A respiratory syncytial virus F protein mutant and its application

Abstract

The present invention belongs to the field of biomedicine, and in particular to a respiratory syncytial virus F protein mutant and its application. The respiratory syncytial virus F protein mutant includes at least one pair of amino acid residues in the F1 subunit and / or F2 subunit of the F protein polypeptide replaced by cysteine, and / or the F protein signal peptide is partially replaced by a human immunoglobulin light chain signal peptide, and / or the alanine A at position 107 of the polybasic sequence is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R, which can expose more neutralizing antibody epitopes, while maintaining a stable pre-fusion conformation, ensuring that it can induce effective neutralizing antibody reactions and antibody binding reactions to RSV subtypes A and B, and is suitable for various vaccine forms with RSV F protein as an antigen.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a respiratory syncytial virus F protein mutant and application thereof in preparing a respiratory syncytial virus vaccine. Background Art

[0002] Respiratory Syncytial Virus is one of the important pathogens that causes severe lower respiratory tract infections in infants and young children worldwide. According to statistics, the number of children under 5 years old infected with RSV is as high as 34 million worldwide. Since they cannot obtain sustained immunity after RSV infection, they may be repeatedly infected. Almost 100% of infants and young children have been infected more than once within 2 years of age, resulting in 66,000-199,000 deaths. In the analysis of single pathogen infection factors for all-cause mortality in children aged 1 to 1 year, RSV infection caused a mortality rate of 6.7% in children under 1 year old, second only to malaria, causing a serious disease burden worldwide. In addition to infants and young children, adults and the elderly with low immune function are also at high risk of RSV infection. Every year, 3% to 10% of adult colds are caused by RSV. The disease burden caused by RSV infection in the elderly is comparable to that caused by colds in non-epidemic seasons. RSV infection can lead to increased mucus secretion and inflammation, causing heart failure and secondary bacterial pneumonia and other serious complications. RSV is also an important nosocomial infection pathogen, especially for premature infants, infants with congenital heart disease, bronchopulmonary dysplasia and all immunosuppressed patients, and has a huge impact on human health, global medicine and economy. Therefore, WHO lists RSV vaccine as one of the vaccines that should be given priority development in the world in the 21st century, and the development of a safe and effective RSV vaccine is an urgent need.

[0003] RSV belongs to the genus Pneumovirus of the family Paramyxoviridae and is a single-stranded negative-strand RNA virus. Its genome is 15.2 kb in length and transcribes ten genomes, encoding a total of 10 major proteins, including three transmembrane proteins (G, F and SH), two matrix proteins (M and M2), three nucleocapsid proteins (N, P and L) and two non-structural proteins (NS1 and NS2). Among them, adhesion protein G and fusion protein F are the two most important viral proteins for RSV to stimulate the body to produce protective antibodies. G protein mediates the binding of the virus and determines the diversity of RSV antigens. RSV is divided into two subtypes, A and B, based on their antigenic differences; F protein is mainly responsible for the fusion of virus and host cell membranes. Its protein sequence is highly conserved among different subtypes and plays an important role in inducing immune protection and high levels of serum neutralizing antibodies. Neutralizing antibodies induced by F protein can simultaneously inhibit RSV virus infection of both subtypes A and B, and can induce cellular immune CD8+ toxic T cell responses to eliminate pathogens in the body. Therefore, F protein is an important antigen target for RSV vaccine development.

[0004] RSV F protein is the main target protein of neutralizing antibodies. It belongs to type I integral membrane protein. Its precursor protein F0 is composed of 574 amino acids, and three F0s form trimers through hydrophobic interaction. During the virus invasion-mediated membrane fusion process, the precursor F0 will be cleaved by Furin protease and release a short peptide P27 (109~127aa) containing 27 amino acids. The remaining two segments F2 and F1 are connected by two disulfide bonds to form a mature F protein, which is displayed on the cell membrane or the surface of the virion by budding. At this time, the F protein is in a high-energy metastable state and is very unstable, which is called pre-F. After infecting cells, it will spontaneously change into a low-energy post-fusion conformation post-F. A large number of preclinical and clinical data show that the sites with high neutralizing activity mainly exist in the pre-fusion conformation. Vaccines with RSV F pre-fusion conformation as antigen can stimulate the body to produce higher levels of virus neutralizing antibodies. Although RSV vaccines based on pre-F show good application prospects, maintaining the pre-fusion conformation of F protein has also become a key difficulty in vaccine research and development. So far, only two recombinant protein vaccines targeting RSV F protein from GSK and Pfizer have been approved for marketing in the world. Both products use the pre-fusion conformation of the F protein and are used in the United States for people aged 60 and above. However, there are no related preventive vaccine products on the market in China.

[0005] Therefore, developing a pre-fusion conformation F protein antigen with good safety, high expression level and stability has become a technical problem that technicians in this field need to solve urgently. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention has developed a mutant of the pre-fusion F protein of respiratory syncytial virus, which can have a stable pre-fusion conformation and high expression level, and can form a stable trimer, so that it can be used for the treatment of RSV infection. In 2013, McLellan's team and others first developed the first F protein "DS-Cav1" with a stable pre-fusion conformation by point mutation: intramolecular disulfide bonds were obtained by point mutations of S155C and S290C in the F1 fragment, and S190F and V207L ​​point mutations were added to enhance the binding activity of key neutralizing epitopes (DOI: 10.1126 / science.1243283). The protein was confirmed to stimulate significantly higher levels of neutralizing antibody titers. At present, the stable pre-fusion conformation F protein vaccine developed based on structural biology has also entered the clinical stage. Therefore, the recombinant RSV F protein of the present invention introduces mutations based on the amino acid sequence of DS_Cav1 (called wild type), and the mutations include amino acid substitutions, deletions and insertions.

[0007] The first aspect of the present invention provides a respiratory syncytial virus F protein, wherein the F protein comprises at least one mutation selected from the following group:

[0008] (a) at least one pair of amino acid residues is substituted by cysteine;

[0009] (b) the F protein signal peptide is partially replaced by the human immunoglobulin light chain signal peptide;

[0010] (c) At least two polynucleotide sequence sites are replaced.

[0011] In some embodiments, at least one pair of amino acid residues in the F1 subunit and / or the F2 subunit of the F protein polypeptide is substituted with cysteine.

[0012] In some embodiments, the cysteine ​​substitution forms a non-native disulfide bond connection between the F1 subunit and the F2 subunit of the F protein, and the non-native disulfide bond includes disulfide bonds other than Cys69-Cys212 and Cys37-Cys439 formed between the F1 subunit and the F2 subunit.

[0013] In the pre-fusion conformation of RSV F protein, the β2 and β4 chains are close to each other, promoting the spatial folding of the two discontinuous epitopes of the 0 epitope; the addition of non-natural disulfide bonds can relatively stabilize the spatial position between the β2 and β4 chains to enhance the structural stability between the pre-fusion F1 subunit and the F2 subunit, prevent conformational changes in the HRA and HRB regions of the heptapeptide repeat region, stabilize the Pre-F protein structure well, maintain or enhance the binding activity of important sites such as the 0 epitope and the V epitope, and improve the pre-fusion F protein-specific immunogenicity of the mutant antigen.

[0014] In some embodiments, the F protein includes at least one pair of cysteine ​​mutations among 102C+147C, 102C+149C, 102C+150C, 101C+147C, 101C+146C, and 100C+145C, and the mutation sites refer to the sites of the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:01.

[0015] In some embodiments, the F protein signal peptide portion replaced by the human immunoglobulin light chain signal peptide includes at least amino acid residues 1 to 25 of the sequence shown in SEQ ID NO:1.

[0016] In some embodiments, the human immunoglobulin light chain signal peptide is as shown in SEQ ID NO:2.

[0017] The wild-type F protein first forms the F0 precursor protein, which is then cleaved by furin to release a 27-amino acid polypeptide pep27. The remaining two segments F2 and F1 are connected by two disulfide bonds to form the mature F protein-pre-F, which is displayed on the cell membrane or the surface of the virion by budding.

[0018] In some embodiments, in order to improve the removal efficiency of the p27 polypeptide inside the F protein, two polybasic sites on the F protein are replaced.

[0019] In some embodiments, the p27 polypeptide within the F protein includes at least amino acid residues 110 to 136 of the wild-type respiratory syncytial virus F protein.

[0020] In some embodiments, compared with the amino acid sequence of the wild-type respiratory syncytial virus F protein as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0021] In some embodiments, the F protein includes cysteine ​​mutations 102C+147C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0022] In some embodiments, the F protein includes cysteine ​​mutations 102C+149C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0023] In some embodiments, the F protein includes cysteine ​​mutations 102C+150C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0024] In some embodiments, the F protein includes cysteine ​​mutations 101C+147C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0025] In some embodiments, the F protein includes cysteine ​​mutations 101C+146C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0026] In some embodiments, the F protein includes cysteine ​​mutations 100C+145C, the F protein signal peptide portion is replaced by the human immunoglobulin light chain signal peptide as shown in SEQ ID NO:2, and compared with the wild-type respiratory syncytial virus F protein amino acid sequence as shown in SEQ ID NO:1, the alanine A at position 107 in the polybase sequence RARR and KKRKRR of the F protein is mutated to arginine R, and the lysine K at position 134 is mutated to arginine R.

[0027] In some embodiments, the F protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 3-8.

[0028] In some embodiments, the F protein includes a protease cleavage site sequence, such as an HRV 3C enzyme cleavage site (LEVLFQGP) or a protein tag, such as a 6×His-tag (HHHHHH) and a Strep tag-II tag (WSHPQFEK), which is not required for the function of the RSV F protein (e.g., inducing an immune response).

[0029] Another aspect of the present invention provides a recombinant nucleic acid comprising a nucleotide sequence encoding the respiratory syncytial virus F protein as described above.

[0030] In some embodiments, the recombinant nucleic acid is codon optimized for expression in a selected prokaryotic or eukaryotic host cell.

[0031] Another aspect of the present invention provides an expression vector comprising the recombinant nucleic acid as described above.

[0032] Another aspect of the present invention provides a host cell, comprising the recombinant nucleic acid or the expression vector as described above.

[0033] In some embodiments, the host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells. Preferably, the mammalian cells are selected from ExpiCHO, VERO and Expi293.

[0034] Another aspect of the present invention provides a method for preparing the respiratory syncytial virus F protein as described above, the method comprising:

[0035] S1: culturing the host cell as described above under conditions suitable for the expression of the respiratory syncytial virus F protein;

[0036] S2: collecting the expression product and purifying the expression product to obtain the respiratory syncytial virus F protein.

[0037] Another aspect of the present invention also provides the use of the respiratory syncytial virus F protein in the preparation of a vaccine for preventing respiratory syncytial virus infection.

[0038] The present invention also provides a vaccine for preventing respiratory syncytial virus infection, comprising the respiratory syncytial virus F protein as described above, or the respiratory syncytial virus F protein prepared by the preparation method as described above, and a pharmaceutically acceptable carrier or excipient.

[0039] In some embodiments, the single dose of human vaccine contains 60 to 120 ug of the recombinant RSV F protein. In some embodiments, the single dose of human vaccine contains preferably 60 ug of the recombinant RSV F protein. In some embodiments, the single dose of human vaccine contains preferably 120 ug of the recombinant RSV F protein.

[0040] In some embodiments, the carrier or excipient comprises a buffer. Pharmaceutically acceptable carriers and excipients are well known in the art and can be selected by those skilled in the art. Including but not limited to: pH regulators (such as phosphate buffer), surfactants (such as cationic, anionic or non-ionic surfactants), adjuvants, solubilizers, stabilizers, media for containing or administering therapeutic agents, and any combination thereof. Those skilled in the art can select suitable excipients and carriers to produce formulations suitable for administration to subjects via a selected route of use. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including oils derived from petroleum, animals, plants or synthesis. Suitable excipients include but are not limited to glycerol, polyethylene glycol, and calcium ions, magnesium ions, zinc ions and other divalent cation-related salts, etc.

[0041] In some embodiments, the vaccine further comprises an adjuvant.

[0042] In some embodiments, the adjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG and QS-21.

[0043] Generally, an adjuvant should be selected to enhance a Th1 biased immune response in a subject or subject population to which the vaccine is administered and to be safe and effective in the subject or subject population.

[0044] Optionally, the vaccine may also contain at least one other antigen of a pathogenic organism different from RSV, for example, a virus different from RSV, such as herpes zoster virus, human papillomavirus, hepatitis B virus, coronavirus or influenza virus. Alternatively, the pathogenic organism may be a bacterium, such as diphtheria, tetanus or pneumococcus.

[0045] The present invention also provides a method for preparing the vaccine for preventing RSV infection, which specifically comprises: packaging the purified F protein and adjuvant separately or mixing them thoroughly according to a certain proportion.

[0046] In some embodiments, the method further comprises additional steps such as gene synthesis, expression vector construction, purified protein lyophilization, etc. The expressed recombinant RSV F protein can be recovered and purified from the recombinant cell culture by any one or more of many methods known in the art, including ammonium sulfate precipitation, filtration, ultrafiltration, affinity chromatography, anion / cation exchange chromatography, hydrophobic interaction chromatography, etc.

[0047] Compared with the prior art, the advantages and positive effects of the present invention are:

[0048] The present invention obtains a pre-fusion conformation of RSV F protein that can expose more neutralizing antibody epitopes by replacing and deleting multiple amino acids in wild-type RSV F protein, and ensures that it can cause effective neutralizing antibody reactions and antibody binding reactions to RSV subtypes A and B while maintaining a stable pre-fusion conformation. In addition, the present invention uses recombinant RSV F protein as an immunogen combined with an adjuvant to obtain a stronger immune induction efficacy, which not only improves the humoral immune response, but also strongly stimulates Th1 type immunity, greatly improving the immunogenicity of RSV antigens. The mutation method disclosed in the present invention is applicable to other virus strains of human RSV; it is applicable to various vaccine forms with RSV F protein as an antigen, such as recombinant protein vaccines, nucleic acid vaccines, virus-like particle vaccines, and vector vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0050] Figure 1This is the pCAGGS plasmid map.

[0051] Figure 2 The expression results of F protein mutants were detected by Western blot.

[0052] Figure 3 This is the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified F protein mutant HN108-05 and the control protein Ds_Cav1 under reducing conditions.

[0053] Figure 4 The co-expression results of F protein and Furin protease were detected by Western blot. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with specific embodiments:

[0055] To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0056] Example 1: Design and gene synthesis of mutants that introduce disulfide bonds to stabilize the pre-fusion conformation of F protein by cysteine ​​substitution:

[0057] By observing the spatial structure of the prefusion F protein of respiratory syncytial virus, amino acid mutations that can stabilize the prefusion conformation were designed according to the principles of structural biology. Using the amino acid sequence (SEQ ID No. 01) of the wild-type prefusion F protein of respiratory syncytial virus (Ds_Cav1) as a template, a disulfide bond was introduced by cysteine ​​substitution to enhance the structural stability between the prefusion F1 subunit and the F2 subunit to prevent the conformational transition of the HRA and HRB regions of the heptad repeat region, while maintaining or enhancing the binding activity of important sites such as the Φ epitope and the V epitope, thereby improving the prefusion F protein-specific immunogenicity of the mutant antigen. In order to promote the secretory expression of the F protein, the F protein signal peptide (1-25aa) was replaced with the human immunoglobulin light chain signal peptide "MRVPAQLLGLLLLWLRGARC (SEQ ID No .02)". In order to improve the removal efficiency of the p27 polypeptide inside the F protein (110-136aa), two polybasic sites on the F protein (RARR109 and KKRKRR134) were replaced by (RRRR107 and KKRRRR134). Based on the original sequence DS_Cav1, a total of 6 protein variants were designed, and the amino acid sequences are shown in SEQ ID NO:3, 4, 5, 6, 7 and 8 respectively.

[0058] The F protein variant nucleotide sequence designed according to the table was submitted to Beijing Qingke Biotechnology Co., Ltd., and the nucleic acid sequence was determined by codon optimization according to the host Expi293F cells, and inserted into the eukaryotic expression pCAGGS (plasmid map see Figure 1 , purchased from Beijing Qingke Biotechnology Co., Ltd., the sequence is shown in SEQ ID NO.9) sequence, and the whole gene synthesis was performed.

[0059] Table 1 Design of mutants for introducing disulfide bonds and polybasic site replacement by cysteine ​​substitution, detailed sequences are shown in SEQ ID No. 03 to No. 08.

[0060] Table 1. Design of mutants for cysteine ​​substitution to introduce disulfide bonds and polybasic site replacement

[0061]

[0062] Example 2: Expression and preliminary screening of recombinant F protein:

[0063] 1. Cell transfection and growth curve monitoring:

[0064] The expression vector synthesized by genetic engineering technology was cloned and transformed, and the plasmid was extracted and then used to transfect Expi293 cells for recombinant protein expression. Expi293 cells in the logarithmic growth phase were passaged at high density one day before transfection, and the viable cell density was 2-3×106 / mL. After the cells grew overnight, the cell density was diluted to 3×106 / mL. 2.5mL of cell suspension was taken from each well for transfection. 2ul DNA was used for each well, diluted with Opti-MEM medium and gently mixed, and then 8ul PEI40000 was gently added to the diluted DNA immediately, and the DNA complex was allowed to stand at room temperature for 10-20min to prepare the DNA complex. After the incubation, the DNA complex was added drop by drop to the cell solution to be transfected, and the cells were transferred to a shaker at 37℃ with 8% CO2 and cultured at 225rpm. 500ul of feed was added 18-22h after transfection and culture continued. Table 2 shows the changes in cell viability and density during transfection of different protein variants.

[0065] Table 2. Changes in cell viability and density during transfection of different protein variants

[0066]

[0067] Expression and identification of different F protein mutants:

[0068] 2.1 Western Blot method to detect the expression of different protein variants:

[0069] (1) On the third day of culture, collect the cell culture supernatant by centrifugation at 4700 rpm for 40 min at 4°C and sterilize by filtration using a 0.22 μm filter membrane.

[0070] (2) β-Mercaptoethanol and loading buffer were added to the sample, and the sample was heat denatured at 100°C for 10 min. The sample loading volume of the cell supernatant electrophoresis sample was 25 μl / well. The purified DS_Cav1 protein of the control group was loaded with 5 μg. The voltage was 120 V and the electrophoresis was performed for 60 min.

[0071] (3) After electrophoresis, the protein was transferred to a PVDF membrane using the dry transfer method and blocked with TBS solution containing 5% skim milk powder for 2 hours.

[0072] (4) His mouse monoclonal antibody was diluted 1:2000 with TBS buffer. The PDVF membrane was transferred to the diluted antibody and incubated at 4°C overnight.

[0073] (5) Wash three times with TBST every other day, 5 min each time.

[0074] (6) Dilute HRP-Goat anti-Mouse secondary antibody with TBS containing 5% skim milk powder, transfer the PVDF membrane to the diluted secondary antibody, and incubate on a shaker at room temperature for 1 h.

[0075] (7) Wash three times with TBST, 10 min each time, add substrate color development solution, develop for 10 min in the dark, and take pictures.

[0076] (8) Figure 2 As shown, Western Blot analysis of the expression level of F protein mutants in Expi293 cells. The detection antibody is His tag. The first lane in the figure is Marker, the second lane is the control pre-fusion F protein Ds_Cav1, and the third to eighth lanes are F protein mutants HN108-01 to HN108-06.

[0077] 2.2 Double sandwich ELISA to detect the expression of different protein variants:

[0078] Based on the results of Western Blot, D25 (D25 antibody can specifically recognize the F protein of respiratory syncytial virus before fusion conformation) was used as the coating antibody and MPE8 was used as the labeled antibody detection pair to further detect the expression of each mutant. The specific steps are as follows:

[0079] Antibody coating: dilute D25 antibody to 100 ng / ml with coating solution, add 100 μl / well into the ELISA plate, and coat overnight at 4°C.

[0080] Blocking: Wash the plate with 300 μl / well, rinse twice and pat dry, add 300 μl / well of blocking solution and incubate at 37°C for 2 hours.

[0081] Sample addition: Wash the plate twice, add serially diluted standards and samples to be tested, 100 μl / well, and incubate at room temperature for 1.5 h.

[0082] Primary antibody incubation: After washing the plate 5 times, add the detection antibody MPE8 at a final concentration of 100 ng / mL, 100 μl / well, and incubate at room temperature for 1.5 h.

[0083] Secondary antibody incubation: Wash the plate 5 times, add 100 μl / well of HRP-labeled anti-IgG antibody, and incubate at room temperature for 1 h.

[0084] Color development: Add 100 μl / well of color development solution and react at room temperature in the dark for 15 minutes.

[0085] Reading: Open the microplate reader in advance, add 50 μl / well of stop solution to the reaction wells of the microplate after color development is complete, place it in the microplate reader (450nm) for reading, and the result is expressed as OD value. The results are shown in Table 3:

[0086] Table 3 ELISA results for detecting the expression of different mutants

[0087]

[0088] After comprehensive consideration of the expression level, the cysteine-substituted mutants pHN108-03, pHN108-04, pHN108-05, and pHN108-06 performed better, with expression levels comparable to those of the wild-type DS_Cav1 protein. When the other mutation designs were the same, pHN108-05 with the 101C-146C mutation introduced can improve protein expression to a certain extent, and can be used for the next step of screening and evaluation.

[0089] Example 3: Preparation of recombinant preF protein and detection of protein stability:

[0090] Protein high expression:

[0091] Recombinant proteins DS_Cav1 and pHN108-05 were expressed by transient transfection in Expi293F cells using PEI40000. The day before transfection, Expi293F cells in logarithmic growth phase were passaged at high density at 2.5×10 6 / ml cell density was inoculated in a 1L shake flask. On the day of transfection, the cell density was diluted to 3×10 6 / ml, dilute the expression plasmid DNA with Opti-MEM, then immediately add the transfection reagent PEI40000 to the mixed DNA and mix well, and let it stand at room temperature for 15 minutes. Add the DNA-PEI complex to the cells and culture them in a carbon dioxide shaker at 37°C, 8% CO2, and 125rpm. Centrifuge at 4700rpm for 40 minutes on the fourth day after transfection, and collect the cell culture supernatant. After 0.22um filtration and sterilization, purify the protein by StrepII affinity chromatography.

[0092] Protein purification:

[0093] Prepare a 30 mL gravity column and filter the cell supernatant to remove impurities to prevent clogging of the column. Use one-step affinity chromatography to purify the recombinant protein expressed in large quantities.

[0094] Column loading: Take an empty column, press the lower gasket to the bottom of the column and compact it, rinse the gasket with deionized water, and close the lower outlet immediately after the water flows out from the lower outlet; suspend the resin, use a gun to take an appropriate amount of slurry and add it to the column (the ratio of preservation liquid to filler is 1:1), open the lower outlet to drain the protective liquid; add an appropriate amount of deionized water to rinse the column material, and close the lower outlet after the column material has drained; install the upper gasket, ensure that there is no gap between the gasket and the column material, and be careful not to use force to remove the gasket to prevent damage to the column material.

[0095] Equilibration: Use 5 column volumes of equilibration solution to equilibrate the filler so that it is in the same buffer system as the target protein.

[0096] Binding: Take an appropriate amount of sample to suspend the balanced filler, and transfer the sample-filler mixture to all samples, and combine on a shaker at 4°C overnight.

[0097] Loading: Add the sample to the equilibrated column. You can load the sample multiple times to increase the binding efficiency and collect the flow-through.

[0098] Equilibration / washing: Use 10 column volumes of washing solution to wash the column to remove non-specifically bound proteins and collect the washing solution.

[0099] Elution: Use 5 times the column volume of eluent to elute the target protein and collect in separate tubes.

[0100] Concentration and fluid replacement: The collected protein samples were concentrated and fluid replaced using a 30kDa ultrafiltration tube.

[0101] Quantification: After the concentrated protein is mixed, it is quantified by UV280nm, and a small amount of protein is taken out for electrophoresis detection. The test results are as follows: Figure 3 The rest were aliquoted and stored at -80℃.

[0102] Double antibody sandwich ELISA to detect the stability of purified protein expression:

[0103] The purified protein was divided into 50ul / tube, and 2 tubes were taken from each group, which were placed at 4°C in the dark for 0d, 7d, 14d and 28d, respectively. Then, the double antibody sandwich ELISA method was used, with D25 as the coating antibody and MPE8 as the labeled antibody detection pair, to detect the expression changes of pDs-Cav1 and mutant pHN108-05 purified proteins after incubation at 4°C for different times. Among them, the expression amount is expressed as the concentration of epitope-specific neutralizing antibody detection activity, and the results are shown in Table 4:

[0104] Table 4 Comparison of expression levels between mutants and wild type

[0105]

[0106] SEC-HPLC detection of purified protein structural stability:

[0107] In order to analyze the stability of the designed protein mutant structure, the present invention adopts size exclusion chromatography (SEC-HPLC) to separate and detect the purified samples, and calculates the relative proportion of the main peak according to the peak area of ​​the chromatographic peak. The SEC purity of the control sample pDs_Cav1 and the mutant pHN108-05 purified samples after being placed at 4°C for different times was determined respectively. The specific implementation method is: the chromatographic column is TSKgel G3000SWXL (7.8*300mm 5μm), the detection wavelength is 280nm, the mobile phase is phosphate buffer, the flow rate is 0.6ml / min, the injection volume is 12μg, and the isocratic elution is 15min. The preparation of the test solution is as follows: blank solution: take 0.5ml Buffer to the injection bottle and set aside; take 200μl sample and add it to the liner tube in the injection bottle, remove the bubbles and set aside. The mutant of the present invention was confirmed to be a trimer conformation by testing, and the antigen chromatography detection after purification showed a single protein peak, which met the purity requirements. The results of the relative proportion of the trimer are shown in Table 5.

[0108] Table 5 Trimer detection results of mutants and wild type

[0109]

[0110] After comprehensive consideration of expression and structural stability, the cysteine-substituted mutant pHN108-05 performed better. Compared with the wild-type DS-Cav1, the D25 activity concentration did not decrease significantly or did not decrease after storage at 4°C for 28 days, and the pre-fusion conformation retention rate was comparable to that of DS-Cav1 or had higher stability.

[0111] Example 4: Mouse immunization and neutralizing antibody detection:

[0112] Animal Immunization:

[0113] Female BALB / C mice aged 6-8 weeks were selected for immunization and randomly divided into groups of 6 mice / group, namely negative control group, HN108-05+HA201 group, HN108-05+HA208 group, Ds-Cav1+HA201 group, and Ds-Cav1+HA208 group. The specific groups and numbers are shown in Table 6. Animals in the negative control group were given PBS buffer; the test groups were given 10μg of the target protein with 25μL HA201 or HA208 adjuvant. The immunization method was intramuscular injection of the hind limbs, and the injection volume was 100ul / dose / mouse. Each mouse was immunized twice, with an immunization interval of 3 weeks. Blood was collected from mice in each group two weeks after the second immunization, and the collected serum was stored at -20°C for subsequent neutralizing antibody detection.

[0114] Table 6 Grouping and numbering of immunized mice

[0115]

[0116] Neutralizing antibody detection:

[0117] The specific implementation steps are as follows: (1) the immunized serum is incubated in a 56°C water bath for 30 min; (2) the inactivated serum sample is diluted 3-fold in a 96-well plate with DMEM medium containing 2% FBS, starting from an appropriate multiple, for a total of 6 gradients, with 4 replicates for each sample; (3) 50-100 PFU of RSV A2 and RSV B strains are added to the wells respectively, mixed and incubated at 37°C, 5% CO2 for 1 h; (4) the virus and serum mixture is transferred to a 96-well plate pre-inoculated with Hep-2 cells and cultured at 37°C, 5% CO2 for 5-10 days; (5) the cytopathic effect is observed and the neutralizing antibody titer is calculated using Reed-Muench method. The neutralizing antibody titer is defined as the highest serum dilution with more than 50% intact Hep-2 cells.

[0118] As shown in Tables 7 and 8, compared with the negative control PBS group, the sera of mice in each experimental group two weeks after the second immunization can induce a higher level of neutralizing antibodies, and the sera of mice in each experimental group three weeks after the second immunization can effectively neutralize the replication of type A and type B wild-type live viruses in in vitro cells. The geometric mean titer of neutralizing antibodies induced by the candidate antigen HN108-05+adjuvant HA201 of the present invention is equivalent to that of HN108-05+adjuvant HA208, and HN108-05+HA208 is slightly higher than HN108-05+HA201, but there is no significant difference, but the geometric mean titer of neutralizing antibodies induced by the two is significantly higher than that of DS-Cav1+HA201 and DS-Cav1+HA208.

[0119] Table 7 Serum neutralizing antibody titer test results (type A)

[0120]

[0121] Table 8 Serum neutralizing antibody titer test results (type B)

[0122]

[0123] In summary, the RSV F protein mutant HN108-05 provided by the present invention can induce a higher level of neutralizing antibody titer than DS-Cav1, and when used as a vaccine product, it can induce cross-protection against both A and B strains.

[0124] Example 5: Co-expression of furin protein promotes F protein expression:

[0125] Cell transfection and growth curve monitoring:

[0126] Furin protease is an accelerator of pre-F maturation. Integrating the Furin expression cassette into the vector during cell development can further increase the yield of F protein. The study found that it may be due to the use of different promoters. If Furin protease and F protein are co-expressed, a higher level of expression can be achieved. Based on this study and the screening results of the above mutants, Furin protease was co-transfected with the control protein Ds_Cav1 and the mutant pHN108-05 into Expi293 cells. The specific implementation plan is shown in Table 9:

[0127] Table 9 Implementation scheme for co-expression of furin

[0128]

[0129] 2. Effect of co-expression of furin on the expression of recombinant F protein:

[0130] 2.1 Western Blot detection of F protein expression in co-expression supernatant samples:

[0131] (1) On the 3rd to 4th day of culture, collect the cell culture supernatant by centrifugation at 4700 rpm for 40 min at 4°C and sterilize by filtration using a 0.22 μm filter membrane.

[0132] (2) β-mercaptoethanol and loading buffer were added to the sample, and the sample was heat denatured at 100°C for 10 min. The sample loading volume of the cell supernatant electrophoresis sample was 10 μl / well. The purified DS_Cav1 protein of the control group was loaded with 3 ug, and the voltage was 120 V for 60 min.

[0133] (3) After electrophoresis, the protein was transferred to a PVDF membrane using the dry transfer method and blocked with TBS solution containing 5% skim milk powder for 2 hours.

[0134] (4) Anti-RSV F protein monoclonal antibody was diluted 1:2000 with TBS buffer, and the PDVF membrane was transferred to the diluted antibody and incubated at 4°C overnight.

[0135] (5) Wash three times with TBST every other day, 5 min each time.

[0136] (6) Dilute HRP-Goat anti-Rabbit secondary antibody with TBS containing 5% skim milk powder, transfer the PVDF membrane to the diluted secondary antibody, and incubate on a shaker at room temperature for 1 h.

[0137] (7) Wash three times with TBST, 10 min each time, add substrate color development solution, develop for 10 min in the dark, and take pictures.

[0138] (8) Figure 4 As shown, Western Blot analysis of the expression level of F protein mutants on Expi293 cells. The detection antibody is anti-RSV F protein. The first lane in the figure is a 235KDa protein marker, the second lane is a negative control empty pCAGGS, the third lane is the control sample purified protein Ds_Cav1, and the fourth to seventh lanes are the expression of HN108-05 and Ds_Cav1 without and with Furin protease.

[0139] 3. Double antibody sandwich ELISA to detect the expression of F protein in co-expression supernatant samples:

[0140] Based on the results of Western Blot, D25 (D25 antibody can specifically recognize the F protein of respiratory syncytial virus pre-fusion conformation) was used as the coating antibody and MPE8 was used as the labeled antibody detection pair to further detect the changes in the expression level of pre-F protein after the control sample DS_Cav1 and the mutant pHN108-05 were co-expressed with Furin. The results are shown in Table 10.

[0141] Table 10 Expression levels of mutants and wild type after co-expression with Furin:

[0142]

[0143] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0144] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A respiratory syncytial virus F protein, characterized in that The amino acid sequence of the F protein is shown in SEQ ID NO:

7.

2. A recombinant nucleic acid comprising a nucleotide sequence encoding the respiratory syncytial virus F protein as claimed in claim 1.

3. An expression vector comprising the recombinant nucleic acid according to claim 2.

4. A host cell comprising the recombinant nucleic acid according to claim 2 or the expression vector according to claim 3.

5. The host cell according to claim 4, characterized in that The host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells.

6. A method for preparing the respiratory syncytial virus F protein according to claim 1, characterized in that: The method comprises: S1: culturing the host cell according to claim 4 or 5 under conditions suitable for the expression of the respiratory syncytial virus F protein; S2: collecting the expression product and purifying the expression product to obtain the respiratory syncytial virus F protein.

7. Use of the respiratory syncytial virus F protein according to claim 1, or the respiratory syncytial virus F protein prepared by the preparation method according to claim 6 in preparing a vaccine for preventing respiratory syncytial virus infection.

8. A vaccine for preventing respiratory syncytial virus infection, comprising the respiratory syncytial virus F protein according to claim 1, or the respiratory syncytial virus F protein prepared by the preparation method according to claim 6, and a pharmaceutically acceptable carrier or excipient.

9. The vaccine according to claim 8, characterized in that The vaccine further comprises an adjuvant, and the adjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG and QS-21.

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