Varicella-zoster virus gB protein mutant and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a varicella-zoster virus gB protein mutant and application thereof. According to the gB protein mutant of the varicella-zoster virus, provided by the invention, proline mutation, shearing of N-terminal and C-terminal structural domains, release of electrostatic repulsive force in the protein, mutation of a Frelin enzyme cutting site and the like are carried out in a process of converting a gB protein fusion precursor into a fusion afterbody conformation, so that the gB protein mutant of the varicella-zoster virus is obtained. The conformation before fusion can be stabilized, and the expression quantity and the stability of the antigen protein are improved. As a vaccine component, the mutant has good immunogenicity, activates autoimmunity of animals and plays a role in prevention or treatment of vaccines.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a varicella-zoster virus gB protein mutant and an application thereof. Background Art

[0002] Varicella-zoster virus (VZV) belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, and is an enveloped double-stranded DNA virus. Primary infection can cause chickenpox, and latent infection reactivation can lead to herpes zoster. There is no specific treatment for herpes zoster in clinical practice, and there are complications such as postherpetic neuralgia (PHN).

[0003] There is no specific treatment for herpes zoster in clinical practice. Vaccination is the most economical and effective measure to prevent herpes zoster caused by VZV. The successful development of preventive biological products depends on the selection of appropriate viral proteins as antigens. The genome of VZV includes 71 ORFs, which can encode 13 glycoproteins. At least six glycoproteins, gE (gp I), gB (gp II), gH (gp III), gI (gp IV), gC (gp V) and gL (gp VI), have been found to coexist on the surface of the viral envelope and the membrane surface of infected cells. In vitro studies have shown that these six glycoproteins can induce the production of neutralizing antibodies. Shingrix, a preventive vaccine with gE protein as antigen, has been proven to be effective, while vaccine products with other glycoproteins as antigens are still in a very early stage of research and development. Among them, gB is the most conserved glycoprotein among all herpes viruses. Preventive vaccines with gB as antigen are expected to stimulate a broader spectrum of immune protection, which is a good research and development direction. As disclosed in US Patent US20210163542A1, the vaccine composition of the combination of herpes virus envelope proteins that induces immune response comprises gB, gH / gL heterodimer and gH. Prior art "Wang Zhiyu. Immunogenicity of purified herpes simplex virus glycoprotein gB [J]. International Journal of Biologicals, 1987(05):40-41. DOI:CNKI:SUN:GWYC.0.1987-05-035." verified the feasibility of using purified gB and appropriate adjuvants as vaccine components in mice.

[0004] At present, the research of existing multiple herpesvirus proves that gB is a membrane fusion protein. All enveloped viruses, including herpesvirus, must be fused to the host membrane by membrane fusion protein to transfer their genome into the target cell to start infection, and the protein changes from the initial conformation (before fusion) to the final, more stable conformation (after fusion). Antibodies and vaccines that can block the above steps are often excellent in effect. As demonstrated by the I-class membrane fusion protein F of respiratory syncytial virus (RSV), the pre-fusion conformation provides an attractive target for drug and vaccine development, and the most effective neutralizing antibodies are only directed to this conformation. However, the natural state gB protein of herpesvirus is difficult to realize soluble expression and conformational instability, even on the natural membrane, the full-length gB trimer mixture existing in the pre-fusion or post-fusion conformation has also been found, and gB only presents the post-fusion conformation after purification by a specific method, so people know very little about the pre-fusion conformation of gB, and it is more challenging to prepare preventive vaccines with the pre-fusion conformation gB protein as antigen. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a gB protein mutant of varicella-zoster virus, and provides a mutant that can stably present a pre-fusion conformation, providing a new direction for clinical application.

[0006] The present invention focuses on the study of the pre-fusion conformation of the VZV membrane fusion protein gB. Based on the guidance of structural biology and bioinformatics, specific mutants that can stably present the pre-fusion conformation are designed and expressed at high levels in mammalian cell lines, in order to lay the foundation for the development of new products to prevent and block VZV infection.

[0007] In one aspect, the present invention provides a mutant of the gB protein of varicella-zoster virus.

[0008] In the mutant, the amino acid that undergoes alpha helix penetration during the transformation of the gB protein fusion precursor to the fusion postform is modified with proline or a proline analog.

[0009] The proline analogues may generally be proline derivatives.

[0010] The types of proline derivatives include, but are not limited to, chemical group-substituted proline derivatives, racemates, diastereomers, and proline salts.

[0011] The chemical group can be: a straight chain or branched alkyl group, a phenyl group, a substituted phenyl group, a naphthyl group, a substituted naphthyl group, a hydroxyl group, a benzyl group, a substituted benzyl group, an alkoxyalkyl group, an acyl group (such as a formyl group, an acetyl group, a chloroacetyl group, a trifluoroacetyl group, a substituted or unsubstituted benzoyl group, a substituted or unsubstituted phenylacetyl group), an alkoxycarbonyl group (such as a methoxyformyl group, an ethoxyformyl group, a tert-butyloxycarbonyl group, a fluorenylmethoxycarbonyl group) or an aralkyloxycarbonyl group (such as a benzyloxycarbonyl group).

[0012] The proline salt may be a D-type proline salt or an L-type proline salt.

[0013] Specifically, the modification may be a substitution or insertion of proline or a proline analog.

[0014] Specifically, the sequence of the gB protein is SEQ ID NO.1, and the amino acid whose modification occurs to penetrate the alpha helix may be the amino acid between 454-466.

[0015] The modification may be any one or more mutations to proline or a proline analog, and / or any insertion of one or more proline or a proline analog.

[0016] Preferably, the modification is any one mutation to proline or a proline analog.

[0017] Specifically, the feline protease cleavage site in the mutant is inactivated or does not exist.

[0018] Preferably, the inactivation or absence is achieved by deleting or replacing the fragment containing the ferin protease cleavage site in SEQ ID NO.1 with a linker.

[0019] Specifically, the deletion may be the deletion of 1-23 amino acids between positions 415-440; the replacement with a linker may be the replacement of amino acids between positions 428 and 431 with a linker of 1-10 amino acids in length.

[0020] Preferably, the mutant further comprises mutation of one or more charged amino acids into polar amino acids, hydrophobic amino acids or aromatic amino acids.

[0021] Specifically, the charged amino acid is the lysine at position 670 of SEQ ID NO.1 mutated into a polar amino acid, a hydrophobic amino acid or an aromatic amino acid.

[0022] Preferably, the charged amino acid is lysine at position 670 of SEQ ID NO.1, which can be mutated to any one of tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine, phenylalanine, valine, leucine, isoleucine, proline, alanine, and arginine; more preferably, it is one of tryptophan, alanine, arginine, and asparagine.

[0023] More preferably, the charged amino acid is lysine at position 670 of SEQ ID NO.1 mutated to asparagine.

[0024] The gB protein mutant also includes a C-terminal transmembrane and intracellular region deleted and connected to a heterologous trimerization domain that helps maintain a trimer conformation.

[0025] Specifically, amino acids 680-690 and thereafter at the C-terminus of SEQ ID NO.1 may be deleted, preferably amino acids after 685 are deleted.

[0026] The heterologous trimerization domain includes, but is not limited to, any one or more of a collagen trimerization domain, a glycoprotein trimerization domain, and a fibrin trimerization domain.

[0027] Specifically, the heterologous trimerization domain may be the amino acid sequence shown in SEQ ID NO.2 or an optimized sequence thereof.

[0028] The optimization type may be: increasing interchain electrostatic forces, hydrophobic forces (van der Waals forces) or disulfide bonds through point mutations.

[0029] In another aspect, the present invention provides a nucleic acid molecule encoding the aforementioned gB protein mutant.

[0030] The present invention also provides an expression vector comprising the nucleic acid molecule.

[0031] The types of the expression vector include, but are not limited to, any one or more of CMV vector, pcDNA3.1 vector, and pSV2 expression vector.

[0032] Preferably, the expression vector is pKS001 vector.

[0033] The present invention also provides a cell comprising the aforementioned gB protein mutant or nucleic acid molecule or expression vector.

[0034] The cells are generally genetically engineered cells with expression function, including but not limited to: any one or more of CHO, 293, and SF9 cells.

[0035] The present invention also provides a preparation method or a purification method for the above gB protein mutant, nucleic acid molecule, expression vector, and genetic engineering cell.

[0036] Specifically, by methods already disclosed in the prior art, such as synthesizing nucleic acid molecules encoding gB protein mutants, preparing expression vectors by enzyme ligation, transfecting cells with expression vectors to prepare genetically engineered cells, column purification, etc., but the present invention is not limited to specific preparation methods, and all preparation methods that can prepare the gB protein mutants, nucleic acid molecules, expression vectors, and genetically engineered cells of the present invention obtained according to the prior art are within the scope of protection of the present invention.

[0037] The site selected for the enzyme cleavage may be one that has been disclosed in the art or one that has not been disclosed, and any enzyme cleavage site that can achieve the expression of a gB protein mutant by the corresponding nucleic acid molecule may be used.

[0038] Preferably, the encoding nucleic acid sequence of the gB protein mutant is codon-optimized and synthesized using CHO as a host, cloned into a pKS001 vector using HindIII and NotI, and then transformed into CHO for expression.

[0039] Preferably, the purification method of the gB protein mutant can be column purification, and the column purification can be one-step or two-step.

[0040] Specifically, the column purification includes but is not limited to using UniGel-80SP (-(CH 2 ) 3 SO 3 -ligand) column or HiLoad 16 / 600Superdex 200 TM Purification was performed using a PG column.

[0041] Preferably, UniGel-80SP (-(CH 2 ) 3 SO 3 -ligand) columns and HiLoad 16 / 600Superdex 200 TM Purification was performed using a PG column.

[0042] The UniGel-80SP (-(CH 2 ) 3 SO 3-ligand) chromatography column purification conditions can be: 4-6mL / min loading, 5-15% Buffer B washing, 20-25% Buffer B elution, wherein Buffer A is 15-25mmol / L Hcit-Nacit, pH 4.0-6.0, and Buffer B is 15-25mmol / L Hcit-Nacit, 0.5-1.5mol / L NaCl, pH 4.0-6.0.

[0043] In yet another aspect, the present invention provides the use of the aforementioned gB protein mutant or nucleic acid molecule or expression vector or cell in the preparation of a vaccine or medicine for varicella or herpes zoster.

[0044] The vaccine or drug includes the aforementioned gB protein mutant or nucleic acid molecule or expression vector or cell; or the vaccine or drug is prepared by preparing the gB protein mutant through genetic engineering of the aforementioned nucleic acid molecule or expression vector or cell and then preparing the vaccine or drug.

[0045] The vaccine may also include other pharmaceutically acceptable carriers, such as any one or more of aluminum adjuvant, CpG, AS01 adjuvant, AS03 adjuvant, and MF59 adjuvant.

[0046] The aluminum adjuvant includes, but is not limited to, any one or more of aluminum hydroxide, aluminum phosphate, aluminum sulfate, ammonium alum, and potassium alum.

[0047] The vaccine of the present invention can be a multi-component vaccine, and on the basis of the gB protein mutant of the present invention, can also include other vaccine types, including but not limited to: any one or more of influenza vaccine, pneumonia vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, measles vaccine, mumps vaccine, and rubella vaccine.

[0048] The present invention also provides a pharmaceutical composition comprising the aforementioned gB protein mutant or nucleic acid molecule or expression vector or cell.

[0049] The present invention also provides the use of the aforementioned gB protein mutant or nucleic acid molecule or expression vector or cell in preparing a kit for detecting varicella-zoster virus infection.

[0050] The application may be as a quality control product or a standard product.

[0051] The present invention also provides the use of the aforementioned gB protein mutant or nucleic acid molecule or expression vector or cell in preparing a kit for detecting gB protein antibodies, such as a magnetic particle chemiluminescence kit and a colloidal gold kit.

[0052] Beneficial effects of the present invention: The gB protein mutant of varicella-zoster virus provided by the present invention can stably present the pre-fusion conformation. After the mutation design described in the present invention, the expression levels of multiple VZV gB mutants measured by binding to mab-93K antibody are higher than those of undesigned control molecules, and most VZV gB protein mutants can be stably stored. After being placed at 37°C for 7 days, most VZV gB protein mutants can still maintain a high binding activity with mab-93K monoclonal antibody. The mutation design provided by the present invention can help to increase the antigen expression level and increase the antigen stability. When the gB protein mutant provided by the present invention is used as a key active ingredient in a vaccine, it has good immunogenicity, can activate animal autoimmunity, and play a preventive or therapeutic role. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a template amino acid sequence for constructing the VZV gB protein mutant described in certain embodiments; the template amino acid sequence includes the heterologous signal peptide sequence "MRGGGLICALVVGALVAAVASA" (underlined), "|…|" (deleted N-terminal signal peptide and flexible region, residues 1-26), a linker sequence (lowercase, italicized, residues 685-689), a fibritin T4-derived trimerization domain (black bold font, residues 690-720), and a fusion tag (uppercase, italicized, residues 721-738); the amino acid positions are the corresponding positions relative to the wild-type VZV gB sequence described in SEQ ID NO: 1.

[0054] Figure 2 The expression level of gB protein was measured using the gB protein-specific antibody mab-93K.

[0055] Figure 3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis under non-reducing conditions were performed for the preferred fusion gB mutants (lanes 1-14, respectively: YK024Ag003, YK024Ag008, YK024Ag017, YK024Ag018, YK024Ag019, YK024Ag020, YK024Ag021, YK024Ag022, YK024Ag023, YK024Ag024, YK024Ag025, YK024Ag029, YK024Ag031 and YK024Ag032).

[0056] Figure 4 The chromatogram and SDS-PAGE analysis results of the purified representative mutant YK024Ag030 are shown.

[0057] Figure 5 These are the results of evaluating the immunogenicity (binding antibodies) of the vaccine composition prepared with the YK024Ag030 mutant protein as the core component. DETAILED DESCRIPTION

[0058] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0059] The present invention discloses varicella-zoster virus fusion gB protein mutant and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0060] the term:

[0061] In the present application, the term "varicella-zoster virus" or "VZV" belongs to the Herpesviridae family, alphaherpesvirinae subfamily, and the virus can cause highly contagious chickenpox upon primary infection and herpes zoster upon reactivation of latent infection.

[0062] In the present application, the term "protein mutant" may generally refer to a protein whose amino acid sequence or protein structure has one or more changes compared to the wild-type protein. The changes may include: deletion, insertion, substitution, truncation and / or deletion of one or more amino acids, processing or cutting of the protein structure. In the present application, the protein mutant refers to a varicella-zoster virus (VZV) gB protein mutant.

[0063] In the present application, the term "amino acid mutation" generally refers to the modification of an amino acid in a wild-type amino acid sequence. For example, the modification may include replacement, insertion and / or deletion of one or more amino acids. In the present application, the amino acid mutation may include deletion or replacement of at least one amino acid residue at a specified position of an amino acid sequence. In certain embodiments, the amino acid mutation can optimize the conformation of a protein composed of the amino acid sequence. Amino acid mutations may be produced using genetic or chemical methods well known in the art. For example, genetic methods may include site-directed mutagenesis, PCR, and gene synthesis, etc.

[0064] In the present application, the term "penetration of an alpha helix structure" means that an alpha helix structure with two bends changes into a linear alpha helix structure that no longer bends under certain circumstances.

[0065] In the present application, the term "codon optimization" generally refers to replacing one or more codons in a parent polypeptide encoding nucleic acid by codons encoding the same amino acid residue with different relative usage frequencies to improve the expression of the nucleic acid encoding the polypeptide. In the present application, as long as the mutation of the amino acid is the same as the present application, all possible codons encoding the mutant amino acid are within the scope of the present application.

[0066] In the present application, the term "trimer" generally refers to a protein structure composed of three protein subunits of the same type or different types, and the protein trimer can be connected together by a special chemical structure. In the present application, the protein trimer can be the gB protein of varicella-zoster virus.

[0067] In the present application, the term "conformational change" generally refers to a change in the spatial structure of a protein molecule. For example, the conformational change may include a change in the chemical bonds in the protein molecule or a change in the folding mode of a polypeptide.

[0068] In the present application, the term "signal peptide" generally refers to an amino acid sequence present at the N-terminus of a transmembrane protein as a signal when it crosses the membrane. For example, the transmembrane protein may include a secretory protein or a cell membrane protein. For example, the signal peptide can be synthesized at the N-terminus of the transmembrane protein in the form of a precursor polypeptide.

[0069] In the present application, the term "before fusion" generally refers to the conformation presented by the structural protein of the virus before the infection host cell when membrane fusion has not yet occurred. Generally speaking, the wild-type VZV gB protein is a metastable protein before the fusion, and its conformation is discontinuous after the virus infects the host cell and membrane fusion occurs, and gradually and irreversibly changes into a conformation of a low-energy stable state (conformation after fusion). In the present application, a series of transformations are carried out to the VZV gB protein so that the gB protein mutant can stably be in the fusion precursor state.

[0070] In this application, the expression of protein mutation sites is usually expressed as "amino acid + amino acid position + amino acid after mutation". In this application, the mutation may include but is not limited to the addition, replacement, deletion and / or deletion of amino acids. For example, the term "K670N" usually refers to the mutation of lysine (K) at position 670 to asparagine (N).

[0071] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

[0072] In the application, the term "vector" refers to a nucleic acid carrier into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries are expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication start site. The vector may also include components that assist it to enter the cell or assist the target element to be integrated into the host cell, such as virus particles, liposomes, protein coats or integrase, but not limited to these substances.

[0073] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the VZV gB protein mutant described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are generally non-toxic to the recipient at the dosages and concentrations used.

[0074] In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium, and / or nonionic surfactants.

[0075] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0076] This application is based on structural biology and combined with immunological principles to design a new type of protective vaccine with high safety, good stability, near-natural conformation, and reliable preparation process. The VZV gB protein mutant provided in this application can be stably maintained in a fusion precursor state, and can induce a higher level of binding antibodies in animal immune tests when used alone or with a suitable adjuvant.

[0077] In the present application, the term "adjuvant" generally refers to any substance that assists or regulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen. In the present application, the adjuvant can be used to enhance the antigenicity of the VZV gB protein mutant. In certain embodiments, the adjuvant can include a mineral (e.g., alum, aluminum hydroxide, or phosphate) suspension. In certain embodiments, the adjuvant can include an oil-in-water emulsion. In certain embodiments, the adjuvant can include a liposome. In certain embodiments, the adjuvant can include an immunostimulant such as MPL, CpG, Poly:IC.

[0078] In this application, the term "vaccine" refers to a pharmaceutical composition comprising an immunogen capable of eliciting a prophylactic or therapeutic immune response in an individual. Typically, a vaccine elicits an antigen-specific immune response against a pathogen, such as a viral pathogen.

[0079] In the present application, the term "specific antibody" refers to an antibody that specifically binds to VZV gB glycoprotein. An exemplary specific antibody is mab93K antibody.

[0080] In the present application, the term "mab-93K" refers to the antibody described in the document "Oliver, SL, et al. (2020). "Aglycoprotein B-neutralizing antibody structure at 2.8Auncovers a criticaldomain for herpesvirus fusion initiation." Nat Commun 11(1):4141.", which has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:5 of this patent and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:6.

[0081] Without intending to be bound by any theory, the following examples are merely intended to illustrate the VZV gB protein mutants, preparation methods, quality characterization and uses of the present application, and are not intended to limit the scope of the present invention.

[0082] Example 1 Design and expression of VZV gB protein mutants

[0083] The present invention has evaluated dozens of VZV gB protein mutants in total. This example summarizes the design and expression of 17 representative VZV gB protein mutants (each of which is identified by a unique identifier, such as YK024Ag008, YK024Ag017, etc.), and the representative VZV gB protein mutants include a wild-type control molecule (YK024Ag003) without mutation design and 16 preferred mutants, the preferred mutants including the fibritin folding sub-trimerization domain and the introduced amino acid mutations. The design and expression of exemplary VZV gB mutants are provided in Table 1. Each of the 16 preferred mutants is designed and prepared based on the amino acid sequence described in SEQ ID NO: 1. The methods for expressing and purifying these exemplary VZV gB mutants are described in Examples 2 and 3, and the method for determining the expression of VZV gB mutants is described in Example 4. The expression of the VZVgB mutant is the target protein content in the cell transient expression supernatant described in Example 2 of the present invention. Compared with the wild-type control molecule, the expression levels of the 16 preferred molecules were significantly increased, indicating that the mutation designs provided by the present invention can increase the expression level of the pre-fusion conformation VZV gB protein mutants.

[0084] Table 1 Sequence design and expression level of exemplary VZV gB protein mutants

[0085]

[0086]

[0087] Example 2 Construction of expression vector and small volume expression of VZV gB protein mutant

[0088] The nucleic acid sequence encoding the VZV gB protein mutant was codon optimized and synthesized using CHO as the host, cloned into the pKS001 vector (purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number: A13201) using HindIII and NotI, and the plasmid was extracted for further transfection.

[0089] Expi 293 cells (purchased from Thermo fisher, catalog number: A14527) with fast growth and good cell morphology were used as transfection hosts for large-volume transfection. The viable cell density of the seed cells was adjusted to 3 million per milliliter before transfection, and 20 mL of cell solution was used for transfection for each project. Each project used 20 μg of plasmid and 100 μg of PEI MAX transfection reagent, which were diluted with 600 μL of OPM-293CD05 culture medium (purchased from Shanghai Aopuma Biotechnology Co., Ltd., catalog number: 81075-001), gently mixed, and then the diluted transfection reagent was gently added to the diluted plasmid, and incubated at room temperature for 8 minutes to prepare the plasmid complex. After the incubation, the prepared plasmid complex was slowly dripped into the cell solution to be transfected, and the cell solution was placed at 37°C, 8% CO 2 The cells were cultured in a shaker at 200 rpm. 18-24 hours after transfection, 10% culture volume of Balan CD CHO Feed4 (purchased from FUJIFILM Irvine Scientific, Inc, catalog number: 94134-10L) (0.8X) (V / V) was added to each bottle of transfected cells, and the cell status was monitored every day. The transfection cycle was usually 4 days. On the 4th day, the cell culture supernatant was harvested to measure the expression level and transferred to the purification section for purification.

[0090] Example 3 Purification of VZV gB protein mutants

[0091] The clear solution harvested by centrifugation was adjusted to pH 5.0 ± 0.1 with 5M HCl, and purified water was added to adjust the conductivity to no more than 8.0 mS / cm. After adjustment, it was filtered with a 0.22 μm filter and filtered using UniGel-80SP (-(CH 2 ) 3 SO 3 -ligand) column for purification, the purification process is 5mL / min loading, 5-15% Buffer B washing, 25% Buffer B elution, wherein Buffer A is 20mmol / L Hcit-Nacit, pH 5.0, Buffer B is 20mmol / L Hcit-Nacit, 1mol / L NaCl, pH5.0. Collect the fractions containing the target protein after the first step of purification, and use HiLoad 16 / 600Superdex 200 TM The PG column was further purified, and the mobile phase buffer system was Buffer C (1xPBS, pH 7.4). The target fragment elution component was selected and concentrated to 1 mg / mL using a 30 kD ultrafiltration tube for subsequent quality research of the target protein.

[0092] The purification results of the selected representative VZV gB mutant YK024Ag030 (SEQ ID NO.3, coding sequence SEQ ID NO.4) are shown in Figure 4 As shown, the target protein with high electrophoretic purity can be obtained after purification.

[0093] Example 4 Expression and stability determination of VZV gB protein mutants (monoclonal antibody mab-93K was used to detect the target protein content of VZV gB protein mutants)

[0094] The cell supernatant harvested in Example 2 of the present invention was filtered through a disposable 0.22 μm sterile filter and then packaged. The sample was directly tested for the expression of the target protein in the supernatant or placed in a 37°C constant temperature box for stability study under high temperature conditions. The sampling points for stability study were 0 days, 1 day, 3 days, and 7 days. The gator based on the principle of biomembrane optical interference was used. TM The molecular interaction detector calculates the content of the target protein in the cell expression supernatant and stability test samples by testing the binding rate of the specific recognition monoclonal antibody mab-93K and the VZV gB mutant.

[0095] The specific test parameters are as follows: after the protein A probe is balanced for 50 seconds, the mab-93K monoclonal antibody (concentration diluted to 20μg / mL) is captured to about 4 nanometers, and after 120 seconds of equilibrium, the binding reaction with the cell expression supernatant of the VZV gB protein mutant is carried out for 200 seconds, and then a 120-second dissociation reaction is carried out to test the binding of the designed VZV gB protein mutant with the mab-93K monoclonal antibody. The content of the target protein is calculated based on the standard curve established based on the standard sample with known concentration.

[0096] In some embodiments, the expression level of the selected VZV gB protein mutants is measured as shown in Tables 1 and Figure 2 As shown in Table 2, the results of stability investigation are shown in Table 2. After the amino acid mutation design described in the present invention, the expression levels of multiple VZV gB mutants measured by binding to mab-93K antibody are higher than those of the undesigned control molecules, and most VZV gB protein mutants can be stably stored. After being placed at 37°C for 7 days, most VZV gB protein mutants can still maintain a high binding activity with mab-93K monoclonal antibody, proving that the mutation design provided by the patent of the present invention can help to increase the expression level of the antigen and increase the stability of the antigen.

[0097] Table 2 Stability study of exemplary VZV gB protein mutants

[0098]

[0099]

[0100] " / " indicates no activity; "*" indicates no detection

[0101] Example 5 Analysis of the molecular weight of target proteins of selected VZV gB protein mutants using immunoblotting

[0102] In order to determine the content and molecular weight of the target protein in the supernatant of the transiently transfected cells, the supernatant of the transiently transfected cells was subjected to protein gel electrophoresis (SDS-PAGE) and immunoblotting analysis.

[0103] Sample preparation: Take an appropriate amount of sample, dilute 5× non-reducing SDS-PAGE loading buffer to 1×, and heat in a 100°C metal bath for 15 minutes.

[0104] Electrophoresis: After the sample is cooled to room temperature, use 4%-20% precast gel electrophoresis to separate the protein sample, with a sample load of 3 μg. If less than 3 μg, load 40 μL. Electrophoresis parameters: voltage 180V, time 50min.

[0105] Transfer: Soak the PVDF membrane in methanol for 15 minutes to activate it. Rinse the SDS-PAGE gel after electrophoresis with deionized water. From the negative electrode to the positive electrode, form a transfer sandwich with a transfer pad, transfer filter paper, SDS-PAGE gel, PVDF, transfer filter paper, and transfer pad. In an ice bath, transfer the membrane at a current of 400mA for 60 minutes to transfer the protein to the PVDF membrane.

[0106] Blocking: The transferred PVDF membrane was rinsed with Western washing solution (PBST) and then placed in blocking solution (5% skim milk powder-PBST solution) to block the hydrophobic binding sites on the membrane and incubated on a shaker at room temperature for 2 hours.

[0107] Primary antibody incubation: HRP-labeled mouse anti-His Tag antibody was diluted 1000 times with blocking solution as the primary antibody. After the PVDF membrane was washed with PBST, the diluted primary antibody was immediately added and incubated on a shaker at room temperature for 2 hours. The PVDF membrane was washed with PBST to remove the unbound primary antibody, and washed three times, 5 minutes each time.

[0108] DAB color development: After DAB color development and incubation at room temperature for 30 minutes, wash twice with distilled water to terminate the color development reaction and take photos for archiving.

[0109] In some embodiments, the results of immunoblot analysis of selected VZV B protein mutants are as follows: Figure 3 As shown, the VZVB protein mutants all contain target proteins with expected molecular weights.

[0110] Example 6 Structural thermal stability of selected pre-fusion VZV gB protein mutants

[0111] In order to investigate the thermodynamic stability of the designed VZV gB protein mutant, this experiment evaluated the particle size and thermal denaturation of the antigen by Prometheus Panta. The specific implementation method is: take 50 μL of the test sample, centrifuge at 8℃ 15000rcf for 10 minutes, load it into a capillary (manufacturer: Nano Temper, item number: PR-C002), load it into the tray, and each test sample has 3 replicates. Select the Thermal Unfolding module in the equipment software, start at 25℃, end at 95℃, and heat at a rate of 1℃ / min for temperature detection.

[0112] The results of the investigation on the structural thermal stability of representative VZV gB protein mutants are shown in Table 3. The initial denaturation stability (Tm1) of each mutant was higher than 70°C, proving that each mutant protein had excellent structural thermal stability.

[0113] Table 3 Structural stability data of exemplary VZVgB protein mutants

[0114]

[0115] * indicates the mean, σ * Variance

[0116] Example 7 Immune response of VZV gB protein mutants in mice

[0117] In order to study the immunogenicity of the VZV gB protein mutant provided by the present invention, the inventors of the present invention prepared a vaccine composition (0.5mL / dose) of a representative VZV gB protein mutant YK024Ag030 and different adjuvants, and the group information is specifically shown in Table 4. The immunogenicity of each group of vaccine compositions was investigated in SPF female 6-8 week old BALB / c mice, with 8 mice in each group, for a total of 6 groups. The immunization method was intramuscular injection of the inner thigh of the mouse, and the injection volume was 50 μL / dose / mouse (1 / 10 of the intended dose for a person). Each mouse was immunized 2 times in total, with an immunization interval of 3 weeks. Blood was collected from each group of mice two weeks after the second immunization, and serum was separated. The binding antibody titer for gB protein in the serum was detected by ELISA.

[0118] Table 4 Vaccine composition formulations of VZV gB protein mutant YK024Ag030 and different adjuvants

[0119]

[0120] Note: AS01 adjuvant and AS03 adjuvant in Table 4 are fixed-ingredient adjuvants commonly used in the art. AS01 adjuvant is a liposome adjuvant containing monophosphoryl lipid A and saponin QS-21; AS03 adjuvant is an oil-in-water emulsifier containing α-tocopherol, squalene, and Tween 80.

[0121] The results are as follows Figure 5 As shown, compared with the blank group (Group 1), high levels of binding antibodies against B protein were detected in the sera of mice in each experimental group two weeks after the second immunization, indicating that the VZV B protein mutant provided by the present invention has excellent immunogenicity.

[0122] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A gB protein mutant of varicella-zoster virus, characterized in that: The amino acid that undergoes alpha helix penetration during the transformation of the gB protein precursor to the post-fusion conformation is modified with proline or a proline analog.

2. The gB protein mutant according to claim 1, characterized in that The modification is replacement or insertion of proline or a proline analog.

3. The gB protein mutant according to claim 2, characterized in that The sequence of the gB protein is SEQ ID NO.1, and the amino acids that cause alpha helix penetration are amino acids between 454-466.

4. The gB protein mutant according to claim 3, characterized in that The modification is any one or more mutations to proline or a proline analog, and / or any insertion of one or more proline or a proline analog.

5. The gB protein mutant according to claim 4, characterized in that The modification is any mutation to proline or a proline analog.

6. The gB protein mutant according to claim 1, characterized in that The feline cleavage site is inactive or absent.

7. The gB protein mutant according to claim 6, characterized in that The fragment containing the ferin protease cleavage site in SEQ ID NO.1 is deleted or replaced with a linker.

8. The gB protein mutant according to claim 7, characterized in that The deletion is to delete 1-23 amino acids between positions 415-440; the replacement is a linker to replace the amino acids between positions 428 and 431 with a linker with a length of 1-10 amino acids.

9. The gB protein mutant according to claim 1, characterized in that It also includes mutating one or more charged amino acids into polar amino acids, hydrophobic amino acids or aromatic amino acids, preferably mutating the lysine at position 670 of SEQ ID NO.

1.

10. The gB protein mutant according to claim 9, characterized in that: The charged amino acid is lysine at position 670 of SEQ ID NO.1, which is mutated to asparagine.

11. The gB protein mutant according to claim 1, characterized in that It also includes a C-terminal transmembrane and intracellular region deleted and connected to a heterologous trimerization domain that helps maintain the trimer conformation.

12. The gB protein mutant according to claim 11, characterized in that The amino acids 680-690 and thereafter at the C-terminus of SEQ ID NO.1 are deleted.

13. The gB protein mutant according to claim 11, characterized in that The heterologous trimerization domain is the amino acid sequence shown in SEQ ID NO.

2.

14. A nucleic acid molecule encoding the gB protein mutant according to any one of claims 1 to 13.

15. An expression vector comprising the nucleic acid molecule of claim 14.

16. A cell comprising the gB protein mutant according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, or the expression vector according to claim 15.

17. Use of the gB protein mutant according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the expression vector according to claim 15, or the cell according to claim 16 in the preparation of a vaccine or medicine for varicella or herpes zoster.

18. A vaccine comprising the gB protein mutant according to any one of claims 1 to 13.

19. A pharmaceutical composition comprising the gB protein mutant according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the expression vector according to claim 15, or the cell according to claim 16.

20. Use of the gB protein mutant according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the expression vector according to claim 15, or the cell according to claim 16 in the preparation of a kit for detecting varicella-zoster virus infection.

21. Use of the gB protein mutant according to any one of claims 1 to 13, the nucleic acid molecule according to claim 14, the expression vector according to claim 15, or the cell according to claim 16 in preparing a kit for detecting gB protein antibodies.

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