A method for preparing a respiratory syncytial virus bivalent antigen and uses thereof

Abstract

The present application belongs to the field of biological medicine, and particularly relates to a bivalent antigen for preventing respiratory syncytial virus infection and application thereof. The present application is based on F protein sequences of RSVA and B subtypes, and by removing post-F specific I and IV epitope sequences and pre-F and post-F common III epitope partial sequences, new F bivalent antigen sequences of A and B subtypes are obtained respectively; then the new F bivalent antigen sequences of A and B subtypes are connected together to form a new F bivalent antigen dimer, which is maintained in a pre-F conformation in vivo and in vitro, so as to stimulate the body to produce antibodies specific to the pre-F epitope and having high neutralization activity. The bivalent dimer antigen has the advantages of stimulating the body to produce more neutralizing antibodies and high yield.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to proteins for preventing respiratory syncytial virus infection and their applications. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the most common pathogens causing lower respiratory tract infections in infants and young children worldwide. Almost all children will be infected with RSV at least once before the age of two, leading to pneumonia or bronchitis, and even death. In immunocompromised and weakened elderly populations, RSV infection can cause severe lower respiratory tract clinical symptoms, thus increasing the risk of death. Therefore, developing a safe, effective, and inexpensive vaccine to prevent RSV infection has become one of the most urgent problems to be solved worldwide.

[0003] RSV belongs to the Paramyxoviridae family and the Pneumovirus genus. Its genome is a single-stranded negative-sense virus, 15.2 kb in length, encoding 11 viral proteins. Based on the RSV genome sequence, it is divided into two subtypes, A and B. Due to the high degree of conservation of the RSV F protein, with only about 10% difference between different RSV subtypes, the F protein is the main antigen for the design and development of broad-spectrum RSV vaccines.

[0004] During the fusion of the RSVF protein with the host cell membrane, the protein changes from a pre-F conformation with higher potential energy and lower stability to a post-F conformation with lower potential energy and relative stability. During this conformational change, pre-F specific epitopes... The V epitope is blocked, preventing the body from producing antibodies with high neutralizing activity; therefore, the development of RSV vaccines based on pre-F has become a hot research topic in the prevention of RSV infection. Currently, there are clinically available RSV vaccines, all of which are modified based on the monomeric structure of the F protein [Krarup A., et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nature Communications, 2015, 6:8143; Crank MC., et al. A proof of concept for structure-based vaccine design targeting RSV in humans. Science, 2019, 365, 505-509.], so that they can maintain the pre-F conformation. However, according to literature reports, these protein vaccines all have problems such as low yield (below 6 mg / L) [Joyce MG., et al. Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV. Nature Structural & Molecular biology, 2016, 23(9): 811-820.] and inability to maintain the pre-F conformation [Crank MC., et al. Science, 2019, 365, 505-509.]. Summary of the Invention

[0005] To address the problems existing in the prior art, the inventors of this invention completed the present invention by modifying the RSVF protein sequence. Specifically, the key design of this invention is as follows: First, based on the F protein sequences of RSVA and B subtypes respectively, new F antigen sequences are obtained by removing the post-F-specific I and IV epitope sequences and the III epitope portion shared by pre-F and post-F; Second, two or more new F antigen sequences are tandemly linked to form new F divalent antigen dimers. These two design features ensure that the new F divalent antigen dimers maintain the pre-F conformation in vivo and in vitro, thereby stimulating the body to produce antibodies specific to the pre-F epitopes of subtypes A and B and possessing high neutralizing activity.

[0006] This invention provides a bivalent respiratory syncytial virus antigen, which is a respiratory syncytial virus antigen obtained by tandemly combining the F protein monomers of RSV A and RSV B subtypes, which have removed the I and IV epitopes specific to post-F and a portion of the III epitope common to pre-F and post-F. Preferably, the respiratory syncytial virus antigen is obtained by tandemly combining one or more (e.g., three, four, five, or six) F protein monomers of the RSV A subtype with one or more (e.g., three, four, five, or six) F protein monomers of the RSV B subtype.

[0007] Preferably, based on the F protein sequence of the RSV A2 strain (preferably its amino acid sequence as shown in SEQ ID NO: 1), the N104-V147 segment (specifically as shown in SEQ ID NO: 2) and the V308-S552 segment (specifically as shown in SEQ ID NO: 3) are removed; a linker sequence (e.g., the two amino acids GS) is inserted between T103 and G148, and C37 is mutated to A, resulting in a V protein containing the characteristics specific to pre-F. The amino acid sequence of the RSV A2F protein monomer, which contains the common II epitope of pre-F and post-F and the III epitope containing only the V40-L45 (VSKGYL) amino acid sequence;

[0008] Based on the F protein amino acid sequence of RSV B9320 strain (preferably as shown in SEQ ID NO: 4), the N104-V147 segment (specifically as shown in SEQ ID NO: 5) and the V308-S552 segment (specifically as shown in SEQ ID NO: 6) were removed; A103 and G143 were directly linked, and C37 was mutated to A, T97 to M, S197 to N, Q202 to R, and K226 to M, resulting in the pre-F-specific V and The amino acid sequence of the RSV B9320F protein monomer, which contains the common II epitope of pre-F and post-F and the III epitope containing only the V40-L45 (VSKGYL) amino acid sequence;

[0009] The newly obtained RSVA2F protein monomer and RSV B9320F protein monomer are tandemly linked, or multiple of the two protein monomers are tandemly linked together, with the protein following the tandem link not including the M1-Q26 segment (specifically as shown in SEQ ID NO: 9), to obtain the respiratory syncytial virus bivalent antigen.

[0010] Preferably, the amino acid sequence of the post-F-specific I epitope is removed as F387-D392 (FNPKYD) and the amino acid sequence of the post-F-specific I epitope is removed as K427-S436 (KNRGIIKTFS); and the portion of the III epitope common to pre-F and post-F, except for the amino acid sequence V40-L45 (VSKGYL), is removed.

[0011] More preferably, the amino acid sequence of the respiratory syncytial virus bivalent antigen obtained by tandemly combining two or more monomers is shown in SEQ ID NO: 7.

[0012] In addition, a tag sequence and a stop codon sequence are added to the 3' end; preferably, there is a linker peptide between each monomer.

[0013] More preferably, its amino acid sequence is as shown in SEQ ID NO: 7, or further includes a tag sequence, preferably as shown in SEQ ID NO: 8.

[0014] The present invention also provides the encoding gene of the respiratory syncytial virus bivalent antigen; preferably, the encoding nucleotide sequence of the tag sequence is a nucleic acid sequence encoding 6 histidines, preferably, as shown in SEQ ID NO: 8; further, it also includes the encoding nucleotide sequence of the stop codon as a single stop codon.

[0015] Preferably, its nucleotide sequence is as shown in SEQ ID NO: 10.

[0016] The present invention also provides a recombinant expression vector or recombinant host cell containing the aforementioned coding gene. Specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as the pCAGGS vector, and the starting host cell of the recombinant host cell is a mammalian cell such as 293F cell.

[0017] Furthermore, the encoding gene of the respiratory syncytial virus antigen is provided.

[0018] The present invention also provides a recombinant expression vector containing the aforementioned coding gene and a recombinant host cell. Specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as the pCAGGS vector, and the host cell is a mammalian cell such as 293F cell.

[0019] The present invention also provides a method for preparing the respiratory syncytial virus (RSV) bivalent antigen, characterized in that the RSV antigen encoding gene is expressed in host cells using a recombinant expression vector to obtain the RSV antigen; specifically, the starting vector of the recombinant vector is a mammalian cell expression vector such as pCAGGS vector, and the host cell is a mammalian cell such as 293F cell; optionally, the method further includes the steps of collecting cell supernatant and purifying the RSV antigen after expression.

[0020] The present invention also provides an antigen composition consisting of the respiratory syncytial virus antigen as the active ingredient and an adjuvant, preferably an aluminum adjuvant and an AddaVax adjuvant.

[0021] The present invention also provides the use of the respiratory syncytial virus antigen in the preparation of reagents for immunizing animals to obtain antibodies.

[0022] The dimer antigen provided by this invention not only maintains its pre-F conformation in vitro and in vivo, thereby stimulating the body to produce antibodies specific to the pre-F epitope and possessing high neutralizing activity, but also has the advantage of stimulating the body to produce more neutralizing antibodies. Furthermore, experiments have demonstrated that the F antigen dimer of this invention also has the advantage of high yield (exceeding 30 mg / L in experiments). Attached Figure Description

[0023] Figure 1 Superdex 200 Increase 10 / 300 (GE) molecular sieve chromatography and electrophoresis of RSV new F divalent antigen dimer.

[0024] Figure 2 Schematic diagram of the immunization strategy.

[0025] Figure 3 Results of RSVF-specific IgG antibody titers induced in cotton rats after immunization with RSVF new divalent antigen dimer.

[0026] Figure 4 The results of in vivo conformational analysis of the protein after rat immunization with the new F divalent RSV antigen dimer (using ELISA to infer whether the antigen is pre-F or post-F in vivo, and the relatively high neutralizing activity of V and V in the specific antibody). (labeled as antibody level).

[0027] Figure 5 Results of RSVA2 virus neutralizing antibody titers induced after rats were immunized with RSV neoF divalent antigen dimer.

[0028] Figure 6 Results of RSVB9320 virus neutralizing antibody titers induced after rats were immunized with RSV new F divalent antigen dimer.

[0029] Figure 7 Results of live virus titers in various tissues after re-challenge with RSV new F divalent antigen dimer in cotton rats immunized with RSV. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but this should not be construed as limiting the invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods well known to those skilled in the art. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] Example 1: Expression and purification of RSV novel F antigen dimer

[0032] Based on the F protein sequence of RSVA2 strain (as shown in SEQ ID NO: 1), the F sequence of RSV B9320 strain (as shown in SEQ ID NO: 3), and the amino acid sequence of RSV F protein monomers that do not contain the I and IV epitopes specific to post-F and the III epitope shared by pre-F and post-F, the amino acid sequences of the new RSVF protein monomers were obtained by tandemly combining two sequences (as shown in SEQ ID NO: 7), adding 6 histidine tags to the 3' end (as shown in SEQ ID NO: 8), and after codon optimization, the respiratory syncytial virus antigen encoding gene (SEQ ID NO: 10) was obtained.

[0033] The obtained respiratory syncytial virus antigen-encoding gene sequence was then cloned into the pCAGGS vector to obtain the pCAGGS-new F divalent antigen dimer plasmid. The plasmid was then transfected into a 293F cell expression system for expression, and the cell supernatant was collected and purified after expression.

[0034] Chromatography was performed using Superdex 200 Increase 10 / 300 (GE) molecular sieves, and the elution peak around 13.5 mL was collected (e.g., Figure 1 SDS-PAGE analysis was performed on the protein sample (shown in the image). The protein in the elution volume of approximately 13.5 ml was about 54 kDa under both non-reducing conditions (without DTT) and reducing conditions (with DTT), confirming that the protein obtained from this elution peak is a new F divalent antigen dimer.

[0035] Example 2: Affinity analysis of RSV new F divalent antigen dimer antibody

[0036] Using the Biacore 8K biomolecular interaction analysis system, the new RSV F antigen dimer obtained in Example 1 was immobilized on an SM chip. Then, various antibody Fab fragments targeting different epitopes of the F protein were flowed onto the chip surface, and the affinity between the antigen and various antibodies was measured.In this example, Epitope I 4D7 [Flynn JA., et al. Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein. PLoS ONE, 2016, 11(10): e0164789.], Epitope II Palivizumab [The IMPACT-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics, 1998, 102, 531-537], Epitope III MPE8 [Gilman MS. et al. Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein. PLoS Pathogen, 2015, 11: e1005035.], Epitope IV 101F [McLellan JS. et al. Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F. Journal of Virology, 2010, 84: 12236-12244.], Epitope V CR9501 [Gilman MS. et al. Transient opening of trimeric prefusion RSV F proteins. Nature Communications, 2019, 10: 2105.], are selected. Epitope Am22 [McLellan JS. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody.Scinece,2013,340(6136):1113-1117.]. Biacore 8K analysis yielded the binding and affinity of the novel RSV F divalent antigen dimer to the antibody (as shown in Table 1). Blank cells indicate that the antigen and antibody do not bind.

[0037] Table 1. Binding and affinity of RSV novel F divalent antigen dimer to antibodies

[0038]

[0039] Example 3: Immunization of cotton rats with RSV new F divalent antigen dimer

[0040] MF59 (AddaVax used below is an MF59-like adjuvant) and aluminum adjuvant are two commonly used adjuvants approved by the SFDA. This invention selects these two adjuvants as vaccine adjuvants for animal experiments, which will have direct guiding significance for subsequent clinical trials. Therefore, this invention dilutes the antigen obtained in Example 1 with PBS solution to the required concentration, then emulsifies it with Alhydrogel aluminum adjuvant and AddaVax adjuvant respectively, and then immunizes 6-8 week old female cotton rats in groups. The immunization grouping, immunogen dosage, and adjuvant information for each group are shown in Table 2, with blank cells indicating "none". The immunization strategy is as follows: Figure 2 As shown, each cotton mouse received three immunizations via intramuscular injection in the thigh on days 0, 21, and 42, with each injection consisting of 200 μl, 100 μl in each thigh. Blood samples were collected from the orbital fossa on days 19, 40, and 56. After standing, the blood samples were centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then inactivated at 56°C for 30 minutes and stored at -80°C for specific antibody titer detection and RSV neutralization experiments.

[0041] Table 2. Immune grouping, immunogen dosage, adjuvant, and challenge strain for each group.

[0042] 1 6 PBS-Al A 2 6 PBS-Al B 3 6 PBS-Add A 4 6 PBS-Add B 5 7 3μg-Add A 6 7 3μg-Add B 7 7 10μg-Al A 8 7 10μg-Al B

[0043] Example 4: ELISA assay for detecting antigen-induced specific antibody titers

[0044] The RSV novel F divalent antigen dimer protein obtained in Example 1 was diluted to 3 μg / ml with ELISA coating buffer and added to each well of a 96-well ELISA plate (Coring, 3590). After incubation at 4°C for 12 hours, the coating buffer was discarded, and the plate was washed twice with PBS. Then, 100 μl of 5% skim milk powder prepared with PBS was added to each well as the blocking buffer, and the plate was blocked at 37°C for 1 hour. After blocking, the blocking buffer was discarded, and 100 μl of the serially diluted serum sample obtained in Example 3 was added. Serum samples were serially diluted 4-fold starting from 50-fold with the blocking buffer, for a total of 12 dilutions. 100 μl of blocking buffer was added directly to the negative control wells. After incubation at 37°C for 1 hour, the supernatant was discarded, and the plate was washed 3 times with PBST. Then, chicken anti-mouse IgG secondary antibody diluted 1:5000 with the blocking buffer was added, and the plate was incubated at 37°C for 1 hour, followed by washing 4 times with PBST. Add 1:5000 dilution of HRP-conjugated goat anti-chicken IgG triple antibody to blocking buffer, incubate at 37°C for 0.5 hours, then wash the plate four times with PBST. Add TMB chromogenic buffer for color development, and stop the reaction with 2M hydrochloric acid after an appropriate reaction time. Detect OD on a microplate reader. 450 Reading value. The antibody titer value is defined as the highest serum dilution where the reaction value is greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:25.

[0045] The results are as follows Figure 3 As shown, throughout the immunization process, the level of specific antibodies in the serum of mice in each immunization group increased significantly with the increase of the number of immunizations, with the highest antibody level after 3 immunizations. This result indicates that the RSV new F divalent antigen dimer can effectively activate the antibody response in mice.

[0046] Example 5: ELISA assay for the conformation of the novel F divalent antigen dimer in vivo.

[0047] The RSV novel F divalent antigen dimer protein and RSV post-F protein obtained in Example 1 were diluted to 3 μg / ml with ELISA coating buffer and added to each well of an ELISA 96-well plate (Coring, 3590), 100 μl per well. After incubation at 4°C for 12 hours, the coating buffer was discarded, and the plates were washed twice with PBS. Then, 100 μl of 5% skim milk powder prepared with PBS was added to each well as blocking buffer, and the plates were blocked at 37°C for 1 hour.

[0048] Add 100 μl of 3 μg / ml V epitope antibody CR9501Fab protein solution to each well of a 96-well plate containing the new RSV F divalent antigen dimer. Epitope antibody Am22Fab protein solution was incubated at 37°C for 1 hour. The supernatant was then discarded, and the plate was washed three times with PBST. Serum samples obtained in Example 3 were added, and the serum samples were serially diluted 4-fold with blocking buffer, starting at 50-fold, for a total of 12 dilutions. 100 μl of blocking buffer was added directly to the negative control wells. After incubation at 37°C for 1 hour, the supernatant was discarded, and the plate was washed three times with PBST. Chicken anti-mouse IgG secondary antibody diluted 1:5000 with blocking buffer was added, and the plate was incubated at 37°C for 1 hour, followed by washing four times with PBST. Goat anti-chicken IgG triple antibody conjugated with HRP was added, and the plate was incubated at 37°C for 0.5 hours, followed by washing four times with PBST. TMB chromogenic buffer was added for color development, and the reaction was stopped by adding 2M hydrochloric acid after an appropriate reaction time. OD was measured using a microplate reader. 450 Reading value. The antibody titer value is defined as the highest serum dilution where the reaction value is greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:25.

[0049] Serum samples collected after the triple immunization obtained in Example 3 were serially diluted 4-fold starting from 50-fold with blocking buffer, for a total of 12 dilutions. These were then incubated directly at 37°C with 96-well plates coated with RSVpost-F protein and washed with PBS. After 1 hour, the supernatant was discarded, and the plates were washed 3 times with PBST. Then, chicken anti-mouse IgG secondary antibody diluted 1:5000 with blocking buffer was added, and the plates were incubated at 37°C for 1 hour, followed by 4 washes with PBST. Next, goat anti-chicken IgG tertiary antibody conjugated with HRP was added, and the plates were incubated at 37°C for 0.5 hours, followed by 4 washes with PBST. TMB chromogenic buffer was added for color development, and the reaction was terminated by adding 2M hydrochloric acid after an appropriate reaction time. OD was measured using a microplate reader. 450 Read value.

[0050] The antibody titer calculation method is the same as in Example 3. The data for specific antibody binding in the triple-immune serum (triple-immune) in Example 3 (black), the data for direct binding between serum and post-F (white), and the data for serum and blocking... Comparison of data on specific antibody binding in serum after epitope formation (light gray) and data on specific antibody binding in serum after blocking V epitope formation (dark gray) (e.g.) Figure 4 (As shown). The data for the three exemptions were significantly higher than the data directly combined with the post-F data by 10. 2 This indicates that, in vivo, the RSV neo-F antigen dimer protein exists primarily in the pre-F conformation. (Data from the three-immune treatment and blocking...) Compared to the epitope data, there is a difference of approximately 10. 1-2 The difference between the two is the amount of RSV neoF antigen dimer produced in vivo. Epitope antibodies. Similarly, the data from the three-immune vaccination program differed by approximately 10% compared to the data from the blocked V epitope. 1.5-2 The difference between the two is the V epitope antibody produced in vivo by the RSV neo-F antigen dimer. Because... Antibodies against epitopes and V epitopes are the most potent neutralizing antibodies against RSV. This result indicates that the new RSV F antigen dimer can effectively stimulate the production of antibodies with good virus-neutralizing activity in vivo, thereby achieving immune protection against the virus.

[0051] Example 6: Virus neutralization experiment with immune serum

[0052] The serum obtained in Example 3 was initially diluted 1:5, and then serially diluted 4-fold seven times, each diluted with an equal volume of 200 TCID50. 50 RSV A2 and RSV B9320 were mixed. After co-incubating at 37°C for 1 hour, 100 μl of the mixture was added to a 96-well plate with approximately 70% HEp-2 cell coverage. After incubating at 37°C for 2 hours, the mixture was discarded, and 100 μl of DMEM medium containing 2% FBS was added. After incubating at 37°C for 4 days, the medium was discarded, the cells were washed twice with PBS, and fixed with ice-cold 80% acetone solution for 20 minutes. The supernatant was discarded, and the cells were washed twice with PBS. 100 μl of 5% skim milk powder prepared with PBS was added to each well, and the cells were blocked at 37°C for 1 hour. After blocking, the blocking solution was discarded, and 100 μl of Palivazumab primary antibody solution with a concentration of 3 μg / ml prepared with the blocking solution was added to each well. After incubating at 37°C for 1 hour, the plate was washed three times with PBST. Add HRP-conjugated goat anti-human IgG secondary antibody diluted 1:5000 with blocking buffer, incubate at 37°C for 1 hour, then wash the plate 4 times with PBST. Add TMB chromogenic buffer for color development, and stop the reaction by adding 2M hydrochloric acid after an appropriate reaction time. Detect OD on a microplate reader. 450 Reading value. The antibody titer value is defined as the highest serum dilution where the reaction value is greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) is still less than 2.1 times the negative control value, the sample titer is defined as half of the lowest dilution, i.e., 1:25.

[0053] The results are as follows Figure 5 , Figure 6 As shown, during the entire immunization process, after three immunizations, the neutralizing antibody levels reached 2 at a dose of 10 μg aluminum adjuvant and 3 μg AddaVax adjuvant. 10 and 2 8 The above antibody titer levels indicate that the new RSV F divalent antigen dimer can effectively stimulate mice to produce neutralizing antibodies, thereby achieving immune protection.

[0054] Example 7: Cotton Mouse Virus Challenge Protection Experiment

[0055] The cotton mice from Example 3 were infected with intranasal drops on day 63, and 10 mice were infected with these methods. 5 TCID 50 RSV A2 virus and 10 5 TCID 50 RSVB9320 virus (such as) Figure 2 As shown in Table 2, the cells were sacrificed after 5 days. Lungs, trachea, and nasal cheek bones were removed. After homogenization, the tissues were serially diluted 3-fold. 100 μl of the homogenate was added to a pre-prepared 96-well plate of HEp-2 cells with approximately 70% cell coverage. The plate was incubated at 37°C with 5% CO2 aeration for 1 hour. The supernatant tissue homogenate was then discarded, and 100 μl of DMEM medium containing 2% fetal bovine serum was added to each well. The plate was incubated at 37°C with 5% CO2 aeration for 4 days. The viral titer (TCID50) was then measured. 50 The result is as follows: Figure 7 As shown, the results of virus cultures from the lungs, nasal cheekbones, and trachea all indicated that, regardless of whether RSVA2 or RSV B9320 virus was used, and regardless of whether AddaVax or aluminum adjuvant was used, the viral load in all tissues of the vaccine-treated mice was near the detection line (dashed line in the figure), approximately 2. 2.3 The viral titer was 2, while the protein-free adjuvant-only group (PBS) had an average viral titer of 2. 7-10 This result fully demonstrates that the new RSV F divalent antigen dimer has a good protective effect on the body.

Claims

1. A bivalent respiratory syncytial virus antigen, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

7.

2. The respiratory syncytial virus bivalent antigen as described in claim 1, characterized in that, A tag sequence and a stop codon sequence are also added to the 3' end.

3. The respiratory syncytial virus bivalent antigen as described in claim 2, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

8.

4. The encoding gene of the respiratory syncytial virus bivalent antigen as described in any one of claims 1 to 3.

5. The encoding gene for the respiratory syncytial virus bivalent antigen as described in claim 4, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

10.

6. A recombinant expression vector containing the coding gene as described in claim 4 or 5.

7. The recombinant expression vector as described in claim 6, characterized in that, The starting vector is a mammalian cell expression vector.

8. A recombinant host cell containing the encoding gene as described in claim 4 or 5.

9. The recombinant host cell as described in claim 8, characterized in that, The recombinant host cell originating from the host cell is a mammalian cell.

10. The method for preparing respiratory syncytial virus bivalent antigen according to any one of claims 1 to 3, characterized in that, The respiratory syncytial virus antigen encoding gene is expressed in host cells via a recombinant expression vector to obtain the respiratory syncytial virus antigen.

11. The method as described in claim 10, characterized in that, The recombinant expression vector originates from a mammalian cell expression vector, and the host cell is a mammalian cell; the method also includes the steps of collecting cell supernatant after expression and purifying the respiratory syncytial virus antigen.

12. An antigen composition comprising the respiratory syncytial virus bivalent antigen as described in any one of claims 1 to 3 as the active ingredient and an adjuvant.

13. The antigen composition according to claim 12, characterized in that, The adjuvant is an aluminum adjuvant or an AddaVax adjuvant.

14. The use of the respiratory syncytial virus bivalent antigen as described in any one of claims 1 to 3 in the preparation of a reagent for immunizing animals to obtain antibodies.

Images