A plant-expressed varicella-zoster virus gB recombinant antigen, and preparation method, product and application thereof

Abstract

The application discloses a plant-expressed varicella-zoster virus gB recombinant antigen and a preparation method, product and application thereof, and belongs to the technical field of plant molecular biology and vaccine engineering. The technical problems to be solved are that the existing VZV gB protein has low heterologous expression, poor stability and insufficient antigen activity; the existing VZV vaccine has high production cost, is difficult to popularize and has a single antigen with an immune escape risk; and the plant expression system is not mature in VZV gB antigen production, and lacks a complete preparation scheme and related products. The technical solution is that the natural VZV gB protein is modified, a furin protease cleavage site is knocked out, and a H527P mutation is introduced, related nucleic acid molecules, vectors and plant source cells are constructed, high-activity antigens are obtained through culture and purification, and the high-activity antigens are applied to the preparation of VZV drugs / vaccines, and have the advantages of low cost, high safety and strong immunogenicity.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biotechnology and vaccine engineering technology, specifically relating to a plant-expressed varicella-zoster virus gB recombinant antigen, its preparation method, product, and application. Background Technology

[0002] Varicella-zoster virus (VZV) belongs to the Alphaherpesviridae family and is a highly contagious double-stranded DNA virus that can spread widely worldwide. Infection can cause two different clinical manifestations: primary infection manifests as chickenpox, which is more common in children; latent infection followed by reactivation manifests as shingles, which is more common in middle-aged and elderly people and those with weakened immune systems. Shingles is characterized by severe neuralgia, and approximately 10%-20% of patients develop postherpetic neuralgia (PHN), a prolonged and difficult-to-cure condition that significantly reduces patients' quality of life and places a heavy burden on public health.

[0003] Currently, vaccination is the core means of preventing VZV infection and related diseases. In recent years, the Shingrix subunit vaccine, which has been marketed, has become an important option for preventing shingles due to its high protective efficacy. However, the vaccine still has obvious limitations: (1) The vaccine is expensive to produce and relies on mammalian cell expression systems for production, resulting in high vaccine prices and making it difficult to popularize in developing countries and low-income groups; (2) The vaccine uses only VZV glycoprotein E (gE) as a single antigen, and the antigen type is relatively simple. Long-term use may pose a risk of immune escape, affecting the long-term protective effect of the vaccine.

[0004] VZV glycoprotein B (gB) is a key membrane fusion protein on the viral envelope, playing a central role in viral adsorption and entry into host cells. It synergistically interacts with the gH / gL complex on the viral envelope to mediate the fusion of the viral envelope with the host cell membrane, a necessary step in VZV infection of host cells. Simultaneously, the gB protein possesses abundant antigenic epitopes, capable of inducing specific humoral and cellular immune responses, effectively inhibiting viral replication and spread. Therefore, it is considered a highly promising core antigenic candidate target for VZV subunit vaccine development.

[0005] Despite the promising prospects of gB protein in vaccine applications, its use in heterologous expression systems is subject to several limitations: natural gB protein has furin protease cleavage sites, making it easily cleaved and degraded, resulting in insufficient protein stability; at the same time, the complex folding process of gB protein makes it prone to misfolding during heterologous expression, forming inactive inclusion bodies, which not only leads to low protein expression levels but also reduces its antigenic activity and immunogenicity, seriously hindering the industrial application of gB protein in vaccine development.

[0006] In recent years, plant molecular farming, as a novel recombinant protein production technology platform, has gradually become a research hotspot in the biopharmaceutical field due to its unique advantages. This technology uses plants such as tobacco and Arabidopsis thaliana as bioreactors, enabling low-cost, rapid, and large-scale production of recombinant proteins. Furthermore, the plant expression systems do not carry human pathogens, eliminating concerns about viral contamination and exhibiting extremely high biosafety. In addition, the unique glycosylation modification system of plant cells may provide new pathways for regulating the immunogenicity of recombinant antigens.

[0007] However, current technologies still have significant gaps and shortcomings in the application of VZV gB protein in plant expression systems: on the one hand, there is no rational design and modification of gB protein to solve the problems of low expression level, poor stability, and insufficient antigenic activity in plants; on the other hand, existing plant vaccine research focuses on optimizing the expression level of recombinant proteins, and research on the role of plant-specific glycosylation structures in gB antigen delivery and immunogenicity regulation is relatively scarce, failing to fully utilize the unique advantages of plant expression systems.

[0008] Relevant patent documents retrieved: This document, published in China (CN119306808A) on January 14, 2025, discloses a mutant of the varicella-zoster virus gB protein and its applications. By modifying the amino acids (positions 454-466) of the alpha helix during the conformational transition from the gB protein fusion precursor to the fusion precursor with proline or its analogues, knocking out the furin protease cleavage site, optionally deleting the C-terminal transmembrane and intracellular regions and connecting a heterotrimeric domain, and mutating lysine at position 670 to asparagine or other amino acids, a gB protein mutant that stably exhibits the pre-fusion conformation can be obtained. The document describes the construction of its encoding nucleic acid molecule and expression vectors such as pcDNA3.1 and pKS001, which are then transfected into mammalian cell lines such as CHO and 293 to achieve high-level expression. High-activity mutants are obtained after column purification and applied to the preparation of varicella or shingles vaccines, drugs, and diagnostic kits, demonstrating improved antigen expression levels and stability, and enhanced immunogenicity.

[0009] Relevant non-patent literature retrieved: The journal or book title is "Journal of Virology," and the article title is "Mutagenesis of varicella-zoster virus glycoprotein B: putative fusion loop residues areessential for viral replication, and the furin cleavage motif contributes to pathogenesis in skin tissue in vivo," volume number 83, no. 15, published on May 27, 2009. This article discloses a study on the mutation of VZV gB protein, focusing on the function of the gB protein fusion loop and furin protease cleavage motif. The study found that point mutations at W180G and Y185G in the putative fusion loop of the VZV gB protein prevent VZV from replicating, confirming that amino acid residues in this region are crucial for VZV replication in vitro. Meanwhile, deletion mutations (Δ491RSRR494) and point mutations (491GSGG494) at positions 491-494 of the furin protease recognition motif in the gB protein do not affect VZV replication in vitro, viral particle morphology, or protein localization, but they do weaken VZV's replication ability in human skin xenografts in vivo. This study is the first to demonstrate that the cleavage of herpesvirus fusion proteins plays a crucial role in the in vivo pathogenic mechanism of the virus.

[0010] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Existing VZV gB protein mutations and expression rely on mammalian cell expression systems, resulting in high production costs, difficulty in large-scale deployment, and a lack of exploration into the application potential of plant expression systems. Current technologies only focus on the impact of gB protein mutations on viral replication, pathogenicity, and conformational stability, failing to address the specific characteristics of plant expression systems and thus the problems of low gB protein expression levels, poor stability, and insufficient antigenic activity in plants. Current research does not address the regulatory role of plant cell-specific glycosylation modification systems on the immunogenicity of gB antigens, failing to fully leverage the unique advantages of plant expression systems in recombinant antigen production. Furthermore, existing VZV vaccines either rely on high-cost mammalian cell expression systems or have a single antigen type, posing a risk of immune escape. There is a lack of a low-cost, high-safety, and highly immunogenic VZV gB recombinant antigen and related preparation methods based on plant expression systems, which cannot meet the needs for the development and widespread adoption of novel VZV vaccines. The relevant evidence is as follows: Chinese patent CN119306808A explicitly uses mammalian cell lines such as CHO and 293 to express gB protein mutants, without involving plant expression systems; the literature in the Journal of Virology only discusses the impact of gB protein mutations on VZV replication and pathogenicity, without mentioning plant expression-related content; none of the existing technologies have designed gB protein modification schemes specifically for the characteristics of plant expression systems, nor have they studied the regulatory role of plant glycosylation on the immunogenicity of gB antigens, and existing vaccines suffer from high production costs or single antigens, and the above problems have not been effectively solved.

[0011] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: Screening for mutation sites is challenging. Screening for mutation sites in VZV gB protein requires simultaneously addressing three core requirements: ensuring the mutated gB protein maintains correct conformational stability, preserving its natural antigenic epitopes and immunogenicity; adapting to the expression characteristics of plant cells, enabling efficient transcription, translation, and correct folding of the mutant within plant cells; and ensuring the mutated gB recombinant antigen possesses good immunogenicity, effectively inducing a specific immune response. These three requirements are interconnected and mutually restrictive, with no clear screening rules. Furthermore, the combined effects of different mutation sites vary significantly, making it difficult to quickly screen for the optimal mutation site suitable for the plant expression system. Extensive and repeated mutation, expression, and activity verification experiments are necessary, greatly increasing the difficulty and time required for research and development. Summary of the Invention

[0012] The purpose of this invention is to provide: A plant-expressed varicella-zoster virus gB recombinant antigen, its preparation method, products, and applications, as well as related technologies, are disclosed to address the following technical issues: low heterologous expression levels, poor stability, and insufficient antigenic activity of existing VZV gB proteins; high production costs and limited availability of existing VZV vaccines, with single antigens posing a risk of immune escape; and the immature application of plant expression systems in VZV gB antigen production, lack of complete preparation protocols and related products, or combinations thereof.

[0013] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0014] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0015] The definition of the standard chemical term can be found in the reference "Genetic Engineering (4th Edition), Higher Education Press, 2022-11-28".

[0016] Unless otherwise stated, conventional methods within the scope of the art, such as double enzyme digestion subcloning, cell culture, cell transfection, etc., shall be used.

[0017] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0018] The terms "optional" or "arbitrarily" mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: "The dosage form of the cell preparation includes any one or more of solid dosage forms, semi-solid dosage forms, and liquid dosage forms"; this means: the dosage form of the cell preparation may be a solid dosage form; or the dosage form of the cell preparation may be a semi-solid dosage form; or the dosage form of the cell preparation may be a liquid dosage form; or the dosage form of the cell preparation may be both a semi-solid and a liquid dosage form.

[0019] The term "VZV" used in this article refers to varicella-zoster virus, which belongs to the Alphaherpesviridae family. It is a highly contagious double-stranded DNA virus that can cause both chickenpox and shingles.

[0020] The term "gB" used in this article refers to varicella-zoster virus glycoprotein B, a key membrane fusion protein on the VZV envelope. It has abundant antigenic epitopes, can induce a specific immune response in the body, and is a core antigenic candidate target for the development of VZV subunit vaccines.

[0021] The term "vector backbone" as used in this article refers to the essential functional DNA segments in a genetic engineering vector that maintain its replication, amplification, and selection within the host cell. It typically contains core elements such as the origin of replication, selection marker genes (e.g., antibiotic resistance genes), and multiple cloning sites (MCS), forming the basic framework for constructing recombinant expression vectors.

[0022] The term "infection" as used in this article refers to the process of introducing exogenous genes or gene-containing vectors into plant cells or tissues using microbial or physical methods.

[0023] The term "recombinant antigen" as used in this article refers to a protein product with antigenic activity that is expressed in host cells after a natural antigen (such as VZV gB protein) has been modified by genetic engineering techniques through mutation, truncation, or other means.

[0024] The term "furin protease cleavage site" as used in this article refers to a specific amino acid sequence in the natural VZV gB protein that can be recognized and cleaved by the furin protease (specifically, the specific amino acid site region of the gB protein involved in relevant literature and this invention). Cleavage leads to the degradation of the gB protein and affects its stability.

[0025] The term "plant molecular pharmaceutical" as used in this article refers to a novel pharmaceutical technology that uses plant cells, tissues, or whole plants as bioreactors to express recombinant proteins (such as antigens, drug proteins, etc.) through genetic engineering techniques. It has advantages such as low cost, scalability, and high biosafety.

[0026] The term "heterologous expression" as used in this article refers to the process of introducing a gene or gene fragment from one organism into another different host organism (such as introducing the VZV gB gene into plant cells) to enable it to express the corresponding protein.

[0027] The term "immunogenicity" as used in this article refers to the ability of an antigen to stimulate the body's immune system to produce a specific immune response (including humoral immunity and cellular immunity), which is a core characteristic of vaccine antigens.

[0028] The term "conformational stability" used in this article refers to the ability of a protein molecule to maintain its specific spatial structure (conformation). The conformational stability of gB protein directly affects the integrity of its antigenic epitopes and its immune activity.

[0029] The term "genetically engineered cell" as used in this article refers to engineered cells that can stably express the target protein by introducing a foreign target gene (such as the VZVgB recombinant antigen encoding gene) into a host cell through genetic engineering technology.

[0030] The term "glycosylation modification" used in this article refers to a type of post-translational modification of proteins, which is the process of linking sugar chains to specific amino acid residues of proteins under the action of enzymes. Glycosylation modification in plant cells has its own specificity and can affect the immunogenicity and stability of recombinant antigens.

[0031] The term "inclusion body" used in this article refers to inactive, insoluble protein aggregates formed during heterologous protein expression due to misfolding and aggregation, which can lead to a decrease in the expression level and activity of the target protein.

[0032] In a first aspect, the present invention provides: a plant-expressed VZV gB recombinant antigen.

[0033] Among them, the technical feature is: VZV gB recombinant antigen.

[0034] The VZV gB recombinant antigen has the amino acid sequence shown in SEQ ID NO.2.

[0035] The VZV gB recombinant antigen was prepared by modifying the VZV gB amino acid sequence shown in SEQ ID NO.1 as follows: (1) Knock out furin protease cleavage sites; (2) Introduce the H527P mutation at position 527.

[0036] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred embodiment: The VZV gB recombinant antigen has the amino acid sequence shown in SEQ ID NO.2. This technical solution, based on solving the technical problem of "natural VZV gB protein being easily degraded by enzymes and having poor stability in heterologous expression systems", further solves the technical problem of "low expression level of gB protein, low folding efficiency, and insufficient antigenic activity in plant cells", while enabling the recombinant antigen to be efficiently expressed in a stable trimer form in plant expression systems, while retaining the native conformation and good immunogenicity.

[0037] Secondly, the present invention provides: a nucleic acid molecule.

[0038] This includes the technical feature: nucleic acid molecules.

[0039] The nucleic acid molecule encodes the VZV gB recombinant antigen of the present invention.

[0040] Preferably, the nucleic acid molecule has the nucleotide sequence as described in SEQ ID NO.12.

[0041] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the second aspect of the present invention includes: The first preferred option has a nucleotide sequence as described in SEQ ID NO.12. This technical solution, in addition to solving the technical problem of "low transcription and translation efficiency and poor codon fit of the natural VZV gB gene in plant cells", further solves the technical problem of "ensuring that the encoded VZV gB recombinant antigen has higher conformational stability, antigenic activity and immunogenicity".

[0042] Thirdly, the present invention provides: a gene engineering vector.

[0043] Among its technical features is the use of gene engineering vectors.

[0044] The genetic engineering vector contains the nucleic acid molecules of the present invention.

[0045] Specifically, the genetic engineering vector includes an expression cassette for driving the expression of the nucleic acid molecule in plant cells.

[0046] Preferably, the expression cassette comprises: a plant-derived promoter, a signal peptide coding sequence, a nucleic acid molecule, a tag coding sequence, an endoplasmic reticulum retention signal coding sequence, and a plant-derived terminator.

[0047] More preferably, the plant-derived promoter includes any one of the Arabidopsis MacT promoter, CaMV 35S promoter, Arabidopsis RD29A promoter, and maize Ubiquitin promoter.

[0048] More preferably, the plant-derived promoter is the Arabidopsis thaliana MacT promoter.

[0049] More preferably, the plant-derived terminator includes any one of the following: Arabidopsis thaliana RD29B terminator, CaMV 35S terminator, Arabidopsis thaliana NOS terminator, and rice tms terminator.

[0050] More preferably, the plant-derived terminator is the Arabidopsis thaliana RD29B terminator.

[0051] More preferably, the signal peptide coding sequence is the endoplasmic reticulum signal peptide (SP) coding sequence of Arabidopsis thaliana BIP1 protein, and its nucleotide sequence is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.4.

[0052] More preferably, the tag encoding sequence is a histidine tag encoding sequence; in addition, any one of the HA tag or Myc tag encoding sequences may also be used.

[0053] More preferably, the endoplasmic reticulum retention signal coding sequence is an HDEL signal peptide coding sequence, the nucleotide sequence of which is shown in SEQ ID NO.11 and the amino acid sequence of which is shown in SEQ ID NO.10.

[0054] Preferably, the genetic engineering vector is constructed by inserting the expression cassette into the multiple cloning site of the vector backbone.

[0055] More preferably, the carrier skeleton includes any one or more of the pTEX series carriers, pBI121 series carriers, and pCAMBIA series carriers.

[0056] In a further preferred embodiment, the carrier skeleton is a pTEX1 carrier.

[0057] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes: The first preferred option: The genetic engineering vector comprises an expression cassette, wherein the plant-derived promoter is the Arabidopsis thaliana MacT promoter, the plant-derived terminator is the Arabidopsis thaliana RD29B terminator, the signal peptide coding sequence is the endoplasmic reticulum signal peptide coding sequence of the Arabidopsis thaliana BIP1 protein, the tag coding sequence is the His tag coding sequence, the endoplasmic reticulum retention signal coding sequence is the HDEL coding sequence, and the vector backbone is the pTEX1 vector. This technical solution, in addition to solving the technical problem that "conventional genetic engineering vectors cannot be adapted to plant expression systems, resulting in low expression efficiency of target nucleic acid molecules and insufficient recombinant antigen yield," further addresses the technical problem that "recombinant antigens are misfolded, easily degraded, have poor stability, and have complex purification processes in plant cells."

[0058] Fourthly, the present invention provides: a genetically engineered cell.

[0059] Among its technical features is genetically engineered cells.

[0060] The genetically engineered cells described herein include the genetically engineered vector of the present invention.

[0061] Preferably, the genetically engineered cells include, but are not limited to, any one of the following: tobacco Benzodiaceae cells, Arabidopsis thaliana cells, tobacco BY-2 cells, tomato cells, corn cells, and rice cells.

[0062] More preferably, the genetically engineered cells are tobacco Benzovia cells.

[0063] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first priority option is to use *Nicotiana benthamiana* cells for genetic engineering. This technical solution addresses the technical problem that "conventional host cells cannot be adapted to plant-derived genetic engineering vectors, resulting in low expression efficiency and insufficient yield of recombinant antigens." Furthermore, it solves the technical problem of "achieving efficient and stable expression of recombinant antigens while reducing culture and preparation costs, providing reliable cellular support for the industrial application of recombinant antigens."

[0064] Fifthly, the present invention provides: a cell preparation.

[0065] Among them, the technical feature is: cell preparation.

[0066] The dosage form of the cell preparation includes any one or more of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0067] In a sixth aspect, the present invention provides a method for preparing VZV gB recombinant antigen.

[0068] This includes the technical feature: preparation method.

[0069] The preparation method includes using the VZV gB recombinant antigen, nucleic acid molecule, genetic engineering vector, genetic engineering cell or cell preparation of the present invention.

[0070] Preferably, the preparation method includes the following steps: S1. Culture genetically engineered cells and induce the expression of VZV gB recombinant antigen in the nucleic acid molecules of the genetically engineered cells; S2. Collect the cultured genetically engineered cells or cell cultures and extract proteins; S3. The protein is separated and purified to obtain the VZV gB recombinant antigen.

[0071] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the sixth aspect of the present invention includes: The first preferred solution: the specific next steps of the preparation method. This technical solution, while solving the technical problems of "complex preparation process, low yield, and insufficient purity of VZV gB recombinant antigen", further solves the technical problems of "easy degradation and loss of activity of recombinant antigen during preparation, and high preparation cost and difficulty in large-scale production".

[0072] In a seventh aspect, the present invention provides the use of the VZV gB recombinant antigen, nucleic acid molecule, genetic engineering vector, genetic engineering cell or cell preparation of the present invention in the preparation of drugs for the prevention or treatment of varicella-zoster virus.

[0073] Eighthly, the present invention provides: a medicament for the prevention or treatment of varicella-zoster virus.

[0074] Among them, the technical feature is: drug.

[0075] The drug comprises the VZV gB recombinant antigen, nucleic acid molecule, genetic engineering vector, genetic engineering cell or cell preparation of the present invention.

[0076] Specifically, the dosage form of the drug includes any one or more of the following: injection, oral preparation, and topical preparation.

[0077] Preferably, the drug is a vaccine.

[0078] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0079] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or more combinations of wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solutes, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0080] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the eighth aspect of the present invention includes: The first preferred option: the drug is a vaccine. This technical solution, while addressing the technical problems of "high production cost and insufficient immunogenicity of existing varicella-zoster virus vaccines," further solves the technical problem of "poor vaccine stability."

[0081] Examples 1-5 of this invention at least support the protection scope of the technical feature “VZV gB recombinant antigen”.

[0082] The technical feature “VZV gB recombinant antigen” is derived from the aforementioned explanation and / or the corresponding technical features in Examples 1-5, such as knocking out the furin protease cleavage site and introducing the H527P mutation at position 527, through the common feature “modifying the VZV gB amino acid sequence shown in SEQ ID NO.1”. Therefore, those skilled in the art can reasonably infer that the technical feature VZV gB recombinant antigen, the subordinate concept of VZV gB recombinant antigen, the technical means that are essentially equivalent to VZV gB recombinant antigen, and the technical means that can replace VZV gB recombinant antigen based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing VZV gB recombinant antigen with recombinant antigen or recombinant VZV gB while keeping other technical features unchanged still falls within the protection scope of this invention.

[0083] Examples 1-5 of this invention at least support the protection scope of the technical feature "nucleic acid molecule".

[0084] The technical feature "nucleic acid molecule" is derived from the DNA fragment encoding gBΔF H527P, recombinant genes, etc., as explained above and / or in Examples 1-5, through the common feature "nucleic acid molecule encoding VZV gB recombinant antigen". Therefore, those skilled in the art can reasonably infer that the technical feature nucleic acid molecule, the subordinate concept of nucleic acid molecule, the technical means that are basically equivalent to nucleic acid molecule, and the technical means that can replace nucleic acid molecule based on the existing technical level within the scope of conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing nucleic acid molecule with encoding gene, gene, etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0085] Examples 1-5 of this invention at least support the protection scope of the technical feature "genetic engineering vector".

[0086] The technical feature “genetic engineering vector” is summarized from the corresponding technical features pTEX1-SP-gBΔF H527P-FD-His-HDEL plasmid, pTEX1-SP-gBΔF H527P-mCor1-His-HDEL plasmid, pTEX1-SP-gBΔF H527P-hCor-His-HDEL plasmid, etc., in the foregoing explanation and / or Examples 1-5, by the common feature “containing a nucleic acid molecule encoding the VZV gB recombinant antigen”. Therefore, those skilled in the art can reasonably presume that the technical features of gene engineering vectors, the subordinate concepts of gene engineering vectors, the technical means that are basically equivalent to gene engineering vectors, and the technical means that can replace gene engineering vectors within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of this invention. For example, if other technical features remain unchanged, replacing gene engineering vectors with pTEX1-SP-gBΔF H527P-mCor1-HA-HDEL plasmids, pTEX-SP-gBΔF H527P-mCor1-HA-HDEL plasmids, etc., still falls within the protection scope of this invention.

[0087] Examples 1-5 of this invention at least support the protection scope of the technical feature "genetically engineered cells".

[0088] The technical feature "genetically engineered cell" is derived from the common feature "genetically engineered cell" as explained above and / or from the corresponding technical feature of *Tobacco Benzoinus* cells in Examples 1-5. Therefore, those skilled in the art can reasonably infer that the technical feature "genetically engineered cell," its subordinate concepts, the fundamentally equivalent technical means of "genetically engineered cell," and technical means that can replace "genetically engineered cell" within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of this invention. For example, replacing "genetically engineered cell" with engineered cells, reconstituted cells, etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0089] Examples 1-5 of this invention at least support the protection scope of the technical feature "cell preparation".

[0090] The technical feature "cell preparation" is derived from the aforementioned explanations and / or the corresponding technical features HEK293-gB, Nb-gB, etc., in Examples 1-9, summarized by the common feature "preparation containing genetically engineered cells". Therefore, those skilled in the art can reasonably infer that the technical feature "cell preparation", the subordinate concept of "cell preparation", the basically equivalent technical means of "cell preparation", and the technical means that can replace "cell preparation" based on the existing level of technology and conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing "cell preparation" with pharmaceutical preparations, formulations, etc., while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0091] Examples 1-5 of this invention at least support the protection scope of the technical feature "preparation method".

[0092] The technical feature "preparation method" is summarized from the common feature "preparation of VZV gB recombinant antigen" in the foregoing explanation and / or the corresponding technical features in Examples 1-5, such as recombinant gene construction, transient transgenic plants, and preparation of plant-derived gB protein (NB-gB). Therefore, those skilled in the art can reasonably infer that the technical feature "preparation method," its subordinate concepts, the basically equivalent technical means of the preparation method, and the technical means that can replace the preparation method based on the existing technical level within the scope of conventional technical means and common knowledge should all fall within the protection scope of this invention. For example, replacing the preparation method with a method, or a method for preparing gB protein, while keeping other technical features unchanged, still falls within the protection scope of this invention.

[0093] Examples 8-9 of this invention at least support the protection scope of the technical feature "the application of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus".

[0094] The technical feature “Application of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus” is summarized from the aforementioned explanation and / or the corresponding technical features in Examples 8-9, such as gBΔF H527P enhancing macrophage uptake and gBΔF H527P increasing antibody titers in immunized mice, through the common feature “Effectiveness in the prevention or treatment of varicella-zoster virus”. Therefore, those skilled in the art can reasonably presume that the application of the technical features of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus, the subordinate concept of the application of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus, the technical means that are substantially equivalent to the application of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus, and the technical means that can replace the application of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus based on the existing level of technology and conventional technical means and common knowledge, should all fall within the scope of protection of this invention. For example, replacing the application of the product in the preparation of drugs for the prevention or treatment of varicella-zoster virus with the application of the product in the preparation of vaccines for the prevention or treatment of varicella-zoster virus, or the application of the product in the preparation of oral solutions for the prevention or treatment of varicella-zoster virus, while keeping other technical features unchanged, still falls within the scope of protection of this invention.

[0095] Examples 8-9 of this invention at least support the protection scope of the technical feature "drug".

[0096] The technical feature "drug" is derived from the aforementioned explanation and / or the corresponding technical features in Examples 8-9, such as gBΔFH527P enhancing macrophage uptake and gBΔF H527P increasing antibody titers in immunized mice, through the common feature "products with preventive or therapeutic effects against varicella-zoster virus." Therefore, those skilled in the art can reasonably presume that the technical feature "drug," its subordinate concepts, essentially equivalent technical means, and technical means that can replace the drug within the scope of conventional and common knowledge based on existing technology should all fall within the protection scope of this invention. For example, replacing the drug with an injection or vaccine while keeping other technical features unchanged still falls within the protection scope of this invention.

[0097] The beneficial effects of this invention are as follows: This invention offers significant advantages over existing technologies. By knocking out the furin protease cleavage site and introducing the H527P mutation, it effectively prevents gB protein degradation, stabilizes the trimer conformation, and fully preserves the antigenic epitope. Simultaneously, it adapts to plant expression systems to construct dedicated expression vectors and expression elements, combined with molecular chaperone-assisted folding, significantly improving the expression level and preparation efficiency of recombinant antigens, filling the gap in VZV gB plant expression technology. This invention utilizes the unique modification characteristics of plant cells to obtain highly uniform high-mannose glycosylation modification, significantly enhancing the antigen uptake and presentation capabilities of macrophages. The prepared plant-derived gB recombinant antigen exhibits stronger immunogenicity, inducing high-titer, long-lasting specific antibodies, thus overcoming the deficiency of single antigens in existing vaccines and reducing the risk of viral immune escape. Furthermore, the plant expression system boasts high biosafety, low production cost, and ease of large-scale mass production, overcoming the problems of high price and limited availability associated with traditional mammalian cell expression vaccines. Furthermore, this invention forms a complete technical system from modifying antigens, encoding nucleic acids, engineering vectors, host cells to preparation methods and vaccine drug applications. The solution is mature and controllable, and can be made into a variety of pharmaceutical dosage forms, possessing both clinical practicality and industrialization value.

[0098] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: This invention provides a method for preventing or treating varicella-zoster virus (VZV) infection, characterized in that the method includes using the VZV gB recombinant antigen, nucleic acid molecule, genetic engineering vector, genetic engineering cell, cell preparation, or drug of this invention.

[0099] Specifically, the method includes administering to the subject an effective amount of VZV gB recombinant antigen, nucleic acid molecule, genetically engineered vector, genetically engineered cell, cell preparation, or drug.

[0100] Preferably, the subjects include mammals.

[0101] More preferably, the subject is a human being. Attached Figure Description

[0102] Figure 1 This figure shows the screening of trimerized domains in plant expression systems; A in the figure is a schematic diagram of the construction of the FD fusion group and the mCor1 fusion group; B is the SDS-PAGE and Western blot analysis results of the FD fusion group and the mCor1 fusion group; C is the SDS-PAGE and Western blot analysis results of the hCor fusion group and the mCor1 fusion group; ns in the figure represents no significant difference.

[0103] Figure 2This diagram illustrates the high-level expression of the gBΔF H527P trimer in plants. Figure A shows the construction process; BC represents the high-level expression of the gBΔF H527P trimer in plants, with SDS-PAGE and Western blot analysis results in B; C shows the quantitative analysis results; DE shows the enhancement of gBΔF H527P expression through molecular chaperone co-expression, with SDS-PAGE and Western blot analysis results in D; and E shows the quantitative analysis results. Figure 3 To ensure the gBΔF H527P trimer maintains its binding ability with monoclonal antibody mAb 93k; A in the figure represents DS-PAGE and CBB staining analysis of recombinant mAb 93k; B represents the results of the Co-IP experiment.

[0104] Figure 4 The figures show plant-derived / HEK293 cell-derived gB protein; A in the figure represents SEC analysis of plant-derived gB protein; B represents SDS-PAGE and CBB staining analysis of plant-derived gB protein; C represents SDS-PAGE and CBB staining analysis of HEK293-gB purified and concentrated under non-reducing conditions; and D represents SDS-PAGE and CBB staining analysis of HEK293-gB purified and concentrated under non-reducing conditions.

[0105] Figure 5 To compare the amino acid sequences of Nb-gB and HEK293-gB using Geneious Prime software.

[0106] Figure 6 The cryo-electron microscopy structure of the gBΔF H527P trimer is shown in the figure. A is the two-dimensional classification average map of the gBΔF H527P trimer, scale bar = 140 Å; B is the cryo-electron microscopy (Cryo-EM) structure, with each monomer represented by a different color; C is the comparison of the full-length sequence structure of plant-derived gBΔF H527P with the reported gB structure; D is the comparison of the DIV structure of plant-derived gBΔF H527P with the reported gB structure.

[0107] Figure 7Highly uniform high-mannose glycosylation was achieved for gBΔF H527P expressed in the endoplasmic reticulum of plant cells; Figure A shows the N-glycosylation of HEK293-gB and NB-gB analyzed by PNGase F digestion; Figure B shows the N-glycosylation of HEK293-gB and NB-gB analyzed by Endo H shows the N-glycosylation type of HEK293-gB and NB-gB by enzyme digestion analysis; C is a schematic diagram of the experimental procedure for analyzing the N-glycosylation structure of HEK293-gB and NB-gB by mass spectrometry; D shows the N-glycosylation sites and their modification ratios of HEK293-gB and NB-gB identified by mass spectrometry analysis; E shows the different types of N-glycans and their relative abundance in HEK293-gB and NB-gB identified by mass spectrometry analysis; F shows the relative proportions of high-mannose and other types of N-glycans at each glycosylation site of HEK293-gB and NB-gB; G shows the main N-glycan structure type at each glycosylation site of HEK293-gB; H shows the main N-glycan structure type at each glycosylation site of NB-gB.

[0108] Figure 8 Plant-derived gBΔF H527P can enhance macrophage uptake; Figure A shows a representative confocal micrograph of macrophages after 48 hours of IL-4 treatment, where CD206 is shown in red, the nucleus in blue, and the cytoskeleton (actin) in green; B shows flow cytometry analysis of CD206 levels; C shows M2 macrophages (F4 / 80). + CD206 + The ratio of ) is shown in the figure; D represents the uptake efficiency of Cy5-labeled gBΔF H527P of HEK293-gB and Nb-gB in macrophages; ***P<0.001; ****P<0.0001 in the figure.

[0109] Figure 9 Plant-derived gBΔF H527P can increase antibody titers in immunized mice; Figure A is a schematic diagram of the immunization procedure; B is the serum ELISA analysis of immunized HEK293-gB and Nb-gB mice at 14 DPI; C is the serum ELISA analysis of immunized HEK293-gB+Alum and Nb-gB+Alum mice at 14 DPI; D is the serum ELISA analysis of immunized HEK293-gB and Nb-gB mice at 28 DPI; E is the serum ELISA analysis of immunized HEK293-gB+Alum and Nb-gB+Alum mice at 28 DPI; ns in the figure represents no significant difference; * indicates P<0.05. Detailed Implementation

[0110] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0111] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0112] Example 1: Construction of a stably expressed gB protein variant A rational design was carried out for the varicella-zoster virus (VZV) gB protein: the protein stability was improved by knocking out the furin protease cleavage site (ΔF); at the same time, the H527P stable mutation was introduced at position 527 to enhance its trimer conformation stability, and finally the modified gB variant gBΔF H527P was obtained.

[0113] The amino acid sequence of the VZV gB protein is shown in SEQ ID NO.1: MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVVSVSPSSFYESLQVEPTQSEDITRSAHLGDGDEIREAIHKSQDAETKPTFYVCPPPTGSTIVRLEPPRTCPDYHLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAWAGSSYTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVRNNHKVEAFNEDKNPQDMPLIASKYNSVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGLRDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVGWNWKPKRTEVCSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQCVKEEARAIINRIYTTRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRENTNHSPQKHPTRNTRSRRSVPVELRANRTITTTSSVEFAMLQFTYDHIQEHVNEMLARISSSWCQLQNRERALWSGLFPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRIILQNSMRVSGSTTRCYSRPLISIVSLNGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYFLFGHHYVYYEDYRYVREIAVHDVGMISTYVDLNLTLLKDREFMPLQVYTRDELRDTGLLDYSEIQRRNQMHSLRFYDIDKVVQYDSGTAIMQGMAQFFQGLGTAGQAVGHVVLGATGALLSTVHGFTTFLSNPFGALAVGLLVLAGLVAAFFAYRYVLKLKTSPMKALYPLTTKGLKQLPEGMDPFAEKPNATDTPIEEIGDSQNTEPSVNSGFDPDKFREAQEMIKYMTLVSAAERQESKARKKNKTSALLTSRLTGLALRNRRGYSRVRTENVTGV。

[0114] The amino acid sequence of the said gBΔFH527P is shown in SEQ ID NO.2: TKPTFYVCPPPTGSTIVRLEPPRTCPDYHLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAWAGSSYTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVRNNHKVEAFNEDKNPQDMPLIASKYNSVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGLRDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVGWNWKPKRTEVCSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQCVKEEARAIINRIYTTRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRENTNHSPQKHPTRNTGSGGSVPVELRANRTITTTSSVEFAMLQFTYDHIQEPVNEMLARISSSWCQLQNRERALWSGLFPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRIILQNSMRVSGSTTRCYSRPLISIVSLNGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYFLFGHHYVYYEDYRYVREIAVHDVGMISTYVDLNLTLLKDREFMPLQVYTRDELRDTGLLDYSEIQRRNQMHSLRFYDIDKVVQ。

[0115] The VZV gB protein without knocking out the furin protease cleavage site (ΔF) was used as a control, named gB H527P, and its amino acid sequence is shown in SEQ ID NO.3: MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVVSVSPSSFYESLQVEPTQSEDITRSAHLGDGDEIREAIHKSQDAETKPTFYVCPPPTGSTIVRLEPPRTCPDYHLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAWAGSSYTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVRNNHKVEAFNEDKNPQDMPLIASKYNSVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGLRDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVGWNWKPKRTEVCSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQCVKEEARAIINRIYTTRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRENTNHSPQKHPTRNTRSRRSVPVELRANRTITTTSSVEFAMLQFTYDHIQEPVNEMLARISSSWCQLQNRERALWSGLFPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRIILQNSMRVSGSTTRCYSRPLISIVSLNGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYFLFGHHYVYYEDYRYVREIAVHDVGMISTYVDLNLTLLKDREFMPLQVYTRDELRDTGLLDYSEIQRRNQMHSLRFYDIDKVVQYDSGTAIMQGMAQFFQGLGTAGQAVGHVVLGATGALLSTVHGFTTFLSNPFGALAVGLLVLAGLVAAFFAYRYVLKLKTSPMKALYPLTTKGLKQLPEGMDPFAEKPNATDTPIEEIGDSQNTEPSVNSGFDPDKFREAQEMIKYMTLVSAAERQESKARKKNKTSALLTSRLTGLALRNRRGYSRVRTENVTGV。

[0116] Example 2 Screening of Trimerization Domains in a Plant Expression System 1. Comparison between the FD fusion group and the mCor1 fusion group 1.1 Recombinant Gene Construction Using the pTEX1 binary vector as a backbone, two types of gBΔF H527P recombinant expression cassettes with different trimerization domains were designed: FD fusion group (gBΔF H527P-FD): The DNA fragment encoding gBΔF H527P is fused with a histidine tag (His-tag) and an endoplasmic reticulum retention signal (HDEL) at the C-terminus of the trimerization domain of FD.

[0117] mCor1 fusion group: (gBΔF H527P-mCor1): The DNA fragment encoding gBΔF H527P is fused with a histidine tag (His-tag) and an endoplasmic reticulum retention signal (HDEL) at the C-terminus of the trimerization motif of mouse crown protein 1A (mCor1).

[0118] All expression vectors were fused to the N-terminus of the Arabidopsis thaliana BIP1 protein endoplasmic reticulum signal peptide (SP) to achieve endoplasmic reticulum-targeted localization of the protein. SP-gBΔF H527P-FD-His-HDEL expression cassettes and SP-gBΔF H527P-mCor1-His-HDEL expression cassettes were constructed, as illustrated in the diagrams below. Figure 1 As shown in A in the figure. Pro in the figure MacT RD29BT is the Arabidopsis thaliana MacT promoter; RD29BT is the Arabidopsis thaliana RD29B terminator.

[0119] The SP-gBΔF H527P-FD-His-HDEL expression cassette and the SP-gBΔF H527P-FD-His-HDEL expression cassette were artificially synthesized and then subcloned into the pTEX1 vector via BamHI / XmaI double digestion, resulting in the pTEX1-SP-gBΔFH527P-FD-His-HDEL plasmid and the pTEX1-SP-gBΔF H527P-mCor1-His-HDEL plasmid, respectively.

[0120] The sequence used in the expression box is shown below: The amino acid sequence of the endoplasmic reticulum signal peptide (BIP1 SP) of Arabidopsis thaliana BIP1 protein is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.5. SEQ ID NO. 4: MARSFGANSTVVLAIIFFGCLFALSSAIEEATKL.

[0121] SEQ ID NO.5: ATGGCTCGCTCGTTTGGAGCTAACAGTACCGTTGTGTTGGCGATCATCTTCTTCGGATGTTTATTTGCGTTGTCCTCTGCAATAGAAGAGGCTACGAAGTTA.

[0122] The amino acid sequence of FD is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.7. SEQ ID NO.6: GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0123] SEQ ID NO.7: GATATATTCCAGAAGCTCCACGAGATGGCCAAGCTTATGTTAGAAAGGACGGCGAATGGGTGTTGTTGTCAACATTCTTG.

[0124] The amino acid sequence of mouse corona protein 1A (mCor1) is shown in SEQ ID NO. 8, and the nucleotide sequence is shown in SEQ ID NO. 9. SEQ ID NO. 8: VSRLEEDVRNLNAIVQKLQERLDRLEETVQAK.

[0125] SEQ ID NO.9: GTGTCCAGGCTGGAGGAGGACGTGCGCAACCTCAACGCGATCGTCCAGAAGCTGCAGGAGAGGTTGGACAGGCTGGAGGAGACCGTGCAGGCCAAG.

[0126] The amino acid sequence of the endoplasmic reticulum retention signal (HDEL) is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.11. SEQ ID NO.10: HDEL.

[0127] SEQ ID NO. 11: CACGATGAGTTG.

[0128] The DNA fragment encoding gBΔF H527P is shown in SEQ ID NO.12:

[0129] 1.2 Preparation of transient transgenic plants The expression vector was transformed into Agrobacterium GV3101 strain using the heat shock method. Single clones of Agrobacterium containing the target vector were picked and inoculated into YEP liquid medium, and cultured overnight at 28°C with shaking. Nicotiana benthamiana (Nicotiana benthamiana) strains grown for 4 weeks in a greenhouse (25°C, 16h light / 8h dark) were selected. Nicotiana benthamiana Agrobacterium was infiltrated using a needleless syringe. Leaves were collected 3 days after infiltration to detect the expression level of the target protein.

[0130] 1.3 SDS-PAGE and Western blot analysis Leaf samples were homogenized in protein extraction buffer (TBS buffer containing 1 mM EDTA, 0.2% (v / v) Tween 20, and 1 mM PMSF). Total protein extract or purified protein was separated by 8% or 10% SDS-PAGE. Western blots were performed using mouse anti-His-tagged primary antibody (1:10000 dilution, CWBIO, Cat# CW0286M); secondary antibodies were horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:5000 dilution, Abcam, Cat#ab6789) and HRP-labeled goat anti-human IgG Fc (1:5000 dilution, Abcam, Cat# M212104S). High-sensitivity ECL chemiluminescence assay kit (Tanton) was used. TM High-sig ECL Western Blotting Substrate (Cat# 180-501) was developed, and images were acquired using a Tanon 5200 multicolor luminescence imaging system.

[0131] The SDS-PAGE and Western blot analysis results of the recombinant gB variant (gBΔF H527P) transiently expressed in *Nymphaea benthamiana* are as follows: Figure 1 As shown in B in the figure. The above results indicate that both domains can mediate the expression of gBΔF H527P in plants.

[0132] 2. Comparison between hCor fusion group and mCor1 fusion group 2.1 Recombinant Gene Construction hCor fusion group: (gBΔF H527P-hCor): The DNA fragment encoding gBΔF H527P is fused with a histidine tag (His-tag) and an endoplasmic reticulum retention signal (HDEL) at the C-terminus of the trimerization motif of human coronaprotein 1A (hCor).

[0133] All expression vectors were fused with the endoplasmic reticulum signal peptide (SP) of Arabidopsis thaliana BIP1 protein at the N-terminus to achieve endoplasmic reticulum-targeted localization of the protein, constructing the SP-gBΔF H527P-hCor-His-HDEL expression cassette.

[0134] The SP-gBΔF H527P-hCor-His-HDEL expression cassette was synthesized and then subcloned into the pTEX1 vector via BamHI / XmaI double digestion to obtain the pTEX1-SP-gBΔF H527P-hCor-His-HDEL plasmid.

[0135] The amino acid sequence of human coronaprotein 1A (hCor) is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14. SEQ ID NO. 13: VSRLEEEMRKLQATVQELQKRLDRLEETVQAK.

[0136] SEQ ID NO.14: GTGTCAAGACTTGAAGAAGAGATGAGGAAACTCCAAGCTACCGTTCAGGAACTTCAAAAGCGACTTGATCGGCTTGAAGAGACAGTCCAAGCCAAA.

[0137] 2.2 Preparation of transient transgenic plants Transgenic plants were instantaneously produced using the methods described above.

[0138] 2.3 SDS-PAGE and Western blot analysis SDS-PAGE and Western blot analysis were performed using the methods described above, and the results are as follows: Figure 1 As shown in C, Clear and singular gBΔF H527P target protein bands were detected in both the mCor1 and hCor1 fusion groups, with molecular weights consistent with expectations, demonstrating successful expression of both fusion proteins in *Nycium benzoate*. Gray-scale quantification of the relative density of the two protein bands showed that the relative expression level of gBΔF H527P in the mCor1 fusion group was slightly higher than that in the hCor1 fusion group, but the difference was not statistically significant. This confirms that both the mCor1 and hCor1 trimerization domains can effectively mediate the expression of gBΔF H527P in *Nycium benzoate*.

[0139] Example 3: High-level expression of gBΔF H527P trimer in plants 1. Recombinant gene construction DNA fragments encoding VZV gB protein, gBΔF H527P, and gB H527P were fused with a histidine tag (His-tag) and an endoplasmic reticulum retention signal (HDEL) to the C-terminus of the trimerization motif of mouse crown protein 1A (mCor1). All expression vectors were fused with the endoplasmic reticulum signal peptide (SP) of Arabidopsis thaliana BIP1 protein at the N-terminus to achieve endoplasmic reticulum-targeted localization of the protein. SP-gB-mCor1-His-HDEL expression cassettes, SP-gBΔF H527P-mCor1-His-HDEL expression cassettes, and SP-gB H527P-mCor1-His-HDEL expression cassettes were constructed, as illustrated in the diagrams below. Figure 2 As shown in the figure.

[0140] The SP-gB-mCor1-His-HDEL expression cassette was synthesized and subcloned into the pTEX1 vector via BamHI / XhoI double digestion. The SP-gBΔF H527P-mCor1-His-HDEL and SP-gB H527P-mCor1-His-HDEL expression cassettes were also subcloned via BamHI / XhoI double digestion. The plasmids pTEX1-SP-gB-mCor1-His-HDEL, pTEX1-SP-gBΔF H527P-mCor1-His-HDEL, and pTEX1-SP-gB H527P-mCor1-His-HDEL were obtained, respectively.

[0141] 2. Transgenic plants Transgenic plants were instantaneously produced according to the method described in Example 2.

[0142] 3. SDS-PAGE and Western blot analysis SDS-PAGE and Western blot analysis were performed according to the method described in Example 2. The analysis results are as follows: Figure 2 As shown in B, the quantitative results are shown in [the table]. Figure 2 The results showed that all three recombinant gB proteins were successfully expressed in *Nicotiana benthamiana*, and all existed in intact trimer form without obvious degradation bands. Among them, the expression level of gBΔF H527P protein mediated by the SP-gBΔF H527P-mCor1-His-HDEL expression cassette was significantly higher than that of wild-type gB (SP-gB-mCor1-His-HDEL group) and the gBH527P variant (SP-gB H527P-mCor1-His-HDEL group). This indicates that the gBΔF H527P mutant has the best expression efficiency in the *Nicotiana benthamiana* expression system and can achieve high-level expression.

[0143] 4. Co-expression of molecular chaperones enhances the expression level of gBΔF H527P. 4.1 Construction of recombinant vectors for co-expression of molecular chaperones A recombinant expression vector for the calreticulin (CRT) molecular chaperone was constructed using the pTEX1 binary vector as a backbone. The DNA fragment encoding CRT was fused with a human influenza virus hemagglutinin tag (HA-tag) and an endoplasmic reticulum retention signal (HDEL) at its C-terminus. The expression vector was fused with the endoplasmic reticulum signal peptide (SP) of Arabidopsis thaliana BIP1 protein at its N-terminus to achieve endoplasmic reticulum-targeted localization of the protein, thus constructing the SP-CRT-His-HDEL expression cassette. After artificial synthesis, the SP-CRT-His-HDEL expression cassette was subcloned into the pTEX1 vector via BamHI / XhoI double enzyme digestion to obtain the pTEX1-SP-CRT-His-HDEL plasmid.

[0144] 4.2 Transient co-expression of Tobacco Benzoviae The pTEX1-SP-gBΔF H527P-mCor1-His-HDEL plasmid constructed in Example 3, and the above-mentioned CRT expression plasmid (pTEX1-SP-CRT-His-HDEL plasmid) were transformed into Agrobacterium GV3101 strain by heat shock. They were then inoculated into YEP liquid medium and cultured overnight at 28°C with shaking.

[0145] Agrobacterium containing the gBΔF H527P vector was mixed with Agrobacterium containing the CRT vector at a volume ratio of 4:1 and used for leaf infiltration in *Nicotiana benthamiana* (+CRT group); a control group containing only Agrobacterium containing the gBΔF H527P vector was also set up (-CRT group). Four-week-old *Nicotiana benthamiana* plants were selected, and Agrobacterium was infiltrated using a needleless syringe. Leaf samples were collected three days after infiltration for protein expression analysis.

[0146] 4.3 SDS-PAGE and Quantitative Detection Take 0.1g of soaked tobacco leaves, grind them with liquid nitrogen, add 1mL of extraction buffer (50mM Tris-HCl pH7.5, 150mM NaCl, 1mM EDTA, 0.02% (v / v) Tween 20, 1mM PMSF) to resuspend, homogenize, centrifuge and collect the supernatant, separate by SDS-PAGE, and quantify using BSA as the standard gradient.

[0147] The results are as follows Figure 2 As shown in DE, the quantitative results indicate that after co-expression of molecular chaperone CRT, the expression level of gBΔF H527P was significantly increased compared with the control group. Molecular chaperone CRT can effectively assist the correct folding of gBΔF H527P protein in Nicotiana benthamiana, reduce protein degradation, and significantly increase the expression level of recombinant protein.

[0148] Example 4: gBΔF H527P trimer maintains its binding ability to monoclonal antibody mAb 93k. 1. Preparation of recombinant monoclonal antibody mAb 93k The antibody was expressed via transient transfection into HEK293T cells. The variable regions of the heavy and light chains, after design and mammalian cell codon optimization, were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. The optimized gene was cloned into the pSecTag2A vector containing the human IgG1 constant region, and after sequence verification, transfection-grade plasmids were prepared in large quantities. The heavy chain sequence of the recombinant monoclonal antibody mAb 93k is shown in SEQ ID NO.15, and the light chain sequence is shown in SEQ ID NO.16.

[0149] SEQ ID NO.15: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNFAISWVRQAPGQGLEWMGRIMPLFVSTYAQKFQGRVTISADASTSTAYMELSSLRSRDDTAMYYCARDITAPGAAPTLNFYGM DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0150] SEQ ID NO.16: DIQMTQSPSTLSASVGDRVTITCRASQTISTWLAWYQQTPRKAPKLMIYKASILENGVPSRFGSGSGTEFTLTISSLQPEDFATYYCQQYKSYPWTFGQGTKVEI.

[0151] Take 293F cells in logarithmic growth phase and dilute them to 2×10⁻⁶ with KOP293 serum-free medium. 6Cells / mL, suspended culture at 37℃, 5% CO2, 120 rpm. In a 200 mL culture system, 200 μg of total plasmid (heavy chain:light chain = 1:2) was mixed with 10 mL of KPM buffer, and 1 mL of TA-293 transfection reagent was diluted with 10 mL of KPM. The mixture was incubated at room temperature for 10 min to form a complex, and then added dropwise to the cell culture medium. 24 h after transfection, 1.2 mL of HKE-293 expression enhancer and KT-Feed were added to enhance antibody expression.

[0152] On day 4 after transfection, the culture supernatant was collected by centrifugation at 3500 rpm for 15 min, clarified, and loaded onto a Protein A affinity chromatography column equilibrated with PBS. The column was washed with 50 mL of 20 mM phosphate buffer and eluted with glycine buffer (pH 3.0) until no obvious protein signal was detected by G250 dye. The eluted antibody was dialyzed against 1×PBS overnight, concentrated by ultrafiltration, and used for subsequent experiments.

[0153] SDS-PAGE and Coomassie Brilliant Blue (CBB) staining analysis of recombinant monoclonal antibody mAb 93k are as follows: Figure 3 As shown in A, the results indicate that the recombinant monoclonal antibody mAb 93k has high purity and can be used for subsequent verification of gB protein binding activity and functional experiments.

[0154] 2. Co-immunoprecipitation (Co-IP) experiment of gB protein and monoclonal antibody mAb 93k The co-immunoprecipitation (Co-IP) experiment was used to verify whether the gB and gBΔF H527P proteins expressed in *Nicotiana benthamiana* retained their binding ability to the specific monoclonal antibody mAb 93k, thereby confirming the native conformation and antigenic activity of the recombinant protein.

[0155] Take tobacco leaves expressing gB and gBΔF H527P after soaking, homogenize them in extraction buffer, centrifuge to remove insoluble matter, and the supernatant is the total soluble protein (TSP).

[0156] The total soluble protein was mixed with purified monoclonal antibody mAb 93k and gently incubated overnight at 4°C with rotating motion to allow gB protein to fully bind with m93K antibody, forming an antigen-antibody complex. Protein A+G agarose beads (Beyotime, Cat#P2012) were then added to the reaction system, and incubation was continued at 4°C with rotating motion. The agarose beads were washed several times with TBS containing 0.05% (v / v) Tween 20 to remove non-specific binding proteins. SDS-PAGE loading buffer was added, and the bound proteins were eluted by boiling. After SDS-PAGE separation, Western blot analysis was performed: anti-His antibody detected His-tagged gB protein, and anti-human IgG antibody detected m93K antibody. Samples of total soluble protein (non-recombinant protein, non-treat) or His-tagged SARS-CoV-2 Spike protein from tobacco leaves served as negative controls.

[0157] The above results confirm that the gB and gBΔF H527P proteins expressed in *Nicotiana benthamiana* can bind efficiently and specifically to the specific monoclonal antibody m93K, retaining the natural antigen conformation and antibody binding activity.

[0158] Example 5 Plant-derived / HEK293 cell-derived gB protein 1. Preparation of plant-derived gB protein (NB-gB) Plant-derived gB proteins were prepared according to the research methods described in "Song, S.-J., Diao, H.-P., Moon, B., Yun, A., and Hwang, I.(2022a). The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants. Front. PlantSci. 13:878677." Brief procedure: Agrobacterium containing the pTEX1-SP-gBΔF H527P-mCor1-His-HDEL plasmid prepared in Example 3 was transfected into Nicotiana benthamiana. Plant tissues were thoroughly ground in liquid nitrogen, and extraction buffer (PBS, 300mM NaCl, 5mM imidazole, 0.2% Tween 20, 1mM PMSF) was added for complete lysis. The tissues were centrifuged 2-4 times to remove as much tissue residue as possible. The supernatant was filtered through a 0.22μm sterile filter and then mixed with Ni 2+-NTA magnetic beads were incubated at 4°C for 15 min. After thorough washing with washing buffer (PBS, 300 mM NaCl, 20 mM imidazole, 0.2% (v / v) Tween 20), the bound protein was eluted with elution buffer (PBS, 300 mM NaCl, 300 mM imidazole, 0.2% (v / v) Tween 20). The elution product was dialyzed through a 50 kDa pore size concentrator (Merck, UFC9050) to remove imidazole. The concentrated protein was further purified using a Superose 6 Increase 5 / 150 GL gel filtration column. Figure 4 As shown in AB.

[0159] 2. Preparation of HEK293 cell-derived gB protein (HEK293-gB) HEK293 cells were cultured in suspension in serum-free CD medium (SMM 293-TI, Sinocare), with a shaker speed of 150-175 rpm and a CO2 incubator concentration of 5%-8%. Cells were passaged three times before transfection. On the day of transfection, the seed cell volume was adjusted to 2 × 10⁶ cells / year. 6 Cells / mL were transfected using TF1 transfection reagent (Sinochem, Cat. No. STF02) at a DNA:reagent ratio of 1:10 (w / v) to the expression plasmid (pTEX1-SP-gBΔF H527P-mCor1-His-HDEL plasmid), following the manufacturer's instructions. The day of transfection was designated as day 0. Feed medium (293 Feed, Sinochem, Cat. No. M293-SUPI-100) was added on days 1, 3, and 5 post-transfection. Cells were cultured for 5-7 days, after which the supernatant was collected for protein purification.

[0160] After centrifugation and filtration to remove cells and debris, the culture supernatant was processed using Ni... 2+ -NTA affinity chromatography was used to purify recombinant gB protein. Protein concentration was determined by 280 nm UV absorption, and purity was identified by SDS-PAGE combined with Coomassie brilliant blue staining. Figure 4 As shown in CD.

[0161] The amino acid sequences of Nb-gB and HEK293-gB were aligned using Geneious Prime software. The alignment results are shown below. Figure 5 Compared to HEK293-gB, the Nb-gB protein retains additional amino acid residues at the N-terminus, including: (1) a residual sequence from the BIP1 signal peptide (IEEATKL); (2) a translation sequence introduced by the BamHI restriction endonuclease site; and (3) the C-terminal endoplasmic reticulum retention signal HDEL.

[0162] Example 6: Cryo-electron microscopy structural analysis of gBΔF H527P trimer 1. Sample preparation and imaging for electron microscopy Cryo-electron microscopy samples were prepared in a Vitrobot Mark IV (FEI) sample preparation system at 8°C and 100% humidity. 4 μL of VZV gBΔF H527P protein at a concentration of 4 mg / mL was added to a glow discharge-treated Quantifoil R1.2 / 1.3 porous carbon grid. After standing for 5 seconds, the sample was filtered for 2–5 seconds and then rapidly immersed in liquid ethane for freezing. Initial grid screening was performed on a 200 kV Vecnai Arctica transmission electron microscope (FEI) equipped with a Falcon II direct electron detector. Final data acquisition was performed on a 300 kV Titan Krios electron microscope (FEI) equipped with a Gatan GIF Quantum LS energy filter (20 eV slit width). Images were acquired in super-resolution mode using a Gatan K3 Summit direct electron detector at a nominal magnification of 81000× and a corrected pixel size of 0.48 Å. Data acquisition was performed automatically in cinematic mode using EPU software. Each data stack contains 32 images, with a total exposure time of 2.56 s (0.08 s per frame) and a cumulative radiation dose of approximately 50 e / Å. 2 Frame alignment and dose weighting were performed using MotionCor2, and the final pixel size after secondary merging was 0.96 Å.

[0163] 2. Image Data Processing and 3D Reconstruction A total of 1283 microscopic images were acquired and processed in cryoSPARC software. After importing the images, contrast transfer function (CTF) correction was performed using Patch CTF to remove low-quality images. 200 images were selected for initial particle selection using a speckle picking method. After two-dimensional classification, the average classification map corresponding to the VZV gBΔF H527P trimer projection was used as a template for automatic particle selection across the entire dataset, yielding 2,589,276 particles from the 1283 images. After 3x downsampling, the particles underwent three rounds of two-dimensional classification, resulting in approximately 1,065,000 high-quality particles used for initial model building and heterogeneous optimization. The classification map was evaluated in Chimera, and particles of the optimal category were re-extracted at their original pixel size (0.96 Å) for further initial modeling and heterogeneous optimization to remove low-quality particles. After two rounds of optimization, 64,299 particles were finally selected. Three-dimensional reconstruction was performed using non-uniform (NU) optimization combining global and local CTF correction, with C3 triple symmetry constraints applied, achieving a final resolution of 2.9 Å. The local resolution was calculated and visualized in ChimeraX.

[0164] 3. Structural Modeling Based on the 2.9 Å resolution cryo-electron microscopy density map of the VZVgB trimer, de novo modeling was performed in COOT software. The full-length VZVgB structure predicted by AlphaFold was docked to the density map in Chimera, and then manually corrected in COOT to construct the VZVgB trimer atomic model. The main chain structure was optimized in PHENIX using the real_space_refine module under secondary structure and geometric constraints to avoid overfitting; after further manual adjustments in COOT, the final refinement was completed in PHENIX.

[0165] After multiple rounds of particle picking and two-dimensional classification, the average two-dimensional classification image of the gBΔF H527P trimer was obtained as follows: Figure 6 As shown in Figure A, the results indicate that the protein particles are homogeneous and conformally stable, with a resolution of approximately 6.0 Å, laying the foundation for high-resolution three-dimensional reconstruction. Based on the high-quality particles obtained through screening, the initial model was constructed using ab initio, followed by heterogeneity optimization and non-uniformity (NU) refinement. C3 triple symmetry constraints were then applied, ultimately yielding the cryo-electron microscopy three-dimensional structure of the gBΔF H527P trimer with a resolution of 2.9 Å. The results are shown in Figure A. Figure 6 As shown in Figure B, the structure reveals that the three monomers are marked with different colors, clearly presenting the overall spatial conformation of the gB trimer.

[0166] The plant-derived gBΔF H527P structure (green) obtained in this invention was compared with the reported full-length gB structure of VZV-infected cells (PDB: 6VN1, salmon red) in terms of both full length and domains. The full-length structure comparison showed that the overall conformation of the plant-derived gBΔFH527P was highly consistent with that of the natural gB, and the C-terminal fused mCor1 trimerized domain was clearly visible, without affecting the natural folding of gB. Figure 6 The local alignment results of the C); IV domain (DIV) confirmed that the conformation of the key antigenic region of plant-derived gBΔF H527P was a perfect match with that of the natural protein, preserving the complete antigenic epitope (C). Figure 6 (D in the middle).

[0167] Example 7: Highly uniform high-mannose glycosylation of gBΔF H527P expressed in the endoplasmic reticulum of plant cells. 1. PNGase F and Endo H enzyme digestion analysis The N-glycosylation modification pattern of gB protein expressed in HEK293 cells and Nicotiana benthamiana was analyzed using peptide N-glycosidase F (PNGase F, Beyotime, Cat# P2318L) and endoglycosidase H (Endo H, NEB, Cat# P0702S). Approximately 2 μg of purified protein (NB-gB and HEK293-gB) was denatured at 95°C for 10 min in 1× glycoprotein denaturation buffer (0.5% DS, 1% β-mercaptoethanol). After cooling to room temperature, 1× G7 reaction buffer (50 mM sodium phosphate, pH 7.5) containing 1% NP-40 was added, followed by 500 U of PNGase F or 500 U of Endo H, for a total volume of 20 μL. The mixture was incubated at 37°C for 1 h. Undigested samples were used as controls. The digested products were mixed with SDS-PAGE loading buffer, boiled for 10 min, and then subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining for imaging.

[0168] 2. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) The N-glycosylation modification of HEK293-gB and NB-gB was analyzed by in-gel enzymatic digestion combined with high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS). Approximately 1 mm samples were cut from the SDS-PAGE gel. 3 Protein bands were decolorized with 50% acetonitrile (dissolved in 50mM ammonium bicarbonate), dehydrated with acetonitrile, and air-dried at room temperature. Disulfide bonds were reduced with 10mM DTT at 56℃ for 1 h, followed by alkylation with 20mM iodoacetamide at room temperature in the dark for 1 h. After further dehydration of the gel, trypsin (0.025μg / μL, 37℃) and chymotrypsin (0.05μg / μL, 30℃) were added, and the mixture was enzymatically digested at 37℃ for 16 h. Peptides were extracted three times at 37℃ with an extraction buffer (trifluoroacetic acid:acetonitrile:water = 5%:50%:45%), the extracts were combined, vacuum dried, desalted using a C18 column, and reconstituted in 0.1% formic acid for LC-MS / MS analysis.

[0169] Separation was performed using a 50 μm inner diameter × 170 mm Reprosil-Pur 120 C18-AQ column at 60 °C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-80% acetonitrile aqueous solution. The flow rate was 0.6 μL / min, with gradient elution. Analysis was performed on a Vanquish Neo-Orbitrap Exploris 480 system: primary mass spectrometry resolution was 60,000 m / z, with a mass-to-charge ratio range of 400–1200 m / z; secondary mass spectrometry was performed in data-dependent mode with a resolution of 15,000 m / z and a step-normalized collision energy of 30 m / z. Glycosylation sites were identified using BioPharma Finder 5.1 software. Enzyme digestion modes were set to trypsin, chymotrypsin, and pepsin. Variable modifications included deamidation, oxidation, and glycosylation, with iodoacetamide alkylation as the fixed modification. The mass deviation tolerance was 20 ppm.

[0170] The measurement results are as follows Figure 7 As shown, gBΔF H527P expressed in the endoplasmic reticulum of plant cells achieves highly uniform high-mannose glycosylation.

[0171] Example 8: Plant-derived gBΔF H527P can enhance macrophage uptake. 1. Isolation and M2 polarization of mouse bone marrow-derived macrophages Bone marrow-derived macrophages (BMDM) were isolated from 5-week-old male C57BL / 6 mice. Mouse bone marrow cells were seeded into 6-well plates and cultured for 6 days at 37°C and 5% CO2 saturated humidity in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 20 ng / mL macrophage colony-stimulating factor (M-CSF; MCE, HY-P7085). Adherent cells were collected as BMDM. M2 polarization: BMDM were seeded into confocal culture dishes and stimulated with 40 ng / mL interleukin-4 (IL-4; MCE, HY-P70644) for 48 h. Cells were stained with FITC-labeled phalloidin (UElandy, YP0114S), APC-labeled anti-mouse CD206 antibody (BioLegend, clone C068C2), and DAPI (Solarbio, C0065), and imaged using a confocal laser scanning microscope (STELLARIS STTED, Leica). Flow cytometry analysis: Cells were stained with FITC-labeled anti-mouse CD80 antibody (BioLegend, clone 16-10A1), PE-labeled anti-mouse F4 / 80 antibody (BioLegend, clone BM8), and APC-labeled anti-mouse CD206 antibody (BioLegend, clone C068C2), and detected by flow cytometry (B5R3V6, Saijing Biotechnology).

[0172] 2. Real-time quantitative reverse transcription PCR (RT-qPCR) The mRNA level of CD206 in BMDM was detected using a LightCycler 480 II system (Roche). Total RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596018CN), and its concentration and purity were determined using a NanoDrop spectrophotometer. 1 μg of total RNA was reverse transcribed into cDNA using the TransScript® One-Step cDNA Synthesis Kit (TRANS, AT341-02). Quantitative PCR was performed using the PerfectStart® Green qPCR Kit (TRANS, AQ601-02-V2).

[0173] Primer sequences: CD206 forward (SEQ ID NO.17): 5′-GTGGGGACCTGGCAAGTATC-3′; CD206 reverse (SEQ ID NO.18): 5′-CACTGGGGTTCCATCACTCC-3′.

[0174] Using β-actin as an internal reference gene: β-actin positive (SEQ ID NO.19): 5′-TCTGTGTGGATTGGTGGCTCTA-3′; β-actin reverse (SEQ ID NO.20): 5′-CTGCTTGCTGATCCACATCTG-3′.

[0175] The relative expression level is calculated based on the expression ratio of the target gene to the internal reference gene.

[0176] 3. Macrophage uptake experiment Macrophage uptake efficiency of gB protein prepared using different expression systems was evaluated using BMDM. BMDM was isolated from the femur and tibia of C57BL / 6 mice as described above, and then... 5 Cells were seeded in confocal culture dishes and stimulated with IL-4 for at least 48 hours to regulate CD206 expression. HEK293-gB and NB-gB proteins were labeled using a Cy5 labeling kit (Elabscience, E-LK-C003B). BMDM was co-incubated with 300 μg of Cy5-labeled gB protein for 6 hours, followed by FITC phalloidin and DAPI staining, and then imaged using a confocal laser scanning microscope. The fluorescence intensity of Cy5-labeled gB was quantified using ImageJ software.

[0177] Figure 8The plant-derived gBΔF H527P enhances macrophage uptake. Figure A shows that after 48 hours of IL-4 treatment, macrophage CD206 expression (M2 marker, red) was significantly upregulated, and the cytoskeleton (Actin, green) and nucleus (DAPI, blue) were clearly stained, indicating successful construction of the M2 macrophage model. Quantitative analysis by flow cytometry (…) Figure 8 (BC) shows that M2 macrophages (F4 / 80) in the IL-4 treated group + CD206 + The proportion of M2 macrophages was significantly higher than that of the control group (Control), and the difference was statistically significant, further confirming that the M2 macrophage model was successfully constructed and can be used for subsequent antigen uptake experiments.

[0178] Figure 8 The D-values ​​in the HEK293-gB group showed weaker red fluorescence (Cy5-labeled antigen) intensity in macrophages, indicating low antigen uptake efficiency; while the Nb-gB group showed significantly enhanced red fluorescence intensity in macrophages, indicating efficient uptake of plant-derived gBΔFH527P by macrophages. Quantitative fluorescence results (…) Figure 8 The results showed that the relative fluorescence density of Cy5 in macrophages of the Nb-gB group was about 4 times that of the HEK293-gB group, and the difference was statistically significant, confirming that the macrophage uptake efficiency of plant-derived gBΔF H527P was much higher than that of HEK293 cell-derived protein.

[0179] Example 9: Plant-derived gBΔF H527P can increase antibody titers in immunized mice. 1. Mouse Immunization Experiment Twenty female BALB / c mice, aged 6-8 weeks, were randomly divided into 4 groups (n=5 per group) and immunized twice, on day 0 and day 14 respectively: HEK293-gB group: 2μg HEK293-gB; Nb-gB group: 2μg Nb-gB; Group emulsified with alum adjuvant: 2μg HEK293-gB + 50μL alum adjuvant; Nb-gB emulsification group with alum adjuvant: 2μg Nb-gB + 50μL alum adjuvant; Blood samples were collected before immunization (baseline), on day 14 after the first immunization (14 DPI), and on day 28 after the first immunization (277 DPI). Serum antibody titers were measured by ELISA. Antigen-specific IgG responses were detected using a 96-well high-binding-strength ELISA plate. 100 μL of 0.5 μg / mL recombinant gB protein (Essential Biotech, Cat. No. 40958-VNAS) coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.0) was added to each well and incubated overnight at 2–8°C. After washing twice, 200 μL of blocking buffer (TBS containing 0.1% Tween 20 and 3% BSA, pH 8.0) was added to each well and blocked at 37°C for 1 h. After washing twice, mouse serum was serially diluted 6-fold in the blocking buffer, with 200 μL added to each well and incubated at 37°C for 1 h. After washing three times, 100 μL of HRP-labeled goat anti-mouse IgG (Abcam, Cat. No. ab6789) diluted 1:5000 was added to each well and incubated at 37°C for 1 h. Wash the plate three times, add TMB chromogenic solution (Beyotime, Cat# P0209), incubate at 37℃ for 10 min, add 50 μL of stop solution to each well to stop the reaction, and measure the absorbance at 450 nm wavelength using an ELISA reader.

[0180] The measurement results are as follows Figure 9 As shown, aluminum adjuvants can significantly enhance the immunogenicity of gB from both sources, and the immunomodulatory effect of plant-derived gBΔFH527P is far superior to that of HEK293 cell-derived gBΔF H527, which can induce mice to produce higher titers and more durable specific antibodies.

[0181] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A plant-expressed VZV gB recombinant antigen, characterized in that, The VZV gB recombinant antigen has the amino acid sequence shown in SEQ ID NO.

2.

2. The VZV gB recombinant antigen according to claim 1, characterized in that, The VZV gB recombinant antigen was prepared by modifying the VZV gB amino acid sequence shown in SEQ ID NO.1 as follows: (1) Knock out furin protease cleavage sites; (2) Introduce the H527P mutation at position 527.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the VZV gB recombinant antigen as described in any one of claims 1-2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecule has the nucleotide sequence as described in SEQ ID NO.

12.

5. A gene engineering vector, characterized in that, The genetic engineering vector comprises the nucleic acid molecule as described in any one of claims 3-4.

6. The gene engineering vector according to claim 5, characterized in that, The genetic engineering vector comprises an expression cassette for driving the expression of the nucleic acid molecule in plant cells; the expression cassette comprises: a plant-derived promoter, a signal peptide coding sequence, a nucleic acid molecule, a tag coding sequence, an endoplasmic reticulum retention signal coding sequence, and a plant-derived terminator.

7. The gene engineering vector according to claim 6, characterized in that, The genetic engineering vector is constructed by inserting the expression cassette into the multiple cloning site of the vector backbone.

8. The gene engineering vector according to claim 7, characterized in that, The vector skeleton includes any one or more of the pTEX series vectors, pBI121 series vectors, and pCAMBIA series vectors.

9. A genetically engineered cell, characterized in that, The genetically engineered cells comprise the genetically engineered vector as described in any one of claims 5-8.

10. A cell preparation, characterized in that, The cell preparation described herein comprises the genetically engineered cells of claim 9.

11. The cell preparation according to claim 10, characterized in that, The dosage form of the cell preparation includes any one or more of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

12. A method for preparing VZV gB recombinant antigen, characterized in that, The preparation method includes using the VZV gB recombinant antigen according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the gene engineering vector according to any one of claims 5-8, the gene engineering cell according to claim 9, or the cell preparation according to any one of claims 10-11.

13. The preparation method according to claim 12, characterized in that, The preparation method includes the following steps: S1. Culture genetically engineered cells and induce the expression of VZV gB recombinant antigen in the nucleic acid molecules of the genetically engineered cells; S2. Collect the cultured genetically engineered cells or cell cultures and extract proteins; S3. The protein is separated and purified to obtain the VZV gB recombinant antigen.

14. The use of the VZV gB recombinant antigen according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the genetic engineering vector according to any one of claims 5-8, the genetic engineering cell according to claim 9, or the cell preparation according to any one of claims 10-11 in the preparation of drugs for the prevention or treatment of varicella-zoster virus.

15. A drug for the prevention or treatment of varicella-zoster virus, characterized in that, The drug comprises the VZV gB recombinant antigen as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the genetically engineered cell as described in claim 9, or the cell preparation as described in any one of claims 10-11.

16. The medicament according to claim 15, characterized in that, The dosage form of the drug includes any one or more of the following: injection, oral preparation, and topical preparation.

17. The medicament according to claim 16, characterized in that, The drug mentioned is a vaccine.

18. The medicament according to claim 15, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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