Respiratory syncytial virus pre-fusion F protein modified antigen polypeptide and application thereof

Abstract

The invention relates to the technical field of protein engineering, in particular to a respiratory syncytial virus pre-fusion F protein modified antigen polypeptide and application thereof. The antigen polypeptide is obtained by any one or more of the following mutations on the basis of RSV F protein: A74C, E218C, V157C, N183C, A153C, K61C, K75G, N496A and E487P. Mutation sites capable of improving the stability of RSV F protein and the binding capacity of the RSV F protein and an antibody are obtained through research and screening, and antigen polypeptides based on the mutation sites are further provided. The antigen polypeptide provided by the invention can be correctly folded into a conformation before fusion of the F protein of the respiratory syncytial virus in an expression process, is stably maintained, has higher neutralizing antibody titer aiming at respiratory syncytial virus infection after immunization, and has important application value in the field of prevention and treatment of the respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the technical field of protein engineering, and in particular to a respiratory syncytial virus pre-fusion F protein modified antigen polypeptide and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is the main virus of lower respiratory tract infections (LRTI), often causing pneumonia and bronchiolitis. The age curve of human infection with RSV is U-shaped, with the highest incidence in infants and the elderly. Most adults have been infected with RSV in childhood, but the immune protection effect after infection cannot be maintained continuously and completely.

[0003] RSV belongs to the family Paramyxoviridae and the genus Pneumovirus. The virus surface has an envelope and the genome is a single-stranded negative-sense RNA. There are three proteins on the surface of RSV virus particles, namely small hydrophobic protein (SH), attachment glycoproteins (G) and fusion glycoproteins (F). The main function of G protein is to bind to the host cell surface receptor, and it is the structural protein with the highest mutation rate in RSV isolates. RSV is divided into two subtypes, RSV A and RSV B, based on the mutation and evolution of RSV G protein sequences. In recent years, the infection incidence of RSV A and RSV B has tended to be similar, and the two can circulate together in the same area. F protein mainly mediates the fusion of viral membrane and cell membrane, and the neutralizing antibodies produced by humans and animals after RSV infection are mainly targeted at F protein. F protein is the key target of RSV vaccine, but F protein tends to change from the metastable prefusion (pre-F) conformation to the postfusion conformation (post-F) after fusion, and the neutralizing activity of antibodies against post-F is low. Therefore, the key point in RSV vaccine design is to enable the F protein to exist in the form of pre F and maintain the stable state of pre F. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a respiratory syncytial virus pre-fusion F protein modified antigen polypeptide and application thereof.

[0005] In a first aspect, the present invention provides a polypeptide, which is obtained by comprising any one or more of the following mutations based on RSV F protein: A74C, E218C, V157C, N183C, A153C, K61C, K75G, N496A and E487P.

[0006] The expression of mutation used in the present invention is a universal expression in the art. Taking A74C as an example, the first A is alanine and the last C is cysteine, that is, the 74th amino acid is mutated from alanine to cysteine.

[0007] Furthermore, the polypeptide is obtained by including any one or more of the following mutations on the basis of RSV F protein: (1) A74C and E218C; (2) A153C and K461C; (3) V157C and N183C; (4) E487P; (5) K75G; (6) N496A.

[0008] Furthermore, the polypeptide has the following mutations simultaneously based on RSV F protein: A74C, E218C, V157C, N183C and E487P.

[0009] Furthermore, the following gene editing methods are also included: i) replacing the amino acid sequence at positions 04-144 with the linker GS; ii) adding the T4 fibritin motif after the 513th amino acid sequence; The T4 fibritin motif includes: GYIPEAPRDGQAYVRKDGEWVLL; iii) adding the linker SAIG ​​between the extracellular region and the T4 fibritin motif; iv) Connecting Twin-Strep-tag Ⅱ to the C-terminus of the T4 fibritin motif, and adding a linker GA and an enterokinase cleavage site DDDDK in the middle, wherein the Twin-Strep-tag Ⅱ includes: SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK.

[0010] Furthermore, the RSV F protein comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids, and having the same functional protein.

[0011] Furthermore, the polypeptide comprises the amino acid sequence shown in SEQ ID NO.1.

[0012] In a second aspect, the present invention provides a nucleic acid for encoding the polypeptide. Preferably, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO.2.

[0013] In a third aspect, the present invention provides a vaccine comprising the polypeptide and an adjuvant; the adjuvant comprises: one or more of aluminum adjuvant, MF59, AS01 adjuvant or CpG adjuvant, preferably MF59.

[0014] In a fourth aspect, the present invention provides a kit comprising the polypeptide, or the nucleic acid, or the vaccine.

[0015] In a fifth aspect, the present invention provides a preparation method, comprising: The coding gene of the polypeptide is constructed on an expression vector to obtain an expression plasmid, and the expression plasmid is transfected into an expression cell line to express the polypeptide; The expression cell line is preferably a CHO cell.

[0016] In a sixth aspect, the present invention provides use of the polypeptide in preparing a diagnostic or preventive agent for respiratory syncytial virus.

[0017] The present invention has the following beneficial effects: The present invention is based on multiple mutation methods to transform the respiratory syncytial virus pre-fusion F protein modified antigen polypeptide. Compared with the respiratory syncytial virus pre-fusion F protein before the transformation, its binding activity with monoclonal antibodies D25 and AM14 is better, and the stability is good. The binding activity with neutralizing monoclonal antibodies D25 and AM14 is not reduced after being placed at 4°C or 37°C for 30 days, and repeated freezing and thawing for 5 times will not affect it. In addition, after the antigen polypeptide is prepared into a vaccine, it can induce a higher level of neutralizing antibody titer against RSV strain A2 and RSV strain 18537 in mice compared with sc9-10 DS-Cav1, which is of great significance for the prevention and treatment of respiratory syncytial virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 These are the ELISA test results of RSV pre F protein modified antigen polypeptides with different mutation site combinations provided in Example 2 of the present invention; wherein, the left figure shows the binding activity with monoclonal antibody D25, and the right figure shows the binding activity with monoclonal antibody AM14.

[0020] Figure 2 This is the SDS-PAGE comparison result of DS2-2 and sc9-10 DS-Cav1 provided in Example 3 of the present invention.

[0021] Figure 3 The stability results of DS2-2 detected by ELISA provided in Example 3 of the present invention are shown; wherein, the left figure shows the binding activity with monoclonal antibody D25 under different conditions, and the right figure shows the binding activity with monoclonal antibody AM14 under different conditions.

[0022] Figure 4 It is a comparison chart of serum neutralizing antibody titers after mice were immunized with DS2-2 and the control sc9-10 DS-Cav1 provided in Example 3 of the present invention; the left figure is the experimental result of RSV strain A2, and the right figure is the experimental result of RSV strain 18537. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0025] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0026] In the following examples, the RSV F protein inactive precursor F0 (unmutated wild-type sequence, SEQ ID NO.1) is 574 amino acids in length and contains two furin cleavage sites. It is cut into four polypeptides during the maturation process, namely, the signal peptide (amino acids 1-25), F2 (amino acids 26-109), p27 (amino acids 110-126) and F1 (amino acids 127-574). Subsequently, F2 and FI are non-covalently bound to form F protein monomers, and three F protein monomers are bound to form a trimer. Among them, the N-terminus of polypeptide F2 contains a hydrophobic fusion peptide (amino acids 137-157), and the C-terminus contains an intracellular region (amino acids 551-574) and a transmembrane region (amino acids 530-550).

[0027] The term "D25" refers to the antibody described in CN101778866B, and its specific amino acid sequence refers to sequence 1-8 in the specification. Monoclonal antibody D25 specifically binds to the ф epitope of pre F, but not to post F.

[0028] The term "Mota" or "Motavizumab" refers to the antibody described in WO2007002543A2, and its specific sequence refers to the amino acid sequence of antibody A4B4L 1FR-S28R (Motavizumab) in its patent specification (sequence numbers 48 and 11). Motavizumab binds to both pre F and post F.

[0029] The term "AM14" refers to the antibody described in CN101778866B, and the specific sequence refers to the amino acid sequence of AM14 described in Example 4 of the patent specification. AM14 specifically binds to pre F trimer.

[0030] Example 1 Screening of stable mutation sites for RSV pre F modified antigen

[0031] 1. Structural characteristics of sequence template sc-F0: In order to make RSV F soluble and stable trimer, the amino acid sequence of RSV F0 precursor protein (sequence such as GenBank: AAB86664.1) was initially transformed into sc-F0, and further point mutation was used as a control template to screen mutation sites that are conducive to the formation of stable pre-fusion trimer. The sc-F0 transformation method is as follows: The amino acids 1-513 of the F0 precursor were truncated to promote protein secretion and expression; The amino acid sequence at positions 104-144 in the F0 sequence (sequence NNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGV) was replaced with a linker (sequence GS) to make F0 form a single chain that can fold correctly; A T4 fibritin motif (sequence GYIPEAPRDGQAYVRKDGEWVLL, sequence source NCBI Reference Sequence: WP_371335556.1, 458G-480L) was added after the amino acid sequence at position 513 of F0, which was conducive to the formation of trimers, and a linker (sequence SAIG) was added between the F0 extracellular region and the T4 fibritin motif; Twin-Strep-tag Ⅱ (sequence SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK) was connected to the C-terminus of the T4 fibritin motif for subsequent purification, and a linker (sequence GA) and enterokinase cleavage site (sequence DDDDK) were added in the middle.

[0032] 2. Plasmid construction and transfection expression: (1) Using sc-F0 as a template, mutation sites were screened, soluble F protein mutant genes were synthesized and cloned into the pEE12.4 plasmid, and protein was expressed using the CHO-K1 cell expression system.

[0033] (2) CHO-K1 cells were cultured in suspension using Ex-Cell Advanced CHO Fed-batch medium containing 4 mM glutamine at 37°C, 5% CO2, 70% relative humidity, and 120 rpm. The cells were passaged one day before transfection and cultured for 12-16 h before transfection.

[0034] (3) During transfection, adjust the cell density to 1×10 7 viable cells / mL, plasmid and PEI 40000 transfection reagent were added successively and mixed quickly. After the cells were cultured in a carbon dioxide shaking incubator for 1 h, Ex-Cell Advanced CHO Fed-batch medium containing 4 mM glutamine was added to dilute the cells to 3×106 cells / mL and continue to culture. One day after transfection, Ex-Cell Advanced CHO Fed-batch medium containing 4 mM glutamine was added to continue to culture the cells. Two days after transfection, Cell Boost 7a and Cell Boost 7b feeding medium were used for feeding culture. After continuing to culture for 3 days, the cell culture fluid was harvested and centrifuged at 800×g for 10 min to remove the cells. The obtained supernatant was used to detect the expression level of each plasmid and the stability of the pre-fusion conformation by ELISA.

[0035] 3. ELISA detection: Dilute the capture antibody Motavizumab to 1 μg / mL with PBS, add 100 μL per well to a 96-well flat-bottom transparent high-adsorption ELISA plate, seal the plate with a sealing film and incubate overnight at 4°C. Discard the coating solution in the plate, add 300 μL PBST to each well for washing, and repeat 3 times. Add 100 μL of blocking solution to each well, incubate at 37°C for 1 h, and then wash the plate. Dilute each harvested supernatant 10 times and add it to the ELISA plate. Set up 3 replicate wells for each sample, 100 μL per well, incubate at 37°C for 1 h, and then wash the plate. Dilute the detection antibodies D25-HRP, AM14-HRP, and α-Strep tag pAb-HRP and add them to the ELISA plate, 100 μL per well, incubate at 37°C for 1 h, and then wash the plate. Add 100 μL TMB single-component colorimetric solution to each well and react in the dark at room temperature for 5-10 min. Then add 50 μL of ELISA stop solution to each well to terminate the reaction. Use a microplate reader to read the OD of each well. 450 The average value of each supernatant sample was taken for comparison.

[0036] 4. The test results are shown in Table 1. Mutant M1 (A74C, E218C) can improve the binding with monoclonal antibody AM14, and can also improve the binding with monoclonal antibody D2 and antibody α-Strep tag, indicating that this mutation can not only effectively improve the correct folding efficiency of the trimeric pre F protein, but also improve the total expression efficiency of the F protein. Mutant M2 (A153C, K461C) only improves the binding with monoclonal antibody AM14, and greatly reduces the binding with monoclonal antibody D2 and antibody α-Strep tag. Mutants M4 (V157C, N183C), M8 (E487P), M10 (K75G) and M11 (N496A) simultaneously improve the binding with monoclonal antibody D2 and antibody α-Strep tag, that is, simultaneously improve the correct folding efficiency of pre F protein and the total expression efficiency of F protein. In the future, the mutations that are conducive to the stability of pre F conformation will be combined for further evaluation.

[0037] Table 1 Stable mutation sites and expression results of RSV pre F protein modified antigen polypeptide

[0038] Example 2 Screening of stable mutation site combinations for RSV pre F modified antigen

[0039] The present invention combines the positive cloning sites in Example 1, constructs a new expression plasmid, and then transfects CHO-K1 cells for expression. After cell transfection, the cell supernatant is collected and tested by ELISA after 7 days of feeding culture. During ELISA testing, each supernatant is diluted 3 times continuously and added to the ELISA plate for testing. The OD450 Values, plot sample dilution-OD 450 The constructed plasmids with stable mutation site combinations of RSV pre F protein are shown in Table 2, and the ELISA test results are shown in Figure 1 The results showed that the RSV F protein expressed by the combination DS2-2 had stronger binding to D25 and AM14, indicating that DS2-2 could more effectively fold into the pre F conformation. DS2-2 will be further evaluated in the future.

[0040] Table 2. Combinations of stable mutation sites in RSV pre F protein modified antigen peptides

[0041] Example 3 Related research verification of DS2-2

[0042] 1. Large-scale expression and purification of DS2-2

[0043] The present invention transfects CHO-K1 cells with plasmid pEE12.4-DS2-2 to express DS2-2 in large quantities. Two days after transfection, fed-batch culture is performed using Cell Boost 7a, Cell Boost 7b feed medium and 45% glucose solution. After 14 days of culture, the cell culture fluid is harvested, centrifuged at 800×g for 10 min to remove cells, and the supernatant is centrifuged at 10,000×g for 10 min to remove cell debris, and the obtained supernatant is used for subsequent affinity chromatography purification.

[0044] The StrepTrap HP affinity chromatography column was installed on the GE AKTA protein purifier. The chromatography column was equilibrated with 10 column volumes of equilibration buffer, and the supernatant was filtered through a 0.45 μm filter membrane and slowly loaded into the chromatography column. After eluting the impurities with 10 column volumes of equilibration buffer and equilibrating the chromatography column again, the target protein was eluted with 6 column volumes of eluent. The eluate containing the target protein was concentrated using an ultrafiltration tube with a molecular weight cutoff of 50 kD, and the solution was replaced with PBS. sc9-10 DS-Cav1 (Joyce, Zhang et al. 2016) was expressed and purified at the same time as a control. The purified protein was quantified using NanoDrop and then identified by SDS-PAGE. The electrophoresis results are shown in Figure 2 As shown, the monomeric F protein of DS2-2 under reducing conditions is consistent with the reported band size of sc9-10 DS-Cav1.

[0045] 2. DS2-2 Storage Stability Study

[0046] In the present invention, DS2-2 is placed at 4° C., 25° C. and 37° C. for 30 days or repeatedly frozen and thawed for 5 times, and the binding status of DS2-2 with monoclonal antibody D25 is detected by ELISA.

[0047] The ELISA test method is as follows: DS2-2 after each condition treatment was diluted to 1 μg / mL with PBS, and 1 μg / mL and 0.5 μg / mL freshly thawed DS2-2 were set as stable control and unstable control respectively. 100 μL was added to each well of a 96-well flat-bottom transparent high-adsorption ELISA plate, and the plate was sealed with a sealing film and incubated at 4°C overnight. The coating solution in the plate was discarded, and 300 μL PBST was added to each well for washing, and repeated 3 times. 100 μL of blocking solution was added to each well, incubated at 37°C for 1 h, and then washed. Monoclonal antibody D25 or AM14 was diluted 3 times and added to the ELISA plate, 100 μL per well, incubated at 37°C for 1 h, and then washed. The secondary antibody Rabbit Anti-Human IgG H&L (HRP) was diluted and added to the ELISA plate, 100 μL per well, incubated at 37°C for 1 h, and then washed. After adding 100 μL of TMB single-component colorimetric solution to each well, react in the dark at room temperature for 5-10 min. Then add 50 μL of ELISA stop solution to each well to terminate the reaction. Use an ELISA reader to read the OD value of each well. 450 Then plot the mAb concentration-OD 450 Value antigen-antibody binding curve.

[0048] The results are as follows Figure 3 As shown in the results of the binding curve with monoclonal antibody D25, the curve obtained by the detection of freshly thawed DS2-2 at 37℃ or 4℃ for 30 days basically overlaps; after DS2-2 was repeatedly frozen and thawed 5 times at -80 / 37℃, its binding curve also basically overlaps with the freshly frozen and thawed DS2-2 curve; after being placed at room temperature for 30 days, its result was lower than the binding curve of freshly thawed DS2-2, but still significantly higher than the binding curve of 0.5 μg DS2-2 (i.e. 50%) coating detection. The results of monoclonal antibody AM14 and D25 detection are basically consistent. DS2-2 placed at room temperature for 30 days affects its binding with AM14, and other conditions have no effect. The results show that DS2-2 can be stably stored at 37℃ or 4℃ for more than 30 days, and repeated freezing and thawing 5 times will not affect its pre F conformation. After being placed at room temperature for 30 days, it can still retain more than 50% of its activity. In summary, the DS2-2 protein has good stability and can keep its pre F conformation stable.

[0049] 3. Mouse immune evaluation

[0050] The present invention selects 6-week-old female BALB / c mice, 5 mice per group. The DS2-2 and sc9-10 DS-Cav1 obtained above were diluted to 200 μg / mL with PBS, respectively, and mixed with an equal volume of MF59 adjuvant (Avituo, C01001). Mice were immunized by intramuscular injection, with 100 μL (10 μg antigen, 50 μL MF59) injected per mouse, and the immunization time was day 0 and day 21, respectively. Blood was collected from mice on the 35th day, and the blood was allowed to stand at room temperature for 4 h. After the blood was stratified, the upper serum was taken for neutralizing antibody detection after centrifugation at 8,000×g for 10 min.

[0051] The neutralizing antibody titer detection method is as follows: Vero cells were inoculated in a 96-well plate and the cells were cultured to a monolayer. The serum was incubated in a 56°C water bath for 30 min to inactivate complement, and then graded diluted with MEM medium containing 2% FBS, and RSV strain A2 or strain 18537 virus solution was added and mixed, and the mixture was incubated at 37°C for 1 h. The virus-serum mixture was added to the Vero cell culture plate and cultured at 37°C for 48 h. The cell culture supernatant was discarded, and 50 μL of 4% paraformaldehyde solution was added to each well for fixation at room temperature for 30 min, and the cells were washed 3 times with PBS. 50 μL of 0.1% TritonX-100 was added to each well for permeabilization for 10 min, and washed 3 times with PBS. 50 μL of 5% BSA was added to each well for blocking for 1 h, and washed 3 times with PBS. Subsequently, RSV F protein-specific antibody Motavizumab-FITC was added and incubated at room temperature for 2 h, and washed 3 times with PBS. Add 100 μL PBS to each well and store the cell culture plate away from light. Use a fluorescence microscope to observe and record the fluorescence of each well in the cell culture plate, and count and calculate the results.

[0052] The results are shown in Table 1 and Figure 4 As shown in the results of neutralizing antibody titer detection of RSV strain A2, the neutralizing antibody titer induced by DS2-2 was significantly higher than that of the control sc9-10 DS-Cav1, and the geometric mean titer (GMT) of neutralizing antibodies of DS2-2 was 2.4 times that of sc9-10 DS-Cav1. In the neutralizing antibody titer detection results of RSV strain 18537, the neutralizing antibody titer induced by DS2-2 was also higher than that of sc9-10 DS-Cav1, and the GMT of DS2-2 was 2.4 times that of sc9-10 DS-Cav1. The results of in vivo immune evaluation in mice showed that DS2-2 could induce higher titer neutralizing antibody levels against RSV strain A2 and RSV strain 18537 at the same time compared with sc9-10 DS-Cav1, and had a more ideal structure and immunogenicity.

[0053] Table 3 Neutralizing antibody titer

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide, characterized in that: The polypeptide is obtained by including any one or more of the following mutations on the basis of RSV F protein: A74C, E218C, V157C, N183C, A153C, K61C, K75G, N496A and E487P.

2. The polypeptide according to claim 1, characterized in that The polypeptide is obtained by including any one or more of the following mutations on the basis of RSV F protein: (1) A74C and E218C; (2) A153C and K461C; (3) V157C and N183C; (4) E487P; (5) K75G; (6) N496A.

3. The polypeptide according to claim 1, characterized in that The following gene editing methods are also included: i) replacing the amino acid sequence at positions 04-144 with the linker GS; ii) adding the T4 fibritin motif after the 513th amino acid sequence; The T4 fibritin motif includes: GYIPEAPRDGQAYVRKDGEWVLL; iii) adding the linker SAIG ​​between the extracellular region and the T4 fibritin motif; iv) Connecting Twin-Strep-tag Ⅱ to the C-terminus of the T4 fibritin motif, and adding a linker GA and an enterokinase cleavage site DDDDK in the middle, wherein the Twin-Strep-tag Ⅱ includes: SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK.

4. The polypeptide according to any one of claims 1 to 3, characterized in that The RSV F protein comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO.1; (2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 obtained by replacing, inserting or deleting one or more amino acids, and having the same functional protein.

5. The polypeptide according to claim 1, characterized in that The polypeptide comprises the amino acid sequence shown in SEQ ID NO.

1.

6. A nucleic acid, characterized in that The nucleic acid is used to encode the polypeptide according to any one of claims 1 to 5.

7. A vaccine, characterized in that include: The polypeptide and adjuvant according to any one of claims 1 to 5; The adjuvant includes: one or more of aluminum adjuvant, MF59, AS01 adjuvant or CpG adjuvant, preferably MF59.

8. A kit, characterized in that: The kit comprises the polypeptide according to any one of claims 1 to 5, or the nucleic acid according to claim 6, or the vaccine according to claim 7.

9. The method for preparing the polypeptide according to any one of claims 1 to 5, characterized in that: include: The coding gene of the polypeptide is constructed on an expression vector to obtain an expression plasmid, and the expression plasmid is transfected into an expression cell line to express the polypeptide; The expression cell line is preferably a CHO cell.

10. Use of the polypeptide according to any one of claims 1 to 5 in the preparation of a diagnostic or preventive agent for respiratory syncytial virus.

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