed in claim 4 in the preparation of respiratory syncytial virus antibodies.

6. Use of the respiratory syncytial virus prefusion F protein as claimed in claim 1 or 2, or the respiratory syncytial virus prefusion F protein prepared by the preparation method as claimed in claim 3, or the biomaterial as claimed in claim 4 in the preparation of a medicament for preventing and / or treating respiratory syncytial virus infection.

7. Use of the respiratory syncytial virus prefusion F protein as claimed in claim 1 or 2, or the respiratory syncytial virus prefusion F protein prepared by the preparation method as claimed in claim 3, or the biomaterial as claimed in claim 4 in the preparation of a diagnostic reagent for respiratory syncytial virus.

8. Use of the respiratory syncytial virus prefusion F protein as described in claim 1 or 2, or the respiratory syncytial virus prefusion F protein prepared by the preparation method as described in claim 3, or the biological material as described in claim 4 in the preparation of nucleic acid vaccines, protein vaccines, virus-like particle vaccines or vector vaccines of respiratory syncytial virus.

9. A respiratory syncytial virus vaccine, characterized in that: It comprises the respiratory syncytial virus pre-fusion F protein as described in claim 1 or 2, or the respiratory syncytial virus pre-fusion F protein prepared by the preparation method as described in claim 3, or the biological material as described in claim 4.

10. A respiratory syncytial virus vaccine according to claim 9, characterized in that: Pharmaceutically acceptable adjuvants and / or excipients are also included.

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