RSV F protein soluble fragment, autotrimer structural domain and application of RSV F protein soluble fragment and autotrimer structural domain

Abstract

The invention relates to an RSV F protein soluble fragment, an autotrimer structural domain and application thereof, and belongs to the technical field of recombinant protein. Based on an amino acid sequence of a wild RSV F protein, a soluble and rigid sequence is screened out, a soluble fragment is constructed, disulfide bond mutation pair modification is performed on the basis of the soluble fragment to obtain a self-derived trimer structural domain sequence, and the self-derived trimer structural domain sequence is used for replacing a transmembrane region and an intracellular region of the RSV F protein. Therefore, trimerization can be realized, stable pre-fusion conformation can be maintained, and soluble expression in cells can be realized at the same time. The invention also provides an RSV F protein mutant containing an autotrimer structural domain sequence, the generation and the stability of the pre-F conformation trimer are proved, a representative is selected from the pre-F conformation trimer for animal experiments, and it is proved that the pre-F conformation trimer has good immunogenicity and can be used for developing RSV vaccines and preparing RSV specific antibodies or RSV detection reagents.

Description

Technical Field

[0001] The present application relates to a soluble fragment of RSV F protein, an autologous trimer domain thereof, and their applications, belonging to the technical field of recombinant proteins. Background Art

[0002] Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus belonging to the Paramyxoviridae family, mainly divided into type A and type B. The virus is transmitted through direct contact and can cause various symptoms such as bronchitis, pneumonia, asthma, and respiratory failure after infection. RSV is the main cause of respiratory tract infections in children globally and also causes severe respiratory and pulmonary infections in infants, the elderly, and those with weakened immune systems. The pathogenic mechanism of RSV involves its surface glycoprotein, especially the F protein (fusion protein), which plays a key role in the virus invasion process. The F protein exists in the form of a precursor (F0), which consists of 574 amino acids. During the process of virus-host cell membrane fusion, the F0 protein is cleaved by furin protease, releasing two subunits: F1 (amino acids 137 to 574) and F2 (amino acids 26 to 109). These two subunits are linked by disulfide bonds to jointly form the mature F protein trimer structure. During this process, the trimeric F protein undergoes a conformational change from the pre-fusion state (Pre-F) to the post-fusion state (Post-F). The RSV F protein exhibits different antigenicity in the Pre-F and Post-F states. The F protein in the Pre-F state has higher immunogenicity, while in the Post-F state, it helps the virus enter the host cell. Therefore, using the native trimeric Pre-F protein as a vaccine antigen can induce an effective immune response in the body, prevent virus invasion, and reduce the severity of infection.

[0003] In recent years, three RSV vaccines with trimeric Pre-F as the antigen have been successfully launched internationally, namely Arexvy from GSK, Abrysvo from Pfizer, and mRESVIA from Modena. Currently, there is no RSV preventive vaccine on the market in China. However, the currently launched RSV vaccines all face the problem of insufficiently durable immune protection. The protection rates of the three vaccines in the first RSV epidemic season were 82.6% (LRTD, Arexvy), 66.7% (LRTD2, Abrysvo), and 83.7% (LRTD2, mRESVIA) respectively, while in the second epidemic season, the protection rates were 67.2% (LRTD, Arexvy), 55.7% (LRTD2, Abrysvo), and 50.3% (LRTD2, mRESVIA) respectively. And the booster dose did not show a stronger immune protection rate (67.1%, LRTD, Arexvy).

[0004] Currently, recombinant protein vaccines on the market (such as COVID-19 vaccines and RSV vaccines) use heterotrimeric tags (such as the Foldon sequence from bacteriophage T4, the trimerization motif from human collagen, etc.) to replace the C-terminal transmembrane region and intracellular region in order to achieve soluble expression and maintain the native trimeric structure. However, the heterotrimeric domain is not the self-sequence of the antigen, which may cause problems such as immune pre-existence and safety. Repeated injections of vaccines containing heterotrimeric domains may produce antibodies against the heterotrimer, affecting the immune protection effect of the vaccine and resulting in insignificant booster immune effects after multiple injections. Therefore, in order to provide continuous immune protection to the body, the present invention aims to design a pre-fusion conformation RSV antigen protein containing a self-trimeric domain, that is, using the F protein's own sequence as the trimerization domain sequence, so that the antigen does not contain any heterologous sequences, while still maintaining a stable trimeric pre-fusion conformation and showing good immunogenicity in animal models, and can potentially be further developed as a core antigen for RSV vaccines. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention aims to study the RSV F protein and design specific mutants that can stably maintain the pre-fusion conformation through structural biology and bioinformatics methods. First, the present invention provides the amino acid sequence (soluble fragment) from the soluble expression region of the RSV F protein itself, and further creates a self-trimeric domain of the RSV F protein on this basis; then, the present invention provides a mutant of the RSV F protein containing the self-trimeric domain, which uses the self-trimeric domain to replace the transmembrane region and intracellular region at the C-terminus of the F protein. The three mutant sequences can form trimers using the self-trimeric domain without introducing a heterologous trimeric domain (such as Foldon). Its conformation is similar to the native pre-fusion conformation F protein, so it has a stable trimeric structure and good immunogenicity, which is helpful for the prevention, diagnosis, and treatment of RSV.

[0006] To solve the above technical problems, the specific technical solutions of the present invention are as follows:

[0007] (1) In the first aspect, the present invention provides a soluble fragment of the RSV F protein:

[0008] The soluble fragment is an amino acid sequence from the soluble expression region of wild-type RSV F protein (SEQ ID NO: 1), and is a rigid amino acid sequence with a length not exceeding 70 amino acids. In an alternative embodiment, the rigid amino acid sequence comprises amino acids at positions 74-96 (F74-96), 149-158 (F149-158), 150-206 (F150-206), 215-238 (F215-238), 268-283 (F268-283) or 491-509 (F491-509) of the wild-type RSV F protein amino acid sequence. The specific sequences of the above six segments are as follows:

[0009] F74-96: AKVKLIKQELDKYKNAVTELQLL (SEQ ID NO: 3).

[0010] F149-158: ASGIAVSKVL (SEQ ID NO: 4);

[0011] F150-206: SGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPI (SEQ ID NO: 5);

[0012] F215-238: SNIETVIEFQQKNNRLLEITREFS (SEQ ID NO: 6);

[0013] F268-283: NDQKKLMSSNVQIVRQ (SEQ ID NO: 7);

[0014] F491-509: SISQVNEKINQSLAFIRKS (SEQ ID NO: 8).

[0015] The soluble fragment may be selected from one of the above six sequences (F74-96, F149-158, F150-206, F215-238, F268-283, F491-509). Alternatively, the soluble fragment is selected from the derivative sequences of the above six sequences: based on SEQ ID NO: 1, on the basis of any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, the N-terminal side is truncated or extended by x1 adjacent amino acid residues, and / or the C-terminal side is truncated or extended by x2 adjacent amino acid residues to obtain a sequence, where 0 ≤ x1 ≤ 10, 0 ≤ x2 ≤ 10 (that is, the value of x1 and / or x2 can be any integer between 0 and 10), x1 and x2 are not both 0 at the same time, and the length of the derivative sequence obtained by the truncation or extension is more than 5 amino acids (at least 5 amino acids); among them, preferably, 0 ≤ x1 ≤ 5, 0 ≤ x2 ≤ 5; more preferably, 0 ≤ x1 ≤ 2, 0 ≤ x2 ≤ 2; still more preferably, 0 ≤ x1 ≤ 1, 0 ≤ x2 ≤ 1.

[0016] Preferably, the soluble fragment is selected from: F74-96, F149-158, F215-238 or F491-509.

[0017] (2) In the second aspect, the present invention provides a RSV F protein autologous trimer domain:

[0018] It is further modified from the aforementioned soluble fragment, and the structure is: N-terminal-(X-Y) m -soluble fragment-C-terminal, where X and Y represent any amino acid residues (for example, X-Y are two adjacent amino acids from outside the transmembrane region and intracellular region of the RSV F protein), m represents its quantity and m = 0 or 1; optionally, on the basis of the above structure, a disulfide bond mutation pair is further introduced to make it more stable after forming a trimer structure: taking the first amino acid at the N-terminal of the soluble fragment as the 1st amino acid site, the downstream amino acids are sequentially numbered starting from 2, and the upstream amino acids are numbered in reverse starting from -1, replacing the original amino acids at site A (-2nd and -1st sites), site B (6th and 7th sites), site C (13th and 14th sites) and / or site D (20th and 21st sites) with cysteine (Cys, C), thereby obtaining the autologous trimer domain sequence. Preferably, site A, site C, site AB, site AC, site ABC, site ACD, site BCD or site ABCD are selected for the disulfide bond mutation pair modification.

[0019] Preferably, the present invention also provides the following 16 specific autologous trimer domain sequences:

[0020] F74-96+A: CCAKVKLIKQELDKYKNAVTELQLL (SEQ ID NO: 9);

[0021] F74-96+AB: CCAKVKLCCQELDKYKNAVTELQLL (SEQ ID NO: 10).

[0022] F149-158+A: CCASGIAVSKVL (SEQ ID NO: 11);

[0023] F149-158+AB: CCASGIACCKVL (SEQ ID NO: 12);

[0024] F150-206: SGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPI (SEQ ID NO: 5);

[0025] F215-238+A: CCSNIETVIEFQQKNNRLLEITREFS (SEQ ID NO: 13);

[0026] F215-238+ABCD: CCSNIETCCEFQQKCCRLLEICCEFS (SEQ ID NO: 14);

[0027] F215-238+AC: CCSNIETVIEFQQKCCRLLEITREFS (SEQ ID NO: 15);

[0028] F215-238+ACD: CCSNIETVIEFQQKCCRLLEICCEFS (SEQ ID NO: 16);

[0029] F215-238+BCD: SNIETCCEFQQKCCRLLEICCEFS (SEQ ID NO: 17);

[0030] F215-238+C: SNIETVIEFQQKCCRLLEITREFS (SEQ ID NO: 18);

[0031] F268-283+A: CCNDQKKLMSSNVQIVRQ (SEQ ID NO: 19);

[0032] F268-283+AB: CCNDQKKCCSSNVQIVRQ (SEQ ID NO: 20);

[0033] F491 - 509 + A: CCSISQVNEKINQSLAFIRKS (SEQ ID NO: 21);

[0034] F491 - 509 + AB: CCSISQVCCKINQSLAFIRKS (SEQ ID NO: 22);

[0035] F491 - 509 + ABC: CCSISQVCCKINQSCCFIRKS (SEQ ID NO: 23);

[0036] Among them, "A" represents introducing a disulfide bond mutation pair at site A, "AB" represents introducing disulfide bond mutation pairs at site A and site B respectively, and so on; no modification of introducing disulfide bond mutation pairs is made to F150 - 206.

[0037] Preferably, the autologous trimeric domain is selected from: F149 - 158 + A, F215 - 238 + AC, F491 - 509 + ABC, F74 - 96 + A or F74 - 96 + AB; more preferably from: F149 - 158 + A, F215 - 238 + AC, F491 - 509 + ABC or F74 - 96 + A; even more preferably F74 - 96 + A.

[0038] (III) In the third aspect, the present invention provides an RSV F protein mutant comprising the autologous trimeric domain:

[0039] The RSV F protein mutant uses the autologous trimeric domain to replace the fragment of the RSV F protein at the C - terminus including the transmembrane region and the intracellular region to help the mutant protein form a trimer and achieve soluble expression at the same time. The transmembrane region and the intracellular region of the wild - type RSV F protein are located at amino acids 525 - 574. Therefore, the "fragment including the transmembrane region and the intracellular region" refers to a fragment covering amino acid residues 525 - 574 and may be slightly longer than it, such as the n - th amino acid of the wild - type RSV F protein and its downstream sequence, where 512 ≤ n ≤ 525 (preferably, n = 512 or 514, that is: using the autologous trimeric domain to replace amino acids 512 - 574 or 514 - 574 of the RSV F protein). In an alternative embodiment, replacing the fragment including the transmembrane region and the intracellular region means using the autologous trimeric domain sequence of the RSV F protein to replace the amino acid sequence starting from the 512 - th or 514 - th amino acid. The autologous trimeric domain sequence is selected from or preferably from the sequences listed in the above "second aspect".

[0040] In an alternative embodiment, the mutant of the RSV F protein further includes a modification selected from the following:

[0041] (1)Disulfide bond mutations at positions other than at least one pair of ABCD sites to stabilize the Pre-F conformation:

[0042] In an alternative embodiment, the disulfide bond mutations at the other positions include D486C + E487C: Mutating the amino acids at positions 486 and 487 in the wild-type RSV F protein amino acid sequence to cysteine. In an alternative embodiment, the disulfide bond mutations at the other positions include S / N105C + M370C: Mutating the amino acids at positions 105 and 370 in the wild-type RSV F protein amino acid sequence to cysteine; it should be noted that the purpose of further introducing the new disulfide bond S / N105C + M370C on the basis of D486C + E487C is to replace the proline mutation and obtain a more stable Pre-F conformation, so it can also be regarded as a modification for the purpose of "proline mutation" (see Table 2).

[0043] (2)At least one proline mutation:

[0044] In an alternative embodiment, the mutant of the RSV F protein further includes at least one proline mutation to enhance the conformational stability of the Pre-F protein. In an alternative embodiment, the proline mutation includes mutating the amino acids at positions 210, 211, 211 + 213, or 215 in the wild-type RSV F protein amino acid sequence to P. Preferably, the proline mutation includes mutating the amino acids at positions 210 or 215 in the wild-type RSV F protein amino acid sequence to P.

[0045] (3)At least one cavity filling mutation:

[0046] In an alternative embodiment, the mutant of the RSV F protein further includes at least one cavity filling mutation to enhance the conformational stability of the Pre-F protein. In an alternative embodiment, the cavity filling mutation is mutating the amino acid at position 190 in the wild-type RSV F protein amino acid sequence to the amino acid leucine (S190L) with a larger side chain to fill the structural cavity and enhance the conformational stability of Pre-F.

[0047] (4)At least one electrostatic mutation:

[0048] In an alternative embodiment, the mutant of the RSV F protein further includes at least one electrostatic mutation to enhance the conformational stability of the Pre-F protein. In an alternative embodiment, the electrostatic mutation is mutating the amino acid at position 487 in the wild-type RSV F protein amino acid sequence to threonine (E487T) to reduce the charge repulsion in this region and stabilize the Pre-F conformation.

[0049] (5)Modification of the p27 sequence:

[0050] In an alternative embodiment, the mutant of the RSV F protein further includes a modification to the p27 sequence (aa 110-136), i.e., deletion of the fragment containing p27. In an alternative embodiment, the modification of the p27 sequence refers to deletion of the amino acids between positions 105 and 147 of the wild-type RSV F protein amino acid sequence (S / N105-A147).

[0051] As a preferred embodiment, compared with the wild-type RSV F protein, the mutant of the RSV F protein, in addition to replacing the fragment containing the transmembrane region and the intracellular region at the C-terminus with the self-source trimerization domain, also has the following modifications a), b), c) and d):

[0052] a) Disulfide bond mutation D486C + E487C, or electrostatic mutation E487T;

[0053] b) Proline mutation: selected from S215P, Q210P, S211P or S211P + S213P;

[0054] c) Cavity filling mutation: S190L;

[0055] d) P27 sequence modification: S / N105-A147.

[0056] More preferably, compared with the wild-type RSV F protein, the mutant of the RSV F protein, in addition to replacing the fragment containing the transmembrane region and the intracellular region at the C-terminus with the self-source trimerization domain, also has a modification selected from one of the following groups:

[0057] (1) D486C + E487C, S215P, S190L, S / N105-A147;

[0058] (2) D486C + E487C, Q210P, S190L, S / N105-A147;

[0059] (3) D486C + E487C, S211P, S190L, S / N105-A147;

[0060] (4) S215P, S190L, E487T, S / N105-A147;

[0061] (5) Q210P, S190L, E487T, S / N105-A147;

[0062] (6) S211P, S190L, E487T, S / N105-A147;

[0063] (7) S211P + S213P, S190L, E487T, S / N105-A147.

[0064] More preferably, the RSV F protein mutant has a modification selected from one of the following groups compared to the wild-type RSV F protein:

[0065] (1) D486C+E487C, S215P, S190L, S / N105-A147, F74-96+A;

[0066] (2) D486C+E487C, S215P, S190L, S / N105-A147, F74-96+AB;

[0067] (3) D486C+E487C, S215P, S190L, S / N105-A147, F149-158+A;

[0068] (4) D486C+E487C, S215P, S190L, S / N105-A147, F491-509+ABC;

[0069] (5) D486C+E487C, Q210P, S190L, S / N105-A147, F74-96+A;

[0070] (6) D486C+E487C, S211P, S190L, S / N105-A147, F74-96+A;

[0071] (7) S215P, S190L, E487T, S / N105-A147, F74-96+A;

[0072] (8) S215P, S190L, E487T, S / N105-A147, F215-238+AC;

[0073] (9) Q210P, S190L, E487T, S / N105-A147, F491-509+ABC;

[0074] (10) S211P, S190L, E487T, S / N105-A147, F491-509+ABC;

[0075] (11) S211P+S213P, S190L, E487T, S / N105-A147, F74-96+A.

[0076] Further preferably, on the basis of the wild-type RSV F protein shown in a sequence such as SEQ ID NO: 1 (RSV-A) or SEQ ID NO: 2 (RSV-B), modifications selected from any of the above (1)-(11) are carried out to obtain an RSV F protein mutant, such as BR90, BR91, BR94, BR95, BR98, BR103, BR110, BR111, BR120, BR122, BR123, BR132, BR133, BR134, BR135, BR136, BR137, BR135’, BR136’, BR137’; among them, BR132, BR133, BR135, BR136, BR135’ or BR136’ are preferred.

[0077] In an alternative embodiment, the RSV F protein mutant of the present invention includes an 8*His tag sequence (HHHHHHHH). Such a sequence is not essential for the functions of the RSV F protein, such as inducing an immune response. Those skilled in the art will recognize such sequences and understand, where appropriate, that such sequences may or may not be included in the RSV F protein mutants disclosed in the present invention.

[0078] (IV) Fourth aspect, the present invention provides a biological material, which comprises any one of the following:

[0079] (a) Any nucleic acid molecule encoding the autologous trimeric domain of the RSV F protein or the RSV F protein mutant.

[0080] (b) A recombinant vector containing the above nucleic acid molecule. Preferably, the basic plasmid of the recombinant vector is pOET1.1.

[0081] (c) A recombinant virus containing the above nucleic acid molecule or recombinant vector. Among them, the preferred types of recombinant viruses include baculovirus of insect cells, adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus.

[0082] (d) A transformed cell containing the above nucleic acid molecule, recombinant vector or recombinant virus. The host cell can be a mammalian cell, a bacterium, a yeast, a fungus or an insect cell; preferably, the insect cell is Sf9 cell, Sf21 cell or High-Five cell.

[0083] (V) Fifth aspect, the present invention also relates to a method for preparing the above RSV F protein, the steps of which include: constructing a recombinant vector, extracting the recombinant vector, transfecting the recombinant vector into a host cell and expressing the protein, and purifying the expression product.

[0084] (6) Sixth aspect, the present invention also provides an immunogenic composition comprising any one of the above RSV F protein mutants, nucleic acid molecules, recombinant vectors, recombinant viruses or transformed cells. Preferably, the immunogenic composition contains an RSV F protein mutant (antigenic protein) against RSV type A, such as BR90, BR110 or BR111; alternatively, the immunogenic composition contains an RSV F protein mutant (antigenic protein) against RSV type B, such as BR135’, BR136’ or BR137’. Further preferably, the immunogenic composition contains antigenic proteins against both RSV type A and RSV type B simultaneously, such as BR90 and BR135’, BR110 and BR136’, or BR111 and BR137’, etc.

[0085] (7) Seventh aspect, the present invention also provides any one of the above RSV F protein mutants, nucleic acid molecules, recombinant vectors, recombinant viruses, transformed cells or immunogenic compositions for any one of the following uses:

[0086] (a) For the preparation of RSV-specific antibodies.

[0087] (b) For the development of drugs for the prevention and / or treatment of RSV infection. Preferably, the drug can be a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.

[0088] (c) For the preparation of diagnostic reagents for RSV.

[0089] Term definitions:

[0090] In the present application, the term "respiratory syncytial virus" or "RSV" refers to a single-stranded negative-sense RNA virus that can cause lower respiratory tract disease symptoms (LRTD) in infants, the elderly and immunocompromised adults. This virus belongs to the genus Pneumovirus of the family Paramyxoviridae.

[0091] In the present application, the term "mutant" specifically refers to those proteins that have one or more changes in the amino acid sequence or protein structure compared to the wild-type protein; these changes can be in various forms, including but not limited to the deletion, insertion, substitution, shortening and / or loss of one or more amino acids. In addition, it may also involve the modification or cleavage of the protein structure. Specifically, the "mutant" in the present application mainly refers to mutants of the respiratory syncytial virus (RSV) F protein.

[0092] In the present application, the term "trimer" generally refers to a protein structure composed of three identical or different types of protein subunits that can be linked together through a special chemical structure. The protein trimer described in the present application can be the RSV F protein.

[0093] In the present application, the term "soluble fragment" refers to a rigid amino acid sequence of a soluble expression region derived from wild-type RSV F; preferably, the soluble fragment is selected from the amino acid sequences of any one of SEQ ID NO: 3 to 8.

[0094] In the present application, the term "autologous trimerization domain" refers to a fragment with trimerization function screened or further modified from the naturally occurring amino acid sequence of the target protein, which can enable the target protein to form a trimer. The autologous trimerization domain described in the present application refers to an amino acid fragment with trimerization function screened and modified based on the amino acid sequence of the RSV F protein itself, for example, obtained by mutating and modifying disulfide bonds on the basis of the above-mentioned "soluble fragment".

[0095] In the present application, the term "heterologous trimerization domain" generally refers to amino acid sequences from different species or different sources, which are usually different from the natural amino acid sequence of the target protein. The heterologous trimerization domain described in the present application generally refers to the amino acid sequence of the C-terminal domain of the T4 phage fibritin (Foldon, whose amino acid sequence is: GYIPEAPRDGQAYVRKDGEWVLLSTFL).

[0096] In the present application, the term "disulfide bond mutation" refers to replacing certain amino acids in the wild-type RSV F protein with cysteine, so as to form a disulfide bond between the sulfur atoms of two cysteine residues to stabilize the RSV Pre-F structure. "Disulfide bond mutation pair" refers to introducing a pair (two) of cysteine mutations.

[0097] In the present application, the term "cavity filling mutation" refers to replacing the amino acid residues in the wild-type RSV F protein with amino acids having larger side chain groups (such as large aliphatic amino acids Ile, Leu, and Met or large aromatic amino acids His, Phe, Tyr, and Trp), so as to fill the cavity in the F protein structure and stabilize the RSV Pre-F structure. For example, the amino acid Ser at position 190 of the RSV F protein amino acid sequence is mutated to Leu.

[0098] In the present application, the term "electrostatic mutation" refers to replacing specific amino acid residues in the wild-type RSV F protein with amino acids that can reduce the ionic repulsion between residues approaching each other during protein folding to stabilize the RSV Pre-F structure. For example, the amino acid Glu at position 487 of the RSV F protein amino acid sequence is mutated to threonine (Thr).

[0099] In the present application, the term "proline mutation" refers to the substitution of an amino acid residue in the wild-type RSV F protein with proline to stabilize the RSV Pre-F structure. The amino acid and carboxyl group in proline are linked by an amino group, forming a rigid five-membered ring structure. This ring structure restricts the rotational freedom of proline in the protein, enabling proline to form fixed folds and turns, thereby increasing the protein's stability.

[0100] In the present application, the term "p27" refers to a 27-amino acid short peptide (aa 110-136) composed of amino acid residues at positions 110 to 136 in the wild-type RSV F protein. This amino acid short peptide will be hydrolyzed and released by furin protease, and two subunits, F1 (aa137-574) and F2 (aa 26-109), will be formed.

[0101] In the present application, the term "p27 sequence modification" refers to the deletion of the fragment containing p27 on the basis of the wild-type RSV F protein, that is, the deleted fragment should cover p27 and can be slightly longer than p27 (extended by about 1 to 15 amino acid residues upstream and / or downstream of p27). In the examples of the present application, the specific p27 sequence modification adopted is "S / N105-A147", which refers to the deletion of the fragment between amino acid residues at positions 105 to 147 in the RSV F protein (the deleted fragment does not include the end values of the numerical range, i.e., amino acid residues at positions 105 and 147).

[0102] In the present application, the term "Mota" or "motavizumab" refers to the antibody described in the paper by Herren et al. (Herren Wu, et al., Journal of Molecular Biology, 2007).

[0103] In the present application, the term "AM14" refers to the antibody described in the paper by Morgan et al. (Morgan S A Gilman, et al., PLoSPathog, 2015).

[0104] In the present application, the term "conformational change" generally refers to a change in the spatial structure of a protein molecule. For example, the described conformational changes can include changes in the polypeptide of chemical bonds in the protein molecule.

[0105] In the present application, the term "comprising" generally means including the expressly specified features, but does not exclude other elements.

[0106] In this application, the "transmembrane region and intracellular region" at the C-terminus of the RSV F protein refers to the sequence corresponding to amino acid residues 525 - 574 of the wild-type RSV F protein amino acid sequence (according to Uniprot: https: / / www.uniprot.org / uniprotkb / P03420 / feature-viewer); the "fragment containing the transmembrane region and intracellular region" can be a fragment that contains amino acid residues 525 - 574 but is slightly longer than that, such as aa 512 - 574, aa 514 - 574, etc.

[0107] Beneficial effects:

[0108] (1) First of all, the present invention provides a soluble fragment from the RSV F protein. Through further modification by disulfide bond mutation, a self-source trimeric domain sequence is obtained, which is rigid and has a stable structure. It can be used to replace the exogenous trimeric domain sequence (such as Foldon) to achieve trimerization of the F protein, avoiding the immune pre-existence and safety problems that may be caused by introducing heterologous trimeric domains.

[0109] (2) Secondly, the RSV F protein mutant provided by the present invention uses a self-source trimeric domain to replace the C-terminal transmembrane region and intracellular region, enabling the target protein to be massively soluble expressed in cells and to form a trimeric structure without using a heterologous trimer, maintaining the pre-fusion conformation and showing excellent stability.

[0110] (3) Furthermore, by mutating and deleting the wild-type amino acid sequence, the RSV F protein mutant obtained has a stable Pre-F protein conformation and shows strong immunogenicity, capable of stimulating the body to produce high levels of specific antibodies. Therefore, it has extremely high potential to become an effective component of the RSV vaccine, either used alone or in combination with other pathogen antigens to form a multivalent vaccine for the prevention and / or treatment of RSV infection. In addition, it can also be used as an RSV-specific detection reagent. Brief description of the drawings

[0111] In order to more clearly show the embodiments and technical solutions of the present invention, the drawings used in the embodiments and technical solutions will be briefly introduced below.

[0112] Figure 1 : Reducing gel electrophoresis analysis diagram of the purified RSV F protein mutant.

[0113] Figure 2 : Non-reducing gel electrophoresis analysis diagram and Western Blot analysis diagram after non-reducing gel electrophoresis of the purified RSV F protein mutant.

[0114] Figure 3: Detection results of binding antibodies produced after immunizing mice with RSV F antigen protein. Detailed implementation mode

[0115] In order to understand and master the purpose, technical solution and advantages of the present invention more accurately and clearly, the present invention will be described in detail below based on specific embodiments and their related experimental data.

[0116] Example 1: Development of RSV F protein autologous trimer mutant

[0117] First, through bioinformatics analysis, the F protein sequences of wild-type RSV type A (RSV-A) and RSV type B (RSV-B) were obtained: respectively based on the F protein sequences of thousands of RSV-A and RSV-B strains publicly available in the NCBI database, the amino acid (common amino acid) that appears most frequently at each site was calculated, and thus the common amino acid sequences of RSV F protein of the two subtypes were constructed, specifically shown in SEQ ID NO.1 and SEQ ID NO.2, which are used to represent the F protein sequences of wild-type RSV of type A and type B respectively.

[0118] Then, based on the wild-type RSV F protein sequence, the development of RSV F protein autologous trimer mutant was carried out, and the process included the following operations:

[0119] (I) Design and gene synthesis of RSV F protein autologous trimer mutant

[0120] (1) Design of autologous trimer domain: First, the RSV F protein was analyzed using structural biology software to obtain the amino acid sequence of its soluble expression region (referred to as "soluble fragment"), which was used to replace the C-terminal transmembrane region and intracellular region of the RSV F protein, hoping to help the target protein form a trimer structure. After replacing the C-terminal sequence of RSV F with the soluble fragment, in order to further stabilize the trimer structure formed by the autologous sequence, disulfide bond mutations were carried out around the soluble fragment or its adjacent amino acids to obtain the final autologous trimer domain.

[0121] (2) Overall design of RSV F protein mutant: After designing the autologous trimer domain, it was combined with other modifications (disulfide bond mutation, proline mutation, etc.) of the RSV F antigen protein to obtain the overall design result of the amino acid sequence of the RSV F protein mutant. Then, after codon optimization to adapt to the expression requirements of the host Sf9 insect cells, the nucleic acid coding sequence of the RSVF protein mutant was obtained, and then full gene synthesis was carried out.

[0122] (II) Construction of recombinant plasmid and expression of RSV F antigen protein

[0123] The synthesized target gene was inserted into the baculovirus transfer vector pOET1, and flashBAC TM was used. With the baculovirus expression system kit, the recombinant plasmid and the baculovirus genome were co-transfected into Sf9 insect cells. The transfection operation was carried out according to the kit instructions. Seven days after transfection, the supernatant of Sf9 cells was collected, and at this time, the supernatant contained P0 generation virus. The P0 generation virus was used to infect new Sf9 cells to produce P1 generation virus. Six to seven days after the P1 generation virus infected the cells, the supernatant was collected and the conformation and stability of each antigen were detected by ELISA.

[0124] (III) ELISA detection of RSV F antigen protein

[0125] The conformation and stability of each antigen protein were detected by ELISA. The specific steps were as follows: 100 μl of antibodies such as Motavizumab (referred to as Mota, which can bind both Pre-F and Post-F), AM14 (which only binds to Pre-F) were coated onto a 96-well plate overnight at 4 °C. The next day, after washing 3 times with PBST and patting dry, it was blocked with 5% skim milk prepared with PBST (200 μl / well, blocked at room temperature for 1 hour). The cell supernatant collected in (2) was evenly divided into 4 parts, and each part was treated at 50 °C for 1 h, 65 °C for 1 h, repeatedly frozen and thawed 8 times, and untreated. After the blocked 96-well plate was washed 3 times with PBST and patted dry, 100 μl of the cell supernatant samples untreated and treated under various stress conditions were added to each well, and incubated at room temperature for 1-2 hours. After washing 3 times with PBST, 100 μl of mouse serum (immunized with RSV Pre-F antigen protein) or Rabbit His-tag Antibody was added, and incubated at room temperature for 1 hour. After washing 3 times with PBST, 100 μl of HRP-labeled antibody was added, and incubated at room temperature for 1 hour. After washing 5 times with PBST and patting dry, 100 μl of freshly prepared TMB chromogenic solution was added to each well. After chromogenic reaction for 10-15 minutes, the stop solution was added to terminate the chromogenic reaction, and the absorbance value at wavelengths of 450 nm / 570 nm was read with an enzyme-linked immunosorbent assay instrument. The final absorbance value reading was A450nm - A570nm.

[0126] According to the above process, the development of the RSV F protein autologous trimer mutant was carried out. According to the ELISA detection results, the autologous trimer domain with excellent performance was selected for the next round of screening. At the same time, other amino acid mutations used in combination were screened on this basis, in order to obtain a more stable Pre-F conformation RSV F protein mutant.

[0127] Example 2: The first round of screening for the autologous trimer domain of RSV F protein

[0128] According to the operation steps described in Example 1, the design of the first round of mutants was carried out. The main purpose of this round was to screen for available self-trimeric domains.

[0129] The design of the RSV F protein mutants in this round was carried out based on the consensus sequence of the F protein of wild-type RSV-A (SEQ ID NO: 1). The design of the RSV F protein mutants is as follows:

[0130] (1) In terms of self-trimeric domains:

[0131] 1) Soluble fragment screening: After analysis using structural biology software in this application, 6 amino acid sequences from the soluble expression region of the RSV F protein itself were obtained, namely F149-158, F150-206, F215-238, F268-283, F491-509, and F74-96, to serve as the soluble fragments, and their sequences are shown as follows:

[0132] F74-96: AKVKLIKQELDKYKNAVTELQLL (SEQ ID NO: 3).

[0133] F149-158: ASGIAVSKVL (SEQ ID NO: 4);

[0134] F150-206: SGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPI (SEQ ID NO: 5);

[0135] F215-238: SNIETVIEFQQKNNRLLEITREFS (SEQ ID NO: 6);

[0136] F268-283: NDQKKLMSSNVQIVRQ (SEQ ID NO: 7);

[0137] F491-509: SISQVNEKINQSLAFIRKS (SEQ ID NO: 8).

[0138] 2) Self-trimeric domain: It was further modified from the above soluble fragments, and the structure is: N-terminus-(X-Y) m-Soluble fragment - C-terminal, where X and Y represent two adjacent amino acid residues outside the transmembrane region and intracellular region of the RSV F protein, m represents its quantity and m = 0 or 1; and, on the basis of the above structure, a disulfide bond mutation pair is further introduced to stabilize the trimer structure formed by the self-source sequence. The design idea is as follows: taking the first amino acid at the N-terminal of the soluble fragment as the amino acid site No. 1, the downstream amino acids are sequentially numbered starting from 2, and the upstream amino acids are numbered in reverse starting from -1. Replace the original amino acids at site A (-2 and -1 sites), site B (6 and 7 sites), site C (13 and 14 sites), and / or site D (20 and 21 sites) with cysteine (Cys, C), thereby obtaining the self-source trimer domain sequence. Taking F149-158 as an example, i) a pair of disulfide bond mutations are made at site A to obtain "CCASGIAVSKVL", named "F149-158+A", ii) two pairs of disulfide bond mutations are introduced at its site A and site B to obtain "CCASGIACCKVL", named "F149-158+AB", and so on. This example specifically provides 16 self-source trimer domains, as follows:

[0139] F74-96+A: CCAKVKLIKQELDKYKNAVTELQLL (SEQ ID NO: 9);

[0140] F74-96+AB: CCAKVKLCCQELDKYKNAVTELQLL (SEQ ID NO: 10).

[0141] F149-158+A: CCASGIAVSKVL (SEQ ID NO: 11);

[0142] F149-158+AB: CCASGIACCKVL (SEQ ID NO: 12);

[0143] F150-206: SGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPI (SEQ ID NO: 5);

[0144] F215-238+A: CCSNIETVIEFQQKNNRLLEITREFS (SEQ ID NO: 13);

[0145] F215-238+ABCD: CCSNIETCCEFQQKCCRLLEICCEFS (SEQ ID NO: 14);

[0146] F215 - 238 + AC: CCSNIETVIEFQQKCCRLLEITREFS (SEQ ID NO: 15);

[0147] F215 - 238 + ACD: CCSNIETVIEFQQKCCRLLEICCEFS (SEQ ID NO: 16);

[0148] F215 - 238 + BCD: SNIETCCEFQQKCCRLLEICCEFS (SEQ ID NO: 17);

[0149] F215 - 238 + C: SNIETVIEFQQKCCRLLEITREFS (SEQ ID NO: 18);

[0150] F268 - 283 + A: CCNDQKKLMSSNVQIVRQ (SEQ ID NO: 19);

[0151] F268 - 283 + AB: CCNDQKKCCSSNVQIVRQ (SEQ ID NO: 20);

[0152] F491 - 509 + A: CCSISQVNEKINQSLAFIRKS (SEQ ID NO: 21);

[0153] F491 - 509 + AB: CCSISQVCCKINQSLAFIRKS (SEQ ID NO: 22);

[0154] F491 - 509 + ABC: CCSISQVCCKINQSCCFIRKS (SEQ ID NO: 23);

[0155] Among them, F150 - 206 does not introduce disulfide bond mutations to test whether it can help RSV F form trimerization without introducing any disulfide bonds in the trimer domain.

[0156] When constructing the RSV F protein mutant (antigen protein), the above-mentioned autologous trimer domain will be used to replace the fragment at the C-terminus of the RSV F protein that includes the transmembrane region and the intracellular region (i.e., the fragment including aa 525-574) to help form a trimer structure between RSV F monomer proteins. As a preferred embodiment, the above-mentioned autologous trimer domain is used to replace aa 512-574 or aa 514-574 of the original RSV F protein. Among them, i) the trimer domain without the A-site disulfide bond mutation for modification, such as F150-206, F215-238+BCD or F215-238+C, is used to replace aa 514-574 of the original RSV F protein; ii) the trimer domain with the A-site disulfide bond mutation for modification, that is, the trimer domain starting with "CC" at the N-terminus of the sequence, is used to replace aa 512-574 of the original RSV F protein (essentially also replacing aa 514-574, the difference is only that -CC- at the N-terminus of the trimer domain replaces -LL- originally located at aa 512-513 of the RSV F protein, so it is shown as replacing aa 512-574). The selection basis of this preferred embodiment and the additional beneficial effects it brings are: The amino acid at position 513 is exactly where an α-helix structure ends. Connecting the trimerization domain after the α-helix structure can theoretically help the F protein form a more stable trimer and exclude the interference of other amino acids.

[0157] (2) Other modifications to the RSV F protein mutant are as follows: According to the previous research results, for disulfide bond mutations, D486C+E487C is used, or this mutation is not introduced; for proline mutations, S215P is used; for cavity filling mutations, S190L is used; for electrostatic mutations, E487T is used, or this mutation is not introduced; for the p27 sequence, S / N105-A147 is used (delete the fragment between amino acids 105-147, the "between" does not include the end values, i.e., the 105th and 147th amino acids, the same below). In fact, there are two overall strategies: one group contains the disulfide bond mutation D486C+E487C but does not contain the electrostatic mutation E487T (including: D486C+E487C, S215P, S190L, S / N105-A147), and the other group contains the electrostatic mutation E487T but does not contain the disulfide bond mutation D486C+E487C (including: S215P, S190L, E487T, S / N105-A147), and different autologous trimer domains are added on this basis to compare their effects.

[0158] The specific RSV F protein mutants designed in this round are shown in Table 1. In this table, the left column lists the names of the mutants, and the modifications of each mutant compared to the wild-type RSV F are listed in the same row. The corresponding ELISA test results of each mutant are shown in Table 2.

[0159] Table 1: The First-round Design Scheme of the Homotrimeric Mutants of RSV F Protein

[0160]

[0161] Note: " / " indicates that this item has not been modified.

[0162] Table 2: The Results of the First-round ELISA Detection of the Homotrimeric Mutants of RSV F Protein

[0163]

[0164] After screening with the RSV F trimer-specific antibody AM14, the results showed that:

[0165] Comparing the data in Table 2, it was found that among all the RSV F protein mutants tested in this round, BR89, BR90, BR91, BR94, BR95, BR98, and BR103 had better effects (the Pre-F ratios measured under the three conditions of direct detection, 50°C for 1 h, and freeze-thaw 8 times were all not less than 0.5); at the same time, BR103 also showed good performance (the Pre-F ratios measured under the three conditions of direct detection, 50°C for 1 h, and freeze-thaw 8 times reached or were very close to 0.5, specifically 0.48, 0.48, and 0.5), and especially worth mentioning is that the Pre-F ratio measured under the condition of 65°C for 1 h was still 0.21.

[0166] Combined with the modification methods used in each mutant, further analysis showed that the self-source sequences of the RSV F protein, F74-96 (F74-96+A>F74-96+AB, where ">" means better effect, the same below), F491-509 (F491-509+ABC>F491-509+AB>F491-509+A), F149-158 (F149-158+A>F149-158+AB), F215-238 (F215-238+AC>F215-238+A>F215-238+C>F215-238+BCD>F215-238+ABCD>F215-238+ACD), and F268-283 (F268-283+AB>F268-283+A), etc., could all help the RSV F protein form a relatively stable trimer. F150-206 (BR109) without introducing disulfide bond mutation pairs showed poor performance, indicating that it was necessary to introduce disulfide bond mutation pair modification on the soluble fragment.

[0167] Therefore, the self-source trimeric domains F74-96+A, F491-509+ABC, F149-158+A, and F215-238+AC with better performance were selected for the next round of antigen protein screening.

[0168] Example 3: Second-round screening of the RSV F protein's autologous trimer domain

[0169] The main purpose of this round of RSV F antigen screening is to focus on screening for proline mutation sites that are compatible with the autologous trimer domain.

[0170] The design of the RSV F protein mutants in this round is still based on the consensus sequence of the F protein of wild-type RSV-A (SEQ ID NO:1). The design of the RSV F protein mutants is as follows: (1) In terms of the autologous trimer domain: F74-96+A, F491-509+ABC, F149-158+A, or F215-238+AC are used. (2) Other modifications to the RSV F protein mutants are as follows: For disulfide bond mutations, D486C+E487C is used, or this mutation is not introduced; for proline mutations, Q210P, S211P, S211P+S213P, or S / N105C+M370C are used; for cavity filling mutations, S190L is used; for electrostatic mutations, E487T is used, or this mutation is not introduced; for p27 sequence modification, S / N105-A147 (deleting the fragment between amino acids 105 and 147) is used. In fact, there are two overall strategies: one group contains the disulfide bond mutation D486C+E487C but does not contain the electrostatic mutation E487T (including: D486C+E487C, proline mutations, S190L, S / N105-A147), and the other group contains the electrostatic mutation E487T but does not contain the disulfide bond mutation D486C+E487C (including: proline mutations, S190L, E487T, S / N105-A147). Different autologous trimer domains and different proline mutations are added to compare their effects.

[0171] The specific RSV F protein mutants designed in this round are shown in Table 3. In this table, the left column lists the names of the mutants, and the modifications of each mutant compared to the wild-type RSV F are listed in the same row. The corresponding ELISA test results of each mutant are shown in Table 4.

[0172] Table 3: Second-round design scheme of the RSV F protein's autologous trimer mutants

[0173]

[0174] Table 4: Second-round ELISA test results of the RSV F protein's autologous trimer mutants

[0175]

[0176] After screening with the RSV trimer-specific antibody AM14, the results showed that:

[0177] By comparing the data in Table 4, it is found that among all the RSV F protein mutants tested in this round, BR110, BR111, BR120, BR122, and BR123 have relatively better effects (in the three cases of direct detection, 1 hour at 50°C, and 8 cycles of freeze-thaw, the proportion of Pre-F is not less than 0.5).

[0178] Combined with the modification methods used in each mutant, further analysis shows that:

[0179] (1) In all groups using the disulfide bond mutation D486C + E487C: i) After the combination of the autologous trimer domain F74-96+A and the proline mutations Q210P (BR110) or S211P (BR111), the stability of the Pre-F conformational protein can be significantly improved (where Q210P > S211P > S211P+S213P > S / N105C+M370C); ii) The combination of F491-509+ABC and different proline mutations has the second-best effect (where S211P > Q210P > S211P+S213P > S / N105C+M370C); iii) The combination of F149-158+A (BR126) or F215-238+AC (BR128) with the proline mutation S211P+S213P is less effective than the combination of F74-96+A and S211P+S213P; Therefore, overall, the combination of F74-96+A and Q210P or S211P has the best effect.

[0180] (2) In all groups using the electrostatic mutation E487T: The combination of the autologous trimer domain F74-96+A and S211P+S213P (BR120) has a relatively good effect, but it is still inferior to BR110 or BR111.

[0181] Therefore, in terms of the autologous trimer domain, F74-96+A is selected for the next round of screening. For other modifications, two proline mutations, Q210P and S211P, and S215P, which showed relatively excellent performance in the first round of screening, are selected for the third round of antigen protein screening. The combination with other modifications includes: D486C+E487C, S190L, and S / N105-A147. The third round of antigen protein screening will be carried out on the basis of the amino acid sequences of the wild-type RSV F proteins of type A and type B with the aforementioned modifications to construct mutants, and a His tag will be added after the autologous trimer domain for Pre-F conformational stability testing.

[0182] Example 4: The third round of screening for the autologous trimer domain of RSV F protein

[0183] The main purpose of this round of RSV F antigen screening is to confirm whether the self-derived trimeric domain and related mutations selected can perform excellently in both subtypes A and B, and to add a His tag to the C-terminus of the RSV F protein mutant to facilitate subsequent protein purification. The design of the RSV F protein mutant in this round is based on the consensus sequence of the F protein of wild-type RSV-A or RSV-B (SEQ ID NO: 1 or SEQ ID NO: 2), and the design of the RSV F protein mutant is as follows:

[0184] (1) In terms of the self-derived trimeric domain: F74-96+A is adopted.

[0185] (2) Other modifications to the RSV F protein mutant are as follows: For disulfide bond mutations, D486C+E487C is adopted; for proline mutations, Q210P, S211P or S215P is adopted; for cavity filling mutations, S190L is adopted; no electrostatic mutations are introduced; for the p27 sequence, S / N105-A147 (deleting the fragment between amino acids 105 and 147) is adopted.

[0186] The specific RSV F protein mutants designed in this round are shown in Table 5. In this table, the left column lists the names of the mutants, and the modifications of each mutant compared with the wild-type RSV F protein are listed in the same row. Among them, a) BR132~BR134 are obtained by modifying the consensus sequence of the F protein of wild-type RSV-A (SEQ ID NO: 1), and their amino acid sequences are shown in SEQ ID NO: 70, 71, 72 respectively (note: the mutants that are only different from BR132, BR133, BR134 in that they do not add a His tag are the aforementioned BR90, BR110, BR111, and their amino acid sequences are shown in SEQ ID NO: 30, 50, 51 respectively); b) BR135~BR137 are obtained by modifying the consensus sequence of the F protein of wild-type RSV-B (SEQ ID NO: 2), and their amino acid sequences are shown in SEQ ID NO: 73, 74, 75 respectively (note: the mutants that are only different from BR135, BR136, BR137 in that they do not add a His tag are BR135’, BR136’, BR137’, and their amino acid sequences are shown in SEQ ID NO: 76, 77, 78 respectively). The corresponding ELISA test results of each mutant are shown in Table 6.

[0187] Table 5: The Design Scheme of the Third Round of Self-derived Trimeric Mutants of RSV F Protein

[0188]

[0189] Note:

[0190] (1) An 8×His tag was added after the native trimeric domain in the third-round antigen screening.

[0191] Table 6: Results of the third-round ELISA detection of the native trimeric mutants of RSV F protein

[0192]

[0193] The results of the third-round screening showed that after using the native trimeric domain F74-96+A, the proline mutation Q210P performed the best and could help stabilize the Pre-F conformation in mutants designed using two RSV subtypes (subtype A: BR133; subtype B: BR136). Therefore, antigens containing the Q210P mutation site were selected to design BR133 and BR136 (the modifications they contain are: D486C+E487C, Q210P, S190L, S / N105-A147, F74-96+A+His), as well as BR64 screened out in previous studies (obtained by modifying the subtype A RSV F protein shown in SEQ ID NO: 1, the modifications it contains are: D486C+E487C, Q210P, S190L, S / N105-A147, Foldon, see CN202510136318.0) for subsequent experiments.

[0194] Example 5: Large-scale expression and purification of RSV F antigen protein

[0195] The recombinant baculovirus was used to infect 1 L of High Five cells in the logarithmic growth phase at a ratio of 1:100 (the cell density was about 2×10 6 cells / ml, and the viability was greater than 95%). The infected cells were cultured with shaking at 27°C and 120 rpm for 3-4 days. When it was observed that the cell diameter increased significantly and the viable cell density and cell viability decreased significantly, the cell culture supernatant was recovered by centrifugation at 4000 rpm for 20 minutes. Subsequently, the obtained supernatant was filtered through a 0.22 μm filter membrane, and the filtered liquid was used for purification by a nickel affinity column. The specific purification steps are as follows:

[0196] Equilibration: The nickel column (UniNTA-80Ni) was equilibrated with the binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and the buffer usage was 10 times the column volume.

[0197] Loading: The filtered sample was slowly loaded onto the equilibrated nickel column, and the loading was repeated 3 times to ensure that the target protein was fully bound to the nickel column.

[0198] Washing and impurity removal: Wash with a washing and impurity removal buffer (50 mM NaH2PO4, 300 mM NaCl, 20 - 50 mM imidazole, pH 8.0) at 10 - 20 column volumes to remove non-specifically bound proteins.

[0199] Elution: Elute the target protein twice with an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 - 500 mM imidazole, pH 8.0).

[0200] The collected eluate was placed in a SnakeSkin dialysis bag (Thermo Scientific™), and the PBS dialysis solution was used to replace the salt ions in the eluate. After dialysis, the dialysis solution was aliquoted and stored at -80 °C. At the same time, a small amount of the sample was taken for reducing and non-reducing electrophoresis and Western blot analysis (using the His antibody labeled with HRP from Bioledgend, dilution ratio 1:5000). The experimental results are as Figure 1 and Figure 2 shown: Figure 1 This is the reducing gel electrophoresis analysis diagram of the RSV F protein mutant after purification, which proves that the molecular weight of the RSV F protein monomer conforms to the expectation; Figure 2 This is the non-reducing gel electrophoresis analysis diagram and the Western Blot analysis diagram after non-reducing gel electrophoresis of the RSV F protein mutant after purification, which proves that the molecular weight of the RSV F protein trimer conforms to the expectation, and the band corresponding to the RSV F protein is further confirmed by Western blot detection with the His antibody; Thus, it can be seen that the molecular weights of each antigen protein (monomer and trimer) produced by the insect cell-baculovirus expression system conform to the expectation and are determined to be the RSV F protein, and the purity of each mutant antigen reaches the expected requirements and can be used for subsequent experimental research.

[0201] Example 6: Animal experiment

[0202] Female Balb / C mice at 6 - 8 weeks old were selected and randomly divided into multiple experimental groups with 5 mice in each group. On day 0 (Primary, primary immunization) and day 21 (Boost, booster immunization), the purified RSV F antigen proteins (BR64, BR133, BR136, BR133 + BR136) were immunized by intramuscular injection respectively. The injection dose was 50 μl in each of the left and right legs, totaling 100 μl. The control group was injected with 100 μl of physiological saline as a negative control. Mouse blood was collected on days 14 and 35 respectively, and the serum was separated to detect the binding antibody level. The specific operation method is as follows:

[0203] Coat 100 μl of the purified RSV F antigen proteins (BR64, BR133, BR136) onto a 96-well ELISA plate and let it stand overnight at 4°C. The next day, wash it 3 times with PBST, gently pat dry, and then block the plate with 5% skim milk prepared with PBST (200 μl / well, block at room temperature for 1 hour). After blocking, wash it again 3 times with PBST and pat dry. Dilute the serum samples two-fold with PBST containing 1% skim milk, with the starting dilution factor of 1:100. Add 100 μl of the diluted serum to each well and incubate at room temperature for 1 hour. Subsequently, wash it 3 times with PBST and pat dry, then add 100 μl of Goat Anti-Mouse IgG-HRP antibody and incubate at room temperature for 1 hour. After incubation, wash it 5 times with PBST and pat dry. Add 100 μl of freshly prepared TMB chromogenic solution to each well. After reacting for 10 - 15 minutes, add the stop solution to terminate the chromogenic reaction. Finally, measure the absorbance at a wavelength of 450 nm using an ELISA reader.

[0204] Set the Cut-off value according to 2.1 times the absorbance of the negative control. The maximum dilution factor of the sample with an absorbance ≥ Cut-off value is defined as the binding antibody titer of the sample.

[0205] The results of the binding antibody assay are as Figure 3 shown. It can be seen that:

[0206] (1) The RSV F protein mutants (BR133, BR136) containing the autologous trimer domain provided by the present invention induced the production of specific antibodies against RSV type A or B. Thus, it is proved that the autologous trimer domain of the RSV F protein provided by the present invention can help the RSV F antigen protein form a pre-fusion conformational trimer with immunogenicity, and at the same time, it is applicable to the F proteins of both RSV type A and B. By comparing the amino acid sequences of the RSV F protein of type A shown in SEQ ID NO: 1 and the RSV F protein of type B shown in SEQ ID NO: 2, it can be known that the similarity (Identities) between the two is only 90%. The autologous trimer domain can help the RSV F antigen proteins of different subtypes with 10% differences achieve pre-fusion conformational trimerization, which is sufficient to show that the autologous trimer domain provided by the present invention has wide applicability, and its pre-fusion conformational trimerization ability comes from its own sequence characteristics rather than depending on other structural characteristics of the RSV F antigen protein. The sequence differences between different RSV F antigen proteins within the same subtype are theoretically smaller than those between different subtypes of RSV F antigen proteins (i.e., less than 10%). Therefore, it can be reasonably predicted that when the autologous trimer domain is used to construct other RSV F antigen proteins of subtype A or B other than BR133 and BR136, it can also help their monomers achieve pre-fusion conformational trimerization with immunogenicity.

[0207] (2) Comparative experiments on the RSV F antigen protein of subtype A showed that the effect of using the autologous trimer domain (BR133) was roughly equivalent to that of using the exogenous trimer domain Foldon (BR64). This proved that the autologous trimer domain provided by the present invention can be used to replace heterologous trimer domains such as Foldon to avoid possible pre-existing immunity and safety problems.

[0208] (3) Comparison of experimental groups with different doses (3 μg, 6 μg, 12 μg) of BR133 + BR136 showed that under the three dose conditions, specific antibodies against RSV of type A and type B could be induced, and moreover, a significantly enhanced immune response could be induced after boost immunization; a high antibody titer could be stimulated with a low dose of the antigen protein, which indirectly indicated that the antigen protein provided by the present invention had good immunogenicity and had a cost advantage in the development of vaccine products.

[0209] The above embodiments only represent some application examples of the present invention and do not cover all implementation manners. The feasible implementation manners of the present invention are exemplarily illustrated by the selected embodiments herein, and are not used to limit the scope of application for which the present invention should enjoy protection. According to the embodiments described herein, all other specific implementation manners that can be deduced by those skilled in the same field without additional innovative efforts shall be included within the protection scope of the present invention.

Claims

1. A soluble fragment of RSV F protein, which is a fragment of no more than 70 amino acids from the soluble expression region of the wild-type RSV F protein shown in SEQ ID NO: 1, and the soluble fragment is the following (1) or (2): (1) any one selected from F74-96, F149-158, F150-206, F215-238, F268-283 or F491-509, whose amino acid sequences are shown in SEQ ID NOs: 3, 4, 5, 6, 7, 8, respectively; (2) A derivative sequence selected from any one of F74-96, F149-158, F150-206, F215-238, F268-283 or F491-509: According to SEQ ID NO: 1, based on any one of SEQ ID NOs: 3, 4, 5, 6, 7, 8, the N-terminal side is shortened or extended by x1 adjacent amino acid residues, and / or the C-terminal side is shortened or extended by x2 adjacent amino acid residues, wherein 0≤x1≤10, 0≤x2≤10, and the length of the derivative sequence is more than 5 amino acids.

2. The soluble fragment according to claim 1, characterized in that Said 0≤x1≤5, 0≤x2≤5.

3. The soluble fragment according to claim 2, characterized in that Said 0≤x1≤2, 0≤x2≤2.

4. RSV F protein self-derived trimer domain, which is obtained by transforming the soluble fragment according to any one of claims 1 to 3: the structure is N-terminal-(XY) m -soluble fragment-C-terminus, wherein X, Y represent any amino acid residues, m represents their number and m=0 or 1; Optionally, based on the above structure, disulfide bond mutation pairs are modified for any one, two, three or four groups of sites selected from the following (1) to (4), that is, the original amino acid is replaced with cysteine: (1) A site: amino acid sites -2 and -1; (2) Site B: amino acid sites 6 and 7; (3) C site: amino acid sites 13 and 14; (4) D site: amino acid sites 20 and 21; in, The amino acid site numbering is determined based on the sequence of the soluble fragment: the first amino acid at the N-terminus of the soluble fragment is taken as amino acid site 1, the downstream amino acid sites are numbered sequentially starting from 2, and the upstream amino acid sites are numbered in reverse order starting from -1.

5. The RSV F protein self-derived trimer domain according to claim 4, characterized in that In the self-derived trimer domain, there is a disulfide bond mutation pair selected from one of the following: A site, C site, AB site, AC site, ABC site, ACD site, BCD site or ABCD site.

6. The RSV F protein self-derived trimer domain according to claim 5, characterized in that The self-derived trimer domain is selected from one of the following groups: (1) F74-96+A: comprising or consisting of SEQ ID NO: 9; (2) F74-96+AB: comprising or consisting of SEQ ID NO: 10; (3) F149-158+A: comprising or consisting of SEQ ID NO: 11; (4) F149-158+AB: comprising or consisting of SEQ ID NO: 12; (5) F150-206: comprising or consisting of SEQ ID NO: 5; (6) F215-238+A: comprising or consisting of SEQ ID NO: 13; (7) F215-238+ABCD: comprising or consisting of SEQ ID NO: 14; (8) F215-238+AC: comprising or consisting of SEQ ID NO: 15; (9) F215-238+ACD: comprising or consisting of SEQ ID NO: 16; (10) F215-238+BCD: comprising or consisting of SEQ ID NO: 17; (11) F215-238+C: comprising or consisting of SEQ ID NO: 18; (12) F268-283+A: comprising or consisting of SEQ ID NO: 19; (13) F268-283+AB: comprising or consisting of SEQ ID NO: 20; (14) F491-509+A: comprising or consisting of SEQ ID NO: 21; (15) F491-509+AB: comprising or consisting of SEQ ID NO: 22; (16) F491-509+ABC: comprising SEQ ID NO: 23 or consisting of the sequence.

7. The RSV F protein self-derived trimer domain according to claim 6, characterized in that The self-derived trimer domain is selected from one of the following: F74-96+A, F74-96+AB, F149-158+A, F215-238+AC, F491-509+ABC.

8. The RSV F protein self-derived trimer domain according to claim 7, characterized in that The self-derived trimer domain is selected from F74-96+A.

9. Use of the RSV F protein self-derived trimer domain according to any one of claims 4 to 8 in constructing RSV F antigen protein.

10. The use according to claim 9, characterized in that: The self-generated trimerization domain is used to replace the fragment of RSV F protein including the transmembrane region and the intracellular region.

11. The use according to claim 10, characterized in that: The fragment including the transmembrane region and the intracellular region of the RSV F protein corresponds to the position of the nth amino acid of the wild-type RSV F protein and its downstream sequence, wherein 512≤n≤525.

12. The use according to claim 11, characterized in that: Where n=512 or 514.

13. RSV F protein mutant, characterized in that The C-terminus of the RSV F protein mutant lacks the transmembrane region and the intracellular region, and has an RSV F protein self-derived trimer domain selected from any one of claims 4-8, that is, the self-derived trimer domain is used to replace the fragment including the transmembrane region and the intracellular region.

14. The RSV F protein mutant of claim 13, characterized in that The fragment including the transmembrane region and the intracellular region of the RSV F protein corresponds to the position of the nth amino acid of the wild-type RSV F protein and its downstream sequence, wherein 512≤n≤525.

15. The RSV F protein mutant of claim 14, characterized in that n=512 or 514.

16. The RSV F protein mutant of claim 15, characterized in that The self-derived trimer domain is selected from F74-96+A, F74-96+AB, F149-158+A, F215-238+AC or F491-509+ABC, wherein: F74-96+A: comprising SEQ ID NO: 9, or consisting of the sequence; F74-96+AB: comprising SEQ ID NO: 10, or consisting of the sequence; F149-158+A: comprising SEQ ID NO: 11, or consisting of the sequence; F215-238+AC: comprising SEQ ID NO: 15, or consisting of the sequence; F491-509+ABC: It comprises SEQ ID NO: 23, or consists of this sequence.

17. The RSV F protein mutant of claim 16, characterized in that The self-derived trimer domain selected was F74-96+A.

18. The RSV F protein mutant according to any one of claims 13 to 17, characterized in that The RSV F protein mutant also has the following a), b), c) and d) modifications compared to the wild-type RSV F protein: a) disulfide bond mutation D486C+E487C, or electrostatic mutation E487T; b) Proline mutation: selected from S215P, Q210P, S211P or S211P+S213P; c) Cavity-filling mutation: S190L; d) P27 sequence modification: S / N105-A147.

19. The RSV F protein mutant of claim 18, characterized in that The RSV F protein mutant has a modification selected from one of the following groups relative to the wild-type RSV F protein: (1) D486C+E487C, S215P, S190L, S / N105-A147; (2) D486C+E487C, Q210P, S190L, S / N105-A147; (3) D486C+E487C, S211P, S190L, S / N105-A147; (4) S215P, S190L, E487T, S / N105-A147; (5) Q210P, S190L, E487T, S / N105-A147; (6) S211P, S190L, E487T, S / N105-A147; (7) S211P+S213P, S190L, E487T, S / N105-A147.

20. The RSV F protein mutant of claim 19, characterized in that The RSV F protein mutant has a modification selected from one of the following groups relative to the wild-type RSV F protein: (1) D486C+E487C, S215P, S190L, S / N105-A147, F74-96+A; (2) D486C+E487C, S215P, S190L, S / N105-A147, F74-96+AB; (3) D486C+E487C, S215P, S190L, S / N105-A147, F149-158+A; (4) D486C+E487C, S215P, S190L, S / N105-A147, F491-509+ABC; (5) D486C+E487C, Q210P, S190L, S / N105-A147, F74-96+A; (6) D486C+E487C, S211P, S190L, S / N105-A147, F74-96+A; (7) S215P, S190L, E487T, S / N105-A147, F74-96+A; (8) S215P, S190L, E487T, S / N105-A147, F215-238+AC; (9) Q210P, S190L, E487T, S / N105-A147, F491-509+ABC; (10) S211P, S190L, E487T, S / N105-A147, F491-509+ABC; (11) S211P+S213P, S190L, E487T, S / N105-A147, F74-96+A.

21. The RSV F protein mutant of claim 20, characterized in that The RSV F protein mutant is obtained by performing the modification on the basis of the wild-type RSV F protein having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

22. The RSV F protein mutant of claim 21, characterized in that The RSV F protein mutant is selected from one of the following: BR89, BR90, BR91, BR94, BR95, BR98, BR103, BR110, BR111, BR120, BR122, BR123, BR132, BR133, BR134, BR135, BR136, BR137, and their amino acid sequences are shown below: BR89: SEQ ID NO: 29; BR90: SEQ ID NO: 30; BR91: SEQ ID NO: 31; BR94: SEQ ID NO: 34; BR95: SEQ ID NO: 35; BR98: SEQ ID NO: 38; BR103: SEQ ID NO: 43; BR110: SEQ ID NO: 50; BR111: SEQ ID NO: 51; BR120: SEQ ID NO: 60; BR122: SEQ ID NO: 62; BR123: SEQ ID NO: 63; BR132: SEQ ID NO: 70; BR133: SEQ ID NO: 71; BR134: SEQ ID NO: 72; BR135: SEQ ID NO: 73; BR136: SEQ ID NO: 74; BR137: SEQ ID NO: 75; BR135': SEQ ID NO: 76; BR136': SEQ ID NO: 77; BR137′: SEQ ID NO:

78.

23. The RSV F protein mutant of claim 22, characterized in that The RSV F protein mutant is selected from one of the following: BR90, BR110, BR132, BR133, BR135, BR136, BR135', BR136'.

24. A nucleic acid molecule encoding the RSV F protein self-derived trimer domain described in any one of claims 4-8, or the RSV F protein mutant described in any one of claims 13-23.

25. A biological material comprising the RSV F protein mutant according to any one of claims 13 to 23 and / or the nucleic acid molecule according to claim 24, wherein the biological material is a recombinant vector, a recombinant virus or a transformed cell.

26. An immunogenic composition comprising one of the following: (1) The RSV F protein mutant according to any one of claims 13 to 23; (2) The nucleic acid molecule according to claim 24; (3) The biomaterial according to claim 25.

27. Application of any of the following (1) to (4): (1) The RSV F protein mutant according to any one of claims 13 to 23; (2) The nucleic acid molecule according to claim 24; (3) The biomaterial according to claim 25; (4) The immunogenic composition of claim 26; The application is selected from any one of the following (a) to (c): (a) preparing RSV-specific antibodies; (b) preparing a medicament for preventing and / or treating RSV infection; (c) Preparation of diagnostic reagents for RSV.

28. The use according to claim 27, characterized in that The medicine is a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine for preventing and / or treating RSV.

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