A genetically recombinant varicella-zoster virus fusion protein and its preparation method and application

Abstract

The present invention utilizes gene recombination technology to recombinantly truncate the polypeptide fragment gE1 obtained by truncating the VZVgE protein and the polypeptide fragment gE2, and adds an artificial signal peptide to construct the target fusion gene, insert the pcDNA3.1(+) expression vector, transform Escherichia coli DH5α, and verify the recombinant expression plasmid. Then, the correctly identified recombinant expression plasmid is transfected into CHO cells to express the target fusion gene, and then purified to obtain the gene recombinant VZV fusion protein gE1-gE2, the purity of which can reach more than 95%. Compared with the simple VZVgE protein, the gene recombinant VZV fusion protein can induce a stronger cellular immune response.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, especially the field of genetic engineering vaccines, and particularly relates to a fusion protein constructed by tandem recombination of a polypeptide fragment gE1 obtained by truncating VZV gE protein and a polypeptide fragment gE2, as well as a preparation method and application thereof. Background Art

[0002] Varicella-zoster virus (VZV) belongs to the α subfamily of herpesviruses. It is a double-stranded DNA virus with a length of about 125,000 bp and encodes about 70 open reading frames. The virus nucleocapsid is about 80 to 120 nm in diameter and is icosahedral, surrounded by an inner membrane and an envelope that is partially derived from the cell membrane. VZV primary infection causes chickenpox. After the systemic symptoms and rash of the child subside, a small amount of residual virus will remain dormant in the host's spinal dorsal root ganglia, cranial ganglia, and enteric ganglia nerve cells for life. In the future, when the body's immunity is reduced, the virus lurking in the nerve cells will be reactivated, leading to a painful skin disease - herpes zoster (HZ).

[0003] For varicella and herpes zoster, the main treatment is symptomatic. There is still no specific treatment. Antiviral drugs, such as acyclovir, valacyclovir, and famciclovir, can help patients with varicella and herpes zoster recover quickly, but they cannot stop the virus from being excreted and prevent latent infection. Giving an overdose of immunoglobulin after exposure to VZV is also limited in stopping or reducing the burden of the disease. Therefore, vaccines play an important role in controlling varicella infection. Vaccination is the most effective and reliable means of preventing and controlling varicella.

[0004] The varicella vaccine was first developed by the Takahashi M research group in Japan in 1974 and is called the Oka vaccine (vOka). The live attenuated varicella vaccine is currently the only vaccine approved for the prevention of varicella, but it still has a certain risk of causing latent infection. Compared with the live attenuated vaccine, the inactivated vaccine is relatively safe, but the vaccination dose is larger, and generally 2-3 doses are needed to produce a better protective effect.

[0005] VZV glycoprotein E (gE) subunit vaccine is the mainstream research direction of varicella vaccine at present. gE is encoded by the virus ORF68 gene. This gene consisting of 1872 bases is located in the short fragment region of the VZV genome. gE exists on the surface of virus particles and the surface and cytoplasm of infected cells. It is an essential glycoprotein for the generation of infectious virus particles and the most abundant envelope glycoprotein on the viral envelope and host cell membrane. In the serum of patients with varicella and herpes zoster in the recovery period, VZV antibodies are mainly directed against gE, gB and gH. In particular, cellular immunity and humoral immunity induced by gE as the main target can protect animals from virus attack. Therefore, gE is also an important target of cellular and humoral immune responses. There have been quite a few studies on VZV gE protein: the wild-type or full-length gE protein is generally 623 amino acids. It consists of the main part of the gE protein containing the signal peptide, the hydrophobic anchor region (residues 546-558) and the C-terminal tail. Different studies have slightly different distinctions on the protein molecular structure of gE protein. Some researchers divide the protein molecule into a hydrophilic extracellular region (including a signal peptide), a hydrophobic transmembrane region (residues 545-561) and an intracellular tail (for example, see Grose C. Glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation and intracellular trafficking. Annu Rev Microbil. 1990, 44: 59-80), but the above different distinctions do not produce substantial differences in the application and preparation of gE protein. The protein in a typical pharmaceutical composition will be different from the full-length protein, but a truncated protein. For example, when using modern biomolecular technology to prepare recombinant VZV gE protein, the gE protein is generally truncated to lack a carboxyl-terminal hydrophobic anchor region. At present, there are many literatures and patents reporting the modification and expression of VZV glycoprotein E, such as truncated gE aa1-511 (Vafai et al. 1993, 1994, 1995); truncated gE aa1-546 (Haumont et al. 1996, Jacquet et al. 2002; GlaxoSmithKline EP0405867B1 1990, WO0043527A12000); truncated gE aa1-539 (CN102517302 2011); expression of fusion protein containing gE aa37-161 (CN105906721 2016), etc.

[0006] Some literature has reported the use of gene recombination technology to express gE subunit vaccines, but most of them are still under development, and their effectiveness and safety need to be further determined. Some scholars have also been committed to studying the expression of gE protein by fusion protein, but no substantial progress has been made. Therefore, it is necessary to design and study a fusion protein that can be stably expressed with high quality and can enhance both humoral and cellular immune responses, and apply it to the development of VZV vaccines or therapeutic drugs. Summary of the invention

[0007] The purpose of the present invention is to provide a gene recombinant VZV fusion protein and its preparation method and application based on the fusion gene of two truncated polypeptide fragments of VZV glycoprotein E.

[0008] The present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a genetically recombinant varicella-zoster virus fusion protein, wherein the fusion protein is constructed by tandem recombination of a polypeptide fragment gE1 obtained by truncating VZV gE and a polypeptide fragment gE2.

[0010] In the fusion protein of the present invention, when gE1 is at the N-terminus of the fusion protein, gE2 is at the C-terminus of the fusion protein; when gE1 is at the C-terminus of the fusion protein, gE2 is at the N-terminus of the fusion protein.

[0011] In certain embodiments, gE1 is at the N-terminus of the fusion protein and gE2 is at the C-terminus of the fusion protein.

[0012] The specific amino acid sequence of the varicella-zoster virus gE protein described in the present invention is the amino acid sequence of the varicella-zoster virus Oka strain glycoprotein E with GenBank accession number AAK19946.1 (SEQ ID NO: 27):

[0013] .

[0014] The polypeptide fragment gE1 of the present invention is the amino acids 31-546 of the gE protein obtained by removing the signal peptide (aa1-30) and the carboxyl terminal hydrophobic anchor region (aa547-623) of the gE protein, and its amino acid sequence is shown in SEQ ID NO:1.

[0015] The gE1 described in the present invention includes but is not limited to the content disclosed in the prior art EP0405867B (a suitable VZVgE antigen is a VZV glycoprotein gE truncated to remove the carboxyl terminal anchor region (starting from amino acid position 547)), and the protein sequence generally obtained by expressing the protein in eukaryotic cells will lack a leading sequence (also called a signal peptide, amino acids 1-30).

[0016] The polypeptide fragment gE2 of the present invention contains at least amino acids 31 to 101 of the gE protein, and at most amino acids 31 to 194 of the gE protein.

[0017] The polypeptide fragment gE2 of the present invention is selected from one of amino acids 31-101, 31-119, 31-140, 31-161, 31-181 or 31-194 of VZV gE protein, and the sequence is shown in SEQ ID NO: 3-8.

[0018] In certain embodiments, the genetically recombinant VZV fusion protein is constructed by tandem recombination of: (1) gE1 and (2) gE2, and its amino acid sequence is shown in SEQ ID NO: 15. The gE2 is amino acids 31-101 of the gE protein.

[0019] In certain embodiments, the genetically recombinant VZV fusion protein is constructed by tandem recombination of: (1) gE1 and (2) gE2, and its amino acid sequence is shown in SEQ ID NO: 16. The gE2 is amino acids 31-119 of the gE protein.

[0020] In certain embodiments, the genetically recombinant VZV fusion protein is constructed by tandem recombination of: (1) gE1 and (2) gE2, and its amino acid sequence is shown in SEQ ID NO: 17. The gE2 is amino acids 31-140 of the gE protein.

[0021] In certain embodiments, the genetically recombinant VZV fusion protein is constructed by tandem recombination of: (1) gE1 and (2) gE2, and its amino acid sequence is shown in SEQ ID NO: 18. The gE2 is amino acids 31-161 of the gE protein.

[0022] In certain embodiments, the genetically recombinant VZV fusion protein is constructed by tandem recombination of: (1) gE1 and (2) gE2, and its amino acid sequence is shown in SEQ ID NO: 19. The gE2 is amino acids 31-181 of the gE protein.

[0023] In certain embodiments, the genetically recombinant VZV fusion protein is constructed by tandem recombination of: (1) gE1 and (2) gE2, and its amino acid sequence is shown in SEQ ID NO: 20. The gE2 is amino acids 31-194 of the gE protein.

[0024] The second object of the present invention is to provide a method for preparing a genetically recombinant VZV fusion protein, which comprises the following steps:

[0025] S1. Artificially synthesize the gene sequence of the above fusion protein, and connect the gene of the fusion protein into an expression vector to construct a recombinant expression plasmid.

[0026] S2. The constructed recombinant expression plasmid is transformed into the host bacteria, and the correct recombinant plasmid is selected and identified through double enzyme digestion and DNA sequencing.

[0027] S3. Transfect host cells (preferably Chinese hamster ovary cells (CHO)) with the identified recombinant plasmid to express the gene recombinant VZV fusion protein, and purify it.

[0028] The fusion protein gene sequence was commissioned to be synthesized by WuXi Biologics.

[0029] The construction of the recombinant expression plasmid is as follows:

[0030] Add a signal peptide (amino acid sequence as shown in SEQ ID NO: 29) and add an 8×His tag. Select pcDNA3.1(+) as the vector and perform codon optimization according to the host CHO cells. Recombine the gene of the artificially synthesized fusion protein into the target vector pcDNA3.1(+) to obtain a pcDNA3.1(+)-gE1-gE2-8×His recombinant expression plasmid.

[0031] Preferably, S2 is specifically:

[0032] The recombinant expression plasmid obtained in S1 was transformed into E. coli DH5α competent cells, coated with LB plates containing 100 μg / mL ampicillin, and placed at 37°C overnight. The next day, the transformed colonies were randomly picked, and the fusion protein gE1-gE2 gene fragment was amplified by PCR. The positive single colony plasmid was extracted, double enzyme digestion was performed for identification, and the plasmid containing the exogenous gene was subjected to DNA sequence analysis.

[0033] Preferably, S3 is specifically:

[0034] One day before transfection, CHO cells (EXPICHO from Thermo) were seeded with serum-free cell expression medium OptiPRO at an appropriate concentration. TM The suspension culture was shaken in SFM (37°C, 8% CO2). TM The transfection reagent and pcDNA3.1(+)-gE1-gE2-8×His recombinant expression plasmid complex were prepared in SFM medium, and the recombinant plasmid was transfected, wherein the transfection reagent was ExpiFectamine CHO reagent. The cell supernatant was collected 8 days after transfection, and the protein expression level was detected for subsequent purification. The fusion protein was purified by a combination of immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC)-HPLC to obtain the purified gene recombinant VZV fusion protein gE1-gE2.

[0035] The specific operations of the above experimental steps can be carried out with reference to the existing technology and will not be described in detail here.

[0036] The third object of the present invention is to provide an immunogenic composition comprising at least two of the above fusion proteins.

[0037] In certain embodiments, the immunogenic composition of the present invention comprises the fusion protein gE1-gE2 (aa31-101) and one or more selected from gE1-gE2 (aa31-119), gE1-gE2 (aa31-140), gE1-gE2 (aa31-161), gE1-gE2 (aa31-181) and gE1-gE2 (aa31-194).

[0038] In certain embodiments, the immunogenic composition of the present invention comprises the fusion protein gE1-gE2 (aa31-119) and one or more selected from gE1-gE2 (aa31-101), gE1-gE2 (aa31-140), gE1-gE2 (aa31-161), gE1-gE2 (aa31-181) and gE1-gE2 (aa31-194).

[0039] In certain embodiments, the immunogenic composition of the present invention comprises the fusion protein gE1-gE2 (aa31-140) and one or more selected from gE1-gE2 (aa31-101), gE1-gE2 (aa31-119), gE1-gE2 (aa31-161), gE1-gE2 (aa31-181) and gE1-gE2 (aa31-194).

[0040] In certain embodiments, the immunogenic composition of the present invention comprises the fusion protein gE1-gE2 (aa31-161) and one or more selected from gE1-gE2 (aa31-101), gE1-gE2 (aa31-119), gE1-gE2 (aa31-140), gE1-gE2 (aa31-181) and gE1-gE2 (aa31-194).

[0041] In certain embodiments, the immunogenic composition of the present invention comprises the fusion protein gE1-gE2 (aa31-181) and one or more selected from gE1-gE2 (aa31-101), gE1-gE2 (aa31-119), gE1-gE2 (aa31-140), gE1-gE2 (aa31-161) and gE1-gE2 (aa31-194).

[0042] In certain embodiments, the immunogenic composition of the present invention comprises the fusion protein gE1-gE2 (aa31-194) and one or more selected from gE1-gE2 (aa31-101), gE1-gE2 (aa31-119), gE1-gE2 (aa31-140), gE1-gE2 (aa31-161) and gE1-gE2 (aa31-181).

[0043] The fourth object of the present invention is to provide a genetically recombinant VZV vaccine prepared from the above fusion protein or immunogenic composition.

[0044] The gene recombinant VZV vaccine of the present invention comprises the above-mentioned fusion protein, or immunogenic composition, and optionally a pharmaceutically acceptable adjuvant.

[0045] The pharmaceutically acceptable adjuvant described in the present invention is at least one of aluminum salt adjuvant, Freund's complete adjuvant, propolis adjuvant, oil-water emulsion, cytokine, CpG, DNA, genetically engineered toxin-reducing agent, immunostimulatory complex or liposome.

[0046] In certain embodiments, the liposomal adjuvant is AS01B.

[0047] The formula of AS01B adjuvant is as follows: each dose (0.5 mL) contains MPL 50 μg, QS-21 50 μg, NaCl 4.385 mg, DOPC 1 mg, cholesterol 0.25 mg, KH2PO4 0.54 mg, Na2HPO4 (anhydrous) 0.15 mg, and water for injection.

[0048] In certain embodiments, the adjuvant is a liposome formulation comprising any one of BFA-R837 / PolyI:C, QS-21 / PolyI:C and BFA-MPL / PolyI:C.

[0049] The beneficial effects of the present invention are:

[0050] The present invention utilizes gene recombination technology to tandemly recombine the amino acids 31-546 (i.e., polypeptide fragment gE1) of the VZV gE protein obtained by removing the signal peptide and the C-terminal hydrophobic anchor region with the amino acids 31-161 or 31-140 (i.e., polypeptide fragment gE2) of the VZV gE protein, and adds an artificial signal peptide and an 8×His tag to construct a target fusion gene, insert the pcDNA3.1(+) expression vector, transform Escherichia coli DH5α, and verify the recombinant expression plasmid. Then, the correctly identified recombinant expression plasmid is transfected into CHO cells to express the target fusion gene, and then purified to obtain a recombinant VZV fusion protein, the purity of which can reach more than 95%. Compared with the simple VZV gE protein, the recombinant VZV fusion protein, especially the recombinant VZV vaccine prepared by combining the recombinant VZV fusion protein with the AS01B adjuvant, can induce a stronger humoral immune response and cellular immune response.

[0051] The present invention currently studies the combined vaccination effects of different adjuvants and recombinant antigens. The composite adjuvants all show significantly better adjuvant activity than single MPL, PolyI:C or CpG adjuvants, and the levels of intracellular cytokines IFN-γ and IL-2 stimulated are generally higher. In comparison, the levels of antigen-specific cytokines IFN-γ and IL-2 promoted by the combination of BFA-MPL / Poly I:C composite adjuvant and antigen are the highest. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The results of SDS-PAGE detection of purified fusion proteins gE1-gE2 (aa31-161) and gE1-gE2 (aa31-140) DETAILED DESCRIPTION OF THE INVENTION

[0054] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0055] The term "fusion protein" as used herein generally refers to a protein molecule with biological functional activity obtained by genetic engineering technology. In this article, the fusion protein is a polypeptide fragment gE1 obtained by removing the signal peptide (aa1-30) and the carboxyl terminal hydrophobic anchor region (aa547-623) of the gE protein of the varicella-zoster virus Oka strain, and at least the 31st to 101st amino acids of the gE protein, and at most the 31st to 194th amino acids, that is, the polypeptide fragment gE2 tandemly recombined and constructed to obtain a fusion protein. In some embodiments, the polypeptide fragment gE2 is the 31st to 101st amino acids of the gE protein, and the fusion protein obtained by recombinant is gE1-gE2 (aa31-101). In some embodiments, the polypeptide fragment gE2 is the 31st to 119th amino acids of the gE protein, and the fusion protein obtained by recombinant is gE1-gE2 (aa31-119). In some embodiments, the polypeptide fragment gE2 is the 31st to 140th amino acids of the gE protein, and the recombinant fusion protein is gE1-gE2 (aa31-140). In some embodiments, the polypeptide fragment gE2 is the 31st to 161st amino acids of the gE protein, and the recombinant fusion protein is gE1-gE2 (aa31-161). In some embodiments, the polypeptide fragment gE2 is the 31st to 181st amino acids of the gE protein, and the recombinant fusion protein is gE1-gE2 (aa31-181). In other embodiments, the polypeptide fragment gE2 is the 31st to 194th amino acids of the gE protein, and the recombinant fusion protein is gE1-gE2 (aa31-194).

[0056] The term "include" as used in this article generally means to include, encompass, contain or encompass. In some cases, it also means "for", "consisting of...". DETAILED DESCRIPTION

[0057] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples and drawings. The following examples are only explanations of the present invention and cannot be understood as limiting the present invention. In addition, whether or not there is a His tag or an N-terminal signal peptide in the examples does not affect the protection scope. Those skilled in the art can make non-essential improvements and adjustments to the examples based on the content of the present invention, which still fall within the protection scope of the present invention.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional experimental methods understandable to those skilled in the art. The experimental materials used in the following examples, unless otherwise specified, are all purchased from the market.

[0059] Example 1 Synthesis of fusion gene gE1-gE2 (aa31-161)

[0060] WuXi Biologics was commissioned to artificially synthesize the gene sequence of the fusion protein gE1-gE2 (aa31-161) according to the sequence of SEQ ID NO: 24 described in the present invention.

[0061] Example 2 Construction of pcDNA3.1(+)-gE1-gE2(aa31-161)-8×His recombinant expression plasmid

[0062] Add a signal peptide (amino acid sequence as shown in SEQ ID NO: 29) and add an 8×His tag. Select pcDNA3.1(+) as the vector and perform codon optimization according to the host CHO cells. Recombine the gene sequence of the synthesized fusion protein into the target vector pcDNA3.1(+) to obtain a pcDNA3.1(+)-gE1-gE2(aa31-161)-8×His recombinant expression plasmid. The reaction system is shown in Table 1.

[0063] Table 1. The reaction system of the treated target fragment and the vector ligation

[0064] name volume Enzyme-cut vector 5μL Purified PCR products 5μL Seamless assembly MIX 10μL Total volume 20μL

[0065] The above ligation solution was connected at a constant temperature of 52°C for 30 minutes to obtain the recombinant expression plasmid pcDNA3.1(+)-gE1-gE2(aa31-161)-8×His.

[0066] Example 3 Screening and identification of recombinant plasmid pcDNA3.1(+)-gE1-gE2(aa31-161)-8×His

[0067] Transformation of recombinant expression plasmid:

[0068] (1) Pipette 1-3 μL of the recombinant expression plasmid at a concentration of 100 ng / μL into 100 μL of E. coli DH5α competent cells, gently shake and rotate to mix, and place on ice for 20-30 minutes. (2) Place in a 42℃ water bath for 90 seconds without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μL of 37℃ pre-warmed LB medium to each tube and culture gently at 37℃ with a shaker at 200 rpm for about 40 minutes.

[0069] Verification of recombinant expression plasmid:

[0070] (1) Prepare LB plates containing ampicillin. (2) Take 100 μL of bacterial solution and spread it on an LB plate containing 100 μg / mL ampicillin. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick the bacteria from the plate and shake at 37°C at 250 rpm for 14 hours. Use the bacterial solution for PCR identification and send the positive clone bacterial solution for sequencing.

[0071] Method for identification of cloned plasmids:

[0072] PCR amplified the gE1-gE2 (aa31-161) fragment. The identification primer sequence was synthesized by the company's internal primer department. The PCR reaction used a 20μL system: 0.5μL primer, 2μL template bacterial solution, 0.5μL polymerase buffer, 3μL buffer, and 14μL ddH2O. Cycle parameters: 96℃ pre-denaturation for 3min; 95℃15s, 58℃15s, 72℃20s, 23 cycles, and 72℃ final extension for 1min. The positive clones were screened by bacterial solution PCR method, and the obtained positive bacterial solution was shaken at 37℃ for plasmid sequencing. The correct plasmid was sequenced and double-digested with BamHI-XhoI. The two fragments obtained were consistent with the expected fragment size.

[0073] Recombinant expression plasmid extraction:

[0074] Inoculate 1% E. coli DH5α competent cells containing pcDNA3.1(+)-gE1-gE2(aa31-161)-8×His recombinant expression plasmid in 2mL LB medium and shake culture at 37℃ overnight. After treating the cells, extract 100μg of plasmid using a plasmid extraction kit. Sequencing was performed to verify the target gene, and the sequencing results were consistent with the designed genome sequence.

[0075] Example 4 Cell transfection and gE1-gE2 (aa31-161) protein purification

[0076] Cell transfection:

[0077] CHO cells were cultured and expanded in serum-free cell expression medium in a conical flask at 37°C and 8% CO2. The viable cell density and survival rate were determined before transfection. The cell density should reach 7×10 6 ~10×10 6 / mL, survival rate 95-99% according to ExpiFectamine TM Transfection was performed using the CHO transfection kit. Fresh cell expression medium was preheated to 37°C and the cells were diluted to a final density of 6 × 10 6 / mL, 50mm amplitude incubator, 90rpm shaking culture, 37℃, 8% CO2. TM Prepare transfection reagent and pcDNA3.1(+)-gE1-gE2(aa31-161)-8×His recombinant expression plasmid complex in SFM medium (4°C), i.e., prepare 40 μL OptiPRO for 1 mL CHO cells. TM Add 0.8 μg of recombinant expression plasmid to SFM, mix well and let stand for 5 minutes. Prepare 40 μL OptiPRO TM SFM plus 3 μL ExpiFectamine TM CHO reagent, mix well and place for 5 minutes, then place at room temperature for another 1 to 5 minutes, then slowly transfer the solution to a shake flask and shake it, gently shaking the shake flask during the addition process. Place the cells in a 50mm amplitude incubator at 90rpm for shaking culture, 37°C, 8% CO2. 18-22 hours after transfection, add Enhancer and ExpiCHO Feed according to the kit instructions. Example: Add 6μL Enhancer and 0.24mL ExpiCHO Feed to 1mL of cells, and place the cells in a 50mm amplitude incubator at 90rpm for culture, 37°C, 8% CO2. Collect 8 days after transfection for subsequent purification of the recombinant fusion protein.

[0078] Protein purification:

[0079] A Hi Trap 5ml Chelating HP column loaded with NiSO4 (GE, USA) was used. After pretreatment of the chelating chromatography medium, the column was loaded and washed with 50mmol / L EDTA, 0.2mol / L NaOH, and ultrapure water, respectively. After balancing with more than 5 times the lysis buffer, the cell lysis supernatant was directly loaded onto the column and eluted with solution A (20mmol / L PB, pH7.4, 0.15mol / L NaCl) and solution B (20mmol / L PB, pH7.4, 0.15mol / L NaCl, 0.25mol / L imidazole) as the mobile phase. The target components were collected and continued to be separated by SEC. A HiLoad 26 / 60 Superdex-200 pre-grade gel column (GE, USA) was used. One column volume (about 320mL) was eluted with solution C (20mmol / L PB, pH7.4, 0.15mol / L NaCl) as the mobile phase, and the elution rate was 2.5mL / min. The target protein fractions were collected and the purity was tested by SDS-PAGE. The purified protein of gE1-gE2 (aa31-161) was obtained and analyzed by SDS-PAGE. Figure 1 As shown, the purity of the purified target protein can reach 95.6%.

[0080] Example 5 Synthesis of fusion gene gE1-gE2 (aa31-140)

[0081] WuXi Biologics was commissioned to artificially synthesize the gene sequence of the fusion protein gE1-gE2 (aa31-140) according to the sequence of SEQ ID NO: 23 of the present invention.

[0082] Example 6 Construction of pcDNA3.1(+)-gE1-gE2(aa31-140)-8×His recombinant expression plasmid

[0083] Add a signal peptide (amino acid sequence as shown in SEQ ID NO: 29) and add an 8×His tag. Select pcDNA3.1(+) as the vector and optimize the codons according to the host CHO cells. Recombine the gene sequence of the synthesized fusion protein into the target vector pcDNA3.1(+) to obtain the pcDNA3.1(+)-gE1-gE2(aa31-140)-8×His recombinant expression plasmid. The reaction system is as shown in Table 1. Connect for 30 minutes under the environment to obtain the recombinant expression plasmid pcDNA3.1(+)-gE1-gE2(aa31-140)-8×His.

[0084] Example 7 Screening and identification of recombinant plasmid pcDNA3.1(+)-gE1-gE2(aa31-140)-8×His

[0085] Transformation of recombinant expression plasmid:

[0086] (1) Pipette 1-3 μL of the recombinant expression plasmid at a concentration of 100 ng / μL into 100 μL of E. coli DH5α competent cells, gently shake and rotate to mix, and place on ice for 20-30 minutes. (2) Place in a 42℃ water bath for 90 seconds without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μL of 37℃ pre-warmed LB medium to each tube and culture gently at 37℃ with a shaker at 200 rpm for about 40 minutes.

[0087] Verification of recombinant expression plasmid:

[0088] (1) Prepare LB plates containing ampicillin. (2) Take 100 μL of bacterial solution and spread it on an LB plate containing 100 μg / mL ampicillin. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick the bacteria from the plate and shake at 37°C at 250 rpm for 14 hours. Use the bacterial solution for PCR identification and send the positive clone bacterial solution for sequencing.

[0089] Method for identification of cloned plasmids:

[0090] PCR amplified the gE1-gE2 (aa31-140) fragment. The identification primer sequence was synthesized by the company's internal primer department. The PCR reaction used a 20μL system: 0.5μL primer, 2μL template bacterial solution, 0.5μL polymerase buffer, 3μL buffer, and 14μL ddH2O. Cycle parameters: 96℃ pre-denaturation for 3min; 95℃15s, 58℃15s, 72℃20s, 23 cycles, and 72℃ final extension for 1min. The positive clones were screened by bacterial solution PCR method, and the obtained positive bacterial solution was shaken at 37℃ for plasmid sequencing. The correct plasmid was sequenced and double-digested with BamHI-XhoI. The two fragments obtained were consistent with the expected fragment size.

[0091] Recombinant expression plasmid extraction:

[0092] Inoculate 1% E. coli DH5α competent cells containing pcDNA3.1(+)-gE1-gE2(aa31-140)-8×His recombinant expression plasmid in 2mL LB medium and shake culture at 37℃ overnight. After treating the cells, extract 100μg of plasmid using a plasmid extraction kit. Sequencing was performed to verify the target gene, and the sequencing results were consistent with the designed genome sequence.

[0093] Example 8 Cell transfection and gE1-gE2 (aa31-140) protein purification

[0094] Cell transfection:

[0095] CHO cells were cultured and expanded in serum-free cell expression medium in a conical flask at 37°C and 8% CO2. The viable cell density and survival rate were determined before transfection. The cell density should reach 7×10 6 ~10×10 6 / mL, survival rate 95-99% according to ExpiFectamine TM Transfection was performed using the CHO transfection kit. Fresh cell expression medium was preheated to 37°C and the cells were diluted to a final density of 6 × 10 6 / mL, 50mm amplitude incubator, 90rpm shaking culture, 37℃, 8% CO2. TM Prepare transfection reagent and pcDNA3.1(+)-gE1-gE2(aa31-140)-8×His recombinant expression plasmid complex in SFM medium (4°C), i.e., prepare 40 μL OptiPRO for 1 mL CHO cells. TM Add 0.8 μg of recombinant expression plasmid to SFM, mix well and let stand for 5 minutes. Prepare 40 μL OptiPRO TMSFM plus 3 μL ExpiFectamine TM CHO reagent, mix well and place for 5 minutes, then place at room temperature for another 1 to 5 minutes, then slowly transfer the solution to a shake flask and shake it, gently shaking the shake flask during the addition process. Place the cells in a 50mm amplitude incubator at 90rpm for shaking culture, 37°C, 8% CO2. 18-22 hours after transfection, add Enhancer and ExpiCHO Feed according to the kit instructions. Example: Add 6μL Enhancer and 0.24mL ExpiCHO Feed to 1mL of cells, and place the cells in a 50mm amplitude incubator at 90rpm for culture, 37°C, 8% CO2. Collect 8 days after transfection for subsequent purification of the recombinant fusion protein.

[0096] Protein purification:

[0097] A Hi Trap 5ml Chelating HP column loaded with NiSO4 (GE, USA) was used. After pretreatment of the chelating chromatography medium, the column was loaded and washed with 50mmol / L EDTA, 0.2mol / L NaOH, and ultrapure water, respectively. After balancing with more than 5 times the lysis buffer, the cell lysis supernatant was directly loaded onto the column and eluted with solution A (20mmol / L PB, pH7.4, 0.15mol / L NaCl) and solution B (20mmol / L PB, pH7.4, 0.15mol / L NaCl, 0.25mol / L imidazole) as the mobile phase. The target components were collected and continued to be separated by SEC. A HiLoad 26 / 60 Superdex-200 pre-grade gel column (GE, USA) was used. One column volume (about 320mL) was eluted with solution C (20mmol / L PB, pH7.4, 0.15mol / L NaCl) as the mobile phase, and the elution rate was 2.5mL / min. The target protein fractions were collected and the purity was tested by SDS-PAGE. The purified protein of gE1-gE2 (aa31-140) was obtained and analyzed by SDS-PAGE. Figure 1 As shown, the purity of the purified target protein can reach 96.4%.

[0098] Example 9 Construction of vector and protein expression

[0099] With reference to the above examples, the following target fusion gene to be expressed was constructed, and the purified protein was expressed, as shown in Table 2.

[0100] Table 2 Different target fusion genes

[0101]

[0102]

[0103] Example 10 Preparation of immunogenic components

[0104] 50 μg of recombinant VZV fusion protein gE1-gE2 (aa31-101), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0105] Example 11 Preparation of immunogenic components

[0106] 50 μg of recombinant VZV fusion protein gE1-gE2 (aa31-119), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0107] Example 12 Preparation of immunogenic components

[0108] 50μg of recombinant VZV fusion protein gE1-gE2 (aa31-140), 20mg of sucrose, 0.160mg of sodium dihydrogen phosphate dihydrate, 0.116mg of dipotassium phosphate, and 0.08mg of polysorbate 80 are divided into 2mL vials, 0.5mL per bottle, and freeze-dried in a freeze dryer. The vaccination dose for each person is 1 bottle.

[0109] Example 13 Preparation of immunogenic components

[0110] 50μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-161), 20mg of sucrose, 0.160mg of sodium dihydrogen phosphate dihydrate, 0.116mg of dipotassium phosphate, and 0.08mg of polysorbate 80 are divided into 2mL vials, 0.5mL per bottle, and freeze-dried in a freeze dryer. The vaccination dose for each person is 1 bottle.

[0111] Example 14 Preparation of immunogenic components

[0112] 50 μg of recombinant VZV fusion protein gE1-gE2 (aa31-181), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0113] Example 15 Preparation of immunogenic components

[0114] 50 μg of recombinant VZV fusion protein gE1-gE2 (aa31-194), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0115] Example 16 Preparation of immunogenic components

[0116] 50μg of VZV gE protein, 20mg of sucrose, 0.160mg of sodium dihydrogen phosphate dihydrate, 0.116mg of dipotassium phosphate, and 0.08mg of polysorbate 80 are divided into 2mL vials, 0.5mL per bottle, and freeze-dried in a freeze dryer. The vaccination dose for each person is 1 bottle.

[0117] Example 17 Preparation of immunogenic composition

[0118] 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-161), 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-194), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0119] Example 18 Preparation of immunogenic composition

[0120] 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-161), 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-140), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0121] Example 19 Preparation of immunogenic composition

[0122] 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-101), 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-161), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0123] Example 20 Preparation of immunogenic composition

[0124] 25 μg of recombinant VZV fusion protein gE1-gE2 (aa31-161), 25 μg of recombinant VZV fusion protein gE1-gE2 (aa31-119), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0125] Example 21 Preparation of immunogenic composition

[0126] 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-119), 25 μg of genetically recombinant VZV fusion protein gE1-gE2 (aa31-181), 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0127] Example 22 Preparation of vaccine

[0128] The immunogenic components described in any one of Examples 10-16 and the immunogenic compositions described in any one of Examples 17-21 further comprise AS01B adjuvant. The adjuvant bottle is 0.5 mL, and the vaccination dose per person is 1 bottle, which contains 50 μg of MPL, 50 μg of QS21, 4.385 mg of sodium chloride, 1 mg of DOPC, 0.54 mg of potassium dihydrogen phosphate, 0.25 mg of cholesterol, 0.15 mg of anhydrous disodium phosphate, and water for injection is added to 0.5 mL. Refrigerate at 2-8°C and keep away from light.

[0129] Example 23 Detection of mouse serum specific IgG antibodies

[0130] The specific IgG antibodies in serum were detected by immunizing with the vaccine composed of the immunogenic component gE (1-546aa) described in Example 16, the immunogenic components gE1-gE2 (aa31-140) and gE1-gE2 (aa31-161) described in Examples 12 and 13 and AS01B adjuvant.

[0131] In this study, C57BL / 6 mice were divided into different antigen groups, with 6 mice in each group. They were first vaccinated with 2000 pfu of inactivated varicella vaccine (purchased from Shanghai Rongsheng Bio-Pharmaceutical Co., Ltd.), and then immunized with recombinant herpes zoster vaccine (1 / 10 human dose) on days 35 and 49, respectively. Serum was isolated 14 days (63d) after the second immunization with the recombinant herpes zoster vaccine, and gE protein-specific IgG antibodies were detected by ELISA.

[0132] The results are shown in Table 3. Mice immunized with the recombinant VZV gE1-gE2 vaccine can produce higher titers of specific IgG antibodies, and the recombinant VZV gE1-gE2 (aa31-161) vaccine has more advantages than the recombinant VZV gE1-gE2 (aa31-140) vaccine.

[0133] Table 3 Specific IgG antibody detection results

[0134] Antigen name S1 S2 S3 S4 S5 S6 E140 250 250 250 250 250 250 E161 80000 40000 250 250 320000 250 E 2560000 1280000 1280000 1280000 2560000 1280000 gE1-gE2(aa31-140) 640000 640000 1280000 640000 1280000 1280000 gE1-gE2(aa31-161) 2560000 1280000 1280000 2560000 2560000 2560000

[0135] Example 24 Cellular Immunoassay

[0136] The mice were immunized with the vaccine prepared with the immunogenic component gE (1-546aa) described in Example 16, the immunogenic components gE1-gE2 (aa31-140) and gE1-gE2 (aa31-161) described in Example 12 and Example 13 and the AS01B adjuvant, and the cellular immunity was detected. In this study, C57BL / 6 mice were divided into different antigen groups, and the negative control was a saline group. Each group had 6 mice, and was first vaccinated with 2000pfu of inactivated varicella vaccine (purchased from Shanghai Rongsheng Bio-Pharmaceutical Co., Ltd.), and then immunized with recombinant herpes zoster vaccine (1 / 10 human dose) on the 35th and 49th days, and the humoral immunity and cellular immunity effects of different adjuvants were evaluated on the 63rd day. 14 days after the second immunization with the recombinant herpes zoster vaccine (63d), the mouse spleen was taken to isolate mouse splenic lymphocytes, and the VZV gE (1-546aa) peptide library was used as a stimulus to detect the levels of intracellular cytokines IFN-γ and IL-2 by flow cytometry. Since there was no significant difference in the results of CD8+T cells, the final presentation was the percentage of cells secreting different cytokines in CD4+T cells.

[0137] Table 4 Results of mouse cellular immunity detection

[0138] antigen IL-2+ IL-2+IFN-γ+ IFN-γ+ total Normal saline 0.062 — 0.05706 0.12 gE161 / 5μg 0.43 1 0.6262 2.06 gE140 / 5μg 0.5 1.3 0.736 2.54 gE / 5μg 0.93 1.4 0.452 2.78 gE1-gE2(aa31-161) / 5μg 0.62 1.5 0.7716 2.89 gE1-gE2(aa31-140) / 5μg 0.77 1.9 0.9142 3.58

[0139] The results show that the gene recombinant VZV gE1-gE2 vaccine can induce stronger cellular immunity than the simple VZV gE protein vaccine. These results show that vaccination with the gene recombinant VZV vaccine of the present invention may form a stronger Th1 type cellular immune memory.

[0140] Example 25 Evaluation of adjuvant effect

[0141] The immunogenic components gE (1-546aa) described in Example 16, gE1-gE2 (aa31-140) and gE1-gE2 (aa31-161) described in Examples 12 and 13 were used as immunogens, and the inventors conducted immunogenicity studies using C57BL / 6 mice as an animal model to investigate the immunogenicity of different adjuvants combined with antigens. MPL / Poly I: C composite adjuvant, QS-21 / Poly I: C composite adjuvant, R837 / Poly I: C composite adjuvant and separate MPL, Poly I: C and CpG adjuvants were used as control groups to evaluate the effect of the adjuvant by studying the immunogenicity of the above-mentioned compositions.

[0142] VZV gE antigen was mixed thoroughly with 0.5 ml / dose of adjuvant in an amount of 5 μg. Each adjuvant was in the form of a liposome preparation, and the formulation of CpG preparation (0.5 ml / dose) included: DOPC 1 mg, cholesterol 0.25 mg, CpG 1 mg, Na2HPO4 0.15 mg, KH2PO4 0.54 mg, NaCl 4.385 mg. PolyI:C preparation (0.5 ml / dose) included: DOPC 1 mg, cholesterol 0.25 mg, Poly I:C 1 mg, Na2HPO4 0.15 mg, KH2PO4 0.54 mg, NaCl 4.385 mg. MPL preparation (0.5 ml / dose) included: DOPC 1 mg, cholesterol 0.25 mg, MPL 50 μg, Na2HPO4 0.15 mg, KH2PO4 0.54 mg, NaCl 4.385 mg.

[0143] Preparation of BFA-MPL / Poly I:C composite adjuvant: 4g DOPC, 1g cholesterol and 200mg MPL were dissolved in 20mL isopropanol, and a lipid film was obtained by gradient vacuum rotary evaporation. 50mL of phosphate aqueous solution (50mM PB, containing 100mM NaCl) was added to the lipid film, and the suspension was obtained by rotary hydration in a 25°C water bath. The obtained suspension was diluted to a specific concentration and then homogenized. The liposomes obtained after homogenization were filtered with a 0.22μm polyethersulfone membrane (PES) filter to finally obtain appropriately specific concentrated liposomes. Poly I:C was dissolved in a certain volume of physiological saline to obtain a Poly I:C solution. The concentrated liposomes of a specific concentration were diluted and mixed with a phosphate aqueous solution (10 mM PB, containing 150 mM NaCl) to a certain concentration, and the Poly I:C solution was added and mixed until the concentrations of each component were DOPC 2 mg / ml, cholesterol 0.5 mg / ml, MPL 0.1 mg / ml, and Poly I:C 2 mg / ml, which was the final BFA-MPL / Poly I:C composite adjuvant.

[0144] Preparation of BFA-R837 / Poly I:C composite adjuvant: Weigh 200mg DOPC and 50mg cholesterol, dissolve in 5ml ethanol / isopropanol mixed organic solvent (ethanol isopropanol volume ratio 1:1). 15mg R837 is dissolved in 7.5ml DMSO to obtain R837 mother liquor. 1ml organic phase with DOPC and cholesterol dissolved, add 1ml R837 mother liquor, add 0.5ml ethanol / isopropanol mixed organic solvent (ethanol isopropanol volume ratio 1:1), mix to form alcohol phase, phosphate aqueous solution (10mM PB, containing 150mM NaCl) as water phase, water-to-alcohol ratio 3:1 to prepare sample, remove organic solvent, filter with 0.22μm polyethersulfone membrane (PES) filter, the sample particle size after filtration is 65.01nm, PDI is 0.171. Poly I:C is dissolved in a certain volume of physiological saline to form Poly I:C solution. Take 500 μl of the filtered sample, add Poly I:C solution, add phosphate aqueous solution (10 mM PB, 150 mM NaCl), mix until the content of each component is DOPC 2 mg / ml, cholesterol 0.5 mg / ml, R837 0.1 mg / ml, Poly I:C 2 mg / ml, which is the final BFA-R837 / Poly I:C composite adjuvant.

[0145] Preparation of QS-21 / PolyI:C composite adjuvant: 4g DOPC and 1g cholesterol were dissolved in 20mL isopropanol, and a lipid film was obtained by gradient vacuum rotary evaporation. 50mL phosphate aqueous solution (50mM PB, containing 100mM NaCl) was added to the lipid film, and the suspension was obtained by rotary hydration in a 25°C water bath. The obtained suspension was diluted to a specific concentration and homogenized. The liposomes obtained after homogenization were filtered with a 0.22μm polyethersulfone membrane (PES) filter to finally obtain appropriately specific concentrated liposomes. QS-21 and Poly I:C were dissolved in a certain volume of physiological saline to form a mixed aqueous solution. The concentrated liposomes of a specific concentration were diluted and mixed with a phosphate aqueous solution (10 mM PB, containing 150 mM NaCl) to a certain concentration, and then the mixed aqueous solution was added and mixed until the concentration of each component was DOPC 2 mg / ml, cholesterol 0.5 mg / ml, QS-21 0.1 mg / ml, Poly I: C 2 mg / ml, which was the final QS-21 / Poly I: C composite adjuvant.

[0146] C57BL / 6 mice were vaccinated with 2000pfu of inactivated varicella vaccine (purchased from Shanghai Rongsheng Biopharmaceutical Co., Ltd.), and then immunized with recombinant herpes zoster vaccine (1 / 10HD) on days 35 and 49 after vaccination, and the cellular immune effects of different adjuvants were evaluated on day 63. 14 days after the second immunization with recombinant herpes zoster vaccine (63d), the spleen of mice was taken to separate mouse splenic lymphocytes, and the levels of intracellular cytokines IFN-γ and IL-2 were detected by flow cytometry using VZV gE (1-546aa) peptide library as a stimulus. Because there was no significant difference in the results of CD8+T cells, the final results showed the percentage of cells secreting different cytokines in CD4+T cells. The results of mouse immune detection are shown in Table 5.

[0147] Table 5 Results of cellular immune detection with different adjuvants / antigens

[0148]

[0149]

[0150] The results showed that the composite adjuvants basically exhibited adjuvant activity that was significantly superior to that of single MPL, PolyI:C or CpG adjuvants, and the levels of intracellular cytokines IFN-γ and IL-2 stimulated were generally higher. In comparison, the levels of antigen-specific cytokines IFN-γ and IL-2 promoted by the combination of BFA-MPL / Poly I:C composite adjuvant and antigen were the highest.

[0151] The above embodiments are only illustrative of the present invention and are not intended to limit the protection scope of the present invention. Without departing from the design spirit of the present invention, any non-substantial modifications made by those skilled in the art on the basis of the present invention shall fall within the protection scope of the present invention.

Claims

1. A genetically recombinant VZV fusion protein, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO: 17 or 18.

2. A method for preparing the genetically recombinant VZV fusion protein according to claim 1, characterized in that: The following steps are involved: S1. Artificially synthesizing the gene sequence of the fusion protein according to claim 1, and connecting the gene of the fusion protein into an expression vector to construct a recombinant expression plasmid; S2, transforming the constructed recombinant expression plasmid into the host bacteria, and selecting and identifying the correct recombinant plasmid through double enzyme digestion and DNA sequencing; S3. Use the identified recombinant plasmid to transfect host cells, express the gene recombinant VZV fusion protein, and purify it.

3. The method for preparing the genetically recombinant VZV fusion protein according to claim 2, characterized in that: The expression vector described in S1 is pcDNA3.1(+).

4. The method for preparing the genetically recombinant VZV fusion protein according to claim 2, characterized in that: The host cell is a Chinese hamster ovary cell.

5. An immunogenic composition, characterized in that The immunogenic composition comprises the fusion protein of claim 1.

6. A genetically recombinant VZV vaccine, characterized in that: The gene recombinant VZV vaccine comprises the fusion protein according to claim 1, or the immunogenic composition according to claim 5, and optionally a pharmaceutically acceptable adjuvant.

7. The genetically recombinant VZV vaccine according to claim 6, characterized in that: The adjuvant is at least one of aluminum salt adjuvant, Freund's complete adjuvant, propolis adjuvant, oil-water emulsion, cytokine, CpG-DNA, genetically engineered attenuated toxin, immunostimulatory complex or liposome.

8. The genetically recombinant VZV vaccine according to claim 7, characterized in that: The adjuvant is liposome adjuvant AS01B.

9. The genetically recombinant VZV vaccine according to claim 7, characterized in that: The adjuvant is a liposome preparation selected from any one of BFA-R837 / PolyI:C, QS-21 / PolyI:C and BFA-MPL / PolyI:C, wherein the BFA-R837 / PolyI:C liposome preparation comprises DOPC 2mg / ml, cholesterol 0.5mg / ml, R837 0.1mg / ml, Poly I:C 2mg / ml; the QS-21 / PolyI:C liposome preparation comprises DOPC 2mg / ml, cholesterol 0.5mg / ml, QS-21 0.1mg / ml, PolyI:C 2mg / ml; The BFA-MPL / PolyI:C liposome preparation contained DOPC 2 mg / ml, cholesterol 0.5 mg / ml, MPL 0.1 mg / ml, and PolyI:C 2 mg / ml.

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