Preparation method of rabies virus glycoprotein and application thereof

Abstract

The application discloses a preparation method of rabies virus glycoprotein and application thereof, and belongs to the technical field of biological medicines, and comprises the following steps: expressing a glycoprotein G gene of rabies virus containing an extramembrane region, a transmembrane region and an intramembrane region in yeast; performing cell disruption on the yeast, adding a detergent, and obtaining a solution containing rabies virus glycoprotein G; purifying the solution to obtain full-length rabies virus glycoprotein G; and immunizing the prepared full-length rabies virus glycoprotein G through intramuscular injection, oral administration or atomization inhalation. The scheme provided by the application has the advantages of strong immunogenicity, high safety, low production cost and easiness in scaling, solves the problems of complex traditional vaccine process and high cost, and provides a reliable new type of subunit vaccine solution for rabies prevention.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a method for preparing rabies virus glycoprotein and its application. Background Technology

[0002] Rabies virus (RABV) is the prototype member of this genus and the most common causative agent of rabies in humans. The virus is transmitted through the bite of infected animals (most commonly dogs) and can infect and replicate in the central nervous system, leading to severe neurological disorders. The clinical features of RABV infection typically include multiple neurological dysfunctions, which almost inevitably lead to death. Despite the availability of effective rabies vaccines, the disease remains a significant public health problem, causing an estimated 60,000 deaths globally each year.

[0003] Rabies virus (RABV) contains a single-stranded negative-sense RNA genome encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). Among these viral proteins, the RABV glycoprotein (RABVG) plays a crucial role in the pathogenesis of the virus. It mediates viral recognition and attachment to cell receptors, as well as the fusion of the viral envelope and cell membrane after viral endocytosis and acidification of the endosomal cavity, thereby initiating viral entry into the host cell. Furthermore, as a viral surface protein, RABV-G is also a major target for neutralizing antibodies.

[0004] Current rabies vaccines primarily utilize different cell types, such as human diploid cells and African green monkey kidney cells, to prepare whole-virus inactivated vaccines. These vaccines are produced by culturing these cells, allowing the virus to multiply to a sufficient concentration, and then removing impurities through physical and chemical methods to obtain a high-purity virus suspension. The virus is then inactivated using chemical inactivating agents (mainly β-propiolactone), followed by concentration, dilution, and formulation steps to obtain the final virus. After sterilization, the inactivated vaccine is obtained and administered via intramuscular injection. Although this technology is maturing, it still suffers from problems such as weak immunization efficacy, inconsistent immunogenicity within the same batch of vaccine, and high production costs.

[0005] Yeast expression systems, as mature recombinant protein expression platforms, offer advantages such as high expression levels, mild culture conditions, low production costs, and the ability to modify eukaryotic proteins, and have been widely used in vaccine development. However, no rabies virus G protein recombinant subunit vaccine based on a yeast expression system has yet been successfully marketed, primarily due to technical bottlenecks such as easy degradation after protein expression and insufficient immunogenicity.

[0006] Based on the above background, this invention addresses the shortcomings of existing rabies vaccines by constructing a recombinant expression system for rabies virus G protein using a Pichia pastoris expression system. Through antigen design optimization, expression process improvement, and adjuvant compatibility, a recombinant subunit rabies vaccine with strong immunogenicity, high safety, and simple production is developed, providing a new technical solution for rabies prevention. Summary of the Invention

[0007] Based on the current state of the technology, this invention provides a method for preparing rabies virus glycoprotein and its application, specifically a method for preparing wild-type rabies virus G protein nanoparticles based on a pichiasmoyl engineered yeast expression system.

[0008] According to a first aspect of the present invention, a method for preparing rabies virus glycoprotein is provided, comprising the following steps: expressing the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intracellular region, in yeast; disrupting the yeast cells, adding a detergent to obtain a solution containing rabies virus glycoprotein G; purifying the solution to prepare full-length rabies virus glycoprotein G; and immunizing the recipient with the prepared full-length rabies virus glycoprotein G via intramuscular injection, oral administration, or nebulized inhalation.

[0009] Preferably, the method for expressing the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, in yeast involves transforming yeast with a recombinant expression vector containing the rabies virus glycoprotein G gene, culturing the transformed yeast, and then inducing gene expression; the yeast is Pichia pastoris.

[0010] More preferably, the expression vector is obtained by inserting the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, into a vector containing an AOX promoter. Specifically, the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, is inserted between the NotⅠ and XhoⅠ restriction sites of pPICZα and linearized with BglⅡ. The Pichia pastoris is a Pichia pastoris-engineered yeast strain with the accession number CGMCC No. 19488.

[0011] Furthermore, the glycoprotein G of the rabies virus is the glycoprotein G of the wild-type rabies virus. The glycoprotein G of the wild-type rabies virus is the glycoprotein G of the wild-type CVS-11 strain rabies virus, and its amino acid sequence is selected from: (a1) The amino acid sequence shown in SEQ ID NO.3; (a2) The amino acid sequence shown in SEQ ID NO.3, (a2) has ≥95% homology with (a1).

[0012] Furthermore, the method for cell disruption is a physical, biological, or chemical method; the detergent is a non-ionic or weakly ionic detergent.

[0013] Furthermore, the physical method is glass bead oscillation, high-pressure homogenization, or ball milling; the biological method is enzymatic lysis; the chemical method is alkaline lysis; the nonionic detergent is sodium lauroyl sarcosinate, Triton, Tween, or ethyl phenyl polyethylene glycol; and the weakly ionic detergent is deoxycholate or 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate.

[0014] Preferably, the method for purifying the solution includes cation exchange chromatography and / or anion exchange chromatography and / or gel size exclusion chromatography and / or Ni affinity chromatography.

[0015] According to a second aspect of the present invention, an isolated nucleic acid molecule is provided, said nucleic acid molecule encoding the G protein of the wild-type CVS-11 rabies virus strain prepared by the aforementioned preparation method. Preferably, the sequence of said nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO.2.

[0016] According to a third aspect of the technical solution of the present invention, a rabies virus glycoprotein recombinant subunit vaccine prepared using the aforementioned preparation method is provided. The vaccine is administered via intramuscular injection, and the immunization schedule is a four-day immunization schedule on day 0, day 7, day 14, and day 28. Each animal receives 100 μL of immunization per immunization (containing 10 μg of G protein).

[0017] Preferably, in the prepared rabies virus glycoprotein recombinant subunit vaccine, the neutralizing antibody titer induced by the vaccine is ≥0.5 IU / mL, and the animal protection rate against CVS-11 rabies virus challenge is ≥60%.

[0018] According to a fourth aspect of the present invention, the use of a rabies virus glycoprotein recombinant subunit vaccine prepared according to the aforementioned preparation method in the preparation of a medicament for the prevention of rabies is provided, wherein the medicament is configured as an intramuscular injection formulation and contains AS03 adjuvant.

[0019] Compared with the prior art, the above-mentioned technical solution of the present invention has at least the following beneficial effects: (1) This invention provides a recombinant subunit vaccine for rabies virus glycoprotein. It innovatively utilizes pichiasmic engineered yeast (accession number CGMCC No. 19488) to express the full-length glycoprotein of wild-type CVS-11 rabies virus strain, and employs a two-step purification process (Ni affinity chromatography and Superdex-200 molecular sieve chromatography) to obtain nanoparticle antigens with a purity ≥80%. The core inventive point of this invention lies in the first successful preparation of the full-length rabies virus G protein based on glycosyl engineered yeast, which, when combined with AS03 adjuvant and administered via intramuscular injection, exhibits significant immunoprotective effects, specifically manifested in the following beneficial effects: (2) Strong immunogenicity and reliable safety: This vaccine completely preserves the key neutralizing antibody epitopes of the rabies virus G protein (including the extracellular, transmembrane, and intracellular regions). When mice are immunized by intramuscular injection (four-immunization schedule: days 0, 7, 14, and 28), it can induce a highly efficient specific humoral immune response with a neutralizing antibody titer ≥0.5 IU / mL (up to 2.12 IU / mL), far exceeding the WHO's effective protection standard, and achieving an animal protection rate of over 60% against rabies virus challenge. At the same time, the vaccine contains only purified antigen protein and no viral genetic material, avoiding the infection risk and batch-to-batch inconsistency problems of traditional inactivated vaccines.

[0020] (3) The production process is simplified and the cost is low: The Pichia pastoris expression system has the advantages of mild culture conditions and high expression level (3-8 mg / L fermentation broth). Combined with the two-step purification process, the production cost and complexity are greatly reduced. This process is easy to scale up and solves the technical bottleneck of high production difficulty and high cost of traditional cell vaccines (such as Vero cell vaccines).

[0021] (4) Broad application prospects: The vaccine of this invention has high efficiency, safety and industrial adaptability, providing a new technical solution for the prevention of rabies, and is especially suitable for large-scale immunization and promotion in areas with limited resources. Attached Figure Description

[0022] Figure 1 This is a graph showing the IgG antibody titers of anti-rabies virus in the serum of mice immunized with a mixture of rabies virus, AS03, and MF59 adjuvants two weeks after the second, third, and fourth immunizations. Figure 2 This is a schematic diagram of the structure of the rabies virus G protein; Figure 3 This is a screening diagram of rabies virus G protein positive clones; Figure 4 This is an SDS-PAGE image of rabies virus G protein purified by nickel column chromatography. Figure 5 This is the result of a rapid fluorescent focal inhibition experiment of rabies virus.

[0023] Figure 6 This is a graph showing the animal protection rate results from a rabies virus challenge experiment. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In the following detailed description, reference is made to the accompanying drawings, which form part of this invention and illustrate specific embodiments thereof. In the drawings, similar reference numerals describe substantially similar components in different figures. Specific embodiments of the invention are described in sufficient detail below to enable those skilled in the art to implement the invention. It should be understood that other embodiments may be utilized or structural and logical changes may be made to the embodiments of the invention.

[0026] All expression systems of this invention are available in publicly available literature. Those skilled in the art should understand that even the same bacterium or species may have slightly different growth characteristics due to different sources, but their functions are essentially the same. Therefore, the bacteria or cells mentioned in this invention may also include modified forms of these bacteria or cells.

[0027] The terms used in this invention have the following meanings: In this invention, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "as" or "consisting of".

[0028] The term "around" typically refers to being 0.5% above or below a specified value. Variation within a range of 10%, for example, variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0029] The term "vaccine" or "vaccine composition" refers to a substance or combination of one or more components that, when administered directly or via a carrier to the body via immunization, targets the pathogen of a disease or its associated proteins (peptides, polypeptides), polysaccharides, or nucleic acids. This substance or combination induces specific humoral and / or cellular immunity, thereby providing the body with the ability to prevent the disease.

[0030] Genetically engineered vaccines are vaccines made using recombinant DNA technology to clone and express protective immunogenic gene fragments, utilizing the expressed immunogenic fragment product or the recombinant itself. In preparing genetically engineered vaccines, recombinant DNA biotechnology is used to directionally insert natural or artificially synthesized genetic material into bacterial, yeast, or mammalian cells, allowing for full expression, followed by purification to obtain the vaccine. Genetic engineering technology can be used to produce subunit vaccines without infectious substances, stable attenuated vaccines, and multivalent vaccines that can prevent multiple diseases.

[0031] Glycoprotein vaccines are a type of genetically engineered vaccine. In some embodiments, the glycoprotein vaccine of the present invention includes an immunogenic fragment.

[0032] In this invention, the composition is a composition in which the components have been separated and purified to a purity of at least 50%, 60%, 70%, 80%, or 90% before being mixed to form an antigenic composition. For example, the composition may be an aqueous solution of a water-soluble protein. For example, the composition may contain a detergent. For example, the composition may contain non-... Ionic, zwitterionic, or ionic detergents. For example, subunit compositions may contain lipids. In some cases, immunogenic compositions include immunogenic fragments.

[0033] The term "neutralizing antibody" refers to a soluble protein secreted by B lymphocytes. When pathogenic microorganisms (such as viruses) invade the body, the body produces corresponding antibodies. Pathogenic microorganisms require specific molecules expressed by the pathogen to bind to receptors on the target cell in order to infect the cell and further proliferate. Neutralizing antibodies can bind to immunogenic fragments on the surface of pathogenic microorganisms, thereby preventing the pathogen from adhering to target cell receptors and thus preventing its invasion of the cell.

[0034] The term "glycosylation modification" refers to protein glycosylation, one of the most common post-translational modifications of proteins. It involves the transfer of carbohydrates to specific amino acid residues on the protein by glycosyltransferases, forming glycosidic bonds. Protein glycosylation can be mainly classified into two types: N... Glycosylation and O Glycosylation. Among them, "O" "Glycosylation modification" refers to O The sugar chain is covalently linked to the free OH groups of serine or threonine residues in the protein. Glycosylation sites lack conserved sequences, and glycan chains do not have a fixed core structure; they can be either a monosaccharide or a large sulfonated polysaccharide. The "N" involved in this invention... "Glycosylation modification" refers to N Glycan chains bind to the free radicals of aspartic acid in proteins. NH2-based covalent linkage.

[0035] The term "glycoprotein" refers to the full-length G protein of the CVS-11 strain, including an extracellular domain, a transmembrane domain, and an intracellular domain. Glycoproteins are prepared by expression within a vector. In some embodiments, they can be prepared by recombinant expression in *E. coli*, mammalian cells, or yeast cells. In some embodiments, glycosylated engineered yeast is used to prepare the glycoprotein; preferably, wild-type glycoproteins are prepared using yeast CGMCC No. 19488 (i.e., *Pichia pastoris* genetically modified via a glycosylation pathway, strain deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 19488). This invention does not limit the method of obtaining the glycoprotein; glycoproteins obtained by any other means that have the same structure, function, etc., as those of this invention are also within the scope of protection of this invention.

[0036] In some embodiments, the term "homology" in the above-described proteins refers to the identity of amino acid sequences. In some embodiments, the identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting Gapexistencecost, Perresiduegapcost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0037] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. Vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. Vectors also include vectors having multiple of the above-described functions. A vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0038] In some embodiments of the present invention, the term "recombinant vector" specifically refers to a recombinant vector obtained by cloning the coding genes of the influenza virus hemagglutinin and cholera toxin B subunit into an expression vector, such as the pPICZαA vector (which contains restriction enzyme sites, such as XhoⅠ and SaLⅠ). In other embodiments, the expression vector is selected from one or more of the following vectors: pPIC9, pPIC9K, pPICZαB, pPICZαB vector, pET series vectors, pGEX series vectors, pMAL series vectors, pQE series vectors, pBADmycHis series vectors, pTrcHis series vectors, pTXB series, T series vectors, and other vectors, as well as modified vectors of the above vectors. The present invention does not limit the method of obtaining the recombinant vector; recombinant vectors of the present invention obtained by other means are also within the scope of protection of the present invention.

[0039] The terms "coding gene" and "nucleic acid molecule" refer to ribonucleotide (RNA) or deoxyribonucleotide (DNA) sequences that encode specific amino acid peptide chains. In some embodiments, the coding gene or nucleic acid molecule can be obtained through whole-gene synthesis, PCR amplification, chemical synthesis, etc. This invention does not limit the method of obtaining the coding gene and nucleic acid molecule.

[0040] In some embodiments, the term "more than 95% homology" in the proteins and genes described above may mean at least 96%, 97%, or 98% identity. The term "more than 90% identity" may mean at least 91%, 92%, 93%, or 94% identity.

[0041] The term "host cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains plasmids or vectors including the nucleic acid molecules described in this invention, or that is capable of expressing the antibodies or antigen-binding fragments described in this invention. Host cells may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the fusion protein described in this invention. Host cells can be obtained by in vitro transfection of cells using the vectors described in this invention. The host cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS cells). 1. Cells, NSO cells, or myeloma cells). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell may be a CHO cell.

[0042] In some embodiments of the present invention, the recombinant expression cells are prepared by introducing the encoding gene of influenza virus hemagglutinin into host cells such as *Escherichia coli*, mammalian cells, yeast cells, or *Pichia pastoris* genetically modified via glycosylation through a recombinant vector. The present invention does not limit the method of obtaining the recombinant expression cells; recombinant expression cells or recombinant cells obtained by other means are also within the scope of protection of the present invention.

[0043] The present invention will be described below with reference to examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although similar or equivalent methods and materials described herein may also be used to practice this invention, as will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated herein by reference in their entirety. In case of inconsistency, this specification, including definitions, shall prevail. Materials, methods, and embodiments are illustrative only and not intended to be limiting.

[0045] pPICZαA and GS115 Pichia pastoris are products of Invitrogen. Pichia pastoris strain 19488 is deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 19488.

[0046] The Q5 enzyme, Taq enzyme, dNTPs, restriction endonucleases, and T4 ligase used in the experiment were purchased from NEB. Pfu enzyme, kits, and DH5α competent cells were products of Beijing TransGen Biotech Co., Ltd. Whole-genome synthesis, nucleotide synthesis, primer synthesis, and sequencing were provided by Beijing Qingke Biotechnology Co., Ltd.

[0047] The rabies virus primary antibody (40997-T62) is a product of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; the HIS antibody, goat anti-rabbit IgG secondary antibody (SAB3700885), and enzyme-linked immunosorbent assay (ELISA) coated antigen (LD-PRD-10477) are products of Sigma-Aldrich; the goat anti-mouse IgG secondary antibody (ab205719) is a product of Abcam; and the BglII restriction endonuclease is a product of NEB.

[0048] Nickel ion affinity chromatography (Chelating FastFlow column) is a product of GE Healthcare.

[0049] The specific formulation of YPD liquid medium containing 100 μg / ml Zeocin is as follows: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 100 μg / ml Zeocin. Zeocin is a product of Invitrogen, catalog number ant-zn-1.

[0050] BALB / c (01011) is a product of Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0051] This invention discloses a method for preparing rabies virus glycoprotein and its application, belonging to the field of biomedical technology. It is the first to achieve the preparation of full-length rabies virus glycoprotein (G) based on glycosyl engineered yeast. The full-length sequence of wild-type rabies virus G protein is expressed using Pichia pastoris-based engineered yeast, and nanoparticle antigens with a purity ≥80% are obtained through a two-step purification process (affinity chromatography and molecular sieve chromatography). When combined with AS03 adjuvant, this vaccine, upon intramuscular injection, induces highly effective neutralizing antibodies (titer ≥0.5 IU / mL) and an animal protection rate of over 60%, demonstrating significant immunoprotective effects. The solution provided by this invention has advantages such as strong immunogenicity, high safety, low production cost, and ease of scalability, solving the problems of complex and costly traditional vaccine processes and providing a reliable novel subunit vaccine solution for rabies prevention. The recombinant subunit rabies virus G protein vaccine prepared by this invention can greatly improve vaccine safety. This vaccine contains only purified single antigen protein, does not contain viral genetic material, has no risk of infection, and eliminates the safety hazards caused by incomplete inactivation.

[0052] This invention addresses the technical pain points of existing rabies vaccines, such as complex production processes, high costs, and insufficient immunoprotective efficacy. Through precise gene cloning and construction, efficient induction and expression, multi-step chromatographic purification, and optimized adjuvant compatibility, a novel nanoparticle antigen with good uniformity is prepared. After intramuscular injection, it can efficiently induce the body to produce high-titer protective neutralizing antibodies, achieving a 60% animal protection rate. This provides a novel vaccine solution for rabies prevention with stable immunogenicity, controllable production process, and excellent safety.

[0053] The present invention first provides a method for preparing rabies virus glycoprotein, wherein the core antigen of the vaccine is wild-type CVS-11 strain rabies virus G protein, and the G protein comprises an extracellular region (amino acids 1-439), a transmembrane region (amino acids 440-461), and an intramembrane region (amino acids 462-504).

[0054] In some embodiments, the amino acid sequence of the wild-type CVS-11 rabies virus G protein is selected from: (a1) SEQ ID NO:1 is the amino acid used in this study; In some embodiments, the amino acid sequence is: KFPIYTIPDKLGPWSPIDIHNLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRSAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESVVIISPSVADLDPYDKSLHSRVFPRGKCSGITVSSAYCSTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAIQTSNETKWCPPDQLVNLHDFHSDEIEHLVVEELVEKREECLDALESIMTTNPVSFRRLSPLRKLVPGFGKAYTIFNKTLMEADAHYKSVQTWDEIIPSKGCLRVGARCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHKQVSGVDLGLPNWGKDVLMGAGVLTALMLMIFLMTCCRRTNRAESIQHSLGETGRKVSVTSQSGRVISSWESYKSGGETKL (SEQ ID NO.1).

[0055] In certain embodiments, the nucleotide sequence:

[0056] (a2) AAG34724.1 is the G protein amino acid sequence of the rabies virus CVS-11 strain shown in GeneBank. This sequence has ≥95% homology with the amino acid sequence shown in SEQ ID NO:1 and encodes the full-length G protein.

[0057] In some embodiments, the GenBank is numbered AAG34724.1.

[0058] In some embodiments, the amino acid sequence is: (SEQ ID NO.3) This invention also provides a recombinant subunit vaccine, which is prepared by mixing a core antigen and an AS03 adjuvant at a volume ratio of 1:1. The antigen is 10 μg. The vaccine is administered via intramuscular injection to BALB / c mice at a dose of 10 μg per mouse, with an immunization schedule of four immunizations on days 0, 7, 14, and 28. The vaccine can induce the production of specific IgG antibodies and neutralizing antibodies, with a neutralizing antibody titer ≥0.5 IU / mL (meeting WHO effective protection standards), and a protection rate ≥60% against ERA strain rabies virus challenge.

[0059] The preparation of the core antigen in recombinant subunit vaccines includes the following steps: The G protein-coding gene was amplified from the CVS-11 rabies virus genome using PCR, and a lipid-soluble linker and a 6×His tag coding sequence were fused to the 3' end of the gene. The fusion gene was directionally cloned into the pPICZαA vector to construct a recombinant expression plasmid. After linearization by BglII restriction enzyme digestion, the plasmid was electrotransformed into pichia pastoris-engineered yeast. Positive single colonies were screened, expanded in YPD / Z liquid medium, and then transferred to BMMY medium. Expression was induced for 48 h at pH 6.5 and 25℃. After physical lysis, the colonies were pretreated with membrane proteins and purified sequentially by Ni affinity chromatography and Superdex-200 molecular sieve chromatography to obtain nanoparticle antigens. Pichia pastoris-engineered yeast exhibits glycosylation similar to that of mammalian cells.

[0060] In some embodiments, the G protein is obtained from cultured glycosyl engineered yeast cells via a two-step chromatographic purification process, the purification process including Ni affinity chromatography and Superdex-200 molecular sieve chromatography steps; the vaccine also contains AS03 adjuvant or other pharmaceutically acceptable adjuvants.

[0061] In some embodiments, the glycosylated engineered yeast is a Pichia pastoris strain with accession number CGMCC No. 19488, which can stably express the wild-type CVS-11 rabies virus G protein with correct glycosylation modification.

[0062] This invention also provides an isolated nucleic acid molecule encoding the G protein of the wild-type CVS-11 rabies virus strain described above. In some embodiments, the sequence of the nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO:2. This invention provides the above-described rabies virus glycoprotein recombinant subunit vaccine, which is administered via intramuscular injection in a four-immunization schedule on days 0, 7, 14, and 28, with each animal receiving a dose of 100 μL (containing 10 μg of G protein) per immunization.

[0063] In some embodiments, the neutralizing antibody titer induced by the vaccine is ≥0.5 IU / mL, and the protection rate against CVS-11 rabies virus challenge is ≥60%.

[0064] The present invention provides the use of the above-mentioned rabies virus glycoprotein recombinant subunit vaccine in the preparation of a medicament for the prevention of rabies, wherein the medicament is configured as an intramuscular injection formulation and contains AS03 adjuvant.

[0065] Furthermore, in the following embodiments, unless otherwise specified, the first position of each nucleotide sequence is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0066] The present invention is illustrated using the wild-type rabies virus CVS-11 glycoprotein as an example.

[0067] Example 1. Expression and preparation of rabies virus CVS-11 wild-type recombinant glycoprotein vaccine (I) Cloning and Identification of Core Antigen Genes 1. Antigen target selection: The full-length G protein of wild-type CVS-11 rabies virus (GenBank sequence number AAG34724.1) was selected as the sole immunogenic target. This protein contains an extracellular region (amino acids 1-439), a transmembrane region (amino acids 440-461), and an intracellular region (amino acids 462-504), and retains the neutralizing antibody epitopes completely. Its native conformation can effectively stimulate the body's specific immune response.

[0068] 2. Gene amplification and modification: Based on the CVS-11 rabies virus G protein coding gene sequence, specific primers were designed, and the target gene fragment was amplified from the viral genome using PCR technology; a lipid-soluble linker coding sequence and a 6×His tag coding sequence were fused to the 3' end of the target gene to construct a fusion gene, providing a molecular basis for subsequent protein purification and identification.

[0069] (II) Construction of recombinant expression vectors and screening of engineered bacteria 1. Vector construction and sequencing verification: The modified fusion gene was directionally cloned into the pPICZαA expression vector to construct the recombinant expression plasmid pPICZαA-CVS11-RABV-G-linker-6×His; after linearization by BglII restriction enzyme digestion, the accuracy of the gene sequence was verified by sequencing to ensure that there were no frameshift mutations or base mismatches.

[0070] 2. Yeast transformation and resistance screening: Linearized recombinant plasmids were introduced into Pichia pastoris-engineered yeast host cells using electroporation technology; the transformed bacterial culture was spread on YPD solid medium containing Zeocin resistance (final concentration 100 μg / mL), and incubated upside down at 25℃ for 48-72 h, and single colonies were picked.

[0071] 3. Seed culture preparation: The monoclonal colonies obtained from resistance screening were inoculated into 1.5 ml of YPD / Z liquid medium and cultured at 25°C with shaking at 220 rpm until OD200. 600 =2.0-6.0, to obtain seed liquid.

[0072] (III) Induction of target protein expression and identification of positive clones 1. Optimization of induction expression: The seed culture was transferred to BMMY induction medium at a ratio of 1:10 (v / v), the initial pH was adjusted to 6.5, and the culture was induced in a shaking incubator at 25℃ and 220r / min for 48h. During this period, methanol was added every 24h to a final concentration of 1% to maintain the induction efficiency.

[0073] 2. Cell disruption and protein extraction: After the induction expression is completed, the fermentation cells are collected, and the cells are physically disrupted using disrupting beads. The supernatant is collected by centrifugation to obtain a crude extract containing the target protein.

[0074] 3. Screening of positive clones: The crude extract was identified using Western blot technology. Anti-rabies virus antibody was used as the primary antibody and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody. The expression of the target protein was detected by chemiluminescence colorimetry, and positive clones with high expression of G protein were screened.

[0075] (iv) Large-scale preparation and multi-step chromatography purification of the target protein 1. Large-scale culture: Positive clones were inoculated into 200mL BMMY shake flasks for large-scale culture, maintaining pH 6.5 and temperature 25℃. After the bacterial cell count reached its peak, the target protein was induced to express according to the above induction conditions.

[0076] 2. Membrane protein pretreatment: After fermentation, the cells were collected, crushed, and centrifuged to obtain a crude extract. The membrane protein components were enriched by PEG20000 precipitation method (1% PEG + 0.9% NaCl + 10mm PB7.0, ice bath for 30 min), and dissolved in buffer containing 0.2% sodium lauroyl sarcosinate to achieve solubilization and preliminary separation of membrane proteins.

[0077] 3. Two-step chromatography purification: Step 1: Ni affinity chromatography purification. Load the solubilized sample onto a Ni-FastFlow affinity chromatography column, equilibrate the column with Tris-HCl buffer (pH 7.4) containing 20 mmol / L imidazole, wash with buffer containing 50-80 mmol / L imidazole to remove contaminating proteins, and finally elute the target protein with buffer containing 250-300 mmol / L imidazole. Collect the elution peak fraction.

[0078] Step 2: Superdex-200 molecular sieve chromatography purification. The sample purified by ion exchange chromatography was concentrated to 1-2 mL and loaded onto a Superdex-200 gel filtration chromatography column. The mobile phase was PBS buffer (pH 7.4) containing 150 mmol / L NaCl and 0.1% Tween, with a flow rate of 0.8 mL / min. The final purification of the target protein was achieved based on the molecular weight difference. The target elution peak was collected to obtain uniform wild-type CVS-11 rabies virus G protein nanoparticle antigen. Dynamic light scattering (DLS) detection showed that the particle size was uniform.

[0079] (v) Vaccine formulation preparation and animal immunization experiments 1. Vaccine formulation preparation: The purified nanoparticle antigen and AS03 adjuvant are mixed evenly at 50%. The AS03 adjuvant components are 2.5% squalene, 0.5% Tween 80 and 0.5% Span 85. After thorough emulsification, the vaccine formulation is prepared.

[0080] 2. Animal immunization program: 6-8 week old BALB / c mice were selected as experimental animals and immunized by intramuscular injection. The four-immunization program was implemented (immunization time points: day 0, day 7, day 14, and day 28). The immunization dose for each mouse was 100 μL (containing 10 μg of target protein).

[0081] 3. Serum sample collection: On day 7 after the last immunization (days 14 and 28), orbital venous blood was collected from mice. After standing at 37°C for 60 minutes, the blood was centrifuged at 10,000 r / min for 10 minutes to separate the serum, which was then frozen at -20°C for later use.

[0082] (vi) Comprehensive evaluation of immunization effect Specific IgG antibody detection: The titer of G protein-specific IgG antibodies in serum was detected using an indirect ELISA method. Purchased rabies virus antigen (2 μg / mL) was used as the coating antigen. Serum samples were serially diluted to serve as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. Odulation was measured after TMB colorimetric development. 450 Value, calculate antibody titer (in OD). 450 The highest dilution (≥0.1 and 2.1 times that of the negative control) is the endpoint titer. Results are as follows: Figure 1 As shown, the titer of IgG antibodies in the serum of mice increased significantly over time after immunization.

[0083] The structural integrity of the rabies virus G protein during vaccine preparation is illustrated in the diagram, as shown below. Figure 2 As shown, the key structures of the extramembrane region, transmembrane region, and intramembrane region are clearly presented.

[0084] Positive clones were screened and identified by Western blot, and the results were as follows: Figure 3As shown, high-efficiency expression of the G protein was confirmed. The optimization of the purification process was validated by SDS-PAGE analysis, and the results are as follows. Figure 4 As shown, high-purity nanoparticle antigens were obtained after Ni affinity chromatography and Superdex-200 molecular sieve chromatography.

[0085] Neutralizing antibody titer detection: Serum neutralizing antibody titers were detected using the Rapid Fluorescent Focus Inhibition (RFFIT) assay. Vero cells were used as the host cells, and ERP rabies virus was used as the target virus. The experiment was performed according to WHO standard operating procedures, and the neutralizing antibody titer (IU / mL) was calculated. Results are as follows: Figure 5 As shown, the neutralizing antibody titers were all ≥0.5 IU / mL, meeting the WHO protection standards. Furthermore, rabies virus challenge experiments showed that the vaccine achieved an animal protection rate of over 60% against the CVS-11 rabies virus strain, as shown in the results. Figure 6 As shown, the protective efficacy of the vaccine is confirmed. The above results collectively demonstrate that the vaccine of this invention possesses strong immunogenicity and good protective effect.

[0086] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, it is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing a rabies virus glycoprotein, comprising the following steps: expressing the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intracellular region, in yeast; lysing the yeast cells, adding a detergent to obtain a solution containing rabies virus glycoprotein G; purifying the solution to prepare full-length rabies virus glycoprotein G; and immunizing the recipient with the prepared full-length rabies virus glycoprotein G via intramuscular injection, oral administration, or nebulized inhalation.

2. The method of claim 1, wherein: The method for expressing the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, in yeast involves transforming a recombinant expression vector of the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, into yeast, culturing the transformed yeast, and then inducing gene expression; the yeast is Pichia pastoris.

3. The method of claims 1-2, wherein: The expression vector is obtained by inserting the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, into a vector containing an AOX promoter. Specifically, the rabies virus glycoprotein G gene, which includes an extracellular region, a transmembrane region, and an intramembrane region, is inserted between the NotⅠ and XhoⅠ restriction sites of pPICZα and linearized with BglⅡ. The Pichia pastoris is a glycosyl engineered Pichia pastoris strain with accession number CGMCC No. 19488.

4. The method of claim 3, wherein: The glycoprotein G of the wild-type rabies virus is the glycoprotein G of the wild-type CVS-11 strain rabies virus, and its amino acid sequence is selected from: (a1) The amino acid sequence shown in SEQ ID NO.1; (a2) The amino acid sequence shown in SEQ ID NO.3, (a2) has ≥95% homology with (a1).

5. The method of claim 4, wherein: The physical method is glass bead oscillation, high-pressure homogenization, or ball milling; the biological method is enzymatic lysis; the chemical method is alkaline lysis; the nonionic detergent is sodium lauroyl sarcosinate, Triton, Tween, or ethyl phenyl polyethylene glycol; the weakly ionic detergent is deoxycholate or 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate.

6. An isolated nucleic acid molecule, comprising, The nucleic acid molecule encodes the G protein of wild-type CVS-11 rabies virus prepared by any of the preparation methods described in claims 1-5.

7. The isolated nucleic acid molecule of claim 6, wherein, The sequence of the nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO.

2.

8. A rabies glycoprotein recombinant subunit vaccine prepared using the process as claimed in any one of claims 1 to 5, characterized in that, The vaccine is administered via intramuscular injection. The immunization schedule is a four-day immunization program on days 0, 7, 14, and 28, with each animal receiving a dose of 100 μL (containing 10 μg of G protein) per immunization.

9. The recombinant subunit vaccine of rabies glycoprotein according to claim 8, characterized in that, The vaccine induces neutralizing antibody titers ≥0.5 IU / mL, and provides ≥60% protection against CVS-11 rabies virus challenge in animals.

10. The use of the rabies virus glycoprotein recombinant subunit vaccine prepared by the preparation method of any one of claims 1-5 in the preparation of a medicament for the prevention of rabies, wherein the medicament is configured as an intramuscular injection formulation and contains AS03 adjuvant.

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