Fusion protein complex comprising HMPV and HPIV-3 proteins, and preparation method and application thereof

Abstract

The invention relates to a fusion protein complex, the fusion protein complex comprises a fusion protein M and a fusion protein P. The fusion protein M comprises an F2 region and an F1 region of an F protein of HMPV and an H or L chain of ferritin; the fusion protein P comprises an F2 region and an F1 region of F protein of HPIV-3 and an L or H chain of ferritin; chains in ferritin in the fusion protein M and ferritin in the fusion protein P are different chains, so that a trimer can be formed and used for preparing the vaccine, the vaccine can stimulate an organism to generate neutralizing antibodies aiming at HMPV and HPIV and has broad spectrum, and all protein sequences also have a synergistic effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a fusion protein complex comprising HMPV and HPIV-3 proteins, a preparation method thereof, and an application thereof. Background Art

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

[0003] Human parainfluenza virus (HPIV) mainly causes acute respiratory infections in children under 3 years old. The clinical incidence rate is second only to respiratory syncytial virus infection, and more than half of the cases are caused by HPIV type 3 (HPIV-3 or HPIV3), posing a great threat to the life and health of newborns and infants. Currently, there are no effective therapeutic drugs and prophylactic vaccines for HPIV-3 infection. Most of the designs for human parainfluenza virus type 3 vaccines are mainly live attenuated vaccines. The first step of HPIV-3 infecting host cells is to bind the HN protein on the virus particle to the sialic acid receptor of the host cell membrane protein, and then the F protein mediates the fusion of the virus envelope and the host cell membrane, promoting the virus to enter the host cell. The F protein must be cleaved by host cell hydrolases into subunits F1 and F2 connected by two disulfide bonds before the F protein has fusion activity.

[0004] In view of the large overlap in the infected populations of HMPV and HPIV-3 and the large overlap in their infection windows, it is necessary to develop a combined vaccine against HMPV and HPIV-3. Summary of the Invention

[0005] In order to overcome the above defects, the present invention provides a fusion protein complex comprising HMPV and HPIV-3 proteins, a preparation method thereof, and an application thereof. Specifically,

[0006] In the first aspect of the present invention, a fusion protein complex is provided, and the fusion protein complex comprises a fusion protein M and a fusion protein P, wherein,

[0007] The fusion protein M comprises the F2 region and the F1 region of the F protein of HMPV and the H or L chain of ferritin;

[0008] The fusion protein P comprises the F2 region and the F1 region of the F protein of HPIV-3 and the L or H chain of ferritin;

[0009] The chains in ferritin of the fusion protein M and the fusion protein P are different chains. For example, when the fusion protein M comprises the H chain of ferritin, the fusion protein P comprises the L chain of ferritin, or when the fusion protein M comprises the L chain of ferritin, the fusion protein P comprises the H chain of ferritin.

[0010] Preferably, the F2 region and the F1 region in the fusion protein M and / or the fusion protein P are connected by a cleavage region.

[0011] Preferably, the fusion protein M and / or the fusion protein P further comprises a signal peptide.

[0012] Preferably, the F protein of the fusion protein M and / or the fusion protein P is directly or indirectly connected to the H or L chain of ferritin.

[0013] More preferably, the indirect connection comprises connection through a linker peptide.

[0014] Preferably, the ferritin is derived from any ferritin, such as ferritins of anaerobic and aerobic microorganisms, archaea, bacteria, higher animals and plants. More preferably, the ferritin is derived from insect-ferritin, Helicobacter pylori_Ferritin (HPF).

[0015] More preferably, the fusion protein M comprises the F2 region and the F1 region of the F protein of HMPV and the H chain of ferritin, and the fusion protein P comprises the F2 region and the F1 region of the F protein of HPIV-3 and the L chain of ferritin, or the fusion protein M comprises the F2 region and the F1 region of the F protein of HMPV and the L chain of ferritin, and the fusion protein P comprises the F2 region and the F1 region of the F protein of HPIV-3 and the H chain of ferritin.

[0016] More preferably, the fusion protein M sequentially comprises a signal peptide, the F2 region of the F protein of HMPV, a cleavage region, the F1 region of the F protein of HMPV, a linker peptide and the H chain of ferritin from the N-terminus to the C-terminus;

[0017] The fusion protein P sequentially comprises a signal peptide, the F2 region of the F protein of HPIV-3, a cleavage region, the F1 region of the F protein of HPIV-3, a linker peptide and the L chain of ferritin from the N-terminus to the C-terminus.

[0018] More preferably, the amino acid sequence of the F2 region of the F protein of the HMPV includes:

[0019] (A1) as shown in positions 19-98 of SEQ ID No.1;

[0020] (A2) a protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to any one of (A1);

[0021] (A3) a protein with more than 80% identity to any one of (A1)-(A2) and having the same function.

[0022] More preferably, the amino acid sequence of the F1 region of the F protein of the HMPV includes:

[0023] (B1) as shown in positions 103-489 of SEQ ID No.1;

[0024] (B2) a protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to any one of (B1);

[0025] (B3) a protein with more than 80% identity to any one of (B1)-(B2) and having the same function.

[0026] More preferably, the H chain of the ferritin includes:

[0027] (C1) as shown in positions 499-671 of SEQ ID No.1;

[0028] (C2) a protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to any one of (C1);

[0029] (C3) a protein with more than 80% identity to any one of (C1)-(C2) and having the same function.

[0030] More preferably, the amino acid sequence of the F2 region of the F protein of the HPIV-3 includes:

[0031] (D1) as shown in positions 26-112 of SEQ ID No.3;

[0032] (D2) a protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to any one of (D1);

[0033] (D3) a protein with more than 80% identity to any one of (D1)-(D2) and having the same function.

[0034] More preferably, the amino acid sequence of the F1 region of the F protein of HPIV-3 includes:

[0035] (E1) as shown in positions 149-491 of SEQ ID No. 3;

[0036] (E2) a polypeptide with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues of any of (E1);

[0037] (E3) a protein having more than 80% identity with any of (E1)-(E2) and having the same function.

[0038] More preferably, the amino acid sequence of the L chain of the ferritin includes:

[0039] (F1) as shown in positions 500-682 of SEQ ID No. 3;

[0040] (F2) a polypeptide with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues of any of (F1);

[0041] (F3) a protein having more than 80% identity with any of (F1)-(F2) and having the same function.

[0042] More preferably, the signal peptide includes:

[0043] (G1) as shown in positions 1-18 of SEQ ID No. 1, or as shown in positions 1-18 of SEQ ID No. 3;

[0044] (G2) a polypeptide with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues of any of (G1);

[0045] (G3) a polypeptide having more than 80% identity with any of (G1)-(G2) and having the same function.

[0046] More preferably, the cleavage sequence between the F2 region and the F1 region includes:

[0047] (H1) as shown in positions 99-102 of SEQ ID No. 1, or as shown in positions 113-148 of SEQ ID No. 3;

[0048] (H2) a polypeptide with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues of any of (H1);

[0049] (H3), a polypeptide having more than 80% identity with any one of (H1)-(H2) and having the same function.

[0050] More preferably, the linker peptide connecting the F protein and ferritin includes:

[0051] (I1), GGSGG (SEQ ID No.5), SGSGGGSG (SEQ ID No.6), GSGGGGSG (SEQ ID No.7);

[0052] (I2), a polypeptide having the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to any one of (I1);

[0053] (I3), a short peptide having more than 80% identity with any one of (I1)-(I2) and having the same function.

[0054] In a specific embodiment, the amino acid sequence of the fusion protein M or the fusion protein P includes:

[0055] (J1), SEQ ID No.1 or SEQ ID No.3;

[0056] (J2), a protein having the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to any one of (J1);

[0057] (J3), a protein having more than 80% identity with any one of (J1)-(J2) and having the same function.

[0058] In this article, 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 web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0059] In this article, the identity of more than 80% can 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.

[0060] Preferably, the fusion protein further comprises a tag protein.

[0061] The tag protein refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, facilitating the expression, detection, tracing, and / or purification of the target protein. The tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0062] More preferably, the fusion protein complex self-assembles into nanoparticles after being expressed in cells, more preferably into trimers.

[0063] In a second aspect of the present invention, a nucleotide molecule is provided, which encodes the fusion protein M or the fusion protein P as described above.

[0064] Preferably, the nucleotide molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0065] More preferably, the nucleotide molecule comprises:

[0066] (K1), SEQ ID No. 2 or 4,

[0067] (K2), the complementary, degenerate, or transcribed sequence of (K1),

[0068] (K3), a DNA molecule or mRNA that has more than 75% identity with the DNA molecule or mRNA defined by (K1) or (K2) and encodes the corresponding protein in the fusion protein.

[0069] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the above-mentioned fusion protein of the present invention using known methods, such as directed evolution and site-directed mutagenesis. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence encoding the above-mentioned fusion protein, as long as they encode the above-mentioned fusion protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0070] The identity refers to the sequence similarity with the compared nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher 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.

[0071] The above-mentioned identity of 75% or more can be an identity of 80%, 85%, 90% or 95% or more.

[0072] In the third aspect of the present invention, there is provided a vector, which comprises any one of the above-mentioned nucleotide molecules.

[0073] Preferably, the vector further comprises regulatory factors, such as a promoter that initiates the transcription of the polypeptide or fusion protein coding gene sequence, and may also include a terminator that terminates the transcription of the polypeptide or fusion protein coding gene sequence. Further, the vector may further include enhancer sequences.

[0074] The vector described herein refers to a vector that can transport exogenous DNA, mRNA or a target gene into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of the present invention, the vector is a pUC57 vector and / or a PKS001 (pKS001) vector.

[0075] In the fourth aspect of the present invention, there is provided a host cell, which comprises any one of the above-mentioned nucleotide molecules or vectors.

[0076] The host cells (also referred to as recipient cells) described herein can be plant cells or animal cells. The host cells can be understood to refer not only to specific recipient cells, but also to the progeny of such cells, and due to natural, accidental or intentional mutations and / or alterations, the progeny may not have to be identical to the original parental cells, but are still included within the scope of the host cells. Suitable host cells are known in the art, where: the plant cells can be plant cells such as Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc. but are not limited thereto; the animal cells can be mammalian cells (such as Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human renal epithelial cell lines or their derivatives, such as HEK293, 293T / 17, etc., human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (such as chicken or duck cells), amphibian cells (such as Xenopus laevis cells or 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 are not limited thereto. In one or more embodiments of the present invention, the host cells are CHO-K1 cells, 293T cells.

[0077] Preferably, the host cells can also be microorganisms. The microorganisms described herein can be bacteria, fungi, actinomycetes, protozoa, algae or viruses. Among them, the bacteria can be from the genus Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc. but are not limited thereto. For example, the bacteria can be Escherichia coli, Bacillus subtilis or Bacillus pumilus. In one or more embodiments of the present invention, the microorganism is TOP10 competent cells.

[0078] In the fifth aspect of the present invention, a method for preparing the above-mentioned fusion protein complex is provided. The preparation method includes introducing the above-mentioned nucleotide molecule or vector encoding the fusion protein M and the fusion protein P into a host cell and culturing the host cell.

[0079] Preferably, the nucleotide molecules encoding the fusion protein M and the fusion protein P are introduced into the same vector or different vectors.

[0080] Preferably, the preparation method includes screening monoclonal cell lines with high and stable expression levels.

[0081] More preferably, the screening method includes conventional screening methods in the art, such as adding screening reagents and comparing the bioactivities of clone pools by ELISA method, etc.

[0082] In the sixth aspect of the present invention, an application of any of the above-mentioned fusion protein complexes, nucleotide molecules, vectors or host cells is provided. The application includes any of the following:

[0083] (1) Application in the preparation of products for preventing and / or treating diseases caused by HMPV and HPIV virus infections;

[0084] (2) Application in the preparation of products for inducing immune responses against HMPV and HPIV virus antigens;

[0085] (3) Application in preventing and / or treating diseases caused by HMPV and HPIV virus infections;

[0086] (4) Application in inducing immune responses against HMPV and HPIV virus antigens.

[0087] The products described herein can be reagents, drugs, or vaccines.

[0088] (1) and (2) The products described can be vaccines or antibodies against HMPV and HPIV viruses. 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).

[0089] Furthermore, the HMPV and HPIV antibodies can be neutralizing antibodies that specifically bind to the F proteins of HMPV and HPIV. The neutralizing antibodies can be high-titer neutralizing antibodies against multiple epidemic strains of HMPV and HPIV.

[0090] Preferably, the antibodies include:

[0091] (L1), the protein shown in any of SEQ ID No.8-11;

[0092] (L2), a protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues of any of (L1);

[0093] (L3), a protein with more than 80% identity and the same function as any of (L1)-(L2).

[0094] In the seventh aspect of the present invention, an antibody as described above is provided.

[0095] Preferably, the antibody can be used for treating diseases caused by HMPV and HPIV virus infections or detecting antigens of HMPV and HPIV viruses.

[0096] In the above application or antibody, the diseases caused by HMPV and HPIV virus infections may include respiratory system infections, digestive system infections, cardiovascular system infections, and / or nervous system infections, etc.

[0097] Preferably, the respiratory system infection may include respiratory tract infection and / or lung infection.

[0098] Preferably, the digestive system infection may include intestinal diseases, anorexia, nausea, vomiting, abdominal pain, and / or diarrhea.

[0099] More preferably, the respiratory tract infection may include severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngolaryngitis, tracheitis, and / or bronchitis.

[0100] More preferably, the lung infection may include pneumonia and / or lung injury.

[0101] Preferably, the products for preventing and / or treating diseases caused by HMPV and HPIV virus infections include vaccines.

[0102] Preferably, the HPIV virus includes HPIV types 1-4, and more preferably, the HPIV virus includes type 3 HPIV virus (HPIV-3, or HPIV3).

[0103] In the eighth aspect of the present invention, a vaccine is provided, and the vaccine includes any of the above fusion protein complexes, nucleotide molecules, vectors, host cells, or antibodies.

[0104] Preferably, the vaccine further includes an adjuvant.

[0105] More preferably, the adjuvant can be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the antigen co-administered therewith. The adjuvants described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic component adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte colony-stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant can be a pharmaceutically acceptable adjuvant.

[0106] As is well known to those skilled in the art, in order to enhance the immunogenicity of the antigen protein, in addition to adding a compound with immunopotentiating effects as an adjuvant, the gene combination can also be adjusted to express a particulate structure; or it can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0107] Preferably, the vaccine further comprises a vaccine delivery system.

[0108] The vaccine delivery system can be a class of substances that can carry antigenic substances to the immune system of the body and store and exert their antigenic effects therein for a long time. The vaccine delivery systems described herein can be aluminum salt gel adjuvant vaccine delivery systems, emulsion adjuvant vaccine delivery systems, liposome adjuvant vaccine delivery systems or nano adjuvant vaccine delivery systems.

[0109] Furthermore, the vaccine further comprises one or more pharmaceutically acceptable carriers.

[0110] The pharmaceutically acceptable carriers can be diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants or lubricants but are not limited thereto.

[0111] The vaccine for preventing infection according to 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.

[0112] In a ninth aspect of the present invention, a method for generating an immune response is also provided, which may include administering to a subject any of the above vaccines.

[0113] In the above method, after administering the vaccine to the subject, an immune response against HMPV and HPIV viruses can be induced in the subject. The immune response can be a cellular immune response, or a humoral immune response, or a cellular immune response and a humoral immune response.

[0114] The cellular immune response may include B-cell immune response and T-cell immune response.

[0115] The subjects described herein can be human or non-human animals.

[0116] Furthermore, the non-human animal can be a non-human mammal.

[0117] The non-human mammal can be any one of, but not limited to, mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0118] 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).

[0119] 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, nasal spray or nebulized inhalation.

[0120] Generally speaking, compared with the prior art, the beneficial effects of the present invention are:

[0121] When the insect ferritin heavy chain and light chain assemble into spherical nanoparticles, the two antigens each form a trimer structure, displaying two different antigens on the same ferritin nanoparticle, and B cells recognize these two trimer antigens simultaneously, thus resulting in an immune response with improved neutralization breadth. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 : Construction strategy diagram of eukaryotic expression vector cloning of insect Ferritin protein.

[0123] Figure 2 : Protein purification results of insect iron heavy and light chain nanoparticle antigens.

[0124] Figure 3 : SDS-PAGE analysis results of insect iron heavy and light chain nanoparticle antigen proteins.

[0125] Figure 4 : Electron microscopy observation of the morphology of insect iron heavy and light chain nanoparticles. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0126] The present invention will be further described in detail below in conjunction with the specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0127] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the examples are listed below. Unless otherwise specified, others can be obtained from commercial channels.

[0128] Example 1 Preparation of the fusion protein complex

[0129] Based on the antigen molecule fusion expression and self-assembly into trimers of the nanoparticle structure of the light and heavy chains of insect ferritin (hereinafter simply referred to as insect iron light and heavy chains), specifically,

[0130] I. Sequence description

[0131] Amino acid sequence of HMPV-insectH antigen:

[0132] MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIACGVATAAAVTAGVAIAKCIRLESEVTAIKNCLKKTNECVSTLGCGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISKDLMTDAELARAISNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPCEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSCGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPQDQFNVALDQCFESIENSQALVDQSNRILSSAEKGNTGSGGGGSGRSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV(SEQ ID No.1)

[0133] Among them, positions 1-18: signal peptide region; positions 19-98: F2 region of the F protein of HMPV; positions 99-102: cleavage region; positions 103-489: F1 region of the F protein of HMPV; positions 490-498: flexible linker region; positions 499-671: insect Ferritin - heavy chain region.

[0134] Nucleotide sequence encoding HMPV-insectH antigen:

[0135]

[0136] Amino acid sequence of HPIV-3-insectL antigen:

[0137] MLRGPGPGLLLAVLCLGTAVRCTEAQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQAKSDIEKLKEAIRDTNKAVQSVCSSVGNCIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNCFGDNIGSLQEKGIKLQCIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKVKSDLEESKEWYRRSNQKLSAIGSGGGGSGEYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV(SEQ ID No.3)

[0138] Among them, positions 1-25: signal peptide region; positions 26-112: F2 region of the F protein of HPIV-3; positions 113-148: cleavage region; positions 149-491: F1 region of the F protein of HPIV-3; positions 492-499: flexible linker region; positions 500-682: insect Ferritin-light chain region.

[0139] Nucleotide sequence encoding HPIV-3-insectL antigen:

[0140]

[0141] II. Construction method

[0142] Construct a vector for highly efficient expression of the bivalent HMPV and HPIV-3 vaccines in cells

[0143] Materials: Vector plasmid pKS001, two codon-optimized nanoparticle plasmids respectively fused with HMPV-insect ferritin heavy chain and HPIV-3-insect ferritin light chain

[0144] Method: First, synthesize two antigen molecule plasmids pUC57-HMPV and pUC57-HPIV-3 containing the target design by gene synthesis. Use XbaⅠ and NotⅠ restriction endonucleases to double-digest the fragment containing HPIV-3-insect ferritin light chain and the pKS001 vector, and use T4 DNA ligase to ligate the sequence fragment into the expression vector for restriction digestion and sequencing verification. Then use HindⅢ and EcoRⅠ restriction endonucleases to digest the fragment containing HMPV, and also digest the pKS001-HPIV-3 vector already ligated with the HPIV-3 fragment with HindⅢ and EcoRⅠ restriction endonucleases. After purifying the above fragments by DNA, use T4 DNA ligase to ligate the HMPV sequence fragment into the vector to construct an expression vector, transform the Escherichia coli strain Top10, screen positive clones and extract plasmids for final sequencing (the construction strategy diagram can be seen in Figure 1 ).

[0145] The sequencing results show that a eukaryotic expression plasmid of nanoparticles containing both the fragment of HPIV-3-insect ferritin light chain and the fragment of HMPV-insect ferritin heavy chain is obtained

[0146] Example 2 Transformation, cell culture, protein purification and expression analysis of 293T cells

[0147] Materials: 293T cells, supercoiled plasmid, cell transfection reagent (Polymer Beauty, M5 HiPer SuspensionCell Special Transfection Reagent MF388)

[0148] Method: Construct a successful plasmid and transform 293T cells. Prepare the "Suspension Transfection Reagent-DNA Complex" according to the following method: Dilute 300 μg of plasmid DNA with serum-free medium to a final volume of 5 mL; Dilute 600 μL of "Suspension Transfection Reagent" with serum-free medium to a final volume of 5 mL; Gently mix and incubate at room temperature for 5 min; After incubating for 5 min, add the diluted plasmid DNA to the diluted "Suspension Transfection Reagent" and gently mix. Incubate at room temperature for 20 - 30 min to form the DNA-"Suspension Transfection Reagent" complex. After complete incubation, add 10 mL of the DNA-"Suspension Transfection Reagent" complex to 290 mL of the growth medium containing 293 suspension cells, so that the final cell density is approximately 1×10 6 cells / mL. Culture in a 37 °C, 5% CO2 shaker at a rotation speed of 150 rpm. After 6 days, perform protein purification and expression analysis.

[0149] The purification and expression analysis can be seen in the following steps:

[0150] I. Purification of Insect Ferritin Heavy and Light Chain Nanoparticle Antigen Molecules

[0151] Method: After centrifuging the culture medium of the above-mentioned 293T cells cultured for 6 days at 8000 r / min for 20 minutes, collect the supernatant. Filter it through a 0.45 μm filter membrane (Jinteng, product number: JTSF 025013 / 014) to obtain approximately 300 mL of supernatant cell fluid, and perform protein purification using an AKTA protein purifier.

[0152] The specific steps are as follows: The purification column filler is ECA filler. After connecting the purification column to the AKTA, debug the purification system, and balance the column with 1×PBS balance solution. At this time, the flow rate is set to 3 mL / min. After balancing, set the flow rate to 1 mL / min for loading. After all the samples pass through the purification column, use 1×PBS balance solution to wash at a flow rate of 3 mL / min until the absorption value is stable. Then elute the target protein with 0.2M D-lactose monohydrate, and set the flow rate to 1 mL / min. The samples collected at 285 - 286.5 in the figure are recorded as tube 1, and the samples collected at 286.5 - 288 are recorded as tube 2.

[0153] The results are shown in Figure 2 : In this application, the purified insect ferritin heavy and light chain antigen molecules are initially obtained, with a concentration of 0.4 mg / ml and a volume of 3 ml.

[0154] II. Molecular Sieve

[0155] Method: Concentrate the insect ferro heavy and light chain nanoparticle antigen molecular protein obtained in the previous step using a protein concentrator tube, with a sample loading volume of approximately 600 μL. Separate the protein using a Superose 6 prep grade (Cytiva, catalog number: 10321079) molecular sieve column. First, equilibrate the column with 1×PBS equilibration buffer, and set the flow rate to 1 mL / min at this time. After all the samples enter the separation column, adjust the flow rate to 0.5 mL / min, and collect the samples corresponding to the absorption peaks using centrifuge tubes. The finally obtained absorption peak sample is the I4M4 protein, and this sample is used for SDS-PAGE and animal experiment analysis after concentration.

[0156] Result: High-quality protein corresponding to the molecular sieve was obtained, with a concentration of 0.3 mg / ml and a volume of 1 ml.

[0157] III. SDS-PAGE

[0158] Method: Add 20 μL of 5× protein loading buffer to 80 μL of the fusion protein solution, centrifuge after treatment at 95 °C for 10 min. Take 30 μL of the supernatant for SDS-PAGE analysis, and observe the 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).

[0159] The results are shown in Figure 3 : Lane 1 is the sample collection before eluting the target protein with 0.2M D-lactose monohydrate, and lanes 2 and 3 are the samples of elution tube 1 and elution tube 2. The results show that a protein band with relatively high purity can be observed at 75 kDa on the SDS-PAGE gel, which is consistent with the predicted size of the insect ferro heavy and light chain nanoparticle antigen molecular protein, and the estimated protein purity is above 90%.

[0160] IV. Electron microscopy observation of nanoparticle morphology

[0161] Method: Negatively stain the insect ferro heavy and light chain nanoparticle antigen molecular protein prepared in the molecular sieve.

[0162] The specific negative staining operation is as follows: The ultra-thin carbon film is pre-evacuated for 3 min using a Harrick Basic Plasma Cleaner PDC-32G-2 instrument, then glow-discharged at the medium level for 30 s and taken out. A pipette is used to take a 4-μm sample and drop it onto the carbon film. After placing it horizontally for 1 min, it is blotted dry with filter paper. Then, 7-μm 2% uranyl acetate is dropped, left for 1 min, blotted dry with filter paper, and after leaving it for several minutes, the purified sample after negative staining is observed under a FEI Tecnai Arctica TEM D683 transmission electron microscope.

[0163] The results are shown in Figure 4 : Regular nanoparticles of the insect ferro heavy and light chain nanoparticle antigen molecule protein can be observed in the electron microscope. Through transmission electron microscope analysis, obvious nanoparticle morphology can be seen, and the integrity of the particles is relatively good.

[0164] Synthesis of neutralizing antibodies in Example 3

[0165] Method: The following amino acid sequence was synthesized by GenScript. The vector was double-digested with HindⅢ and EcoRⅠ restriction endonucleases, and the target sequence fragment of the light chain region was inserted using T4 DNA ligase. The PKS001 vector inserted with the light chain region was double-digested with XbaⅠ and NotⅠ restriction endonucleases, and then the target sequence fragment of the heavy chain region was inserted using T4 DNA ligase. An expression vector was constructed, transformed into the Escherichia coli strain Top10, positive clones were screened, plasmids were extracted, and 293T cells were transformed. After culturing for 6 days, the target antibody protein was purified.

[0166] Amino acid sequence of the light chain of the PIVNAbs antibody:

[0167] MLRGPGPGLLLAVLCLGTAVRCTEASSELTQDPAVSVALGQTVRITCQGDILRTYYVSWYQQKPGQAPLLVIYGKNNRPSVIPDRFSGSTSGDTASLTITGAQAEDEAEYYCSSRDRSGNHVLFGGGTKLTVLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID No.8)

[0168] Among them, positions 1 - 25: signal peptide region; positions 26 - 133: PIV neutralizing antibody region; positions 134 - 463: antibody light chain region.

[0169] Amino acid sequence of the heavy chain of the PIVNAbs antibody:

[0170] MLRGPGPGLLLAVLCLGTAVRCTEAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQSPGKGLEWVADISHSAGFLNYADSVKGRFTVSRDNSKSTLHLQMKSLRAEDTAVYYCAKRLAGLPDLEWLLYPNFLDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.9)

[0171] Among them, positions 1-25: signal peptide region; positions 26-153: PIV neutralizing antibody region; positions 154-483: heavy chain region of antibody.

[0172] Amino acid sequence of the light chain of MPVNAbs antibody:

[0173] MLRGPGPGLLLAVLCLGTAVRCTEAQSVLTQPPSVSGAPGQRVTISCTGTNSNIGTGYDVHWYQQLPGTAPKVVLFDNNNRPSGVPDRFSGSKSGTSAALAITGLQAEDEAVYYCQSYDKSLGGWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID No.10)

[0174] Among them, positions 1-25: signal peptide region; positions 26-136: MPV neutralizing antibody region; positions 137-242: light chain region of antibody.

[0175] Amino acid sequence of the heavy chain of MPVNAb

[0176] MLRGPGPGLLLAVLCLGTAVRCTEAEVQVVESGGGLVKPGGSLRLSCAASGFPFSSYKMDWVRQAPGKGLEWVSSISASGSYINYADSVKGRFTISRDNAKNSLYLQMKSLRADDTAVYFCARDGGRELSPFEKWGQGILVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.11)

[0177] Among them, positions 1-25: signal peptide region; positions 26-145: MPV neutralizing antibody heavy chain region; positions 146-475: antibody heavy chain region.

[0178] Result: Two neutralizing antibody proteins, purified PIVNAb and MPVNAb, were obtained and used for subsequent detection. The titer of PIVNAb was 1:100, and the titer of MPVNAb was 1:500.

[0179] Example 4 Antigen Activity Detection

[0180] The cell supernatant during protein purification, the purified protein, and the main peak after molecular sieve were subjected to ELISA analysis. Among them, the primary antibody used was the parainfluenza virus neutralizing antibody PINAbs prepared by the inventors, diluted 1:100, and the metapneumovirus neutralizing antibody MPNAbs, diluted 1:500. The secondary antibody used was Peroxidase-Conjugated Goat anti-human IgG(H+L)(Zhongshan Golden Bridge, ZB-2304), diluted 1:5000. The first row of cell supernatant was the 1-fold stock solution, the purified protein and the peak after molecular sieve were 200 ng / well, and the subsequent dilutions were 10-fold gradients downward.

[0181] Result: The purified protein had binding activity with two virus neutralizing antibodies, and the ELISA titers were all greater than 102400.

[0182] Example 5 obtained a cell line stably and highly expressing a bivalent antigen molecule

[0183] Materials: CHO-K1 cells, supercoiled plasmid, cell electroporator, medium CD04 (containing 25 μM MSX or 4 mM L-Glutamine), cell culture flasks and culture plates.

[0184] Method: Using the cell electroporator of Yida Biotech, set the electroporation parameters as: voltage 200 V, duration 2000 μs, 6 pulses, interval 1000 ms. Add 20 μg of plasmid to 200 μl of CHO-K1 cells, and electroporate the plasmid containing the genes of HMPV and HPIV3 insect iron nanoparticles into CHO-K1 cells.

[0185] Transfer the electroporated CHO-K1 cells into a T25 flask containing 10 ml of CHO CD04 (4 mM L-Glutamine) medium, and culture in an incubator at 37 °C and 5% CO2 for 24 hours. Inoculate into a 96-well plate at a density of 10000 cells / well and 200 μl / well, and statically culture at 37 °C and 5% CO2. Static culture in the 96-well plate for about 14 days, observe the cell well plate under an optical microscope, and mark the wells with good state and monoclonal. When the clone grows to 50 - 70% confluence in the 96-well plate, select single cell clones with good cell state and inoculate them into a 24-well plate. When the cells grow to 95% confluence, batchwise aspirate the culture supernatant as a detection sample, and determine several highly expressing monoclonal cell lines as candidate cell lines.

[0186] According to the fed-batch culture results, 26A was determined as the subsequent spare cell line.

[0187] Example 6 obtained the evaluation of the effect of the mouse immunogenic antigen molecule

[0188] Method: Culture the stably transfected cell line cells in Example 5 to obtain 300 ml of CHO cell culture medium, and purify the antigen molecule protein using the same purification method as in Example 1. Then select 20 6 - 8-week-old female C57BL / 6 mice, randomly divide them into 3 groups, with an interval of 3 weeks for the first, second, and third immunizations, and collect serum for ELISA titer detection two weeks after the third immunization. The immunization protocol is shown in Table 1, and the ELISA immune effect is shown in Table 2.

[0189] Table 1. Immunization protocol of antigen molecule

[0190]

[0191] Table 2. Immune effects of antigen molecules

[0192]

[0193] Results and conclusions:

[0194] The candidate antigens have immune effects against both viruses, and the immune effect of the double adjuvant (0.2 mg AL(OH)3 + CPG) is better than that of the single adjuvant.

[0195] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0196] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0197] In addition, any combination can be made between various different embodiments of the present invention, as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A fusion protein complex, characterized in that: The fusion protein complex includes fusion protein M and fusion protein P, wherein: The fusion protein M includes the F2 region and the F1 region of the F protein of HMPV and the H or L chain of ferritin; The fusion protein P includes the F2 region and the F1 region of the F protein of HPIV-3 and the L or H chain of ferritin; The chains in the ferritin in the fusion protein M and the fusion protein P are different chains.

2. The fusion protein complex according to claim 1, characterized in that The F2 region and the F1 region in the fusion protein M and / or the fusion protein P are connected via a cleavage region, Preferably, the fusion protein M and / or fusion protein P further comprises a signal peptide, More preferably, the F protein of the fusion protein M and / or fusion protein P is directly or indirectly connected to the H or L chain of ferritin.

3. The fusion protein complex according to claim 1 or 2, characterized in that The amino acid sequence of the F2 region of the HMPV F protein includes: (A1), shown at positions 19-98 of SEQ ID No.1, (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), shown at positions 103-489 of SEQ ID No. 1, (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 H chain of the ferritin comprises: (C1), shown at positions 499-671 of SEQ ID No.1, (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 amino acid sequence of the F2 region of the F protein of the HPIV-3 includes: (D1), shown at positions 26-112 of SEQ ID No.3, (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; The amino acid sequence of the F1 region of the HPIV-3 F protein includes: (E1), shown at positions 149-491 of SEQ ID No.3, (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 protein having more than 80% identity with any of (E1)-(E2) and having the same function; The amino acid sequence of the L chain of ferritin includes: (F1), shown at positions 500-682 of SEQ ID No.3, (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 protein having more than 80% identity with any of (F1)-(F2) and having the same function; The signal peptide includes: (G1), as shown in 1-18 of SEQ ID No.1, or, as shown in 1-18 of SEQ ID No.3; (G2) 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 (G1); (G3) a polypeptide having more than 80% identity with any one of (G1)-(G2) and having the same function; The cleavage sequence between the F2 region and the F1 region includes: (H1), as shown in positions 99-102 of SEQ ID No.1, or, as shown in positions 113-148 of SEQ ID No.3; (H2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (H1); (H3) A polypeptide having 80% or more identity with any one of (H1) to (H2) and having the same function. and / or, The connecting peptide of the F protein and ferritin comprises: (I1), GGSGG (SEQ ID No. 5), SGSGGGSG (SEQ ID No. 6), GSGGGGSG (SEQ ID No. 7); (I2) 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 (I1); (I3) A short peptide having 80% or more identity with any one of (I1) to (I2) and having the same function.

4. A nucleotide molecule, characterized in that The nucleotide molecule encodes the fusion protein complex described in 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: (K1), SEQ ID No: 2 or 4, (K2), the complementary, degenerate or transcribed sequence of (K1), (K3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (K1) or (K2) 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 complex 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 complex 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, wherein the use comprises any one of the following: (1) Use in the preparation of products for preventing and / or treating diseases caused by HMPV and HPIV virus infections; (2) Use in the preparation of products for inducing immune responses to HMPV and HPIV virus antigens; (3) Use in the prevention and / or treatment of diseases caused by HMPV and HPIV virus infections; (4) Application in inducing immune response to HMPV and HPIV viral antigens.

10. An antibody, characterized in that The antibodies include: (L1), a protein shown in any one of SEQ ID No.811; (L2) 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 (L1); (L3) A protein having 80% or more identity with any one of (L1) to (L2) and having the same function.

11. A vaccine, characterized in that The vaccine comprises the fusion protein complex described in any one of claims 1-3, the nucleotide molecule described in any one of claims 4-5, the vector described in claim 6, the host cell described in claim 7 or the antibody described in claim 10. Preferably, the vaccine further comprises an adjuvant.

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