RSV f protein soluble fragment, self-source trimerization domain and application thereof

Abstract

The present application relates to RSV F protein soluble fragment, self-source trimer domain and application thereof, and belongs to the technical field of recombinant protein. Based on the amino acid sequence of wild type RSV F protein, the present application screens out a sequence which is soluble and has rigidity, constructs a soluble fragment, and performs disulfide bond mutation on the basis to obtain a self-source trimer domain sequence, which is used to replace the transmembrane region and intracellular region of RSV F protein, so that it can realize trimerization and keep stable pre-fusion conformation, and meanwhile, soluble expression in cells can be realized. The present application also provides a RSV F protein mutant containing the self-source trimer domain sequence, proves the generation and stability of pre-F conformation trimer, and selects a representative therefrom to perform animal experiment, which proves that it has good immunogenicity, and can be used for developing RSV vaccine, preparing RSV specific antibody or RSV detection reagent.

Description

TECHNICAL FIELD

[0001] The present application relates to RSV F protein soluble fragments, self-source trimer domain and application thereof, and belongs to the technical field of recombinant proteins. BACKGROUND

[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 by direct contact, and infection can cause bronchitis, pneumonia, asthma, and respiratory failure and other symptoms. RSV is the main cause of respiratory tract infection in children worldwide, and also causes severe respiratory and pulmonary infection in infants, the elderly, and those with weak immune systems. The pathogenic mechanism of RSV involves its surface glycoprotein, especially the F protein, which plays a key role in the invasion process of the virus. The F protein exists in the form of a precursor (F0), which consists of 574 amino acids. During the fusion of the virus with the host cell membrane, the F0 protein is cleaved by furin, releasing two subunits: F1 (amino acids 137 to 574) and F2 (amino acids 26 to 109). The two subunits are connected by disulfide bonds to form a mature F protein trimer structure. During this process, the trimer F protein undergoes a conformational change from the pre-fusion state (Pre-F) to the post-fusion state (Post-F). RSV F protein exhibits different antigenicity in the Pre-F and Post-F states, with the Pre-F protein having higher immunogenicity, while in the Post-F state, it facilitates viral entry into host cells. Therefore, using the Pre-F protein of the natural trimer as a vaccine antigen can induce the body to produce an effective immune response, preventing viral invasion and reducing the severity of infection.

[0003] In recent years, three RSV vaccines with trimer Pre-F as the antigen have been successfully launched internationally, namely Arexvy by GSK, Abrysvo by Pfizer, and mRESVIA by Modena. Currently, there is no RSV preventive vaccine on the market in China. However, the currently marketed RSV vaccines all face the problem of insufficient long-lasting 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. Moreover, booster shots did not show stronger immune protection (67.1%, LRTD, Arexvy).

[0004] The recombinant protein vaccines currently on the market (such as new crown vaccines and RSV vaccines) replace the C-terminal transmembrane region and intracellular region with a heterologous trimerization tag (such as the Foldon sequence from T4 phage, the trimerization motif from human collagen, etc.) in order to achieve soluble expression and maintain the natural trimerization structure. However, the heterologous trimer domain is not the native sequence of the antigen, which may cause problems such as immune pre-existence and safety. Multiple injections of vaccines containing a heterologous trimer domain may produce antibodies against the heterologous trimer, affecting the immune protection effect of the vaccine, and leading to insignificant booster effect after multiple injections. Therefore, in order to provide sustained immune protection to the body, the present application aims to design a fusion pre-conformation RSV antigen protein containing a self-source trimerization domain, i.e. using the F protein itself sequence as the trimerization domain sequence, so that the antigen does not contain any heterologous sequence, while also maintaining a stable trimerization fusion pre-conformation, and showing good immunogenicity in animal models, which can potentially further develop a vaccine against RSV as a core antigen. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to study the RSV F protein, and through the methods of structural biology and bioinformatics, a specific mutant capable of stably maintaining the fusion pre-conformation is designed. First, the present application provides an amino acid sequence from the soluble expression region of the RSV F protein (soluble fragment), and further creates a self-source trimer domain of the RSV F protein on the basis thereof; then, the present application provides a RSV F protein mutant containing the self-source trimer domain, which replaces the C-terminal transmembrane region and intracellular region of the F protein with the self-source trimer domain, and the three mutant sequences can form a trimer through the self-source trimer domain without introducing a heterologous trimer domain (such as Foldon), which has a similar conformation to the natural fusion pre-conformation F protein, thus having a stable trimer 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 application are as follows:

[0007] (I) In a first aspect, the present application provides a RSV F protein soluble fragment:

[0008] The soluble fragment is a stretch of amino acid sequence from the wild-type RSV F protein (SEQ ID NO: 1) itself soluble expression region, and is a rigid amino acid sequence, which is no more than 70 amino acids in length. In alternative embodiments, the rigid amino acid sequence includes 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, which are specifically 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 can be selected from one of the six sequences (F74-96, F149-158, F150-206, F215-238, F268-283, F491-509) described above. Alternatively, the soluble fragment is selected from a derivative sequence of the six sequences described above: based on any one of SEQ ID NO: 3, 4, 5, 6, 7, 8, a sequence obtained by performing x1 adjacent amino acid residue truncation or elongation on the N-terminal side and / or x2 adjacent amino acid residue truncation or elongation on the C-terminal side of SEQ ID NO: 1, wherein 0≤x1≤10, 0≤x2≤10 (i.e., x1 and / or x2 can be any integer between 0 and 10), x1 and x2 are not 0 at the same time, and the length of the derivative sequence obtained by the truncation or elongation is 5 amino acids or more (at least 5 amino acids); preferably, 0≤x1≤5, 0≤x2≤5; more preferably, 0≤x1≤2, 0≤x2≤2; even 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] (II) In a second aspect, the present application provides a RSV F protein self-source trimer domain:

[0018] which is further modified from the aforementioned soluble fragment, and has the structure: N-terminal-(X-Y) m -soluble fragment-C-terminal, wherein X and Y represent any amino acid residue (for example, X-Y are two adjacent amino acids from the transmembrane region and the intracellular region of the RSV F protein), m represents the number 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 amino acid site No. 1, the downstream amino acids are sequentially numbered from 2, and the upstream amino acids are numbered in reverse order from -1, replacing the original amino acids at A site (No. -2 and No. -1 sites), B site (No. 6 and No. 7 sites), C site (No. 13 and No. 14 sites), and / or D site (No. 20 and No. 21 sites) with cysteine (Cys, C), thereby obtaining a self-source trimer domain sequence. Preferably, A site, C site, AB site, AC site, ABC site, ACD site, BCD site, or ABCD site is selected for disulfide bond mutation pair modification.

[0019] Preferably, the present application also provides the following 16 specific self-source 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] wherein "A" indicates introducing a disulfide pair at position A, "AB" indicates introducing a disulfide pair at position A and B, respectively, and so on; F150-206 is not introduced with a disulfide pair modification.

[0037] Preferably, the self-source trimerization domain is selected from F149-158+A, F215-238+AC, F491-509+ABC, F74-96+A, or F74-96+AB; further preferably from F149-158+A, F215-238+AC, F491-509+ABC, or F74-96+A; more preferably F74-96+A.

[0038] (III) In a third aspect, the present application provides a RSV F protein mutant comprising the self-source trimerization domain:

[0039] The RSV F protein mutant replaces a fragment comprising the transmembrane region and the intracellular region of the RSV F protein C-terminus with the self-source trimerization domain to help the mutant protein form a trimer and achieve soluble expression. The transmembrane region and the intracellular region of the wild-type RSV F protein are located at aa 525-574, so "a fragment comprising the transmembrane region and the intracellular region" refers to a fragment covering aa 525-574 and possibly longer, for example, aa n and the sequence downstream of n of the wild-type RSV F protein, wherein 512≤n≤525 (preferably, n=512 or 514, i.e., aa 512-574 or aa 514-574 of the RSV F protein are replaced with the self-source trimerization domain). In an alternative embodiment, replacing a fragment comprising the transmembrane region and the intracellular region means replacing the sequence of aa 512 or 514 and the sequence downstream thereof with the sequence of the self-source trimerization domain of the RSV F protein. The sequence of the self-source trimerization domain is selected from or preferably from the sequences listed in the "second aspect" above.

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

[0041] (1) at least one disulfide mutation at other positions than the ABCD sites to stabilize the Pre-F conformation:

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

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

[0044] In an alternative embodiment, the mutant of the RSV F protein further comprises at least one proline mutation to enhance the stability of the Pre-F protein conformation. In an alternative embodiment, the proline mutation comprises mutating the amino acids at positions 210, 211, 211+213, or 215 of the wild-type RSV F protein amino acid sequence to P. Preferably, the proline mutation comprises mutating the amino acids at positions 210 or 215 of 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 comprises at least one cavity-filling mutation to enhance the stability of the Pre-F protein conformation. In an alternative embodiment, the cavity-filling mutation is mutating the amino acid at position 190 of the wild-type RSV F protein amino acid sequence to a larger side chain amino acid, leucine (S190L) to fill the structural cavity and enhance the stability of the Pre-F conformation.

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

[0048] In an alternative embodiment, the mutant of the RSV F protein further comprises at least one electrostatic mutation to enhance the stability of the Pre-F protein conformation. In an alternative embodiment, the electrostatic mutation is mutating the amino acid at position 487 of 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 comprises an engineering of the p27 sequence (aa 110~136), i.e. deletion of the fragment containing p27. In an alternative embodiment, the engineering of the p27 sequence refers to deletion of the amino acids between positions 105~147 of the wild type RSV F protein amino acid sequence (S / N105-A147).

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

[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 engineering: S / N105-A147.

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

[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] Further preferred, the RSV F protein mutant has modifications relative to the wild-type RSV F protein selected from one of the following groups:

[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 preferred, on the basis of the wild-type RSV F protein sequence as shown in SEQ ID NO: 1 (RSV-A) or SEQ ID NO: 2 (RSV-B), modifications selected from any one of (1) to (11) above are made to obtain the 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'; wherein, preferably BR132, BR133, BR135, BR136, BR135', or BR136' is selected.

[0077] In alternative embodiments, the RSV F protein mutant of the present application comprises an 8*His tag sequence (HHHHHHHH). The sequence is not essential for the function of the RSV F protein, such as inducing an immune response. The skilled person will recognize such sequences and, where appropriate, understand that the RSV F protein mutant disclosed in the present application can or can not comprise such a sequence.

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

[0079] (a) any nucleic acid molecule encoding the RSV F protein self-source trimer domain or the RSV F protein mutant.

[0080] (b) a recombinant vector comprising the nucleic acid molecule, preferably the base plasmid of the recombinant vector is pOET1.1.

[0081] (c) a recombinant virus comprising the nucleic acid molecule or the recombinant vector, wherein the preferred recombinant virus types include insect cell baculovirus, adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus.

[0082] (d) a transformed cell comprising the nucleic acid molecule, the recombinant vector or the recombinant virus, wherein 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] (Fifth) In a fifth aspect, the present application also relates to a method for preparing the RSV F protein as described above, 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] (VI) In a sixth aspect, the present application also provides an immunogenic composition comprising any of the RSV F protein mutants, nucleic acid molecules, recombinant vectors, recombinant viruses or transformed cells described above. Preferably, the immunogenic composition comprises a RSV F protein mutant (antigenic protein) against RSV type A, such as BR90, BR110 or BR111; or, the immunogenic composition comprises a RSV F protein mutant (antigenic protein) against RSV type B, such as BR135', BR136' or BR137'. Further preferably, the immunogenic composition comprises antigenic proteins against both RSV type A and B, such as BR90 and BR135', BR110 and BR136', or BR111 and BR137', etc.

[0085] (VII) In a seventh aspect, the present application also provides any of the RSV F protein mutants, nucleic acid molecules, recombinant vectors, recombinant viruses, transformed cells or immunogenic compositions described above for use in any of the following:

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

[0087] (b) for the development of a medicament for the prevention and / or treatment of RSV infection, preferably, the medicament can be a recombinant protein vaccine, a vector-based vaccine or a nucleic acid vaccine.

[0088] (c) for the preparation of a diagnostic reagent for RSV.

[0089] Definitions of terms:

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

[0091] In the present application, the term "mutant" is used exclusively to refer to a protein that has one or more changes in the amino acid sequence or protein structure compared to the wild-type protein; the changes can be diverse, including but not limited to deletion, insertion, substitution, shortening and / or loss of one or more amino acids, and in addition, can involve modification or cleavage of the protein structure. Specifically, the "mutant" in the present application refers to a mutant 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 protein subunits of the same or different types connected together by a special chemical structure. The protein trimer described in the present application can be a RSV F protein.

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

[0094] In the present application, the term "self-origin trimerization domain" refers to a fragment with trimerization function based on the naturally occurring amino acid sequence of the target protein, which is screened or further modified from the target protein and can make the target protein form a trimer. The self-origin trimerization domain described in the present application refers to an amino acid fragment with trimerization function screened or modified from the amino acid sequence of RSV F protein itself, for example, which is modified by disulfide bond mutation based on the "soluble fragment" described above.

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

[0096] In the present application, the term "disulfide bond mutation" refers to the replacement of certain amino acids in 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 the introduction of a pair (two) of cysteine mutations.

[0097] In the present application, the term "cavity filling mutation" refers to the replacement of amino acid residues in wild-type RSV F protein with amino acids with 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 the replacement of a specific amino acid residue in wild-type RSV F protein with an amino acid that can reduce the ionic repulsion between residues close to each other during protein folding, so as 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 a proline to stabilize the RSV Pre-F structure. The amino acid and carboxyl group in proline are linked by an amino group to form a rigid five-membered ring structure, which restricts the rotational freedom of proline in the protein, so that proline can form fixed folding and corners to increase the stability of the protein.

[0100] In the present application, the term "p27" refers to a 27-amino acid short peptide (aa 110-136) consisting of amino acid residues 110-136 in the wild-type RSV F protein. The short amino acid peptide is hydrolyzed and released by furin, and forms two subunits F1 (aa 137-574) and F2 (aa 26-109).

[0101] In the present application, the term "p27 sequence modification" refers to the deletion of a fragment containing p27 based on the wild-type RSV F protein, i.e., the deleted fragment should cover p27 and can be slightly longer than p27 (1-15 amino acid residues upstream and / or downstream of p27). In the embodiments of the present application, the p27 sequence modification used is "S / N105-A147", which refers to the deletion of the fragment between amino acid residues 105-147 of the RSV F protein (the deleted fragment does not include the end values of the numerical range, i.e., amino acid residues 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., PLoS Pathog, 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 change can include a change in the polypeptide of a chemical bond in the protein molecule.

[0105] In the present application, the term "comprising" generally refers to including the explicitly specified features, but not excluding other elements.

[0106] In the present application, the "transmembrane region and intracellular region at the C-terminus of the RSV F protein" refers to the sequence corresponding to the amino acid residues at positions 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 containing amino acid residues at positions 525-574 or slightly longer, such as aa 512-574, aa 514-574, etc.

[0107] Advantages:

[0108] (1) First, the present application provides a soluble fragment from the RSV F protein, which is further modified by disulfide bond mutation to replace the original trimerization domain sequence with a rigid and stable structure, which can be used to replace the exogenous trimerization domain sequence (such as Foldon) to achieve F protein trimerization, avoiding the immune pre-existence and safety problems caused by the introduction of heterologous trimerization domain.

[0109] (2) Second, the RSV F protein mutant provided by the present application replaces the C-terminal transmembrane region and intracellular region with the original trimerization domain, so that the target protein can be expressed in a large scale in a soluble form in cells, and can form a trimer structure without using a heterologous trimer, maintaining the pre-fusion conformation and showing excellent stability.

[0110] (3) Third, the RSV F protein mutant obtained by mutating and deleting the wild-type amino acid sequence has a stable Pre-F protein conformation and shows strong immunogenicity, which can stimulate the body to produce high levels of specific antibodies, and therefore has a high potential to become an effective component of RSV vaccine, which can be used alone or in combination with other pathogen antigens to form a multi-vaccine for the prevention and / or treatment of RSV infection, and can also be used as a RSV-specific detection reagent. BRIEF DESCRIPTION OF DRAWINGS

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

[0112] Figure 1 : Analysis diagram of the reduced gel electrophoresis of the RSV F protein mutant after purification.

[0113] Figure 2 : Analysis diagram of the non-reduced gel electrophoresis of the RSV F protein mutant after purification and the Western Blot analysis diagram after non-reduced gel electrophoresis.

[0114] Figure 3Results of detection of binding antibodies produced by mice immunized with RSV F antigen proteins. DETAILED DESCRIPTION

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

[0116] Example 1: Development of RSV F protein self-source trimer mutant

[0117] First, through bioinformatics analysis, the F protein sequences of wild-type RSV-A and RSV-B were obtained: based on the F protein sequences of thousands of RSV-A and RSV-B strains publicly disclosed in the NCBI database, the amino acid with the highest frequency of occurrence at each site (consensus amino acid) was calculated, thereby constructing the consensus amino acid sequence of RSV F protein of each subtype, which is specifically shown in SEQ ID NO. 1 and SEQ ID NO. 2, and which is used to represent the F protein sequence of wild-type RSV of A type and B type, respectively.

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

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

[0120] (1) Design of self-source trimer domain: first, the RSV F protein was analyzed by using structural biology software to obtain the amino acid sequence of the soluble expression region (referred to as "soluble fragment") of itself, which was used to replace the C-terminal transmembrane region and intracellular region of RSV F protein, expecting 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 self-source sequence, disulfide bond mutation was carried out around the soluble fragment or its adjacent amino acids to obtain the final self-source trimer domain.

[0121] (2) Overall design of RSV F protein mutant: after designing the self-source trimer domain, it was used in combination with other modifications (disulfide bond mutation, proline mutation, etc.) for RSV F antigen protein to obtain the overall design result of the amino acid sequence of RSV F protein mutant. Then, codon optimization was carried out to adapt to the expression requirements of host Sf9 insect cells, thereby obtaining the nucleic acid coding sequence of RSV F protein mutant, and then performing whole gene synthesis.

[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 the recombinant plasmid was co-transfected into Sf9 insect cells with the baculovirus genome by flashBAC TM The baculovirus expression system kit was used to co-transfect the recombinant plasmid and the baculovirus genome into Sf9 insect cells. The transfection operation was performed according to the kit instructions. Seven days after transfection, the supernatant of Sf9 cells was collected, and at this time the supernatant was P0 generation virus. The P0 generation virus was used to infect new Sf9 cells to produce P1 generation virus. After 6-7 days of infection of the P1 generation virus, 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 method, and the specific steps were as follows: 100 μl Motavizumab (abbreviated as Mota, Pre-F and Post-F can be combined), AM14 (only combined with Pre-F) and other antibodies were coated in 96-well plates at 4°C overnight. The next day, wash 3 times with PBST and dry, then prepare 5% skim milk with PBST for blocking (200 μl / well, room temperature blocking for 1 hour). The cell supernatant collected in (2) was divided into 4 equal parts, and was treated at 50°C for 1 hour, 65°C for 1 hour, repeated freezing and thawing for 8 times, and not treated. After blocking, the 96-well plate was washed 3 times with PBST and dried, 100 μl of untreated and various stress condition treated cell supernatant samples were added to each well, and incubated at room temperature for 1-2 hours. Wash 3 times with PBST, add 100 μl of mouse serum (immune RSV Pre-F antigen protein) or Rabbit His-tag Antibody, and incubate at room temperature for 1 hour. Wash 3 times with PBST, add 100 μl of HRP enzyme-labeled antibody, and incubate at room temperature for 1 hour. Wash 5 times with PBST and dry, add 100 μl of freshly prepared TMB developing solution to each well. After developing for 10-15 minutes, stop the developing with a stop solution, and read the absorbance value at wavelength 450 nm / 570 nm with a microplate reader. The final absorbance reading is A450 nm-A570 nm.

[0126] According to the above process, the development of RSV F protein self-source trimer mutant was carried out, according to the ELISA detection results, the self-source trimer domain with excellent performance was selected for the next round of screening, and other amino acid mutations were screened on the basis thereof, so as to obtain more stable Pre-F conformation RSV F protein mutant.

[0127] Example 2: First round of screening of RSV F protein self-source trimer domain

[0128] The first round of mutant design was performed according to the operation steps described in Embodiment 1. The main purpose of this round is to screen the available self-source trimer domain.

[0129] The RSV F protein mutant design of this round is based on the F protein consensus sequence (SEQ ID NO: 1) of wild-type RSV-A, and the design of the RSV F protein mutant is as follows:

[0130] (1) Self-source trimer domain:

[0131] 1) Soluble fragment screening: After analysis by structural biology software, the present application obtained 6 amino acid sequences from the soluble expression region of RSV F protein itself, i.e. F149-158, F150-206, F215-238, F268-283, F491-509 and F74-96, to be used as the soluble fragment, and the sequences are 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-source trimer domain: which is further modified from the above soluble fragment, and the structure is: N-terminal-(X-Y) m- soluble fragment-C-terminal, wherein X, Y represent two adjacent amino acid residues from the transmembrane region and the extracellular region of RSV F protein, m represents the number thereof and m = 0 or 1; and, on the basis of the above structure, further introducing a pair of disulfide bond mutation to stabilize the trimer structure formed from the 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 from 2, and the upstream amino acids are numbered in reverse order from -1, replacing the original amino acids at A site (No. -2 and No. -1 sites), B site (No. 6 and No. 7 sites), C site (No. 13 and No. 14 sites) and / or D site (No. 20 and No. 21 sites) with cysteine (Cys, C), thereby obtaining the sequence of the trimer domain from the source. Taking F149-158 as an example, i) a pair of disulfide bond mutations was introduced at the A site to obtain "CCASGIAVSKVL", named "F149-158+A", ii) two pairs of disulfide bond mutations were introduced at the A site and the B site thereof to obtain "CCASGIACCKVL", named "F149-158+AB", and so on. This embodiment specifically provides 16 kinds of trimer domains from the source, which are 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] F150-206 does not introduce disulfide bond mutations in order to test whether not introducing any disulfide bonds in the trimerization domain can help RSV F form a trimer.

[0156] In constructing the RSV F protein mutant (antigen protein), the above self-trimerization domain is used to replace the fragment of the RSV F protein C-terminal containing the transmembrane region and intracellular region (i.e. the fragment containing aa 525~574) to help the formation of trimer structure between RSV F monomer proteins. As a preferred embodiment, the above self-trimerization domain is used to replace aa 512~574 or aa 514~574 of the original RSV F protein. Among them, i) the trimerization domain without A-site disulfide bond mutation pair, 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 trimerization domain with A-site disulfide bond mutation pair, i.e. the trimerization domain with sequence starting with "CC" at the N-terminal, is used to replace aa 512~574 of the original RSV F protein (in essence, also replace aa 514~574, the difference is only that the -CC- at the N-terminal of the trimerization domain replaces the original -LL- at aa 512~513 of the RSV F protein, so it is manifested as replacing aa 512~574). The selection basis of this preferred embodiment and the additional beneficial effects brought by it are: the 513th amino acid is exactly the place where the alpha helix structure stops, and connecting the trimerization domain after the alpha helix structure can theoretically help the F protein to form a more stable trimer, excluding the interference of other amino acids.

[0157] (2) Other modifications for the RSV F protein mutant are as follows: according to the previous research results, in terms of disulfide bond mutation, D486C+E487C is adopted, or the mutation is not introduced; in terms of proline mutation, S215P is adopted; in terms of cavity filling mutation, S190L is adopted; in terms of electrostatic mutation, E487T is adopted, or the mutation is not introduced; in terms of p27 sequence, S / N105-A147 (deleting the fragment between aa 105~147, which does not include the end values, i.e. aa 105 and 147, the same below) is adopted. In fact, there are two groups of strategies: one group contains disulfide bond mutation D486C+E487C but does not contain electrostatic mutation E487T (contains: D486C+E487C, S215P, S190L, S / N105-A147), and the other group contains electrostatic mutation E487T but does not contain disulfide bond mutation D486C+E487C (contains: S215P, S190L, E487T, S / N105-A147), and different self-trimerization domains are added on the basis thereof to compare their effects.

[0158] The specific RSV F protein mutants designed in this round are shown in Table 1, in which the left column is the name of the mutant, and the modifications made to the mutant compared with the wild-type RSV F are listed in the same row. The corresponding ELISA detection results of each mutant are shown in Table 2.

[0159] Table 1: First round design of RSV F protein self-source trimer mutants

[0160]

[0161] Note: " / " means that this item is not modified.

[0162] Table 2: First round ELISA detection results of RSV F protein self-source trimer mutants

[0163]

[0164] After screening by RSV F trimer specific antibody AM14, the results show that:

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

[0166] Further analysis in combination with the modification methods used in each mutant shows that: RSV F protein self-source sequence F74-96 (F74-96+A > F74-96+AB, where ">" means better than, 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) can help RSV F protein form a relatively stable trimer. F150-206 (BR109) without introducing disulfide bond mutation pairs performs poorly, so it is necessary to introduce disulfide bond mutation pairs modification on the basis of soluble fragments.

[0167] Therefore, the self-source trimer domains F74-96+A, F491-509+ABC, F149-158+A and F215-238+AC which perform better are selected into the next round of antigen protein screening.

[0168] Example 3: Second round of screening of RSV F protein self-source trimer domain

[0169] The main purpose of this round of RSV F antigen screening is to focus on screening the proline mutation sites adapted to the self-source trimer domain on the basis of the self-source trimer domain.

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

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

[0172] Table 3: Second round of design scheme of RSV F protein self-source trimer mutants

[0173]

[0174] Table 4: ELISA detection results of RSV F protein self-source trimer mutants in the second round

[0175]

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

[0177] From the data in Table 4, it is found that the effects of BR110, BR111, BR120, BR122, and BR123 are relatively better among all the RSV F protein mutants in this round of testing (direct detection, 50°C for 1 h, and 8 times of freeze-thaw, and the Pre-F ratio is not less than 0.5 in the three cases).

[0178] Further analysis in combination with the modification methods used in each mutant shows that:

[0179] (1) In all groups using disulfide bond mutation D486C+E487C: i) the combination of the original trimer domain F74-96+A with Q210P (BR110) or S211P (BR111) proline mutation can significantly improve the stability of Pre-F conformation protein (among them, Q210P > S211P > S211P+S213P > S / N105C+M370C); ii) the combination of F491-509+ABC with different proline mutations is less effective (among them, S211P > Q210P > S211P+S213P > S / N105C+M370C); iii) the combination of F149-158+A (BR126) or F215-238+AC (BR128) with proline mutation S211P+S213P is less effective than the combination of F74-96+A with S211P+S213P; therefore, the combination of F74-96+A with Q210P or S211P is the best overall.

[0180] (2) In all groups using electrostatic mutation E487T: the combination of the original trimer domain F74-96+A with S211P+S213P (BR120) is relatively good, but still less effective than BR110 or BR111.

[0181] Therefore, in terms of the original trimer domain, F74-96+A is selected for the next round of screening. In terms of other modifications, Q210P and S211P, two proline mutations, and S215P, which performed well in the first round of screening, are selected to enter the third round of antigen protein screening, combined with other modifications including D486C+E487C, S190L, and S / N105-A147. The third round of antigen protein screening will perform the aforementioned modifications on the basis of the amino acid sequences of wild-type RSV F proteins of types A and B to construct mutants, and add His tag after the original trimer domain for Pre-F conformation stability testing.

[0182] Example 4: Third round of screening of RSV F protein original trimer domain

[0183] The main purpose of the third round of screening of RSV F antigens is to confirm whether the screened self-trimerization domain and related mutations can have superior performance in both subtypes A and B, and to add a His tag at the C-terminal end of the RSV F protein mutant to facilitate subsequent protein purification. The design of the RSV F protein mutant is based on the consensus sequence of the wild-type RSV-A or RSV-B F protein (SEQ ID NO: 1 or SEQ ID NO: 2), and the design of the RSV F protein mutant is as follows:

[0184] (1) Self-trimerization domain: F74-96+A is used.

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

[0186] The specific RSV F protein mutants designed in this round are shown in Table 5, in which the left column is the name of the mutant, and the same row lists the modifications made to the mutant compared to the wild-type RSV F protein. Among them, a) BR132-BR134 are obtained by modifying the wild-type RSV-A F protein consensus sequence (SEQ ID NO: 1), and their amino acid sequences are shown in SEQ ID NO: 70, 71, and 72, respectively (Note: the only difference between BR132, BR133, and BR134 is that the mutants without His tag are BR90, BR110, and BR111, respectively, and their amino acid sequences are shown in SEQ ID NO: 30, 50, and 51, respectively); b) BR135-BR137 are obtained by modifying the wild-type RSV-B F protein consensus sequence (SEQ ID NO: 2), and their amino acid sequences are shown in SEQ ID NO: 73, 74, and 75, respectively (Note: the only difference between BR135, BR136, and BR137 is that the mutants without His tag are BR135', BR136', and BR137', respectively, and their amino acid sequences are shown in SEQ ID NO: 76, 77, and 78, respectively). The corresponding ELISA detection results of each mutant are shown in Table 6.

[0187] Table 5: Third round of design of RSV F protein self-trimer mutants

[0188]

[0189] Note:

[0190] (1) The third round of antigen screening all added 8xHis tag after the self-source trimer domain.

[0191] Table 6: Third round ELISA detection results of RSV F protein self-source trimer mutant

[0192]

[0193] The third round of screening results show that after using the self-source trimer domain F74-96+A, the proline mutation Q210P performs best, and can help stabilize the Pre-F conformation in the mutants designed using two RSV subtypes (type A: BR133; type B: BR136). Therefore, the antigens containing the Q210P mutation site BR133 and BR136 (which contain the following modifications: D486C+E487C, Q210P, S190L, S / N105-A147, F74-96+A+His) and BR64 (based on the A type RSV F protein shown in SEQ ID NO: 1, which contains the following modifications: D486C+E487C, Q210P, S190L, S / N105-A147, Foldon, see CN202510136318.0) screened in previous studies were selected for subsequent experiments.

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

[0195] The recombinant baculovirus was used to infect 1L of logarithmic growth phase High Five cells (cell density about 2x10 6 cells / ml, viability greater than 95%) at a ratio of 1:100. After infection, the cells were cultured at 27°C, 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 nickel affinity column purification. The specific purification steps are as follows:

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

[0197] Loading: Slowly load the filtered sample into the equilibrated nickel column, repeat loading 3 times to ensure that the target protein is fully bound to the nickel column.

[0198] Washing: The column was washed with 10-20 column volumes of washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20-50 mM imidazole, pH 8.0) to remove non-specifically bound proteins.

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

[0200] The collected eluate was put into a SnakeSkin dialysis bag (Thermo Scientific™) and dialyzed against PBS to remove salt ions from the eluate. After dialysis, the dialysate was aliquoted and stored at -80°C. A small amount of sample was taken for reducing and non-reducing electrophoresis and Western blot analysis (HRP-labeled His antibody from Bioledgend, dilution ratio 1:5000). The results are shown in Figure 1 and Figure 2 . Figure 1 Figure 6 is a reducing gel electrophoresis analysis chart of RSV F protein mutants after purification, which proves that the molecular weight of RSV F protein monomer is consistent with the expectation; Figure 2 Figure 7 is a non-reducing gel electrophoresis analysis chart and a Western Blot analysis chart after non-reducing gel electrophoresis of RSV F protein mutants after purification, which proves that the molecular weight of RSV F protein trimer is consistent with the expectation, and the bands are further confirmed to correspond to RSV F protein by Western blot detection of His antibody. Thus, it can be seen that the molecular weight of each antigen protein (monomer and trimer) produced by the insect cell-baculovirus expression system is consistent with the expectation and is determined to be RSV F protein, the purity of each mutant antigen reaches the expected requirement, and can be used for subsequent experimental research.

[0201] Example 6: Animal experiment

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

[0203] 100 μl of purified RSV F antigen protein (BR64, BR133, BR136) was coated on a 96-well enzyme-labeled plate at 4°C overnight. The next day, the plate was washed with PBST for 3 times, and then blocked with 5% skim milk prepared in PBST (200 μl / well, room temperature for 1 hour). After blocking, the plate was washed with PBST for 3 times and dried. The serum sample was diluted in PBST containing 1% skim milk by two-fold gradient dilution, and the initial dilution ratio was 1:100. 100 μl of diluted serum was added to each well, and incubated at room temperature for 1 hour. Then, 100 μl of Goat Anti-Mouse IgG-HRP antibody was added, and incubated at room temperature for 1 hour. After incubation, the plate was washed with PBST for 5 times and dried. 100 μl of freshly prepared TMB developing solution was added to each well, and the reaction was allowed to proceed for 10-15 minutes. Then, the color developing reaction was terminated by adding a stop solution. Finally, the absorbance value was measured at 450 nm wavelength by using an enzyme-labeled instrument.

[0204] The cut-off value was set according to 2.1 times of the absorbance value of the negative control. The dilution ratio of the sample with the maximum dilution ratio and the absorbance value ≥ the cut-off value was defined as the binding antibody titer of the sample.

[0205] The binding antibody determination results are shown in Table 1. Figure 3 It can be seen that:

[0206] (1) The RSV F protein mutant (BR133, BR136) containing the self-source trimer domain provided by the application induces specific antibodies against type A or type B RSV. Therefore, it is proved that the self-source trimer domain of the RSV F protein provided by the application can help the RSV F antigen protein to form a fusion pre-conformation trimer with immunogenicity, and it is suitable for the F protein of both type A and type B RSV. As can be seen from the comparison of the amino acid sequences of the type A RSV F protein shown in SEQ ID NO: 1 and the type B RSV F protein shown in SEQ ID NO: 2, the similarity (Identities) of the two is only 90%. The RSV F antigen protein of different subtypes with 10% difference can help it to realize fusion pre-conformation trimerization, which is sufficient to prove that the self-source trimer domain provided by the application has wide applicability, and its fusion pre-conformation trimerization ability comes from its own sequence characteristics, and does not depend on other structural characteristics of the RSV F antigen protein. The sequence difference of different RSV F antigen proteins within the same subtype is theoretically smaller (i.e. less than 10%) than the sequence difference of RSV F antigen proteins between different subtypes. Therefore, it can be reasonably predicted that when the self-source trimer domain is used to construct other A subtype or B subtype RSV F antigen proteins other than BR133 and BR136, it can also help the monomer to realize fusion pre-conformation trimerization with immunogenicity.

[0207] (2) Comparative experiments for the RSV F antigen protein of subtype A show that the effect of using the self-source trimer domain (BR133) is approximately equivalent to that of using the foreign trimer domain Foldon (BR64), thereby proving that the self-source trimer domain provided by the present application can be used to replace the heterologous trimer domain such as Foldon, so as to avoid the immunological pre-existence and safety problems possibly caused by the heterologous trimer domain.

[0208] (3) Comparative experiments of different doses (3 μg, 6 μg, 12 μg) of BR133+BR136 show that under the three dose conditions, specific antibodies against RSV of subtypes A and B can be induced, and after boost, a significantly enhanced immune response can be induced; a low dose of the antigen protein can stimulate a relatively high antibody titer, which shows that the antigen protein provided by the present application has good immunogenicity, and has a cost advantage when used for the development of a vaccine product.

[0209] The above examples only represent some application examples of the present application, and do not include all the embodiments. The selected examples are used to exemplarily illustrate the feasible embodiments of the present application, and are not used to limit the application scope of the present application. According to the embodiments described herein, all the other specific embodiments that can be deduced by the person skilled in the art without additional innovative efforts are all included in the protection scope of the present application.

Claims

1. An RSV F protein self-source trimerization domain, characterized in that, the self-derived trimerization domain is selected from one of the following groups: (1) F74-96+A: the sequence of which is shown as SEQ ID NO: 9; (2) F74-96+AB: the sequence of which is shown as SEQ ID NO:

10.

2. Use of the RSV F protein self-source trimerization domain of claim 1 in the construction of RSV F antigenic proteins, characterized in that, the self-derived trimerization domain is used to replace a fragment comprising the transmembrane region and the intracellular region of the RSV F protein in the RSV F protein; the fragment comprising the transmembrane region and the intracellular region of the RSV F protein is located at a position corresponding to the nth amino acid of a wild-type RSV F protein and the sequence downstream thereof, wherein 512≤n≤525; the sequence of the wild-type RSV F protein is shown as SEQ ID NO:

1.

3. A mutant of RSV F protein characterized in that, the RSV F protein mutant is selected from one of the following: BR90, BR91, BR103, BR110, BR111, BR120, BR132, BR133, BR134, BR135, BR136, BR137, BR135', BR136', BR137', the amino acid sequences of which are shown as follows, respectively: BR90: SEQ ID NO: 30; BR91: SEQ ID NO: 31; BR103: SEQ ID NO: 43; BR110: SEQ ID NO: 50; BR111: SEQ ID NO: 51; BR120: SEQ ID NO: 60; 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.

4. The RSV F protein mutant of claim 3, characterized in that, the RSV F protein mutant is selected from one of the following: BR90, BR110, BR132, BR133, BR135, BR136, BR135', BR136'.

5. A nucleic acid molecule encoding the self-derived trimerization domain of the RSV F protein according to claim 1.

6. A nucleic acid molecule encoding the RSV F protein mutant according to claim 3 or 4.

7. A biological material comprising the RSV F protein mutant according to claim 3 or 4, wherein the biological material is a recombinant virus or a transformed cell.

8. A biological material comprising the nucleic acid molecule according to claim 6, wherein the biological material is a recombinant vector, a recombinant virus or a transformed cell.

9. An immunogenic composition comprising one of the following groups: (1) the RSV F protein mutant according to claim 3 or 4; (2) the nucleic acid molecule according to claim 6; (3) the biological material according to claim 7 or 8.

10. Use of any one of the following (1)-(4): (1) the RSV F protein mutant according to claim 3 or 4; (2) the nucleic acid molecule of claim 6; (3) the biomaterial of claim 7 or 8; (4) the immunogenic composition of claim 9; the use is selected from any one of (a) to (c): (a) the manufacture of an RSV-specific antibody; (b) the manufacture of a medicament for the prevention and / or treatment of RSV infection; (c) the manufacture of a diagnostic reagent for RSV.

11. Use according to claim 10, characterized in that, the medicament is a recombinant protein vaccine, a vector-based vaccine or a nucleic acid vaccine for the prevention and / or treatment of RSV infection.

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