Rabies virus G protein mutant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a rabies virus G protein mutant as well as a preparation method and application thereof. The rabies virus G protein mutant is obtained by carrying out point mutation on an extracellular domain of G protein, the mutant realizes soluble expression of the extracellular domain of the G protein, can induce generation of a high-titer neutralizing antibody, and has more excellent cellular immunity and antibody immune response compared with commercial human rabies vaccines and canine rabies vaccines; the inoculation frequency is expected to be reduced, the immune procedure is shortened, and the immune dosage is reduced.

Description

Technical field

[0001] The present invention relates to the field of biomedical technology, and particularly to rabies virus G protein mutants, preparation methods and applications thereof. Background technology

[0002] Rabies is a zoonotic disease, an acute infectious disease mainly caused by the rabies virus (RABV), and the mortality rate after the onset is almost 100%. Rabies virus belongs to the RNA virus of the genus Rabies virus in the family Rabies virus. The virus particles are bullet-shaped, 100-300nm long and about 75nm in diameter. Their genome encodes five structural proteins, namely RNA polymerase (L protein), nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), and surface glycoprotein (G protein). The outer lipid membrane of rabies virus particles is embedded with G protein, and the trimer formed by G protein constitutes a spike. The spike is the site where the virus binds to the host receptor and contains the main recognition epitope of neutralizing antibodies. At the same time, G protein can be glycosylated. During its expression and secretion, glycosylation modification plays a crucial role in its stability, antigenicity and biological activity. Therefore, G protein is an important target protein for the development of rabies vaccines, antiviral drugs and antibody diagnostic reagents.

[0003] The gene coding region of G protein is 1575 nucleotides in length and encodes 524 amino acids, of which amino acids 1-19 are signal peptides. After the signal peptide is removed, mature proteins that form 505 amino acids include three domains: extracellular domain, transmembrane domain and intracellular domain. Among them, the extracellular domain is the G protein segment exposed to the surface of the virion, the functional segment that performs receptor recognition and membrane fusion, and is also a truly effective antigen segment and target for neutralizing antibodies in vaccine preparation. Studies have shown that the eukaryotic expression of rabies virus G protein is mostly full-length protein. Due to the high hydrophobicity of the transmembrane region, the obtained G protein is very easy to aggregate in solution to form highly uneven protein polymers, so it is difficult to effectively purify. There are literature reports that the G protein ectodomain that expresses only the wild-type CVS-11 strain will form irregular aggregates, and the rabies virus G protein extracellular segment (RABV-G-ecto) is recombinantly and the amino acids 73 to 79 in the first fusion loop of the protein extracellular segment are replaced by the connecting peptide composed of 5 amino acids (GGSGG), and a soluble and uniform recombinant RABV-G-ecto protein in solution is obtained (see "Structural Analysis of Rabies Virus Glycoprotein Reveals pH-DependentConformational Changes and Interactions with a Neutralizing Antibody”, Yang et al., Cell Host & Microbe 27, 441–453, 2020.3.11; CN109627294 B). Another literature reports that purified RABV G protein can produce high titer anti-G protein antibodies, but it cannot induce neutralizing antibodies in mice, indicating that the transmembrane region of rabies virus G protein is crucial for the conformation and biological activity of the extracellular structural region (see "Construction and Identification of RABV G ectodomain Secretory Stable Expression Cell Line", Li Qingqing, Southern Medical University, CNKI, 2018.3.1). It can be seen that it is difficult to obtain a soluble G protein extracellular domain with correct spatial folding and induce the production of neutralizing antibodies. There are still many technical difficulties to overcome using the G protein extracellular domain for the preparation of rabies vaccines.

[0004] At the same time, studies have shown that the conformation of G protein changes during binding to receptors and undergoing membrane fusion, and can switch between the two forms before and after fusion with host cells. Under acidic conditions, G protein is in a post-fusion state and under neutral and alkaline conditions. It is generally believed that the pre-fusion state of G protein can more effectively induce the production of neutralizing antibodies. In addition, G protein may also change from a trimeric structure to a monomeric structure. Therefore, this characteristic of G protein is a difficult and key point in the development and design of protein vaccines and the later process.

[0005] At present, rabies vaccines are mainly purified vaccines cultured by primary gopher kidney cells, chicken embryonic cells, human diploid cells and Vero cells. Among them, the incidence of adverse reactions after human diploid cell vaccination is low, the symptoms are mild, and the immune effect is good, but the cell proliferation is slow, the virus yield is low, and the vaccine cost is high, so it cannot be widely used. In addition to traditional rabies vaccines, new vaccines are also being developed rapidly, such as recombinant protein-based vaccines or mRNA vaccines. In addition, since the rabies vaccines currently used for human use are based on 4- or 5-dose immunization procedures, the time is longer, and the obedience rate of vaccinated people is low, which also causes a heavier economic burden. In April 2022, India launched the world's first new rabies vaccine, and it is also the only recombinant protein vaccine. Its immune program is three injections after exposure, which further shortens the immune program and reduces the immune dose.

[0006] There is currently no rabies recombinant protein vaccine in China, so it is of great significance to develop a rabies vaccine that is more effective, safer and lasting immunity. Contents of invention

[0007] In response to the above technical problems, the present invention transforms the ectodomain of the rabies virus G protein as a target protein to obtain a G protein antigen with stable conformation and strong immunogenicity.

[0008] A. Viral membrane fusion protein

[0009] The rabies virus G protein is a viral membrane fusion protein. A key step in envelope virus infection is the fusion between the viral membrane and the host cell membrane, which is mediated by the viral membrane fusion protein. Among them, viral membrane fusion proteins are classified into Class I (such as membrane fusion proteins of human immunodeficiency virus and respiratory syncytial virus), Class II (such as membrane fusion proteins of dengue viruses) and Class III (such as membrane fusion proteins of rhodarvirus and herpes viruses). Viral membrane fusion proteins are conformational metastable proteins that change from a higher energy pre-fusion conformation to a lower energy post-fusion conformation through an unstable intermediate state after being triggered by certain factors in contact with the host cell. The intermediate state exposes a hydrophobic fusion peptide or fusion loop inserted into the host cell membrane, and the transition to conformation after low-energy fusion pulls the two membranes together, promoting the fusion of the viral envelope and the host cell membrane. Since epitopes produced by stimulating neutralizing antibodies are usually contained in pre-fusion conformations and in some cases are not present in post-fusion forms, immunity of membrane fusion proteins in pre-fusion conformations is clinically desirable because of the potential to induce more efficient neutralizing antibodies than using post-fusion conformations proteins.

[0010] The rabies virus G protein belongs to the III virus membrane fusion protein. Specifically, there are two structural states in the structure of rabies virus G protein (RABV-G for short), the pre-fusion conformation of the bending hairpin structure on the surface of the virus and the post-fusion conformation of mediating membrane fusion extended conformation. Since RABV-G of the natural structure is a pre-fusion conformation under alkaline conditions and a post-fusion conformation under acid conditions, its structure is instable. At the same time, the conformation before fusion will gradually change with the storage time, and will gradually change to the conformation after fusion, reducing its antigenic site, resulting in a decrease in immunogenicity. It can be seen that as the main antigen of rabies virus, its conformation can affect its immunogenicity. Therefore, stabilizing the conformation before G protein fusion can effectively improve its immunogenicity and enhance its immune protection effect.

[0011] B. Wild-type RABV-G

[0012] The present invention provides a wild-type RABV-G, whose full-length amino acid sequence is derived from the G protein of Pasteur Virus (ie, PV strain) (Genbank accession number AAA47218.1).

[0013] The present invention provides a wild-type RABV-G ectodomain whose amino acid sequence is derived from amino acids 20-459 of the G protein of the PV strain (Genbank accession number AAA47218.1).

[0014] In some embodiments, the wild-type RABV-G of the present invention is derived from a strain other than a PV strain.

[0015] Optionally, the wild-type RABV-G of the present invention is derived from the G protein of the following strains: Pitman Moore (GenBank accession number CAI43218.1), HEP-Flury (GenBank accession number BAC53868.1), MOR1-DG (GenBank accession number AAK92057.1), MOR3-HM (GenBank accession number AAK92058.1), NeiMeng1025C (GenBank accession number ABY19509.2), FRA1-FX (GenBank accession number AAK92050.1), CNX8601 (GenBank accession number AAG34722 .1), CHI1-BK (GenBank accession number AAK92060.1), MAU1-CL (GenBank accession number AAK92072.1), Yunnan_Zt07 (GenBank accession number ABX79939.1), RVD (GenBank accession number AAO72525.1), nukovo-32 (GenBank accession number OP642459), CVS-11 (GenBank accession number ADJ29911.1) or CTN (GenBank accession number AY009100) strains.

[0016] In some embodiments, the invention provides a wild-type RABV-G ectodomain whose amino acid sequence has a sequence corresponding to or aligned with amino acids at positions 20-459 of the G protein described above for the PV strain.

[0017] In some embodiments, the amino acid sequence of the wild-type RABV-G ectodomain of the present invention comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0018] Preferably, the amino acid sequence of the wild-type RABV-G ectodomain of the present invention is shown in SEQ ID NO: 1.

[0019] Among them, "corresponding" or "alignment" has a meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding" or "alignment" means that after the reference sequence is aligned with a given sequence by homology or sequence identity, the same residues can be obtained, and the position of the same residue in the given sequence is the position corresponding or aligned with the reference sequence. For example, those skilled in the art can easily compare the G protein of other strains with the G protein of PV strains through common procedures such as Align, so as to obtain the position where the amino acids 20-459 of the G protein of the PV strain are located in the G protein of other strains.

[0020] In some embodiments, the invention provides a wild-type RABV-G ectodomain with an amino acid sequence of at least 90% identity to SEQID NO: 1.

[0021] Preferably, the present invention provides a wild-type RABV-G ectodomain with an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0022] C. RABV-G mutant

[0023] The present invention provides a RABV-G mutant, which comprises a RABV-G ectodomain mutant.

[0024] In some embodiments, the RABV-G mutants provided by the present invention have improved stability in pre-fusion conformation.

[0025] Preferably, the RABV-G mutant of the present invention is specifically a RABV-G ectodomain mutant, which has improved stability in the prefusion conformation.

[0026] As a non-limiting example, the stability of the pre-fusion conformation of the RABV-G mutant can be determined by incubating the RABV-G mutant with a specific antibody in the pre-fusion conformation at neutral and acidic pH and determining antibody binding by ELISA or FACS.

[0027] The present invention provides a RABV-G ectodomain mutant, which has a mutation or substitution of one, two or more amino acids at positions 261, 266, 270, 271, 384 and 424 of the wild-type RABV-G ectodomain, or a mutation or substitution of one, two or more amino acids at sites corresponding to or aligned with the above amino acid sites. The mutation or substitution can improve the stability of the conformation before the G protein fusion.

[0028] In some embodiments, the present invention provides a RABV-G ectodomain mutant that increases the stability of the conformation of the G protein before fusion of the G protein.

[0029] Preferably, the mutation or substitution of the two amino acids mentioned above is selected from the following combinations: H261+H270; D266+H270.

[0030] In some embodiments, the present invention provides a RABV-G ectodomain mutant that presents one, two or more amino acid mutations or substitutions at positions 261, 266, 270, 271, 384 and 424 of SEQ ID NO: 1, which improves the stability of the conformation of the G protein before fusion.

[0031] In some embodiments, the present invention provides a RABV-G ectodomain mutant described in RABV-G ectodomain mutant present in positions 261, 266, 270, 271, 384 and 424 of SEQ ID NO: 1, which improves the stability of the conformation of the G protein before fusion.

[0032] Preferably, the two mutations or substitutions of SEQ ID NO: 1 above are selected from the following combinations: H261+H270; D266+H270.

[0033] Further, the residues to be mutated or substituted at the above-mentioned residues are mutated to proline, or are substituted with proline.

[0034] In some embodiments, the residues to be mutated or substituted at the above-mentioned residues are mutated to leucine, or are substituted with leucine.

[0035] The present invention provides a RABV-G ectodomain mutant, wherein the RABV-G ectodomain mutant has a mutation or substitution of one, two or more amino acids selected from H261L, D266P, H270P, L271P, H384P and H424L in SEQ ID NO: 1, which can improve the stability of the conformation of the G protein before fusion.

[0036] In some embodiments, the RABV-G ectodomain mutant presents a mutation or substitution of two amino acids selected from H261L, D266P, H270P, L271P, H384P and H424L in SEQ ID NO: 1, which improves the stability of the conformation before the G protein fusion.

[0037] Preferably, the two mutations or substitutions in the above SEQ ID NO: 1 are selected from the following combinations: H261L+H270P; D266P+H270P.

[0038] The present invention provides a RABV-G ectodomain mutant, which has an amino acid sequence as shown in any one of SEQ ID NO: 2-9, as shown in Table 1 for details.

[0039] In some embodiments, the amino acid sequence of the RABV-G ectodomain mutant of the present invention is shown in any of SEQ ID NO: 2-9.

[0040] Preferably, the amino acid sequence of the RABV-G ectodomain mutant of the present invention is represented by any one of SEQ ID NOs: 2, 4, 5, 6, 7, 8, and 9.

[0041] Preferably, the amino acid sequence of the RABV-G ectodomain mutant of the present invention is represented by any of SEQ ID NOs: 4, 5, 8, and 9.

[0042] The RABV-G mutant described in the present invention also includes glycosylation modifications, which can be modified by N-glycosylation at the Asn37, Asn247 or Asn319 sites in the ectodomain.

[0043] In some embodiments, the RABV-G mutant of the present invention is modified by N-glycosylation at the Asn319 site in the ectodomain, or further by N-glycosylation at the Asn37 or Asn247 site.

[0044] In some embodiments, the RABV-G mutant of the invention is capable of inducing the production of neutralizing antibodies.

[0045] The neutralizing antibody described in the present invention refers to an antibody that can eliminate the ability of viral infection after binding to a virus. After the enveloped virus surface antigen is bound to the neutralizing antibody, complement can be activated, resulting in the dissolution of the virus.

[0046] Preferably, the RABV-G mutant described in the present invention can induce the production of IgG.

[0047] Preferably, the RABV-G mutant described in the present invention can induce the production of neutralizing antibody IgG.

[0048] Preferably, the titer of neutralizing antibodies induced by the RABV-G mutant of the present invention is higher than that of commercially available rabies vaccines.

[0049] In some embodiments, the RABV-G mutant of the present invention can achieve soluble expression and have correct spatial folding.

[0050] In some embodiments, a signal peptide is introduced at one end of the RABV-G mutant of the invention for extracellular expression of the protein.

[0051] Preferably, a signal peptide is introduced at the N-terminal of the RABV-G mutant of the present invention, and the amino acid sequence of the signal peptide is shown in MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 18).

[0052] In some embodiments, the 6×His tag is introduced at one end of the RABV-G mutant of the present invention for purification of recombinantly expressed proteins.

[0053] Preferably, a 6×His tag is introduced at the C-terminus of the RABV-G mutant described above.

[0054] Optionally, the RABV-G mutant of the present invention is linked to the 6×His tag through a linker.

[0055] Optionally, the linker mentioned above is selected from (GGGGS) n , (GSGGSG) n , (GGS) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 5. In some embodiments, the linker is preferably GSGGSG.

[0056] Table 1: Amino acid sequences of RABV-G ectodomain wild type and mutants of the present invention

[0057] Molecular name Mutation site Amino acid sequence RABV-G1 Wild type KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGA CKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 1) RABV-G6 H270P <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKE SLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIE P LVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 2) <!-- 6 -->]]> RABV-G25 H261L <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTV KTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNL L DFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 3) <!-- 7 -->]]> RABV-G26 D266P <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTT KESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRS P EIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 4) <!-- 8 -->]]> RABV-G28 H384P <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGM SCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQ P MELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 5) <!-- 9 -->]]> RABV-G30 H424L <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETC GFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDV L ERISGVDLGLPNWGKY (SEQ ID NO: 6) <!-- 10 -->]]> RABV-G32 H270P+H261L <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTV KTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNL L DFRSDEIE P LVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 7) <!-- 11 -->]]> RABV-G33 H270P+D266P <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTT KESLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRS P EIE P LVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 8) <!-- 12 -->]]> RABV-G36 L271P <![CDATA[KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKE SLVIISPSVADLDPYDRSLHSRVFPGGNCSGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEH P VVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLADPSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKY (SEQ ID NO: 9)]]>

[0058] D. RABV-G trimers and polymers

[0059] The present invention provides a RABV-G trimer, the trimer consisting of the RABV-G mutant described in the present invention.

[0060] In some embodiments, the RABV-G trimer of the present invention consists of the RABV-G ectodomain mutant of the present invention.

[0061] Preferably, the RABV-G trimer of the present invention is formed by self-assembly of the RABV-G mutant described in the present invention.

[0062] The present invention provides the preparation method of the RABV-G trimer as follows: the RABV-G mutant is recombinantly expressed using a prokaryotic expression system or eukaryotic expression system, and the recombinant RABV-G mutant self-assembles to obtain the RABV-G trimer.

[0063] In some embodiments, the present invention provides a method for preparing the RABV-G trimer as follows: using a prokaryotic expression system or eukaryotic expression system to recombinantly express the RABV-G ectodomain mutant, and the recombinant RABV-G ectodomain mutant self-assembles to obtain the RABV-G trimer.

[0064] The present invention provides a RABV-G multimer, which is composed of the RABV-G trimer of the present invention.

[0065] In some embodiments, the RABV-G multimer of the present invention includes RABV-G hexamers, novolumes, dodecanoes, and the like.

[0066] The present invention provides a preparation method of RABV-G trimer or multimer, the method comprising the following steps:

[0067] (1) Recombinantly expressing the RABV-G mutant described in the present invention in CHO cells through conventional techniques of molecular cloning;

[0068] (2) After expression in the expression culture medium for a certain period of time, the cell supernatant was harvested, and the supernatant was centrifuged and filtered for membrane;

[0069] (3) The filtered supernatant was purified through a nickel column;

[0070] (4) Collect the sample containing the protein of interest, and perform the first molecular sieve purification after ultrafiltration concentration; optionally, the product purified by the first molecular sieve is purified through an anion exchange column, and then undergoes a second molecular sieve purification;

[0071] (5) Use buffer to dilute the purified protein of interest to obtain soluble trimers or multimers with uniform particle size.

[0072] In some embodiments, the buffer used for the first molecular sieve purification in step (4) above is TBS, and the buffer used for the second molecular sieve purification contains a certain concentration of Tween.

[0073] In some embodiments, the buffer in step (5) above is TBS or a buffer containing Tween.

[0074] Further, the content of Tween in steps (4) to (5) is 0.02%-1%.

[0075] Preferably, the content of Tween in steps (4) to (5) is 0.02%, 0.05% or 0.1%, and Tween is selected from Tween-20 or Tween-80, preferably Tween-80.

[0076] E. Immunogenic complex

[0077] The present invention provides an immunogenic complex comprising a protein formed by a covalent binding reaction of the RABV-G antigen component and the granular protein component.

[0078] The RABV-G antigen component of the present invention is selected from the RABV-G mutant, RABV-G trimer or RABV-G multimer described in the present invention.

[0079] In some embodiments, the immunogenic complex of the present invention is a virus-like particle, a structure assembled from a viral protein without any nucleic acid.

[0080] The present invention provides an immunogenic complex comprising:

[0081] (1) Antigen component, which comprises the RABV-G antigen component described in the present invention;

[0082] (2) Particle protein component, which contains nanoparticle protein.

[0083] The present invention provides an immunogenic complex comprising:

[0084] (1) Antigen component, which comprises the RABV-G antigen component and binding peptide 1 described in the present invention;

[0085] (2) Particle protein component, which contains nanoparticle protein and binding peptide 2.

[0086] The present invention provides an immunogenic complex comprising:

[0087] (1) Antigenic component, which comprises the RABV-G antigen component, linking peptide 1 and binding peptide 1 as described in the present invention;

[0088] (2) Particle protein component, which includes nanoparticle protein, linking peptide 2 and binding peptide 2.

[0089] In some embodiments, in any of the immunogenic complexes provided by the invention, the antigen component is formed by fusing the RABV-G antigen component at the C-terminus through the ligation peptide 1 with the binding peptide 1 . Among them, the RABV-G mutant forms a fusion protein with the binding peptide 1 and assembles into a RABV-G trimer or multimer. Alternatively, the RABV-G mutant is assembled into a RABV-G trimer or multimer at the C-terminus by fusing the linking peptide 1 with the binding peptide 1.

[0090] In some embodiments, in any of the immunogenic complexes provided by the invention, the particulate protein component is formed by the N-terminus of the nanoparticle protein by fusing the linking peptide 2 with the binding peptide 2.

[0091] In some embodiments, the immunogenic complex is formed between the antigen component and the particulate protein component by covalent binding peptide 1 and binding peptide 2.

[0092] In some embodiments, in any of the immunogenic complexes provided by the invention, the binding peptide 1 contains an amino acid sequence as indicated by AHIVMVDAYKPTK (SEQ ID NO: 19), referred to as "4T".

[0093] In some embodiments, in any of the immunogenic complexes provided by the invention, the binding peptide 2 comprises, for example, DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKD

[0094] The amino acid sequence shown in FYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 20) is referred to as "4C".

[0095] In some embodiments, both the antigen component and the particulate protein component include a histidine tag.

[0096] In some embodiments, in any of the immunogenic complexes provided by the invention, the particulate protein may be selected from nanoparticle proteins, further from virus-like particulate proteins; both the antigen component and the particulate protein component may self-assemble to form a particle structure by binding.

[0097] In some embodiments, self-assembleable nanoparticles used in the present invention include: NPM particles, ferritin particles, virus-like particles formed by viral structural proteins, I53-50 particles, and the like. Among them, the viral structural protein includes the bacterial phage capsid protein AP205, etc.

[0098] In some embodiments, in any of the immunogenic complexes provided by the invention, the linking peptide 1 comprises (GGGGS) n , (EAAAK) n , (GSGGSG) n , (GGS) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 5. In some embodiments, in any of the immunogenic complexes provided by the invention, the linking peptide 1 is preferably GSGGSG.

[0099] In some embodiments, in any of the immunogenic complexes provided by the invention, the linking peptide 2 comprises (GGS) n , (GGGGS) n , (EAAAK) n , (GSGGSG) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 10. In some embodiments, in any of the immunogenic complexes provided by the invention, the linking peptide 2 is preferably GGSGGSGGSGGS or GGSGGSGGSGGSGGSGGSGGSGGS.

[0100] In some embodiments, in any of the above-mentioned immunogenic complexes provided by the invention, the particle protein component is to form a fusion protein by connecting peptide 2 to the binding peptide 2 at the N-terminus of the nanoparticle protein; preferably, the nanoparticle protein is NPM, AP205 capsid protein3 (AP205) or Ferritin protein. Specifically, in some alternatives, binding peptide 2 (ie "4C") is linked to the coding gene of the nanoparticle protein by connecting peptide 2, inserted into a prokaryotic expression vector (such as pET-28a(+), pET-30a(+)), and expressed in E.coli cells to obtain a fusion protein that binds peptide 2 to the nanoparticle protein. The fusion protein can be purified by chromatography, such as purified by anion exchange chromatography and hydrophobic chromatography to obtain the product. The nanoparticle protein is preferably NPM, AP205 or Ferritin.

[0101] Preferably, in the immunogenic complex provided by the present invention, the amino acid sequence of the antigen component is shown in any one of SEQ ID NO: 2-9, and the amino acid sequence of the granular protein component is shown in SEQ ID NO: 22.

[0102] Specifically, in some alternatives, under suitable reaction conditions, such as at room temperature, any of the above-mentioned antigen components is covalently bound to the granular protein components, and the binding peptide 1 of the antigen component is coupled to the binding peptide 2 of the granular protein components by covalent bonding, thereby forming the immunogenic complex.

[0103] In some embodiments, the present invention provides a method for covalent binding of antigen components and granular protein components, mixing the antigen components with granular protein components, a protein concentration ratio determined by the BCA method, adding 50% sucrose mother liquor to about 25% sucrose final concentration, and adding 1M Tris-HCl (pH 7.4) mother liquor at a total reaction volume of 10% to play a role in stabilizing pH. The binding reaction was carried out at 22°C for 24 hours. The product after purifying the molecular sieve molecule.

[0104] In some embodiments, the invention provides an immunogenic complex comprising:

[0105] (1) Antigenic component, which comprises the RABV-G antigen component, linking peptide 1 and binding peptide 1 as described in the present invention;

[0106] (2) Particle protein component, which includes nanoparticle protein, linking peptide 2 and binding peptide 2;

[0107] The linking peptide 1 is any commonly used linking peptide (eg, flexible linking peptide, rigid linking peptide) including but not limited to (GGS) n , (GSGGSG) n , (GGGGS) n or (EAAAK) n amino acid sequence. The linking peptide 1 is preferably GSGGSG.

[0108] The linking peptide 2 is any commonly used linking peptide (eg, flexible linking peptide, rigid linking peptide) including but not limited to (GGS) n , (GGGGS) n , (EAAAK) n , (GSGGSG) n The amino acid sequence of n can be an integer greater than 0 and less than or equal to 10, preferably GGSGGSGGSGGS or GGSGGSGGSGGSGGSGGSGGS; the nanoparticle protein is NPM, AP205 or ferritin.

[0109] In some embodiments, in any of the immunogenic complexes provided by the invention, the nanoparticle protein NPM comprises an amino acid sequence as shown in SEQ ID NO:21.

[0110] Preferably, in any of the immunogenic complexes provided by the present invention, the particulate protein component comprises NPM-4C, as shown in SEQ ID NO:22, which comprises a fusion protein obtained by ligating the binding peptide 2 with the nanoparticle protein NPM as shown in SEQ ID NO:21.

[0111] Further, the present invention provides a method for preparing an immunogenic complex:

[0112] (1) Connect the RABV-G antigen component and the granule protein component encoding genes into the expression vector separately to construct a recombinant expression plasmid;

[0113] (2) Construct a recombinant host cell that can express the RABV-G antigen component and granular protein component in the host cell;

[0114] (3) Use this recombinant host cell to express the fusion protein and perform purification of the recombinant fusion protein;

[0115] (4) The above-mentioned antigenic components and the particulate protein components are obtained through covalent binding reactions to obtain an immunogenic complex.

[0116] Preferably, the immunogenic complex obtained in step (4) above is purified to obtain a vaccine stock solution.

[0117] Preferably, in the preparation method of the immunogenic complex, in step (1), the plasmid expressing the RABV-G antigen component can be pcDNA3.4, and the nanoparticle vector can be pET-28a(+) and pET-30a(+).

[0118] In some embodiments, the immunogenic complex provided by the invention can induce the production of neutralizing antibodies.

[0119] In some embodiments, the immunogenic complex provided by the present invention is used to prepare neutralizing antibodies.

[0120] The above neutralizing antibodies refer to antibodies that can eliminate the ability of viral infection after binding to the virus. After the enveloped virus surface antigen is bound to the neutralizing antibody, complement can be activated, resulting in the dissolution of the virus, and IgG is preferred.

[0121] In the preparation method of immunogenic complexes described in the present invention (2), the host cell expressing the RABV-G antigen component is CHO, and the host cell expressing the granular protein vector is E.coli.

[0122] In the preparation method of the immunogenic complex described in the present invention (3), the purification method of the granular protein can be referred to patent CN 114395015 B.

[0123] F. Nucleic acid

[0124] The present invention provides the encoding nucleotides of the above-mentioned RABV-G mutants and RABV-G antigen components, and the specific nucleotide sequences are readily obtained by those skilled in the art through conventional means such as codon tables.

[0125] Preferably, the encoding nucleotide of the RABV-G mutant is obtained by codon optimization.

[0126] Preferably, the above-mentioned coding nucleotide sequence is optimized for the preferred codon of the CHO cell line.

[0127] The present invention also provides the above-mentioned particle protein components and the encoding nucleotides of nanoparticle proteins. The specific nucleotide sequences are readily obtained by those skilled in the art through conventional means such as codon tables.

[0128] Preferably, the RABV-G mutant encoding nucleotide sequence provided by the present invention is shown in SEQ ID NO: 10-17, see Table 2 for details.

[0129] The invention also provides vectors comprising nucleotide sequences of the invention, including cloning or expression vectors, and host cells transformed with the vectors described.

[0130] In some embodiments, the vector used in the present invention is selected from pcDNA3.4, pET-28a(+), pET-30a(+).

[0131] In some embodiments, the host cell expressing the antigen component vector is CHO and the host cell expressing the granular protein vector is E.coli.

[0132] Table 2: Nucleotide sequences of RABV-G ectodomain mutants of the present invention

[0133] Molecular name Nucleotide sequence RABV-G6 aag ttccctatct accatcccc agacaagctcggaccttggtcccctatcgacaccaccacccctgtcctatcgacaccaccacctgtcctagtaccaaccccacctgtcctaacaccctggtggaagagggctgccaaccccacctgtctggctggcttc tcctacatggagagctacccctgaaagtgcctgaacggctgaagctaccgctacgctacgccgctta caactggaagag atggctggcgacctgata cgaggaatct ctcccacaaccctatccccgactaccactgg ctgagaaccgttatcaccacaccacacacctcgttatcatctctcctccttccttcgacagatccctgatcttacgacagatccctgcactcttacgacagatccctcgcactctcggtttcccggcggaaactgctctggaaacccaccaccactacactatctggatgcctcgcggaaacggcctctct aagggctccg aaacctgcgg cttcgtggacgagcggggcctgtgtacaagtc tctgaaggggcgcaagctgaagctgtgtgtgg tggcgtgctgggactgagactgatggacggcacctgggtggcctgcagacctgggtggagagagagagagagagagagaatgcctgggtgcctgtgcagagagagagagagaatgcctggtgtgcagagagagagagaatgcctggtgtgcagagaatccacaaaatccgtgtcctt gaatctgcacgtgtggaagagctggtcgtggaagagctggtcaagagagagagagagagagaatgcctgg ccgcagactg agccacctgcggaagctggtgtgtgccttc ggcaaggcctcacacaatcttcaaagacaagaca ttgatggaag ccgacgcccaccaggaagcgtgcggacctggaacgaaat catccccctcctcctcctcctgagaagtcggcggcgatgcdtcttcaat ggcatcacacacatgctgctgtcgtcgtctcgtcgtctcaat ggcacacatggaactgctggtcgtcgtcttcaat ggcacaacgtgctgaacccctgatgcaatc ctccccctgctgctgtcgtct gtgatccctctgatgcaccccccctggccgccgaccctccacctcctaccgtgttcaagaa cggcgacgaggccgaggactttgtcgaagtgcctgcctgcctgcctgcacgagcggatctc cggggtggac ctggcctgcccaattggggcaagtac (SEQ ID NO:10) RABV-G25 AAGTTCCCCATCTACACCATCCCCGACAAGCTGGGCCCTTGGTCTCCTATCGACATCCACCATTTGTCCTGTCCTGTCTAACAACCTGGTCGTGGAGGACGAGGGCTGCACCAATCTGTCCGGTTTCTCCTACATGGAACTCAAAGTGGGCTATATCAGCGCCATCAAG ATGAACGGCTTCACCTGTACCGGCGTCGTGACCGAGGCCGAGACCTACACCACCAACTAACCAACTTCGTGGCTACGTGACGACAACCTTCAAGCGGAAGCACTTCCGGCCACCCCTGATGCTTGCAGAGCCGCTTACAACTGGAAGATGGCCGGATCCTAGATACGAGGAG TCCCTGCACAACCCCTATCCTGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGTCTCTCGTGATCATCTCTCCCTTCTGTGGCTGATCTGGACCCTTACGACAGAAGCCTGCACTCCCAGGGTGTTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCC TCTACCTACTGCTCCACCAACCAACCACAATCTGGATGCCAGAGAACCCTCGGCTGGGCATGTCCTGCGACATCTTCACAAACTCCAGAGGCAAAAGAGCTTCTAAAGGCTCCGAAACCTGCGGCTTTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTAGCCATGCAAACTTCCAACGAGACCAAGTGGTGCCCTCCTGGCCAGCTGGTGAACCTGctgGATTTCCGCTCCGACGAAATCGAGCACCTGGTGGAAGAG CTGGTGAAAAAGAGAGAAGAGTGCCTGGACGCTCTGGAATCCATCATGACCACCAAAAGTCCGTGTCCTTCCGGCGGCTGTCCCACCTGCGGAAGCTGGTCCCCTGGCTTCGGCAAGGCCTACACCATCTTTAACAAGACCCTGATGGAAGCCGATGCCCACTACTACAAG TCCGTGCGGACCTGGAACGATCATCCCCTAGCAAGGGCTGTCTGAGAGTGGGCGCAGATGCCACCCCCACGTGAATGGCGTGTTCTTCAACGGCATCATCCTGGGACCTGATGGCAACGTGCTGATCCCAGAGATGCAGTCTTCTCTGCTGCAGCAGCACATG GAACTGCTGGTCTCTAGTGTGATTCCTCTGATGCACCCTCTGGCCGACCCCCAGCACCGTGTTTAAGAACGGCGACGAGGCTGAGGACTTCGTGGAAGTGCACTTGCCAGATGTTCACGAGAATCAGCGGAGTGGACCTGGGCCTGCCTAATTGGGCAAGTAC (SEQ ID NO:11) RABV-G26 AAGTTCCCCATCTACACCATCCCCGACAAGCTGGGCCCTTGGTCTCCTATCGACATCCACCATTTGTCCTGTCCTGTCTAACAACCTGGTCGTGGAGGACGAGGGCTGCACCAATCTGTCCGGTTTCTCCTACATGGAACTCAAAGTGGGCTATATCAGCGCCATCAAG ATGAACGGCTTCACCTGTACCGGCGTCGTGACCGAGGCCGAGACCTACACCACCAACTAACCAACTTCGTGGCTACGTGACGACAACCTTCAAGCGGAAGCACTTCCGGCCACCCCTGATGCTTGCAGAGCCGCTTACAACTGGAAGATGGCCGGATCCTAGATACGAGGAG TCCCTGCACAACCCCTATCCTGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGTCTCTCGTGATCATCTCTCCCTTCTGTGGCTGATCTGGACCCTTACGACAGAAGCCTGCACTCCCAGGGTGTTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCC TCTACCTACTGCTCCACCAACCAACCACAATCTGGATGCCAGAGAACCCTCGGCTGGGCATGTCCTGCGACATCTTCACAAACTCCAGAGGCAAAAGAGCTTCTAAAGGCTCCGAAACCTGCGGCTTTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTAGCCATGCAAACTTCCAACGAGACCAAGTGGTGCCCTCCTGGCCAGCTGGTGAACCTGCATGATTTCCGCTCCccGAAATCGAGCACCTGGTGGAAGAG CTGGTGAAAAAGAGAGAAGAGTGCCTGGACGCTCTGGAATCCATCATGACCACCAAAAGTCCGTGTCCTTCCGGCGGCTGTCCCACCTGCGGAAGCTGGTCCCCTGGCTTCGGCAAGGCCTACACCATCTTTAACAAGACCCTGATGGAAGCCGATGCCCACTACTACAAG TCCGTGCGGACCTGGAACGATCATCCCCTAGCAAGGGCTGTCTGAGAGTGGGCGCAGATGCCACCCCCACGTGAATGGCGTGTTCTTCAACGGCATCATCCTGGGACCTGATGGCAACGTGCTGATCCCAGAGATGCAGTCTTCTCTGCTGCAGCAGCACATG GAACTGCTGGTCTCTAGTGTGATTCCTCTGATGCACCCTCTGGCCGACCCCCAGCACCGTGTTTAAGAACGGCGACGAGGCTGAGGACTTCGTGGAAGTGCACTTGCCAGATGTTCACGAGAATCAGCGGAGTGGACCTGGGCCTGCCTAATTGGGCAAGTAC (SEQ ID NO:12) RABV-G28 AAGTTCCCCATCTACACCATCCCCGACAAGCTGGGCCCTTGGTCTCCTATCGACATCCACCATTTGTCCTGTCCTGTCTAACAACCTGGTCGTGGAGGACGAGGGCTGCACCAATCTGTCCGGTTTCTCCTACATGGAACTCAAAGTGGGCTATATCAGCGCCATCAAG ATGAACGGCTTCACCTGTACCGGCGTCGTGACCGAGGCCGAGACCTACACCACCAACTAACCAACTTCGTGGCTACGTGACGACAACCTTCAAGCGGAAGCACTTCCGGCCACCCCTGATGCTTGCAGAGCCGCTTACAACTGGAAGATGGCCGGATCCTAGATACGAGGAG TCCCTGCACAACCCCTATCCTGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGTCTCTCGTGATCATCTCTCCCTTCTGTGGCTGATCTGGACCCTTACGACAGAAGCCTGCACTCCCAGGGTGTTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCC TCTACCTACTGCTCCACCAACCAACCACAATCTGGATGCCAGAGAACCCTCGGCTGGGCATGTCCTGCGACATCTTCACAAACTCCAGAGGCAAAAGAGCTTCTAAAGGCTCCGAAACCTGCGGCTTTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTAGCCATGCAAACTTCCAACGAGACCAAGTGGTGCCCTCCTGGCCAGCTGGTGAACCTGCATGATTCCGCTCCGACGAAATCGAGCACCTGGTGGAAGAG CTGGTGAAAAAGAGAGAAGAGTGCCTGGACGCTCTGGAATCCATCATGACCACCAAAAGTCCGTGTCCTTCCGGCGGCTGTCCCACCTGCGGAAGCTGGTCCCCTGGCTTCGGCAAGGCCTACACCATCTTTAACAAGACCCTGATGGAAGCCGATGCCCACTACTACAAG TCCGTGCGGACCTGGAACGATCATCCCCTAGCAAGGGCTGTCTGAGAGTGGGCGCAGATGCCACCCCCACGTGAATGGCGTGTTCTTCAACGGCATCATCCTGGACCTGATGGCAACGTGCTGATCCCAGAGATGCAGTCTTCTCTGCTGCAGcccATG GAACTGCTGGTCTCTAGTGTGATTCCTCTGATGCACCCTCTGGCCGACCCCCAGCACCGTGTTTAAGAACGGCGACGAGGCTGAGGACTTCGTGGAAGTGCACTTGCCAGATGTTCACGAGAATCAGCGGAGTGGACCTGGGCCTGCCTAATTGGGCAAGTAC (SEQ ID NO:13) RABV-G30 AAGTTCCCCATCTACACCATCCCCGACAAGCTGGGCCCTTGGTCTCCTATCGACATCCACCATTTGTCCTGTCCTGTCTAACAACCTGGTCGTGGAGGACGAGGGCTGCACCAATCTGTCCGGTTTCTCCTACATGGAACTCAAAGTGGGCTATATCAGCGCCATCAAG ATGAACGGCTTCACCTGTACCGGCGTCGTGACCGAGGCCGAGACCTACACCACCAACTAACCAACTTCGTGGCTACGTGACGACAACCTTCAAGCGGAAGCACTTCCGGCCACCCCTGATGCTTGCAGAGCCGCTTACAACTGGAAGATGGCCGGATCCTAGATACGAGGAG TCCCTGCACAACCCCTATCCTGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGTCTCTCGTGATCATCTCTCCCTTCTGTGGCTGATCTGGACCCTTACGACAGAAGCCTGCACTCCCAGGGTGTTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCC TCTACCTACTGCTCCACCAACCAACCACAATCTGGATGCCAGAGAACCCTCGGCTGGGCATGTCCTGCGACATCTTCACAAACTCCAGAGGCAAAAGAGCTTCTAAAGGCTCCGAAACCTGCGGCTTTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTAGCCATGCAAACTTCCAACGAGACCAAGTGGTGCCCTCCTGGCCAGCTGGTGAACCTGCATGATTCCGCTCCGACGAAATCGAGCACCTGGTGGAAGAG CTGGTGAAAAAGAGAGAAGAGTGCCTGGACGCTCTGGAATCCATCATGACCACCAAAAGTCCGTGTCCTTCCGGCGGCTGTCCCACCTGCGGAAGCTGGTCCCCTGGCTTCGGCAAGGCCTACACCATCTTTAACAAGACCCTGATGGAAGCCGATGCCCACTACTACAAG TCCGTGCGGACCTGGAACGATCATCCCCTAGCAAGGGCTGTCTGAGAGTGGGCGCAGATGCCACCCCCACGTGAATGGCGTGTTCTTCAACGGCATCATCCTGGGACCTGATGGCAACGTGCTGATCCCAGAGATGCAGTCTTCTCTGCTGCAGCAGCACATG GAACTGCTGGTCTCTAGTGTGATTCCTCTGATGCACCCTCTGGCCGACCCCCAGCACCGTGTTTAAGAACGGCGACGAGGCTGAGGACTTCGTGGAAGTGCACTTGCCAGATGTTctgGAGAGAATCAGCGGAGTGGACCTGGGCCTGCCTAATTGGGGCAAGTAC (SEQ ID NO:14) RABV-G32 AAGTTCCCTATCTACACCATCCCAGACAAGCTCGGACCTTGGTCCCCTATCGACATCCACCACCTGTCCTGTCCTGTCCTTAACAACCTGGTGGTGGAAGATGAGGGCTGCACCAACCTGTCTGGCTTCTCCTACATGGAGCTGAAAGTGGGCTACATCAGCGCCATCAAG ATGAACGGCTTTACCTGTACCGGCGTGGTGACCGAGGCTGAGACATACACCACCAACCAACTTCGTGGGCTACGTGACAACCACCCTTCAAGAGGAAGCACTTCAGACCCTGATGCCTGTAGAGCCGCTTACAACTGGAAGATGGCTGGCGACCCTAGATACGAGGAA TCTCTCCACAACCCCTATCCCCGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGAGCCTCGTTATCATCTCTCCTCCGTGGCTGATCTGGATCCTTACGACAGATCCCCTGCACTCTTCGGGTGTTTCCCGGCGGAAACTGCTCTGGAGTGGCCGTGTCT AGCACCTACTGCTCCACCAACCAACCACTATCTGGATGCCTGAGAACCCTCTCGGCTGGGCATGTCCTGCGACATCTTCACCAACTCCAGAGGCAAACGGGCCTCTAAGGGCTCCGAAACCTGCGGCTTCGTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTGGCCATGCAGACCTCCAACGACAAAAGTGGTGCCCTCCTGGCCAGCTGGTGAATCTGCTGGACTTCCGGAGCGATGAGATTGAGCCTCTCGTCGTGGAAGAG CTGGTCAAGAAGAGAGAGAGAATGCCTGGACGCTCTGGAATCCATCATGACCACAAAATCCGTGTCCTTCCGCAGACTGAGCCACCTGCGGAAGCTGGTGCCTGGCTTCGGCAAGGCCTACACAATCTTCAACAAGACATTGATGGAAGCCGCCCACTACTACAAG AGCGTGCGGACCTGGAACGAAATCATCCCCCTCCAAAGGCTGTCTGAGAGTCGGCGCAGATGCCACCCTCACGTGAACGGCGTGTTCTTCAATGGCATCATCCTGGGCCCTGATGGCAACGTGCTGATCCCAGAGATGCAATCCCTCCCTGCTGCAGCAGCACATG GAACTGCTGGTGTCGTCTGTGATCCCTCTGATGCACCCCCTGGCCGACCCCCTTCTACCGTGTTCAAGAACGGCGACGAGGCCGAGGACTTTGTCGAAGTGCATCTGCCTGCACGAGCGGATCTCCGGGGTGGACCTGGCCTGCCCAATTGGGGCAAGTAC (SEQ ID NO:15) RABV-G33 AAGTTCCCTATCTACACCATCCCAGACAAGCTCGGACCTTGGTCCCCTATCGACATCCACCACCTGTCCTGTCCTGTCCTTAACAACCTGGTGGTGGAAGATGAGGGCTGCACCAACCTGTCTGGCTTCTCCTACATGGAGCTGAAAGTGGGCTACATCAGCGCCATCAAG ATGAACGGCTTTACCTGTACCGGCGTGGTGACCGAGGCTGAGACATACACCACCAACCAACTTCGTGGGCTACGTGACAACCACCCTTCAAGAGGAAGCACTTCAGACCCTGATGCCTGTAGAGCCGCTTACAACTGGAAGATGGCTGGCGACCCTAGATACGAGGAA TCTCTCCACAACCCCTATCCCCGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGAGCCTCGTTATCATCTCTCCTCCGTGGCTGATCTGGATCCTTACGACAGATCCCCTGCACTCTTCGGGTGTTTCCCGGCGGAAACTGCTCTGGAGTGGCCGTGTCT AGCACCTACTGCTCCACCAACCAACCACTATCTGGATGCCTGAGAACCCTCTCGGCTGGGCATGTCCTGCGACATCTTCACCAACTCCAGAGGCAAACGGGCCTCTAAGGGCTCCGAAACCTGCGGCTTCGTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTGGCCATGCAGACCTCCAACGACAAAAGTGGTGCCCTCCTGGCCCTGGCCAGCTGGTGAATCTGCACGACTTCCGGAGCCCTGAGATTGAGCCTCTCGTCGTGGAAGAG CTGGTCAAGAAGAGAGAGAGAATGCCTGGACGCTCTGGAATCCATCATGACCACAAAATCCGTGTCCTTCCGCAGACTGAGCCACCTGCGGAAGCTGGTGCCTGGCTTCGGCAAGGCCTACACAATCTTCAACAAGACATTGATGGAAGCCGCCCACTACTACAAG AGCGTGCGGACCTGGAACGAAATCATCCCCCTCCAAAGGCTGTCTGAGAGTCGGCGCAGATGCCACCCTCACGTGAACGGCGTGTTCTTCAATGGCATCATCCTGGGCCCTGATGGCAACGTGCTGATCCCAGAGATGCAATCCCTCCCTGCTGCAGCAGCACATG GAACTGCTGGTGTCGTCTGTGATCCCTCTGATGCACCCCCTGGCCGACCCCCTTCTACCGTGTTCAAGAACGGCGACGAGGCCGAGGACTTTGTCGAAGTGCATCTGCCTGCACGAGCGGATCTCCGGGGTGGACCTGGCCTGCCCAATTGGGGCAAGTAC (SEQ ID NO:16) RABV-G36 AAGTTCCCCATCTACACCATCCCCGACAAGCTGGGCCCTTGGTCTCCTATCGACATCCACCATTTGTCCTGTCCTGTCTAACAACCTGGTCGTGGAGGACGAGGGCTGCACCAATCTGTCCGGTTTCTCCTACATGGAACTCAAAGTGGGCTATATCAGCGCCATCAAG ATGAACGGCTTCACCTGTACCGGCGTCGTGACCGAGGCCGAGACCTACACCACCAACTAACCAACTTCGTGGCTACGTGACGACAACCTTCAAGCGGAAGCACTTCCGGCCACCCCTGATGCTTGCAGAGCCGCTTACAACTGGAAGATGGCCGGATCCTAGATACGAGGAG TCCCTGCACAACCCCTATCCTGACTACCACTGGCTGAGAACCGTGAAGACCACCAAAGAGTCTCTCGTGATCATCTCTCCCTTCTGTGGCTGATCTGGACCCTTACGACAGAAGCCTGCACTCCCAGGGTGTTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCCTGGAGGCAACTGCTCTGGCGTCGCTGTGTCCC TCTACCTACTGCTCCACCAACCAACCACAATCTGGATGCCAGAGAACCCTCGGCTGGGCATGTCCTGCGACATCTTCACAAACTCCAGAGGCAAAAGAGCTTCTAAAGGCTCCGAAACCTGCGGCTTTGGACGAGCGGGCCTGTACAAGTCTCTGAAG GGCGCCTGCAAGCTGAAGCTGTGGCGTGCTGGGACTGAGACTGATGGACGGCACCTGGGTAGCCATGCAAACTTCCAACGAGACCAAGTGGTGCCCTCCTGGCCAGCTGGTGAACCTGCATGATTTCCGCTCCGACGAAATCGAGCACcctGTGGGAAGAG CTGGTGAAAAAGAGAGAAGAGTGCCTGGACGCTCTGGAATCCATCATGACCACCAAAAGTCCGTGTCCTTCCGGCGGCTGTCCCACCTGCGGAAGCTGGTCCCCTGGCTTCGGCAAGGCCTACACCATCTTTAACAAGACCCTGATGGAAGCCGATGCCCACTACTACAAG TCCGTGCGGACCTGGAACGATCATCCCCTAGCAAGGGCTGTCTGAGAGTGGGCGCAGATGCCACCCCCACGTGAATGGCGTGTTCTTCAACGGCATCATCCTGGGACCTGATGGCAACGTGCTGATCCCAGAGATGCAGTCTTCTCTGCTGCAGCAGCACATG GAACTGCTGGTCTCTAGTGTGATTCCTCTGATGCACCCTCTGGCCGACCCCCAGCACCGTGTTTAAGAACGGCGACGAGGCTGAGGACTTCGTGGAAGTGCACTTGCCAGATGTTCACGAGAATCAGCGGAGTGGACCTGGGCCTGCCTAATTGGGCAAGTAC (SEQ ID NO:17)

[0134] G. Immune Composition

[0135] The present invention provides an immune composition comprising the RABV-G mutant, the RABV-G trimer, the RABV-G multimer or the immunogenic complex in the present invention.

[0136] Optionally, the immune composition of the invention further comprises a pharmaceutically acceptable carrier.

[0137] Preferably, the pharmaceutically acceptable carriers include stabilizers, excipients, surfactants, buffering agents, pH adjusters, the stabilizers are sucrose and arginine, the excipient is mannitol, the surfactant is Tween-80, the buffers are disodium hydrogen phosphate dihydrate, sodium dihydrate dihydrate, and the pH adjusters are hydrochloric acid.

[0138] In some embodiments, the immune composition provided by the invention is an injection or a lyophilized formulation, preferably a lyophilized formulation.

[0139] In some embodiments, the immune composition of the present invention comprises one or more of the above-mentioned RABV-G mutants, RABV-G trimers, RABV-G multimers or immunogenic complexes.

[0140] In some embodiments, the immune composition of the invention can induce the production of neutralizing antibodies.

[0141] In some embodiments, the immune composition of the invention is used to prepare neutralizing antibodies.

[0142] The above neutralizing antibodies refer to antibodies that can eliminate the ability of viral infection after binding to the virus. After the enveloped virus surface antigen is bound to the neutralizing antibody, complement can be activated, resulting in the dissolution of the virus, and IgG is preferred.

[0143] H. Vaccines

[0144] The present invention provides a vaccine containing the immune composition and an adjuvant of the present invention.

[0145] In some embodiments, the vaccine provided by the present invention is a human vaccine.

[0146] In some embodiments, the vaccine provided by the invention is a veterinary vaccine.

[0147] The adjuvants used in the present invention can enhance immune responses through a variety of mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. Compared with the case of immunogen alone, the adjuvant increases the titer of the inducing antibody and / or the binding affinity of the inducing antibody. Preferred adjuvants enhance the intrinsic response to the immunogen without causing conformational changes in the immunogen.

[0148] The adjuvants used in the present invention are selected from the group consisting of: aqueous adjuvants; mineral adjuvants including aluminum salts such as Al(OH) 3 , as well as aluminum phosphate, calcium phosphate, etc.; oil emulsion adjuvant, including oil-in-water emulsion, water-in-oil emulsion, Freund's adjuvant, MF59, white oil span adjuvant, SAF series; microbial adjuvant, including Corynebacterium bacterium, BCG, murayl diphthalate and its synthetic lipophilic derivatives, bacterial lipopolysaccharides, bacterial toxins, and lipid-soluble wax D; liposome adjuvant, including non-phospholipid liposomes, such as Novasomes; cytokine adjuvant, including GM-CSF, IL-2, IL-12; nucleic acid adjuvant, including CpGDNA; Chinese herbal adjuvant, including propolis, saponins, immunostimulatory complex ISCOMS, polysaccharides, glycosides and compound traditional Chinese medicine; chemical adjuvant, including levamisazole, cimetidine erythromycin, etc.

[0149] In some embodiments, the vaccine of the present invention contains an adjuvant selected from Toll-like receptor agonists, including poly(1:C), GLA-LSQ, flagellin, CpG1018.

[0150] In some embodiments, the vaccine of the present invention contains an adjuvant selected from the group consisting of a complex adjuvant, including an AS01, AS03, AS04; Covaxin; AS37; Pickup (PIKACA); a BC adjuvant system such as BCG-CpG-DNA.

[0151] In some embodiments, the polysaccharide adjuvant of the invention is selected from chitosan or inulin; the immunostimulatory complex adjuvant is selected from Matrix-M; the lipid adjuvant is selected from cationic lipid adjuvant such as CAF01, anionic lipid adjuvant such as LPS, monophosphoryl lipid A, liposome nanoparticles (LNP); the saponin adjuvant is selected from Quil A or QS21.

[0152] In some embodiments, the adjuvant of the invention is selected from nanoadjuvants, such as silver nanoparticles, chiral nanoadjuvants.

[0153] In some embodiments, the adjuvant of the present invention is selected from the group consisting of ISA201, ISA206, IMS1313, ISA15A.

[0154] In some embodiments, the adjuvant of the invention is selected from trehalose synthetic derivatives, genetically engineered attenuated toxins, or biodegradable polymerized microspheres. The genetically engineered attenuated toxin adjuvant is selected from cholera toxin, E. coli heat-tolerant toxin or tetanus toxin.

[0155] The adjuvant in the present invention contains 3%-5%, Span 85 0.4%-1%, Tween 80 0.4%-1%, 10 mM citrate or 0.1-0.5% sodium citrate, 0.01-0.05% citric acid (w / v). Squalene is preferably 3.5% to 4.5%, more preferably 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5% (w / v). Squalene adjuvant can be commercial adjuvant MF59 or SWE, where MF59 is an oil-in-water emulsion, which consists of 4.3% squalene, 0.5% Tween-80 and 0.5% Span85. SWE is an oil-in-water emulsion similar to MF59.

[0156] The squalene water oil adjuvant ingredients used in specific embodiments of the present invention are 100% mass: squalene 4.2%, span 85 0.5%, Tween-80 0.5%, citric acid 0.264%, and sodium citrate 0.016%.

[0157] Preferably, the adjuvant used in the present invention is selected from SWE or MF59.

[0158] Preferably, the adjuvant used in the present invention is selected from AS01, AS03 or AS04.

[0159] In some embodiments, the vaccine provided by the invention can induce the production of neutralizing antibodies.

[0160] In some embodiments, the vaccine provided by the invention is used to prepare neutralizing antibodies.

[0161] The above neutralizing antibodies refer to antibodies that can eliminate the ability of viral infection after binding to the virus. After the enveloped virus surface antigen is bound to the neutralizing antibody, complement can be activated, resulting in the dissolution of the virus, and IgG is preferred.

[0162] The present invention provides a method for preparing a vaccine for preventing or treating rabies. The purified antigen component and the granule protein component are covalently bound by binding peptide 1 and binding peptide 2, and mixed with available adjuvants to make an immune composition product. The amount of the immunogenic complex is 0.25-100 μg / agent, preferably 0.5-50 μg / agent, more preferably 0.5 μg / agent, 1 μg / agent, 2 μg / agent, 3 μg / agent, 4 μg / agent, 5 μg / agent, 10 μg / agent, 15 μg / agent, 20 μg / agent, 25 μg / agent, 30 μg / agent, 35 μg / agent, 40 μg / agent, 45 μg / agent, 50 μg / agent.

[0163] The preparation method of a vaccine for preventing or treating rabies according to the present invention, the aqueous and oil adjuvant is squalene, and the saponin adjuvant comprises QS-21. The vaccine stock solution is diluted with buffer (such as TBS) at dose and mixed with the adjuvant at volume ratio 1:1.

[0164] The present invention further provides a complete kit characterized by comprising a rabies vaccine, as well as an appliance and a container for vaccinating the vaccine, and in particular, a needle tube appliance, a powder bearing container, and a solvent bearing container.

[0165] I. Medical uses

[0166] The present invention provides the medical uses of the above-mentioned RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines, including the prevention or treatment of related diseases caused by rabies virus infection.

[0167] Preferably, the RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, vaccines of the present invention are used to prevent pre-exposure and post-exposure prevention of rabies virus infection.

[0168] The present invention provides pharmaceutical preparations of the above-mentioned RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines, which are used to prevent or treat related diseases caused by rabies virus infection.

[0169] The present invention provides vaccination procedures for the above-mentioned RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines. Pre-exposure prevention usually includes two to three visits, two to three intramuscular doses, and is timed to enhance according to the exposure risk. Post-contact prophylaxis usually involve three to five intramuscular doses or four intradermal doses.

[0170] The RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions or vaccines provided by the present invention will generally be administered directly to the subject. Direct delivery can be accomplished by parenteral injection, for example subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, or injection into the space of the tissue. When administered prophylactically, for example, to residents of rabies endemic areas or to travelers to rabies endemic areas, the dosage schedule may consist of three doses, two doses, or one dose. Alternatively or additionally, one dose may be administered prophylactically. When administered therapeutically, eg after rabies exposure, the dosing schedule may consist of five doses, four doses, three doses, two doses, or one dose. In a preferred embodiment, one or two doses are administered therapeutically.

[0171] J. Benefits

[0172] Compared with the prior art, the beneficial effects of the present invention are:

[0173] (1) The RABV-G ectodomain mutant of the present invention realizes soluble expression of the RABV-G ectodomain and can self-assemble into trimers or multimers, which greatly solves the technical difficulties in the art.

[0174] (2) The RABV-G ectodomain mutant vaccine of the present invention has better cellular immunity and antibody immune response than the commercial human rabies vaccine and canine rabies vaccine.

[0175] (3) The RABV-G ectodomain mutant vaccine of the present invention can induce the production of high titer neutralizing antibodies, which is expected to reduce the number of vaccinations, shorten the immune program and reduce the immune dose.

[0176] (4) The RABV-G ectodomain mutant vaccine of the present invention is a recombinant protein vaccine. The entire production process and the final product do not involve complete viruses, are highly safe, and can reduce the complexity that may be brought to the production process from the source, improve production efficiency, and save the cost of large-scale production.

[0177] (5) The present invention also discovered a method for preparing RABV-G ectodomain mutant protein particles of different particle sizes. The method is simple to operate, and the prepared protein particles are uniform and have a uniform particle size, laying the foundation for the preparation of virus-like particles of different types and different particle sizes. Attached description of the drawings

[0178] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the examples will be briefly described below.

[0179] Figure 1 : SDS-PAGE identification results of RABV-G, where A is RABV-G6, B is RABV-G25, C is RABV-G26, D is RABV-G28, E is RABV-G30, F is RABV-G32, G is RABV-G33, and H is RABV-G36;

[0180] Figure 2 : where A-B is the negative electron microscope staining result of G6, C is the particle size and uniformity detection result, and D is the HPLC detection result;

[0181] Figure 3 : AUC test results of G6 samples;

[0182] Figure 4 : A-B is the electron microscope negative staining result of RABV-G25;

[0183] Figure 5 : A-B is the negative electron microscope staining result of RABV-G26;

[0184] Figure 6 : A-B is the electron microscope negative staining result of RABV-G28;

[0185] Figure 7 : A-B is the electron microscope negative staining result of RABV-G30;

[0186] Figure 8 : A-B is the electron microscope negative staining result of RABV-G32;

[0187] Figure 9 : A-B is the electron microscope negative staining result of RABV-G33;

[0188] Figure 10 : A-B is the electron microscope negative staining result of RABV-G36;

[0189] Figure 11 : A-B is the negative electron microscope staining result of G6 after adding 0.02% Tween-80;

[0190] Figure 12 : A-B is the negative electron microscope staining result of G6 after adding 0.05% Tween-80;

[0191] Figure 13 : A-B is the negative electron microscope staining result of G6 after adding 0.1% Tween-80s;

[0192] Figure 14 : SDS-PAGE identification results of G6-NPM virus-like particles. Among them, A is a virus-like particle with 0.2% Tween-80 added, B is a virus-like particle with 0.05% Tween-80 added, C is a virus-like particle with 0.02% Tween-80 added, and D is a virus-like particle without Tween-80 added;

[0193] Figure 15 : The neutralizing antibody detection results of immune serum of mice vaccinated with G1 and G6;

[0194] Figure 16 : The neutralizing antibody test results of two injections of G6 and four immunoselected mice with vaccine;

[0195] Figure 17 : The results of neutralizing antibodies in mice vaccinated with G protein mutants. Specific implementation methods

[0196] The principles and features of the present invention are described below in connection with examples. The examples are for the explanation of the present invention only and are not intended to limit the scope of the present invention. Before further describing specific embodiments of the invention, it should be understood that the scope of protection of the invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the invention are for the purpose of describing specific embodiments, and not for the purpose of limiting the scope of protection of the invention. Test methods that do not specify specific conditions in the following examples are usually subject to conventional conditions or as recommended by each manufacturer. When the embodiment gives a numerical range, it is understood that unless otherwise specified by the invention, the two endpoints of each numerical range and any value between the two endpoints may be optional. Unless otherwise defined, all technical and scientific terms used in the present invention are the same as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the mastery of the prior art and the description of the present invention by those skilled in the art, any methods, equipment and materials of the prior art that are similar or equivalent to those described in the embodiments of the present invention can be implemented. The experimental materials used in the following examples, if not specified, were purchased from conventional reagent companies.

[0197] Example 1: Expression purification of RABV-G ectodomain mutants

[0198] 1. Expression purification of RABV-G ectodomain mutants

[0199] 1. Experimental materials:

[0200] HisTrap excel was purchased from Cytiva, molecular sieve (Superose 6 10 / 300 GL) was purchased from Cytiva, and anion exchange column (Hitrap Q FF) was purchased from Cytiva.

[0201] 2. Experimental method:

[0202] (1) The encoding gene of the signal peptide shown in SEQID NO:18 is introduced at the N-terminal end of the RABV-G ectodomain mutant (SEQ ID NO:2-9) encoding gene shown in Table 1. Optionally, the encoding gene of the 4T sequence (SEQ ID NO:19) and 6×His tag are introduced at the C-terminal end. The fusion gene obtained by conventional molecular cloning methods. The sequence of the fusion gene is optimized for CHO cells, cloned onto the SPB-007 vector, and a stable transfected cell pool was constructed, positive cell pool was screened, and the cell expansion culture was completed. The cell survival rate was higher than 95%.

[0203] (2) The cell supernatant was harvested in expression culture medium for 12 days, collected and centrifuged into a centrifuge tube for 6000g for 20 minutes, and the supernatant was taken and filtered through a 0.22um pore size PES membrane;

[0204] (3) The filtered supernatant was purified through Histrap Excel-5ml nickel column, and the penetration solution was loaded to collect the penetration solution; then eluted with 20mM imidazole, 10-fold volume of TBS buffer, and the samples were collected; then eluted with 500mM imidazole, 10-fold volume of TBS buffer, and the elution sample was collected. All samples were identified by SDS-PAGE;

[0205] (4) Collect the sample containing the protein of interest, concentrate through a 10KD ultrafiltration concentration tube, and further purify it through a Superose6increase 10 / 300 GL molecular sieve, and the buffer is TBS. Samples were collected for SDS-PAGE electrophoresis detection, see the results Figure 1 A. The BCA method determines the protein concentration and maintains the appropriate temperature for subsequent detection. The sample is referred to as G6;

[0206] See the SDS-PAGE identification results for other variants Figure 1 B-H, where B is RABV-G25, C is RABV-G26, D is RABV-G28, E is RABV-G30, F is RABV-G32, G is RABV-G33, and H is RABV-G36.

[0207] (5) Optionally, further purification of G6 is performed. It includes the replacement of G6 protein purified by molecular sieve into 50mM Tris, 50mM NaCl buffer, purified by anion exchange column hitrap Q FF, solution A is 50mM Tris, 50mM NaCl buffer, solution B is 50mM Tris, 1M NaCl buffer, sample is elution through linear gradient, sample is collected, and further purification is carried out through Superose6 increase 10 / 300 GL molecular sieve, buffer is TBS. The protein of interest was collected for SDS-PAGE electrophoresis detection, and the protein concentration was measured by BCA method, and the protein concentration was stored at appropriate temperature for subsequent detection.

[0208] 2. Identification of the properties of G6 protein

[0209] (1) The purified G6 protein was diluted to 0.01 mg / mL with TBS for negative staining observation. It can be observed that the sample formed relatively uniform nanoparticles. See the results. Figure 2 A-B; The particle size and uniformity of the particle were detected at the same time, and the results are shown in Figure 2 C, its particle size is uniform and its size is about 20.36 nm; the purity of G6 protein is detected by HPLC, and the results are shown in Figure 2 D, its main peak-to-peak asymmetry may be caused by glycosylation, with a purity of about 89%.

[0210] (2) The molecular weight and uniformity of G6 protein samples were detected by AUC. See the results Figure 3 , the peak with a molecular weight of about 60.8 kDa on the left side of the figure is a monomer. The self-assembled polymer is a 9-mer with a molecular weight of about 582 kDa as the main component, and a small part can form 6-mer and 12-mer with a molecular weight of about 443 kDa and 754 kDa. It can be seen that G6 can self-assemble into a multimer.

[0211] (3) The samples purified by other RABV-G ectodomain mutants were diluted to 0.01 mg / mL after nickel purification and molecular sieve for negative staining observation, and it was observed that all samples formed relatively uniform nanoparticles. in, Figure 4 A-B is the negative electron microscope staining result of RABV-G25. Figure 5 A-B is the electron microscope negative staining result of RABV-G26. Figure 6 A-B is the negative electron microscope staining result of RABV-G28. Figure 7 A-B is the negative electron microscope staining result of RABV-G30. Figure 8 A-B is the negative electron microscope staining result of RABV-G32. Figure 9 A-B is the negative electron microscope staining result of RABV-G33. Figure 10 A-B is the negative electron microscope staining result of RABV-G36.

[0212] Example 2: Binding and particle characterization of RABV-G ectodomain mutants and NPM

[0213] 1. Preparation of RABV-G ectodomain mutants of different particle sizes

[0214] The purified G6 protein sample was added with 0.02%, 0.05%, and 0.1% Tween-80, respectively, and then negative staining observation was performed. It was observed that the sample was dispersed and the protein particles with uniform particle size were formed. in, Figure 11 A-B is the electron microscope negative staining result of G6 after adding 0.02% Tween-80s. Figure 12 A-B is the electron microscope negative staining result of G6 after adding 0.05% Tween-80s. Figure 13 A-B is the negative electron microscope staining result of G6 after adding 0.1% Tween-80s.

[0215] II. Binding experiments of RABV-G ectodomain mutants and NPM

[0216] The obtained G6 diluted by TBS and G6 dispersed by Tween at different concentrations were mixed with NPM-4C (SEQ ID NO: 22) as part of the particle protein concentration ratio, 6:1, 50% sucrose mother liquor was added to about 25% sucrose final concentration, and 1M Tris-HCl (pH 7.4) mother liquor with a total reaction volume of 10% was added to stabilize the pH. The binding reaction was carried out at 22°C for 24 hours. The bound product was isolated and purified by molecular sieve (purification buffer was 20 mM Tris-HCl, 150 mM NaCl, 25% Sucrose, pH 7.4), and the G6-NPM components were collected. SDS-PAGE identify purified G6-NPM virus-like particles, such as Figure 14 Shown. Among them, A is a virus-like particle formed by adding 0.2% of Tween-80 dispersed G6, B is a virus-like particle formed by adding 0.05% of Tween-80 dispersed G6, C is a virus-like particle formed by adding 0.02% of Tween-80 dispersed G6, and D is a virus-like particle formed by not adding Tween-80. It can be seen that G6 dispersed at different concentrations of Tween-80 can form virus-like particles of different molecular weights after binding to NPM.

[0217] Example 3: Animal Immunity Experiment 1

[0218] (1) Experimental materials

[0219] Mice: 5-6 weeks old female mice of BALB / c line (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.)

[0220] Adjuvant: SWE;

[0221] Fake virus: Chinayan Guobang;

[0222] Cells: 293T cells;

[0223] Control vaccine: lyophilized rabies vaccine for humans (Vero cells, Chengda, Liaoning), and lyophilized rabies vaccine for humans (human diploid cells, Kanghua, Chengdu);

[0224] Other reagents and consumables are commercial conventional reagents and consumables.

[0225] (2) Experimental plan

[0226] As shown in Table 3, one dose of vaccine was immunized on days 0 and 7 respectively. The vaccination method was intramuscular injection, with a dose of 5 μg / dose / substitute, and 25 μg of SWE adjuvant was mixed with each dose of vaccine. Serum was isolated on days 7 and 14 for the detection of neutralizing antibodies.

[0227] Table 3: Animal Immunization Program 1

[0228]

[0229] (3) Neutralizing antibody detection:

[0230] 1. Sample preparation: Mouse serum was placed in a dry thermostat and inactivated at 56°C for 30 min.

[0231] Virus preparation: Remove the fake virus from -80°C, melt it at 4°C, and dilute the virus to 5×10 using DMEM complete medium (DMEM+10% FBS). 4 TCID50 / mL;

[0232] 2. The first wells of the serum sample were diluted with a dilution ratio of 1:100 (i.e., 1.5μL sample + 148.5μL culture medium was diluted, and the final dilution ratio in the well was 1:250). Two complex wells per serum sample were then diluted with a 3-fold gradient, with a total of 7 concentrations (using the last 6 gradients when using the reading);

[0233] 3. Add 50 μL of diluted virus concentration 5×10 per well 4 TCID50 / mL virus, blank holes are sealed with sterile liquid, 200μL / well, 37℃, 5%CO 2 Incubate in the cell incubator for 1 h;

[0234] 4. After the sample and virus are incubated for 1 h, add 100 μL per well and the density is 0.5×10 according to the label. 6 / mL of 293T cells, then put into 37 °C, 5% CO 2 Cell incubator for 24 h;

[0235] 7. Detection: Take out the 96-well white plate and Bio-Lite detection reagent, equilibrate to room temperature, aspirate 150μL / well of liquid in the plate, add 100μL of detection reagent to the plate, let stand for 5 min, then use a fluorescence microplate reader to detect the chemiluminescence value (RLU).

[0236] See the results Figure 15 Compared with G1 (i.e., wild type) and G6 (i.e., wild type) after using SWE adjuvant, G6 was significantly higher than G1 and also higher than Vero cell lyophilized vaccine and human diploid lyophilized vaccine, indicating that G6 has good immunogenicity.

[0237] Example 4: Animal Immunity Experiment 2

[0238] (1) Experimental materials

[0239] Mice: 5-6 weeks old female mice of BALB / c line (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.);

[0240] Adjuvant A: The components are as follows: squalene 4.2%, sipan 85 0.5%, Tween-80 0.5%, citric acid 0.264%, sodium citrate 0.016% (W / W);

[0241] Fake virus: Chinayan Guobang;

[0242] Cells: 293T cells;

[0243] Control vaccine: rabies vaccine for freeze-dried humans (Vero cells, Liaoning Chengda);

[0244] Other reagents and consumables are commercial conventional reagents and consumables.

[0245] (2) Experimental plan

[0246] As shown in Table 4, one dose of RABV-G6 vaccine was immunized on days 0 and 7 respectively. The vaccination method was intramuscular injection, with the dose of 5 μg / dose / substitute. Each dose of vaccine was mixed with 25 μg of adjuvant A. Serum was isolated on days 7 and 14 for the detection of neutralizing antibodies. The control vaccine was vaccinated separately on days 0, 7, 14 and 21. The vaccination method was intramuscular injection, and the dose was 1 / 10 of the human dose / dose / subject. Blood was collected and serum was isolated on days 7, 14, 21 and 35 to detect neutralizing antibodies.

[0247] Table 4: Animal Immunization Program 2

[0248]

[0249] (3) Neutralizing antibody detection:

[0250] 1. Sample preparation: Mouse serum was placed in a dry thermostat and inactivated at 56°C for 30 min.

[0251] Virus preparation: Remove the fake virus from -80°C, melt it at 4°C, and dilute the virus to 5×10 using DMEM complete medium (DMEM+10% FBS). 4 TCID50 / mL;

[0252] 2. The first wells of the serum sample were diluted with a dilution ratio of 1:100 (i.e., 1.5μL sample + 148.5μL culture medium was diluted, and the final dilution ratio in the well was 1:250). Two complex wells per serum sample were then diluted with 3 times gradients, totaling 7 concentrations (use the last 6 gradients when using readings);

[0253] 3. Add 50 μL of diluted virus concentration 5×10 per well 4 TCID50 / mL virus, blank holes are sealed with sterile liquid, 200μL / well, 37℃, 5%CO 2 Incubate in the cell incubator for 1 h;

[0254] 4. After the sample and virus are incubated for 1 h, add 100 μL per well and the density is 0.5×10 according to the label. 6 / mL of 293T cells, then put into 37 °C, 5% CO 2 Cell incubator for 24 h;

[0255] 7. Detection: Take out the 96-well white plate and Bio-Lite detection reagent, equilibrate to room temperature, aspirate 150μL / well of liquid in the white plate, then add 100μL of detection reagent to the plate, let stand for 5 min, then use a fluorescence microplate reader to detect the chemiluminescence value (RLU).

[0256] See the results Figure 16 , the results showed that on days 21 and 35, the neutralizing antibody titers of the RABV-G6 vaccine were higher than those of the control vaccine, indicating that the immunity effect of two shots of RABV-G6 vaccine was better than that of the control vaccine.

[0257] Example 5: Animal Immunity Experiment 3

[0258] (1) Experimental materials

[0259] Mice: 5-6 weeks old female mice of BALB / c line (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.);

[0260] Adjuvant A: The components are as follows: squalene 4.2%, sipan 85 0.5%, Tween-80 0.5%, citric acid 0.264%, sodium citrate 0.016% (W / W);

[0261] Fake virus: Chinayan Guobang;

[0262] Cells: 293T cells;

[0263] Control vaccine: velo cells human rabies vaccine has been launched;

[0264] Other reagents and consumables are commercial conventional reagents and consumables.

[0265] (2) Experimental plan

[0266] As shown in Table 5, one dose of vaccine was immunized on days 0 and 7 respectively. The vaccination method was intramuscular injection, with the dose of 5 μg / dose / substitute. Each dose of vaccine was mixed with 25 μg of adjuvant A. Serum was isolated on days 7 and 14 for the detection of neutralizing antibodies.

[0267] Table 5: Animal Immunization Program 3

[0268]

[0269] (3) Neutralizing antibody detection:

[0270] 1. Sample preparation: Mouse serum was placed in a dry thermostat and inactivated at 56°C for 30 min.

[0271] Virus preparation: Remove the fake virus from -80°C, melt it at 4°C, and dilute the virus to 5×10 using DMEM complete medium (DMEM+10% FBS). 4 TCID50 / mL;

[0272] 2. The first wells of the serum sample were diluted with a dilution ratio of 1:100 (i.e., 1.5μL sample + 148.5μL culture medium was diluted, and the final dilution ratio in the well was 1:250). Two complex wells per serum sample were then diluted with 3 times gradients, totaling 7 concentrations (use the last 6 gradients when using readings);

[0273] 3. Add 50 μL of diluted virus concentration 5×10 per well 4 TCID50 / mL virus, blank holes are sealed with sterile liquid, 200μL / well, 37℃, 5%CO 2 Incubate in the cell incubator for 1 h;

[0274] 4. After the sample and virus are incubated for 1 h, add 100 μL per well and the density is 0.5×10 according to the label. 6 / mL of 293T cells, then put into 37 °C, 5% CO 2 Cell incubator for 24 h;

[0275] 7. Detection: Take out the 96-well white plate and Bio-Lite detection reagent, equilibrate to room temperature, aspirate 150μL / well of liquid in the white plate, then add 100μL of detection reagent to the plate, let stand for 5 min, then use a fluorescence microplate reader to detect the chemiluminescence value (RLU).

[0276] See the results Figure 17 , compared with serum neutralizing antibody titers in immunized mice on day 14, all mutants were better than control vaccines, and G6, G26, G28, G30, G32, G33, and G36 were better than G1 (wild type).

[0277] To sum up, the above-mentioned embodiments and the accompanying drawings are only preferred embodiments of the present invention and do not limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rabies virus G protein mutant, characterized in that: The G protein mutant comprises a mutated wild-type rabies virus G protein extracellular domain, and the mutated wild-type rabies virus G protein extracellular domain can improve the stability of the pre-fusion conformation of the G protein. Preferably, the G protein mutant can induce the production of neutralizing antibodies.

2. The G protein mutant according to claim 1, wherein the wild-type rabies virus G protein is derived from the G protein of the following strains: Pasteur Virus (GenBank accession number is AAA47218.1), Pitman Moore (GenBank accession number is CAI43218.1), HEP-Flury (GenBank accession number is BAC53868.1), MOR1-DG (GenBank accession number is AAK92057.1), MOR3-HM (GenBank accession number is AAK92058.1), NeiMeng1025C (GenBank accession number is ABY19509.2), FRA1-FX (GenBank accession number is AAK92050.1), CNX8601 (GenBank accession number is AAG34722 .1), CHI1-BK (GenBank accession number AAK92060.1), MAU1-CL (GenBank accession number AAK92072.1), Yunnan_Zt07 (GenBank accession number ABX79939.1), RVD (GenBank accession number AAO72525.1), nukovo-32 (GenBank accession number OP642459), CVS-11 (GenBank sequence number ADJ29911.1) or CTN (GenBank accession number AY009100) strains; preferably, the wild-type rabies virus G protein is derived from the G protein of the Pasteur Virus strain (PV strain).

3. The G protein mutant according to claim 2, wherein the amino acid sequence of the extracellular domain of the wild-type rabies virus G protein is derived from amino acids 20-459 of the G protein of the PV strain, or has a sequence corresponding to or aligned with amino acids 20-459 of the G protein of the PV strain; Preferably, the amino acid sequence of the extracellular domain of the wild-type rabies virus G protein is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1; more preferably, the amino acid sequence of the extracellular domain of the wild-type rabies virus G protein is as shown in SEQ ID NO:

1.

4. The G protein mutant according to any one of claims 1 to 3, wherein the G protein mutant has one, two or more amino acid mutations or substitutions at positions 261, 266, 270, 271, 384 and 424 of the extracellular domain of the wild-type rabies virus G protein; Preferably, the mutation or substitution of the two amino acids is selected from the following combinations: H261+H270; D266+H270.

5. The G protein mutant as claimed in claim 4, wherein the residue at the site to be mutated or substituted is mutated to proline, or is substituted by proline; alternatively, the residue at the site to be mutated or substituted is mutated to leucine, or is substituted by leucine.

6. The G protein mutant according to claim 4 or 5, wherein the G protein mutant has one, two or more amino acid mutations or substitutions among H261L, D266P, H270P, L271P, H384P and H424L in SEQ ID NO: 1; Preferably, the two mutations or substitutions present in SEQ ID NO: 1 are selected from the following combinations: H261L+H270P; D266P+H270P.

7. The G protein mutant according to any one of claims 4 to 6, wherein the G protein mutant has an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 9; Preferably, the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2-9; more preferably, the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2, 4-9.

8. The G protein mutant according to any one of claims 1 to 7, wherein a signal peptide or a 6×His tag is introduced at one end of the G protein mutant; preferably, a signal peptide is introduced at the N-terminus of the G protein mutant, and / or a 6×His tag is introduced at the C-terminus; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

18.

9. A rabies virus G protein trimer or polymer, characterized in that: The trimer or polymer comprises the rabies virus G protein mutant according to any one of claims 1-8.

10. The G protein trimer or multimer as described in claim 9, wherein the trimer or multimer is formed by self-assembly of the rabies virus G protein mutant according to any one of claims 1 to 8; preferably, the multimer includes a hexamer, a nonamer and a dodecamer.

11. The G protein trimer or multimer according to any one of claims 9 to 10, wherein the trimer or multimer is prepared by the following method, which comprises the following steps: (1) recombinantly expressing the rabies virus G protein mutant according to any one of claims 1 to 8 in CHO cells by conventional molecular cloning techniques, (2) After expressing in the expression medium for a certain period of time, the cell supernatant is harvested, centrifuged and the supernatant is filtered through a membrane. (3) Purify the filtered supernatant through a nickel column. (4) Collect samples containing the target protein, concentrate by ultrafiltration, and then purify by molecular sieve. (5) Using buffer to dilute the purified target protein, soluble G protein trimers or polymers with uniform particle size can be obtained; in, The buffer used for molecular sieve purification in step (4) is TBS. Optionally, in step (4), the product of the first molecular sieve purification is purified by an anion exchange column and then subjected to a second molecular sieve purification. The buffer in step (5) is TBS or Tween.

12. The G protein trimer or multimer according to claim 11, wherein the step (5) comprises adding 0.02-1% Tween to the purified target protein; Optionally, Tween is selected from Tween-20 or Tween-80; Preferably, the Tween is Tween-80.

13. A method for preparing a G protein trimer or multimer according to any one of claims 9 to 12, comprising the following steps: (1) recombinantly expressing the rabies virus G protein mutant according to any one of claims 1 to 8 in CHO cells by conventional molecular cloning techniques, (2) After expressing in the expression medium for a certain period of time, the cell supernatant is harvested, centrifuged and the supernatant is filtered through a membrane. (3) Purify the filtered supernatant through a nickel column. (4) Collect samples containing the target protein, concentrate by ultrafiltration, and then purify by molecular sieve. (5) Using buffer to dilute the purified target protein, soluble G protein trimers or polymers with uniform particle size can be obtained; in, The buffer used for molecular sieve purification in step (4) is TBS. Optionally, in step (4), the product of the first molecular sieve purification is purified by an anion exchange column and then subjected to a second molecular sieve purification. The buffer in step (5) is TBS or Tween.

14. The preparation method according to claim 13, wherein the step (5) comprises adding 0.02-1% Tween to the purified target protein; Optionally, Tween is selected from Tween-20 or Tween-80; Preferably, the Tween is Tween-80.

15. An immunogenic complex comprising: (1) An antigen component comprising a rabies virus G protein mutant selected from any one of claims 1 to 8, or a rabies virus G protein trimer or multimer according to any one of claims 9 to 12; (2) a granular protein component, which comprises nanoparticle protein; The immunogenic complex formed by the covalent binding reaction between the antigen component and the granule protein component; Preferably, the antigen component further comprises binding peptide 1, the particle protein component further comprises binding peptide 2, the G protein mutant forms a fusion protein with binding peptide 1, the nanoparticle protein forms a fusion protein with binding peptide 2, the antigen component and the particle protein component are covalently bound to each other via binding peptide 1 and binding peptide 2 to form an immunogenic complex; the G protein mutant and binding peptide 1 form a fusion protein and then assemble into a trimer or a multimer; Preferably, the binding peptide 1 contains the amino acid sequence shown in SEQ ID NO: 19, and the binding peptide 2 contains the amino acid sequence shown in SEQ ID NO:

20.

16. The immunogenic complex according to claim 15, characterized in that The antigen component further comprises a connecting peptide 1, and the particle protein component further comprises a connecting peptide 2; the antigen component is formed by fusing a G protein mutant with a binding peptide 1 at the C-terminus through the connecting peptide 1; the particle protein component is formed by fusing a nanoparticle protein with a binding peptide 2 at the N-terminus through the connecting peptide 2; the G protein mutant is assembled into a trimer or a multimer after being fused with the binding peptide 1 at the C-terminus through the connecting peptide 1; Optionally, the connecting peptide 1 is selected from (GGGGS) n , (EAAAK) n 、(GSGGSG) n 、(GGS) n or (GSG) n The amino acid sequence shown, n can be an integer greater than 0 and less than or equal to 5; the connecting peptide 2 is selected from (GGS) n , (GGGGS) n , (EAAAK) n 、(GSGGSG) n An amino acid sequence of, n may be an integer greater than 0 and less than or equal to 10; Optionally, both the antigen component and the particle protein component comprise a histidine tag.

17. The immunogenic complex according to claim 15 or 16, wherein the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2-9; preferably, the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2, 4-9.

18. The immunogenic complex according to any one of claims 15 to 17, wherein the nanoparticle protein is NPM, AP205 or Ferritin protein; preferably, the amino acid sequence of NPM is as shown in SEQ ID NO:

21.

19. A method for preparing an immunogenic complex: (1) The antigen component and granule protein component encoding genes according to any one of claims 15 to 18 are respectively connected into expression vectors to construct expression recombinant plasmids; (2) constructing a recombinant host cell capable of expressing the antigen component and the particle protein component in the host cell; (3) Using recombinant host cells to express the fusion protein and purify the recombinant fusion protein; (4) The antigen component and the particle protein component are subjected to a covalent binding reaction to obtain the immunogenic complex.

20. The preparation method according to claim 19, wherein in step (1), the plasmid for expressing the antigen component is selected from pcDNA3.4, and the vector for expressing the particle protein is selected from pET-28a(+) or pET-30a(+); Optionally, in step (2), the host cell expressing the antigen component is CHO, and the host cell expressing the granule protein is E. coli.

21. An immune composition comprising the rabies virus G protein mutant according to any one of claims 1 to 8, or the rabies virus G protein trimer or multimer according to any one of claims 9 to 12, or the immunogenic complex according to any one of claims 15 to 18; optionally, the immune composition of the present invention further comprises a pharmaceutically acceptable carrier.

22. An immune composition according to claim 21, characterized in that: The pharmaceutically acceptable carrier includes a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster; The stabilizer is preferably sucrose or arginine, the excipient is preferably mannitol, the surfactant is preferably Tween-80, the buffer is preferably disodium hydrogen phosphate dihydrate or sodium dihydrogen phosphate dihydrate, and the pH adjuster is preferably hydrochloric acid.

23. A rabies vaccine, characterized in that: It contains the immune composition according to any one of claims 21-22 and an adjuvant, wherein the adjuvant enhances the immune response through multiple mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages, and the vaccine includes a human vaccine or a veterinary vaccine.

24. The vaccine according to claim 23, wherein the adjuvant is selected from at least one of aqueous adjuvants, mineral adjuvants, oil emulsion adjuvants, microbial adjuvants, liposome adjuvants, cytokine adjuvants, nucleic acid adjuvants, Chinese herbal adjuvants, and chemical adjuvants.

25. According to the vaccine of claim 23, the adjuvant is selected from Toll-like receptor agonists, composite adjuvants, nanoadjuvants, synthetic derivatives of trehalose, genetically engineered toxin-reducing agents or biodegradable polymer microspheres. Preferably, the adjuvant is selected from poly(I:C), GLA-LSQ, flagellin, CpG1018, AS01, AS03, AS04, Covaxin, AS37, PIKACA, BC adjuvant system, SWE, MF59.

26. The vaccine according to claim 23, wherein the component contents (w / w) of the adjuvant are as follows: Squalene 4.2%, Span 85 0.5%, Tween-80 0.5%, Citric acid 0.264%, Sodium citrate 0.016%.

27. A kit, characterized in that: The invention comprises the vaccine according to any one of claims 23 to 26, and the instruments and containers required for inoculating the vaccine, including needle and syringe instruments, powder-carrying containers, and solvent-carrying containers.

28. Use of the rabies virus G protein mutant according to any one of claims 1 to 8, the rabies virus G protein trimer or multimer according to any one of claims 9 to 13, the immunogenic complex according to any one of claims 15 to 18, the immune composition according to any one of claims 21 to 22, the vaccine according to any one of claims 23 to 26, and the kit according to claim 27 for the preparation of drugs for preventing or treating related diseases caused by rabies virus infection, preferably for the preparation of drugs for pre-exposure prevention and post-exposure prevention of rabies virus infection.

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