Fusion protein comprising HMPV-F protein, preparation method and application thereof

Abstract

The invention relates to a fusion protein, the fusion protein comprises an F2 region and an F1 region of an F protein of HMPV and a foldon protein, after the F protein of HMPV is mutated, the stability and expression efficiency can be improved, the F protein of HMPV has a neutralizing effect with an antibody and can be used for preparing drugs, such as vaccines, and the vaccines can stimulate an organism to generate a neutralizing antibody aiming at HMPV and have an immune protection effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a fusion protein comprising HMPV-F protein, a preparation method and application thereof. Background Art

[0002] Human metapneumovirus (HMPV) is a newly discovered human respiratory pathogen in 2001. According to statistics in 2018, more than 14 million children under the age of 5 were infected with HMPV, of which more than 600,000 were hospitalized and more than 16,000 died. Children, the elderly and other immunocompromised people are the main infected populations of human metapneumovirus. Similar to RSV, the stable pre-fusion (pre-F) conformation of the metapneumovirus fusion protein (F) determines the viral epitope and can induce highly neutralizing antibodies. There are currently no effective therapeutic drugs and preventive vaccines for human metapneumovirus infection. The inactivated vaccines, subunit vaccines, recombinant live attenuated vaccines, etc. currently in preclinical research all have weaknesses such as inducing inflammation, insufficient immunogenicity, and difficulty in preparation, and their prospects for clinical application are worrying. Summary of the invention

[0003] In order to overcome the above-mentioned defects, the present invention provides a fusion protein including HMPV-F protein, a preparation method and application thereof, specifically,

[0004] In a first aspect, the present invention provides a fusion protein, wherein the fusion protein comprises the F protein of HMPV (HMPV-F protein) and the foldon protein.

[0005] Preferably, the F protein includes an F2 region and an F1 region.

[0006] More preferably, the F2 region and the F1 region are connected by a cleavage region. Further preferably, the cleavage region comprises a shorter cleavage region relative to the wild type. For example, the cleavage region comprises less than 6 amino acid residues, such as 5, 4, 3, etc. amino acid residues.

[0007] Further preferably, a first connecting peptide is inserted after the cleavage region, and most preferably, the cleavage region is completely deleted and replaced by the first connecting peptide;

[0008] In a specific embodiment, the first connecting peptide includes GS, GSGGSG (SEQ ID No.18), GGGS (SEQ ID No.19), GSGGGGSG (SEQ ID No.22), GGS, GGGGS (SEQ ID No.23), GSGS (SEQ ID No.24), GGSGS (SEQ ID No.25), GSGGS (SEQ ID No.26), GGSGS (SEQ ID No.27), GGGSS (SEQID No.28) or multiple combinations or repeated sequences thereof.

[0009] Preferably, the fusion protein further comprises a signal peptide.

[0010] Preferably, the F2 region and the F1 region comprise mutations relative to the wild type. More preferably, the mutations comprise mutations of amino acids to cysteine ​​and / or proline.

[0011] Further preferably, the mutation comprises mutating one or more pairs of amino acids in positions 84 and 249, 140 and 147, 365 and 455, and 454 and 458 of the F protein to cysteine ​​to form a disulfide bond.

[0012] Further preferably, the mutation comprises mutating the amino acid at position 131, 163 and / or 459 to proline.

[0013] In a specific embodiment, the mutation includes one or more of V84C-V249C, A140C-A147C, T365C-Q455C, T365C-Q455C, D454C-V458C, E131P, R163P and / or A459P.

[0014] The positions of the above-mentioned sites are exemplified by SEQ ID No. 1. Those skilled in the art will appreciate that the above-mentioned sites may include corresponding positions of other wild types (e.g., different serotypes, or even different strains) or fusion proteins, and the correspondence is understood as a corresponding relationship based on amino acid structure and / or functional analysis.

[0015] Preferably, the F protein of the fusion protein is directly or indirectly connected to the foldon protein.

[0016] More preferably, the indirect connection comprises connection via a second connecting peptide. In a specific embodiment, the second connecting peptide comprises GS, GSGGSG (SEQ ID No. 18), GGGS (SEQ ID No. 19), GSGGGGSG (SEQ ID No. 22), GGS, GGGGS (SEQ ID No. 23), GSGS (SEQ ID No. 24), GGSGS (SEQ ID No. 25), GSGGS (SEQ ID No. 26), GGSGS (SEQ ID No. 27), GGGSS (SEQ ID No. 28) or a plurality of combinations or repeated sequences thereof, etc.

[0017] Preferably, the foldon protein includes a restriction site, which is recognized or cleaved by a specific protease; more preferably, the protease is selected from but not limited to one or a combination of two of the following proteases: non-human enterokinase, tobacco etch virus protease, protease from Bacillus subtilis, protease from Bacillus amyloliquefaciens, protease from rhinovirus, papain, homologues of insect papain or homologues of crustacean papain. In a specific embodiment, the protease is derived from the 3C protease of rhinovirus (HRV-3C).

[0018] Preferably, the fusion protein also includes a tag protein, which refers to a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. More preferably, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc. In a specific embodiment, the tag protein includes 8×His and 6×His.

[0019] More preferably, the fusion protein includes, from N-terminus to C-terminus, a signal peptide, the F2 region of the F protein of HMPV, a cleavage region or a first connecting peptide, the F1 region of the F protein of HMPV, a second connecting peptide, and a foldon protein; further preferably, the fusion protein also includes a restriction site and a tag protein after the C-terminus of the foldon protein.

[0020] More preferably, the amino acid sequence of the F2 region of the HMPV F protein comprises:

[0021] (A1), as shown in positions 20-98 of any one of SEQ ID No.1,3-8,

[0022] (A2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (A1);

[0023] (A3) A protein having 80% or more identity with any one of (A1) to (A2) and having the same function.

[0024] More preferably, the amino acid sequence of the F1 region of the HMPV F protein comprises:

[0025] (B1), 103-489 of any one of SEQ ID No. 1, 3-8;

[0026] (B2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (B1);

[0027] (B3) A protein having 80% or more identity with any one of (B1) to (B2) and having the same function.

[0028] More preferably, the foldon protein comprises:

[0029] (C1), as shown in SEQ ID No.20,

[0030] (C2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (C1);

[0031] (C3) A protein having 80% or more identity with any one of (C1) to (C2) and having the same function.

[0032] More preferably, the signal peptide comprises:

[0033] (D1), as shown in SEQ ID No.17,

[0034] (D2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (D1);

[0035] (D3) A protein having 80% or more identity with any one of (D1) to (D2) and having the same function.

[0036] More preferably, the HRV-3C amino acid sequence comprises:

[0037] (E1), SEQ ID No. 21;

[0038] (E2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (E1);

[0039] (E3) A polypeptide having 80% or more identity with any one of (E1) to (E2) and having the same function.

[0040] More preferably, the fusion protein comprises:

[0041] (F1), any one of SEQ ID No. 1, 3-8;

[0042] (F2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (F1);

[0043] (F3) A polypeptide having 80% or more identity with any one of (F1) to (F2) and having the same function.

[0044] Herein, identity refers to amino acid sequence identity. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of amino acid sequences, the value of identity (%) can then be obtained.

[0045] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0046] Preferably, the fusion protein can form a trimer.

[0047] The second aspect of the present invention provides a nucleotide molecule, which encodes any one of the above-mentioned fusion proteins.

[0048] Preferably, the nucleic acid molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0049] More preferably, the nucleotide molecule comprises:

[0050] (G1), any nucleotide molecule shown in SEQ ID No.9, 11-16,

[0051] (G2), complementary, degenerate or transcribed sequence of (G1),

[0052] (G3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (G1) or (G2) and encodes the corresponding protein in the fusion protein.

[0053] Those skilled in the art can easily mutate the nucleotide sequence encoding the above fusion protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the above fusion protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above fusion protein and have the same function.

[0054] The identity refers to the sequence similarity with the compared nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence shown in SEQ ID No: 2 or 4 of the present invention. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0055] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0056] The third aspect of the present invention provides a vector, wherein the vector comprises any one of the above-mentioned nucleotide molecules.

[0057] Preferably, the vector further includes a regulatory factor, such as a promoter for initiating transcription of the above polypeptide or fusion protein encoding gene sequence, and may also include a terminator for terminating transcription of the above polypeptide or fusion protein encoding gene sequence. Further, the vector may also include an enhancer sequence.

[0058] The vector described herein refers to a vector that can carry exogenous DNA, mRNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In one or more embodiments of the present invention, the vector is a pUC57 vector and / or a PKS001 (pKS001) vector.

[0059] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises any one of the above-mentioned nucleotide molecules or vectors.

[0060] The host cell (also referred to as recipient cell) described herein can be a plant cell or an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but is not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), African green monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human kidney epithelial cell line or its derivative strain, such as HEK293, 293T / 17, etc., human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells or Sf-9 cells), etc. but not limited thereto. In one or more embodiments of the present invention, the host cell is CHO-K1 cell, 293T cell.

[0061] Preferably, the host cell may also be a microorganism, and the microorganism described herein may be a bacterium, a fungus, an actinomycete, a protozoa, an algae or a virus. Wherein, the bacterium may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto, for example, the bacterium may be Escherichia coli, Bacillus subtilis or Bacillus pumilus. In one or more embodiments of the present invention, the microorganism is a TOP10 competent cell.

[0062] In a fifth aspect, the present invention provides a method for preparing the above-mentioned fusion protein, the preparation method comprising introducing the above-mentioned nucleotide molecule or vector encoding the fusion protein into a host cell, and culturing the host cell.

[0063] Preferably, the preparation method comprises screening a monoclonal cell line with high and stable expression level.

[0064] More preferably, the screening method includes conventional screening methods in the art, such as adding screening reagents, comparing the biological activity of clone pools by ELISA, and the like.

[0065] In a sixth aspect, the present invention provides an application of any of the above fusion proteins, nucleotide molecules, vectors or host cells, wherein the application includes any of the following:

[0066] (1) Use in the preparation of products for preventing and / or treating diseases caused by HMPV infection;

[0067] (2) Application in the preparation of products for inducing immune response to HMPV virus antigens;

[0068] (3) Use in the prevention and / or treatment of diseases caused by HMPV infection;

[0069] (4) Application in inducing immune response to HMPV virus antigens.

[0070] The product described herein may be a reagent, a drug, or a vaccine.

[0071] Preferably, the preparation further comprises screening, for example, using the above mutant protein or fusion protein as a target to screen candidate preventive or therapeutic drugs or diagnostic agents.

[0072] The products described in (1) and (2) may be vaccines or antibodies against HMPV virus, and the antibodies include full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimum recognition units (MRU), etc. but not limited thereto).

[0073] Furthermore, the HMPV antibody may be a neutralizing antibody that specifically binds to the F protein of HMPV. The neutralizing antibody may be a high-titer neutralizing antibody against multiple epidemic strains of HMPV.

[0074] In a seventh aspect, the present invention provides a drug, which comprises any one of the above-mentioned fusion proteins, nucleotide molecules, vectors or host cells.

[0075] Preferably, the drug is a vaccine, and more preferably, the vaccine further comprises an adjuvant.

[0076] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factor IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0077] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0078] Preferably, the vaccine further comprises a vaccine delivery system.

[0079] The vaccine delivery system can be a type of substance that can carry antigenic substances to the body's immune system, store and exert its antigenic effect therein for a long time. The vaccine delivery system described herein can be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system or a nano adjuvant vaccine delivery system.

[0080] Furthermore, the medicine or vaccine also includes one or more pharmaceutically acceptable carriers.

[0081] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0082] The vaccine for preventing infection of the present invention can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection.

[0083] In an eighth aspect, the present invention also provides a method for producing an immune response, which may include administering any of the above-mentioned drugs or vaccines to a subject.

[0084] In a ninth aspect, the present invention also provides a method for preventing and / or treating an infectious disease caused by HMPV, which may include administering the drug or vaccine to a subject.

[0085] In the above method, after the drug or vaccine is administered to the subject, an immune response against HMPV virus can be induced in the subject. The immune response can be a cellular immune response, a humoral immune response, or a cellular immune response and a humoral immune response.

[0086] The cellular immune response may include a B cell immune response and a T cell immune response.

[0087] The subject described herein can be a human or a non-human animal.

[0088] Furthermore, the non-human animal may be a non-human mammal.

[0089] The non-human mammal may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.

[0090] The subjects described herein include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0091] The administration described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.

[0092] It should be noted that any form of numbering in the present invention, such as 1, 2, I, II, III, A, B, a, b, etc., is only used to distinguish each other and does not indicate the order of time or space, unless otherwise specified.

[0093] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0094] The present invention successfully expressed and purified the metapneumovirus antigen molecule preF protein by designing and using a prokaryotic expression system. The protein molecule can form a trimer structure with a stable structure and can be used for vaccine research and development, providing a reference for the research and application of vaccines designed in other trimer forms.

[0095] The present invention solves the problem of poor stability of wild antigens, and mutates some sites of HMPV F protein so that it can induce antibodies with good binding activity to HMPV F protein after entering the body, thereby giving the body corresponding immune protection. The present invention can form a trimer by fusing the mutated HMPV F protein with the foldon protein, obtain a good immune effect at a low dose, and effectively stimulate the body's cellular immune mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 : Trimer design scheme.

[0097] Figure 2 : Alignment of amino acid sequences of multiple antigens.

[0098] Figure 3 :Construction strategy for cloning the eukaryotic expression vector of HMPV preF trimer antigen protein.

[0099] Figure 4 :Purification of HMPV-His trimer antigen molecules.

[0100] Figure 5 :Molecular screening analysis of HMPV-His trimer antigen molecules.

[0101] Figure 6 : Comparison of neutralization effects of trimer antigen molecules. DETAILED DESCRIPTION

[0102] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0103] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials and reagents used in the examples are listed below, and the others can be obtained from commercial sources unless otherwise specified.

[0104] Example 1 Eukaryotic system expression of hemipulmonary F protein trimer antigen molecule design

[0105] The fusion protein includes, from N-terminus to C-terminus, a signal peptide (MSWKVVIIFSLLITPQHGL, SEQ ID No. 17), the F2 region of the F protein of HMPV, a cleavage region mutated to a first connecting peptide (GSGGSG, SEQ ID No. 18), the F1 region of the F protein of HMPV, a second connecting peptide GGGS (SEQ ID No. 19) flexible linker, followed by a fusion foldon sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID No. 20), then a HRV-3C (LEVLFQGP, SEQ ID No. 21) cleavage site, and finally 8 consecutive histidines (see Figure 1 ). In order to increase the expression level, enhance the structural stability and increase the binding force, multiple mutations were made to the F protein sequence of HMPV. The mutations include:

[0106] 1. Truncate the F2-F1 cleavage region and add a flexible linker to obtain a high-expression molecule design.

[0107] 2. Add and combine multiple groups of disulfide bonds to obtain structurally stable antigen molecules. The disulfide bond combinations include: V84C-V249C, A140C-A147C, T365C-Q455C, and D454C-V458C.

[0108] 3. Proline point mutations are performed to obtain higher neutralizing activity, specifically E131P, R163P, A459P, referred to as: 3P.

[0109] 1. Sequence Structure

[0110] According to the above mutation principle, a fusion protein including a mutated HMPV F protein and a foldon sequence was designed. Specifically, the following fusion protein SEQ ID No. 1-SEQ ID No. 8 (or Seq1-Seq8 for short) can be seen. The nucleotide sequence encoding the above fusion protein is shown in the following SEQ ID No. 9-SEQ ID No. 16 (or Seq9-Seq16 for short):

[0111] Amino acid sequence of seq1 fusion protein:

[0112] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGIAIAKTIRLPSEVNAIKGCLKKTNECV STLGNGVRVLATAVPELKEFVSKNLTSAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPECQFNCPLDQVFESIENSQALVDQSNKILNSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0113] Among them, positions 1-19: signal peptide region; positions 20-88: HPMV antigen F2 region; positions 89-94, mutated cleavage region; positions 95-470, HPMV antigen F1 region; positions 471-474: flexible second connecting peptide linker region; positions 475-501: foldon sequence; positions 503-510: HRV 3C site; positions 512-519: His tag.

[0114] Seq9 nucleotide sequence encoding PM1 fusion protein:

[0115]

[0116] Amino acid sequence of seq2 fusion protein:

[0117] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTCSADQRQSRGATAAAVTAGIAIAKTIRLPSEVNAIKGCLKKTNECV STLGNGVRVLATAVPELKEFVSKNLTSAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNDKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPECQFNCPLDQVFESIENSQALVDQSNKILNSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0118] Seq10 nucleotide sequence encoding Seq2 fusion protein

[0119]

[0120] Amino acid sequence of seq3 fusion protein:

[0121] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGIAIAKTIRLPSEVNAIKGCLKTTNECV STLGNGVRVLATAVPELKEFVSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVPLDQVFESIENSQALVDQSNKILNSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0122] Seq11 nucleotide sequence encoding Seq3 fusion protein

[0123]

[0124] Amino acid sequence of Seq4 fusion protein:

[0125] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGIAIAKTIRLPSEVNAIKGCLKTTNECV STLGNGVRVLATAVPELKEFVSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSCGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDCFNVPLDQVFESIENSQALVDQSNKILNSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0126] Seq12 nucleotide sequence encoding Seq4 fusion protein

[0127]

[0128] Amino acid sequence of Seq5 fusion protein:

[0129] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGIAIAKTIRLPSEVNAIKGCLKTTNECV STLGNGVRVLATAVPELKEFVSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPECQFNCPLDQVFESIENSQALVDQSNKILNSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0130] Seq13 nucleotide sequence encoding Seq5 fusion protein

[0131]

[0132] Amino acid sequence of Seq6 fusion protein:

[0133] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGVAIAKTIRLPSEVTAIKNCLKKTNECV STLGNGVRVLATAVPELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNDPLDQVFENIENSQALVDQSNRILSSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0134] Seq14 nucleotide sequence encoding Seq6 fusion protein

[0135]

[0136] Amino acid sequence of Seq7 fusion protein:

[0137] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGVAIAKTIRLPSEVTAIKNCLKKTNECV STLGNGVRVLATAVPELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDCFNVPLDQVFENIENSQALVDQSNRILSSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0138] Seq15 nucleotide sequence encoding Seq7 fusion protein

[0139]

[0140] Amino acid sequence of Seq8 fusion protein:

[0141] MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELKTCSADQGSGGSGATAAAVTAGVAIAKTIRLPSEVTAIKNCLKKTNECV STLGNGVRVLATAVPELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRCMVRRKGFGILIGVYGSSVIYMVQLPIFGV IDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCS YITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPECQFNCPLDQVFENIENSQALVDQSNRILSSAESAIGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGLEVLFQGPGHHHHHHHHH*

[0142] Seq16 nucleotide sequence encoding Seq8 fusion protein

[0143]

[0144] The amino acid sequence of pre-F_5WB0 fusion protein is referenced by: Hsieh, C.-L., Rush, SA, Palomo, C., Chou, C.-W., Pickens, W., Más, V., and McLellan, JS (2022). Structure-based design of prefusion-stabilized human metapneumovirus fusion proteins. Nature Communications 13.10.1038 / s41467-022-28931-3. The amino acid sequence of post-F_5L1X is referenced by McLellan, JS (2016). Engineering, Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation. PLoS Pathog 12, e1005859.10.1371 / journal.ppat.1005859.

[0145] The sequence alignment of seq1-seq8, pre-F_5WB0 and post-F_5L1X can be found in Figure 2 .

[0146] The structural differences and design principles are shown in Table 1:

[0147] Table 1: Structural differences and design principles

[0148]

[0149]

[0150] 2. Construction Method

[0151] The PKS001 vector plasmid (purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd.) and the plasmid containing the nucleotide encoding the above-mentioned HMPV F protein (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were double-digested with HindⅢ and NotⅠ restriction endonucleases, and the vector and fragments were ligated with T4 ligase and transformed into Escherichia coli strain Top10 (see Figure 3 ), screen positive clones and extract plasmids. After sequencing, extract the correct plasmids in large quantities to obtain high-quality plasmids carrying PKS-HMPV preF antigens.

[0152] Wherein, the nucleotide sequences encoding the fusion proteins of SEQ ID 1-8 are shown as SEQ ID No. 9-16 above, respectively.

[0153] Example 2 Transformation of 293T cells, cell culture, protein purification and expression analysis

[0154] The high-quality plasmid containing nucleotides encoding HMPV preF antigen successfully constructed in Example 1 was transformed into 293T cells, and the "suspension-sensitive" transfection reagent-DNA complex was prepared according to the following method: 300 μg of plasmid DNA was diluted with serum-free medium to a final volume of 5 mL; 600 μL of "suspension-sensitive" transfection reagent (Polymer, M5 HiPerSuspension Cell Special Transfection Reagent MF388) was diluted with serum-free medium to a final volume of 5 mL; gently mix and incubate at room temperature for 5 minutes; after incubation for 5 minutes, the diluted plasmid DNA was added to the diluted "suspension-sensitive" transfection reagent and gently mixed. Incubate at room temperature for 20 minutes to allow the DNA-"suspension-sensitive" transfection reagent complex to form. After complete incubation, 10 mL of the DNA "suspension-sensitive" transfection reagent complex was added to 290 mL of growth medium containing 293 suspension cells, so that the final cell density was approximately 1×10 6 cell / mL.

[0155] The cells were cultured in a shaker at 37°C, 5% CO2, and 150 rpm. Protein purification and expression analysis were performed after 6 days.

[0156] Purification and expression analysis can be seen in the following steps:

[0157] 1. Purification of His-tagged target protein

[0158] Method: After the cell culture fluid cultured for 6 days was centrifuged at 8000r / min for 20 minutes, the supernatant was collected and filtered using a 0.45μm filter membrane (Jin Teng, catalog number: JTSF 025013 / 014) to obtain about 300mL of supernatant cell fluid, which was purified using an AKTA protein purifier. The specific steps are as follows: After connecting the purification column to AKTA, debug the purification system, use the balance solution (20mm PB+0.5M NaCl, PH7.4) to balance the column, and the flow rate is set to 3mL / min. After equilibrium, the flow rate is set to 2mL / min for loading. After all samples have passed through the purification column, use the balance solution at a flow rate of 3mL / min to rinse until the absorption value is stable. Then use the washing solution (20mM PB+0.5M NaCl+30mM imidazole, PH7.4) to rinse the column, and the flow rate is set to 3mL / min. Then, the target protein was eluted with elution buffer (20 mM PB + 0.5 M NaCl + 0.5 M imidazole, pH 7.4) and the flow rate was set to 3 mL / min.

[0159] Results and conclusion: The purified protein with His tag was obtained preliminarily. The concentration of different molecular proteins was different, ranging from 1mg / ml to 5mg / ml, and the volume was about 10ml.

[0160] Take seq1 as an example, its purification diagram is shown in Figure 4 A: Lane 1 is a protein marker, Lane 2 is a control of the supernatant of 293T cell fermentation broth that was not transformed with the corresponding plasmid, Lane 3 is a supernatant of 293T cell fermentation broth transformed with the seq1 plasmid, Lane 4 is the flow-through collected after all samples passed through the purification column, Lane 5 is the solution collected by the washing solution, and Lane 6 is the protein pre-purified sample collected after elution with the eluent. It can be seen that a high-purity protein band can be observed at 65kDa on the SDS-PAGE gel, which is consistent with the predicted size of the antigen molecule protein monomer, and the protein purity is estimated to be above 90%.

[0161] The seq2 protein mutated its cleavage site, and its purification was shown in Figure 4 B: Lane 1 is a protein marker, lane 2 is a control of the supernatant of 293T cell fermentation broth that was not transformed with the corresponding plasmid, lane 3 is the flow-through collected after the 293T cell fermentation broth transformed with the seq2 plasmid passed through the purification column, and lane 4 is the protein pre-purified sample collected after elution with the eluent. It can be seen that two high-purity protein bands can be observed at 70kDa and 45kDa on the SDS-PAGE gel, indicating that the effect of mutating the cleavage site is not as good as directly mutating the cleavage site to a linker.

[0162] Therefore, in the molecular design, seq1, seq3-8 all adopted the construction strategy of mutating the cleavage region into a flexible first connecting peptide linker.

[0163] 2. Molecular sieve

[0164] Method: The collected elution peaks were concentrated and then separated on a Superdex 200 high-resolution agarose gel chromatography column molecular sieve column. The specific steps are as follows: After connecting the chromatography column to AKTA, debug the purification system, use the balance solution (20mMPB+0.15M NaCl) to balance the column, and set the flow rate to 1mL / min. The sample to be separated enters the chromatography column through the sample loop, and the column is continuously rinsed with the balance solution to collect samples with different absorption peaks. After the absorption peak is concentrated, subsequent experiments are carried out.

[0165] Add 20 μL of 5× protein loading buffer to 80 μL of fusion protein solution, treat at 95℃ for 10 min and centrifuge. Take 30 μL of supernatant for SDS-PAGE analysis, and observe protein expression, purification and separation after staining. The protein gel concentration is 10%, and the buffer is 1×Tris-MOPS-SDS Running buffer (MACKLIN, NONE6600).

[0166] Results Figure 5 : Taking Seq1 as an example, after the purified protein passes through the molecular sieve, three specific peaks can be obtained. The peak position of peak 2 is consistent with the size of the trimer. The gel run proves that it is indeed the target protein. After concentration, its protein concentration is about 0.33mg / ml, about 10ml (see Figure 5 A). Figure 5 Lane B is a protein marker, lane 2 is a sample of seq1 protein peak 2 in a non-reduced state, and lane 3 is a sample of seq1 protein peak 2 in a reduced state. It can be seen that the target protein is in the form of a trimer, and the protein purity is estimated to be above 95%.

[0167] Example 3 Antigen Activity Detection

[0168] Methods: References: Hsieh, C.-L., Rush, S. A., Palomo, C., Chou, C.-W., Pickens, W., Más, V., and McLellan, J. S.

[0169] (2022).Structure-based design of prefusion-stabilized humanmetapneumovirus fusion proteins.Nature Communications

[0170] 13.10.1038 / s41467-022-28931-3. Obtain the antibody MPE8 for HMPV pre-F protein and the antibody MPE33 for HMPV post-F protein.

[0171] MPE8 can specifically detect HMPV pre-F protein, and MPE33 can detect HMPV post-F protein. MPE8 and MPE33 proteins were coated with 1X PBS and placed at 4°C overnight, 200ng / well. After the coated plate was taken out from 4°C, the plate was washed 3 times, and the volume of each wash was 300μl / well. If there was residual wash solution in the well after washing, it should be patted dry on absorbent paper. After washing the plate, the purified protein seq1-8, pre-F, post-F was diluted in a 3-fold gradient to 311 (199017) times, and the diluted samples of each concentration were added to the sample wells in turn, 100μl / well, and 100μl of sample diluent was added as a blank control (Blk). Set up duplicate wells and incubate at 37°C for 60min. Add secondary antibody: discard the sample, wash the plate 3 times, each time with a volume of 300μl / well, if there is any residual solution in the well after washing, pat it dry on absorbent paper; add diluted secondary antibody, 100μl / well, cover with film, and incubate at 37℃ for 60min. Wash the 96-well plate 3 times, each time with a volume of 300μl / well, if there is any residual solution in the well after washing, pat it dry on absorbent paper, add single-component TMB colorimetric solution 1 (prepared in advance from Take out, balance to room temperature), 100 μl / well, color development at 25°C in the dark for 15 min. Put the ELISA plate into the ELISA reader, and measure the absorbance at a wavelength of 450 nm. Finally, determine the EC50 value for analysis.

[0172] The results are shown in Table 2:

[0173] Table 2: Binding activity results of fusion protein and antibody

[0174] Fusion Protein MPE8 MPE33 Seq1 4780 78 Seq2 Not detected Not detected Seq3 3245 92 Seq4 4503 75 Seq5 3202 80 Seq6 2986 106 Seq7 3890 63 Seq8 4300 43 pre-F_5WB0 5129 16 post-F_5L1X 70 6210

[0175] Seq1 and Seq3-8 proteins were detected by MPE8 and their titers were found to be above 3000. Seq1 and Seq3-8 proteins were detected by MPE33 and their titers were found to be within 100, indicating that Seq1 and Seq3-8 proteins were mainly in the pre-F form.

[0176] Example 4 Evaluation of the effect of mouse immune antigen molecules

[0177] Method: The corresponding trimer protein purified in Example 2 was used to immunize mice, and 40 C57BL / 6 female mice aged 6-8 weeks were selected and randomly divided into 8 groups, with 5 mice in each group. The first and second immunizations were separated by 3 weeks, and the trimer protein was 10 μg. Serum was collected 21 days after the first immunization and two weeks after the second immunization for ELISA titer detection. The immunization scheme is shown in Table 3, and the ELISA immunization effect is shown in Table 4.

[0178] Table 3. Antigen molecule immunization scheme

[0179]

[0180] Table 4: Antigen molecule immune effect

[0181]

[0182] The above data show that after Seq1, 3-8 form trimers, they can effectively stimulate the body to produce an immune response and form a protective mechanism, serving as a vaccine to prevent HMPV.

[0183] Example 5 Serum neutralization effect detection

[0184] method:

[0185] LLC-MK2 cells grown in T75 bottles were washed twice with 1*PBS, 0.25% trypsin was added, and the cells were resuspended and centrifuged at 37°C for 2min, 4000rpm, 5min. After resuspending the cells with culture medium, the cells were evenly spread in a 96-well cell culture plate and allowed to stand for 1 hour to adhere to the wall. Placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours. After the HMPV virus seed was taken out from -80 degrees, it was melted in a 37-degree water bath, diluted to 40 times, and placed on ice. The mouse serum obtained in Example 5 was diluted with 1*PBS, and the virus was evenly spread on the 96-well plate with diluted mouse serum with a volley gun, and then the mixture of serum and virus was inoculated into the cells and incubated at 37 degrees for 1.5 hours. After that, the mixture of serum and virus was discarded, and a covering layer (MEM+2% serum+1% double antibody+1.5% methylcellulose) of 200ul was added, and it was placed in a 37-degree incubator for 25 hours. Invert and gently remove, shake off the cover layer, add 100ul of tissue fixative to each well and incubate at room temperature for 30 minutes. After discarding the tissue fixative, in order to improve cell permeability, add 1% TRITON X-100 dissolved in PBS, 100ul to each well, and incubate at room temperature for 30 minutes. After discarding the permeabilization solution, add 10% BSA in PBS to block and incubate for 1 hour. After discarding the blocking solution, add 100ul of primary antibody, MPE8 (1:1000) to each well and incubate at 4℃ overnight. After washing the plate 3 times with 1*PBST, add 100ul of secondary antibody, HRP-labeled goat anti-human (1:4000) to each well and incubate at 37℃ for 45 minutes. After washing the plate 5 times with 1*PBST, add 50ul of TrueBlue colorimetric solution to each well and develop for 5 minutes until a good color is produced. Remove the stain and store at room temperature away from light. The blue spots represent infected and pathological cells, which are photographed and analyzed using a CTL enzyme-linked immunospot analyzer.

[0186] Results: See Figure 6 , where 6A is cell culture without virus treatment. 6B is virus infection without serum, 6C-6I is a neutralization experiment after culturing cells and adding serum from mouse immunization in Example 5 after the second immunization, diluted 2000 times. The serum added to 6B-6I is PBS mouse serum, antigen seq1, seq3-seq8 (trimeric protein). According to the lesion map, it can be observed that the trimer antigen has a neutralizing effect after being diluted 2000 times, and Seq1, Seq3, Seq7, and Seq8 have better neutralizing effects.

[0187] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0188] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0189] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein includes F protein and foldon protein of HMPV.

2. The fusion protein according to claim 1, characterized in that The F protein includes an F2 region and an F1 region, Preferably, the F2 region and the F1 region are connected via a cleavage region; More preferably, the cleavage region includes a shorter cleavage region relative to the wild type, further preferably, the first connecting peptide is inserted after the cleavage region, and most preferably, the cleavage region is completely deleted and replaced by the first connecting peptide; Preferably, the fusion protein further comprises a signal peptide; Preferably, the F2 region and the F1 region include mutations relative to the wild type; More preferably, the mutation comprises mutating the amino acid to cysteine ​​and / or proline. Further preferably, the mutation comprises mutating one or more pairs of amino acids in positions 84 and 249, 140 and 147, 365 and 455, and 454 and 458 to cysteine ​​to form a disulfide bond; Further preferably, the mutation comprises mutating the amino acid at position 131, 163 and / or 459 to proline; Preferably, the F protein and foldon protein of HMPV are connected by a second connecting peptide.

3. The fusion protein according to claim 1 or 2, characterized in that The fusion protein includes, from the N-terminus to the C-terminus, a signal peptide, an F2 region of the F protein of HMPV, a first connecting peptide, an F1 region of the F protein of HMPV, a second connecting peptide, and a foldon protein; Preferably, the fusion protein further comprises a restriction site and a tag protein after the C-terminus of the foldon protein; More preferably, the amino acid sequence of the F2 region of the HMPV F protein comprises: (A1), as shown in positions 20-98 of any one of SEQ ID No.1,3-8, (A2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (A1); (A3) a protein having more than 80% identity with any one of (A1)-(A2) and having the same function; The amino acid sequence of the F1 region of the HMPV F protein includes: (B1), 103-489 of any one of SEQ ID No. 1, 3-8; (B2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (B1); (B3) a protein having more than 80% identity with any of (B1)-(B2) and having the same function; The foldon protein comprises: (C1), as shown in SEQ ID No.20, (C2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (C1); (C3) a protein having more than 80% identity with any of (C1)-(C2) and having the same function; The signal peptide comprises: (D1), as shown in SEQ ID No.17, (D2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (D1); (D3) a protein having more than 80% identity with any of (D1)-(D2) and having the same function; and / or, The HRV-3C amino acid sequence comprises: (E1), SEQ ID No. 21; (E2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (E1); (E3), a polypeptide having more than 80% identity with any one of (E1)-(E2) and having the same function; Further preferably, the fusion protein comprises: (F1), any one of SEQ ID No. 1, 3-8; (F2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (F1); (F3) A polypeptide having 80% or more identity with any one of (F1) to (F2) and having the same function.

4. A nucleotide molecule, characterized in that The nucleotide molecule encodes the fusion protein according to any one of claims 1-3.

5. The nucleotide molecule according to claim 4, characterized in that The nucleotide molecule includes DNA and / or RNA. Preferably, the nucleotide molecule includes: (G1), SEQ ID No.9, 11-16, (G2), complementary, degenerate or transcribed sequence of (G1), (G3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (G1) or (G2) and encodes the corresponding protein in the fusion protein.

6. A carrier, characterized in that The vector comprises the nucleotide molecule according to any one of claims 4-5.

7. A host cell, characterized in that The host cell comprises the nucleotide molecule according to any one of claims 4-5 or the vector according to claim 6.

8. A method for preparing the fusion protein according to any one of claims 1 to 3, characterized in that: The preparation method comprises introducing the nucleotide molecule according to any one of claims 4-5 or the vector according to claim 6 into a host cell, and culturing the host cell.

9. A use of the fusion protein according to any one of claims 1 to 3, the nucleotide molecule according to any one of claims 4 to 5, the vector according to claim 6 or the host cell according to claim 7, the use comprising any one of the following: (1) Use in the preparation of products for preventing and / or treating diseases caused by HMPV infection; (2) Use in the preparation of products for inducing immune response to HMPV antigens; (3) Use in the prevention and / or treatment of diseases caused by HMPV infection; (4) Application in inducing immune response to HMPV antigens.

10. A drug, characterized in that The drug comprises the fusion protein of any one of claims 1-3, the nucleotide molecule of any one of claims 4-5, the vector of claim 6 or the host cell of claim 7. Preferably, the drug is a vaccine. More preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

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