Varicella-zoster virus gE fusion protein as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a varicella-zoster virus gE fusion protein as well as a preparation method and application thereof. The varicella-zoster virus gE fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 1 and at least one of the following fragments: a signal peptide, a Foldon sequence, a Ferritin sequence and a His tag sequence, the signal peptide can improve the protein expression quantity, the Foldon sequence can improve the stability of the protein, the particle size of an antigen can be increased, the immunogenicity of the antigen can be improved, and the fusion protein can be used for preparing a medicine for treating the varicella-zoster virus gE fusion protein. The Ferritin sequence can enhance immune response, a vaccine prepared from the gE fusion protein has excellent immunogenicity, and compared with a commercially available vaccine Shingrix, higher humoral immunity and cellular immunity levels can be induced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a varicella-zoster virus gE fusion protein, a preparation method and application thereof in preparing a vaccine for preventing varicella-zoster virus infection. Background Art

[0002] Chickenpox and herpes zoster are two different diseases caused by the varicella-zoster virus. Chickenpox is a self-limiting disease, a common acute infectious disease in childhood, and is highly contagious. Patients develop a systemic rash, accompanied by fever, malaise, and other symptoms, which usually last for 2-4 days. If the virus is not completely eliminated by the body after the symptoms of chickenpox disappear, it will enter the body's dorsal root ganglia or cranial nerve ganglia and remain dormant for life. Herpes zoster is caused by the reactivation of the varicella-zoster virus that has been dormant in the patient's body after chickenpox heals. It is common in the elderly and immunocompromised groups, and can also be seen in young people from time to time. The typical clinical manifestation is painful erythema and blisters on one side of the body, distributed along the skin area innervated by a certain nerve. After the characteristic rash and pain disappear, a persistent neuropathic pain syndrome may develop, called postherpetic neuralgia, which is the most typical sequela of the disease. In addition, the appearance of herpes zoster may be accompanied by complications including myelitis, meningitis, stroke, retinitis, and many gastrointestinal diseases.

[0003] Varicella is caused by VZV infection transmitted through droplets during infancy and has a typical winter and spring seasonality, but herpes zoster is caused by the reactivation of the virus lurking in the patient's body, so the disease has no obvious seasonality. The reactivation of varicella-zoster virus is related to many factors, including: psychological stress, gender, immunosuppression, etc. Among these related factors, the incidence of herpes zoster is most closely related to age. More than half of herpes zoster patients are over 60 years old, and half of them will have complications. The severity of the complications increases with the age of the patients and they cannot be cured, which seriously affects the patients' work and life.

[0004] At present, there are two main countermeasures for herpes zoster: disease prevention and treatment. When acute herpes zoster occurs, broad-spectrum antiviral drugs including acyclovir, valacyclovir and famciclovir are usually taken to inhibit the development of the disease, thereby alleviating the symptoms and preventing secondary infections. The main way to prevent herpes zoster is through vaccination. There are currently only four herpes zoster vaccines, namely: Zostavax from Merck, Shingrix from GlaxoSmithKline (GSK), SkyZoster from SK Chemical Co., Ltd., and Ganwei from Biotech. Among them, SkyZoster is only sold in South Korea. Among these four vaccines, except for Shingrix under GSK, which is a recombinant technology route, the rest are live attenuated technology routes.

[0005] Zostavax (Merck) is a live attenuated vaccine that was approved by the FDA in 2006 for the prevention of herpes zoster and its complications in adults over 50 years old. Clinical trials have shown that Zostavax has a 51.3% preventive effect on herpes zoster in participants aged 60 or above. However, its efficacy against herpes zoster decreases with age (69.8% in adults aged 50 to 59, 51% in people over 60, and only 18% in people over 80). In addition, as a live attenuated vaccine, Zostavax may induce serious diseases in immunocompromised patients, so it cannot be used for immunosuppressed and immunocompromised patients or pregnant women. In addition, a series of adverse reactions may occur after vaccination with Zostavax, mainly pain, erythema and swelling at the injection site. In 2023, the live attenuated shingles vaccine Ganwei developed by Changchun Baike Biopharma in my country was approved for production. It is the world's first live attenuated shingles vaccine suitable for adults over 40 years old. Its biotechnology route is comparable to Zostavax, with similar effects, and the vaccination population will be expanded to those over 40 years old, covering a larger audience group.

[0006] So far, the only recombinant subunit zoster virus vaccine is the adjuvanted zoster subunit vaccine Shingrix developed by GlaxoSmithKline (GSK), which was approved by the FDA in 2017. Shingrix uses VZV gE protein as an antigen and uses the AS01B adjuvant system. The gE protein is abundant on the surface of VZV and has strong immunogenicity. It contains T and B cell epitopes and can induce humoral and cellular immune responses. AS01B is a polysaccharide mixture that contains two components, monophosphoryl lipid A (MBL) and saponin 21 (QS-21), as an adjuvant. The former is a Toll-like receptor 4 agonist prepared from bacterial lipopolysaccharide, and the latter is a common component of adjuvants, but its immune enhancement mechanism is not clear. The two together enhance the immunogenicity of the antigen gE, especially the specific CD4 T cell response. Clinical data show that Shingrix has an efficacy of 97% in people aged 50 to 69 and an efficacy of 91% in people over 70 years old, which is better than the live attenuated vaccine Zostavax. However, Shingrix has a wide range of clinical side effects, mainly including: injection site reactions (pain, redness and swelling), fatigue, headache, fever, nausea, vomiting, diarrhea, etc. In addition, the Shingrix vaccine formula is complex, the antigen needs to be freeze-dried and separated from the adjuvant, the AS01B adjuvant component QS21 cannot be chemically synthesized, the raw materials are scarce, expensive, and the cost is high. Therefore, it is necessary to develop a safe, effective, simple, and low-cost vaccine. Summary of the invention

[0007] The present invention aims at the deficiencies of the prior art (complex preparation process, low yield and poor antigenicity) and provides a novel varicella-zoster virus gE fusion protein and a preparation method thereof; and aims at the deficiencies of the prior vaccine technology (poor immunogenicity, serious side effects and high cost) and provides a novel varicella-zoster virus gE fusion protein vaccine and application thereof.

[0008] The first aspect of the present invention provides a varicella-zoster virus gE fusion protein, characterized in that the gE fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 1 and at least one of the following fragments: a signal peptide, a Foldon sequence, a Ferritin sequence, and a His tag sequence.

[0009] In some embodiments, the gE fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1 and a signal peptide; or, The gE fusion protein comprises the amino acid sequence, signal peptide and Folden sequence as shown in SEQ ID NO: 1; or, The gE fusion protein includes the amino acid sequence shown in SEQ ID NO: 1, a signal peptide and a ferritin sequence.

[0010] In some embodiments, the signal peptide sequence is shown as SEQ ID NO:2; the Foldon sequence is shown as SEQ ID NO:3; and the Ferritin sequence is shown as SEQ ID NO:4.

[0011] In some embodiments, the gE fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5-7.

[0012] Another aspect of the present invention provides a recombinant nucleic acid comprising a nucleotide sequence encoding the varicella-zoster virus gE fusion protein as described above.

[0013] Another aspect of the present invention provides an expression vector comprising the recombinant nucleic acid as described above.

[0014] Another aspect of the present invention provides a host cell, comprising the recombinant nucleic acid or the expression vector as described above.

[0015] In some embodiments, the host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells.

[0016] In some embodiments, the host cell is a CHO cell, and the recombinant nucleic acid is a nucleic acid that has been codon-optimized according to the CHO expression system.

[0017] Another aspect of the present invention provides a method for preparing the varicella-zoster virus gE fusion protein, characterized in that the preparation method comprises the following steps: Step 1: Cultivating the host cell as described above under conditions suitable for the expression of the gE fusion protein; Step 2: Collect the cell supernatant by centrifugation; Step 3: Purify and obtain the gE fusion protein.

[0018] Another aspect of the present invention provides the varicella-zoster virus gE fusion protein, or the use of the varicella-zoster virus gE fusion protein prepared by the preparation method in the preparation of a vaccine for preventing varicella-zoster virus infection.

[0019] Another aspect of the present invention provides a vaccine for preventing varicella-zoster virus infection, comprising the varicella-zoster virus gE fusion protein as described above, or the varicella-zoster virus gE fusion protein prepared by the preparation method as described above, and a pharmaceutically acceptable carrier or excipient.

[0020] In some embodiments, the vaccine further comprises an adjuvant, and the adjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG, cytokine, liposome and QS-21.

[0021] Compared with the prior art, the advantages and positive effects of the present invention are: The recombinant herpes zoster vaccine (CHO cells) of the present invention uses an antigen derived from the gE protein of the human herpesvirus type 3 KV6-3127 (GenBank: KP771902.1) strain, with the original signal peptide (aa1-30), intracellular region, and transmembrane region removed. After retaining part of the extracellular structure (aa31-539), in order to increase the protein expression, a signal peptide (MRVPAQLLGLLLLWLRGARC) was added, and a 10×His tag was added to the C-terminus to facilitate protein purification, thereby constructing the candidate antigen gE1; on the basis of the candidate antigen gE1, a Foldon sequence was added to its C-terminus, and finally a His purification tag was added to construct the candidate antigen gE2 (gE-Foldon); Foldon is a small polypeptide molecule that was originally discovered in the fibrin of T4 phage. It is composed of 27 amino acids (GYIPEAPRDGQAYVRKDGEWVLLSTFL) and can form an α-helical structure. Three identical α-helical structures can form a stable trimer by relying on intermolecular hydrogen bonds and hydrophobic forces. When preparing recombinant proteins, Foldon After being expressed in tandem with the target protein, it can not only form a trimer structure to improve the stability of the protein, but also increase the particle size of the antigen and improve the immunogenicity of the antigen; in addition, based on the candidate antigen gE1, the Ferritin sequence is added to the C-terminus to construct the candidate antigen gE3 (gE-Ferritin); Ferritin itself has certain immune activity and can enhance the immune response by activating immune cells and promoting antibody production. In addition, Ferritin is often used to encapsulate vaccine antigens or adjuvants to protect the antigen from enzymatic degradation and improve its stability and immunogenicity.

[0022] The three candidate antigen sequences were codon optimized, inserted into the expression vector PCAGGS after gene synthesis, and then transfected into mammalian ExpiCHO cells to express fusion proteins, and the supernatant was purified using nickel column affinity chromatography. The production of candidate antigens gE1, gE2, and gE3 was considerable. Animal experiments confirmed that the three candidate antigens combined with HA201 adjuvant had excellent immunogenicity and could induce higher humoral and cellular immunity levels compared with the commercially available vaccine Shingrix. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0024] Figure 1 This is the plasmid map of PCAGGS-gE1.

[0025] Figure 2 This is the plasmid map of PCAGGS-gE2.

[0026] Figure 3 This is the plasmid map of PCAGGS-gE3.

[0027] Figure 4 This is a gel electrophoresis diagram showing the double digestion identification of the expression plasmids PCAGGS-gE1, PCAGGS-gE2, and PCAGGS-gE3 using restriction endonucleases BamHI / PvuI.

[0028] Figure 5 This is the Western Blot detection result of gE1, gE2 and gE3 protein expression. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments: To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0030] Example 1: Recombinant construction of gE1, gE2 and gE3:

[0031] The antigens used in the new varicella-zoster vaccine are in three forms, including antigens gE1 (gE), gE2 (gE-Foldon), and gE3 (gE- Ferritin). They are derived from ORF68 (GenBank: AKG57086.1) of the human herpesvirus type 3 strain KV6-3127 (GenBank: KP771902.1). It is a fragment obtained by removing the original signal peptide (aa1-30) and the carboxyl-terminal hydrophobic anchor region (aa540-623). The amino acid sequence is shown in SEQ ID NO: 1; the Foldon sequence comes from the fibrin of T4 phage (GenBank: KJ477684.1); the Ferritin sequence comes from the partial sequence of ferritin of Pyrococcus furiosus DSM 3638 (GenBank: CP023154.1). The antigen gE proteins gE1 (gE), gE2 (gE-Foldon), and gE3 (gE-Ferritin) described in the present invention are added with a signal peptide and a His tag respectively on the basis of the original gE protein truncated fragment (aa30-539); the signal peptide (sequence is shown in SEQ ID NO:2), Foldon (sequence is shown in SEQ ID NO:3) and the His tag; the signal peptide, Ferritin (sequence is shown in SEQ ID NO:4) and the His tag, the amino acid sequences are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 respectively; the three antigens gE1, gE2, and gE3 carry the His tag, and the theoretical molecular weights are approximately 59kD, 60kD, and 80kD, respectively (the three proteins are modified intracellularly after expression, and the actual molecular weights may be greater than the theoretical molecular weights).

[0032] The three antigen protein target genes were codon-optimized according to the CHO expression system and then delivered to Beijing Qingke Biotechnology Co., Ltd. for synthesis. The synthesized target genes were connected to the PCAGGS expression vector to obtain PCAGGS-gE1 ( Figure 1 )、PCAGGS-gE2( Figure 2 )、PCAGGS-gE3( Figure 3 ). The recombinant plasmid was double-digested for identification to check whether the fragment size after digestion was correct. After confirming that the fragment size was correct, it was used for subsequent plasmid transfection experiments. The digestion identification steps are as follows: The expression plasmids PCAGGS-gE1, PCAGGS-gE2, and PCAGGS-gE3 were double-digested with restriction endonucleases BamHI / PvuI. The enzyme digestion system configuration is shown in Table 1: Table 1 Plasmid restriction enzyme system:

[0033] After digestion in a 37°C water bath for 2 h, nucleic acid gel electrophoresis was performed for 30-40 min and imaged using a gel imager ( Figure 4 ).

[0034] After the expression plasmid PCAGGS-gE1 was digested with restriction endonucleases BamHI / PvuI, three bands appeared, with sizes of 4642bp, 1445bp, and 337bp, respectively. The electrophoresis results were consistent with the theoretical values; after the expression plasmid PCAGGS-gE2 was digested with restriction endonucleases BamHI / PvuI, three bands appeared, with sizes of 4735bp, 1445bp, and 337bp, respectively. The electrophoresis results were consistent with the theoretical values; after the expression plasmid PCAGGS-gE3 was digested with restriction endonucleases BamHI / PvuI, three bands appeared, with sizes of 5197bp, 1445bp, and 337bp, respectively. The electrophoresis results were consistent with the theoretical values. The expression plasmid was correctly digested and identified and can be used for subsequent transfection experiments.

[0035] Example 2: Recombinant expression of gE1, gE2 and gE3:

[0036] The correct expression plasmids PCAGGS-gE1, PCAGGS-gE2, and PCAGGS-gE3 were identified by restriction digestion and transfected into ExpiCHO cells using MetaCell®PEI 40K. The day before transfection, ExpiCHO cells were cultured at 3.5×10 6 The cell density on the day of subculture and transfection reached 8.0-10.0×10 cells / mL 6 cells / mL, and dilute to 6.0×10 cells / mL with pre-warmed fresh culture medium. 6cells / mL. Prepare a polypropylene centrifuge tube. First, add 8% of the final culture volume of culture medium as diluent. Then add DNA below the liquid surface and mix well by inverting 4-5 times. Add transfection reagent to DNA diluent and mix immediately (shake up and down and vortex for 5 seconds). Let the Reagent-DNA mixture stand at room temperature for 15-20 minutes. Slowly add the incubated Reagent-DNA mixture to the shake flask. When adding, gently shake the shake flask to help mix the cell suspension and transfection mixture. 18-22 hours after transfection, add 1% of the starting culture volume of MetaCell™ TiterEnhancer and 10% of the starting culture volume of MetaCell™ CHO TransFeed. Then cool to 32℃ for culture. On the 5th day of transfection, add 10% of the starting culture volume of MetaCell™ CHO TransFeed again. Culture until the 10th day and collect the cell supernatant by centrifugation. Western Blot to detect the expression of recombinant protein.

[0037] 10 days after the expression plasmids PCAGGS-gE1, PCAGGS-gE2, and PCAGGS-gE3 were transfected into ExpiCHO cells, the supernatants were collected and the expression of the target proteins was detected by Western Blot. After reducing electrophoresis, the membranes were transferred and incubated with His rabbit polyclonal antibody (commercially available). The secondary antibody used for detection was HRP-conjugated goat anti-rabbit antibody. The specific steps are as follows: Take 80 μl of cell supernatant, add 20 μl of 5×SDS-PAGE protein loading buffer, mix the cell supernatant and 5×SDS-PAGE protein loading buffer, and heat at 100°C for 5 minutes to fully denature the protein.

[0038] Centrifuge at 5000 rpm for 5 minutes, cool to room temperature, load 10 μl into the SDS-PAGE gel well, and start electrophoresis.

[0039] Electrophoresis can be stopped when the blue dye reaches near the bottom of the gel.

[0040] The gel was taken out for membrane transfer. After membrane transfer, the PVDF membrane was placed in a 5% skim milk solution for blocking for 2 hours.

[0041] The PVDF membrane was washed three times with TBST for 10 minutes each time, and then coated with primary antibody and incubated at 4°C overnight.

[0042] The PVDF membrane was washed three times with TBST for 10 minutes each time, and then coated with secondary antibody and incubated for 1 hour.

[0043] The PVDF membrane was washed three times with TBST for 10 minutes each time, and then washed with TBS for 10 minutes.

[0044] Add color developing solution to develop color.

[0045] Western Blot test results Figure 5 As shown, M: Marker (10-245kd), gE1 molecular weight is about 66kd, gE2 molecular weight is about 66kd, gE3 molecular weight is about 90kd. gE1, gE2, gE3 proteins are expressed in CHO cell supernatant.

[0046] Example 3: Purification of gE1, gE2 and gE3 recombinant proteins:

[0047] After gE1, gE2, and gE3 were transfected into ExpiCHO cells for 10 days, the cell supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and purified using a nickel column. The purified antigen protein was packaged and stored in a -80°C refrigerator for subsequent animal experiments. The purification process is as follows: Add appropriate amount of deionized water to rinse the packing, and close the lower outlet after the packing is dried; install the upper gasket, making sure there is no gap between the gasket and the column material, and be careful not to press the gasket hard to prevent damage to the column material.

[0048] Use 5 column volumes of equilibration solution to equilibrate the filler so that it is in the same buffer system as the target protein.

[0049] Use an appropriate amount of filtered cell culture supernatant to suspend an appropriate amount of filler, add the suspended filler to the cell culture supernatant, and shake at 4°C for 4 hours.

[0050] Add the bound sample to the equilibrated column to ensure full contact between the sample and the medium, and load the sample multiple times to increase the binding efficiency.

[0051] The target protein was eluted with 10 times column volume of eluent and collected in separate tubes. The eluted protein was concentrated using a 30 kDa ultrafiltration tube and the solution was changed by centrifugation at 3000 rpm at 4°C for a certain period of time.

[0052] Finally, concentrate to about 1.5 mL, test the concentration, dilute to 1 mg / ml, and store in aliquots.

[0053] Example 4: Immunogenicity test of gE1, gE2, and gE3 proteins on mice:

[0054] Prepare liposome mixture HA201: dissolve 3D-MLA, DOPC and cholesterol in isopropanol, evaporate and dry to form liposome membrane, add PBS buffer solution to hydrate, homogenize by high-pressure homogenizer until the particle size reaches 90nm~110nm, then add QS-21, dilute, sterile filter and then package.

[0055] Animal experiment immunization procedure: 6-8 week old SPF female BALB / C mice were randomly divided into 5 groups, 6 mice / group, for a total of 30 mice. The immunization dose, immunization route and grouping are shown in Table 2. The dose of recombinant gE protein antigen used in each group was 5ug, with primary immunization on day 0, blood collection on day 14, booster immunization on day 14, whole blood collection for serum separation and spleen collection for leukocyte separation to determine cellular immune indicators on day 28.

[0056] The specific implementation plan is shown in Table 2: Table 2 Mouse immunization scheme:

[0057] Specific IgG antibody detection: Dilute gE antigen to 0.1 g / ml with coating solution, add 100 μl to each well, and coat overnight at 4℃. The next day, wash 5 times with washing buffer, shake off the residual liquid, add blocking solution (2% BSA), and block at 37℃ for 2 hours. Dilute serum sample with sample diluent (10-fold starting dilution). Shake dry the blocked ELISA plate, wash 5 times with washing buffer, and pat dry. Add the sample from the dilution plate to the ELISA plate (100 μl / well) and place at room temperature for 1.5 hours (3 positive quality control wells are made on each plate at the same time, the positive reference is diluted 800 times, 100 μl / well; 3 negative quality control wells are made on each plate at the same time, the negative reference is diluted 100 times, 100 μl / well). Dilute HRP-labeled goat anti-mouse polyclonal antibody (1:5000) with antibody diluent, add secondary antibody, and place at room temperature for 1 hour. Wash the plate 5 times with washing solution, pat dry, and wipe the bottom with absorbent paper. Add colorimetric solution (100 μl / well), develop color at room temperature in the dark for 15 minutes, and add stop solution (50 μl / well) to terminate the reaction. Read the plate and perform data analysis (critical value: this method was used to detect IgG in sera from 120 blank SPF mice. After 100-fold dilution, the average OD450 was 0.071. 2.1 times of this value was set as the cut-off value, that is, OD450 ≥ 0.149 was judged as a positive reaction, and OD450 < 0.149 was judged as a negative reaction).

[0058] ELISA was used to detect VZV gE-specific binding antibodies in serum, and the results are shown in Table 3 : Antibody titers on day 28, gE1 + HA201, gE2 + HA201, and gE3 + HA201 all produced antibody titers that were not lower than those of the control group (Shingrix), while the antibody titer of the gE3 + HA201 group showed a trend of being higher than that of the control group.

[0059] Table 3 Specific IgG antibodies (log10):

[0060] Ultra-micro fluorescent antibody membrane antigen method (FAMA) detection of VZV gE protein antibody detection: Take the cultured MRC-5 cells and subculture them into T25 cell culture flasks. When they grow into a dense monolayer overnight, discard the growth medium, inoculate 0.1ml of herpes zoster virus (titer 3.7lgPFU / ml or more), and then add 5ml of virus culture medium, and observe the cytopathic effect every day. When the cytopathic effect reaches 50% to 75%, discard the culture medium, add trypsin to digest the cells, centrifuge, and discard the supernatant; then resuspend the cells, dilute the cells to a concentration of 1.2×106 / ml, add 5 μl to a 12-well immunofluorescence in situ hybridization slide (6000 cells per well), and then place it in a wet box at 37℃ for 40 min. Add 80% acetone to fix the cells, remove the acetone after 10 minutes, and let it air dry completely. Put the serum sample to be tested in a 56℃ water bath, inactivate it in a water bath for 30 minutes, and then dilute the serum 2-fold in series with PBS (such as 1:16-1:512). Add the diluted serum to the corresponding wells of the multi-well slide, 10 μl per well (make 2 duplicate wells), and set up negative and positive controls (positive serum diluted 1:1000), and incubate in a 4°C wet box overnight. The next day, add 10 μl of fluorescent antibody (dilute the fluorescent-labeled secondary antibody with PBS containing a final concentration of 0.01% Evans blue and set aside for 1:100) to the wells of the slide and incubate in a 37°C wet box for 60 min. Shake off the liquid in the well, wash with PBS 3 times, 5 min each time, and dry. Wash with PBS 3 times, then add 3 μl of 60% glycerol to the slide, cover with a coverslip, press tightly, and place on the stage of a fluorescence microscope to observe the results (FAMA antibody titer = the maximum dilution multiple of the test group cells that appear fluorescent rings / the maximum dilution multiple of the control group cells that do not appear fluorescent rings; FAMA antibody titer ratio ≥4 is judged as positive, and if <4 is judged as negative).

[0061] The FAMA method was used to detect VZV gE protein antibodies. As shown in Table 4, the three gE recombinant proteins of the present invention combined with the HA201 adjuvant can stimulate mice to produce sufficient antibodies.

[0062] Table 4 VZV gE protein antibody detection:

[0063] Specific T cell immune response: Wash the ELISPOT plate (MABTECH: pre-coated with IFN-γ mouse antibody and IL-2 mouse antibody) with sterile PBS, add 200 μl of 1640 medium containing 10% FBS, and block at room temperature for at least 30 min (negative control wells: complete medium 100 μl / well + cells 100 μl / well; antigen stimulation wells: stimulator 100 μl / well + cells 100 μl / well; positive control wells: ConA 100 ul / well + cells 100 μl / well). Adjust the lymphocyte count to 1-5×10 6 pcs / ml, add samples according to the design, then place all plates in a 5% CO2, 37℃ incubator, culture for 42±1 h, and do not move during the culture process. Discard the cell suspension, wash with PBS 5 times, add biotin-labeled detection antibody, and incubate at room temperature for 2 hours. Wash with PBS 5 times, dilute Strep-HRP at 1:1000 with PBS containing 0.5% FBS and add 96-well plate, 100μl per well, incubate at room temperature for 1 hour. Discard the Strep-HRP solution and wash with PBS 5 times. Add TMB substrate, 100 μl / well, color until distinguishable spots appear, rinse with deionized water to stop the color reaction, remove the support and rinse the bottom side of the membrane, let it air dry naturally at room temperature away from light, then place in the ELISPOT plate reader and count.

[0064] Table 5 shows the IFN-γ level on the 28th day, and Table 6 shows the IL-2 level on the 28th day. The three recombinant gE proteins of the present invention are combined with the company's self-developed HA201 adjuvant system to induce a cellular immune response that is superior to Shringrix. For recombinant gE protein, whether it is fused with the trimerization domain Folden (gE2) or the multimerization domain Ferritin (gE3), its immunogenicity is stronger than the monomer design (gE1) without fusion expression, which is manifested in that the designed molecules gE2 and gE3 expressed in a multimeric form induce a cellular immune response that is significantly higher than the monomer design molecule gE1 without fusion expression. At the same time, it was found that the gE protein assembled into a multimeric form using Ferritin as the fusion domain can induce a significantly stronger cellular immune response than Folden.

[0065] Table 5 Specific T cells (IFN-γ):

[0066] Table 6 Specific T cells (IL2):

[0067] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0068] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A varicella-zoster virus gE fusion protein, characterized in that: The gE fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 1 and at least one of the following fragments: a signal peptide, a Foldon sequence, a Ferritin sequence, and a His tag sequence.

2. The varicella-zoster virus gE fusion protein according to claim 1, characterized in that: The gE fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1 and a signal peptide; or, The gE fusion protein comprises the amino acid sequence, signal peptide and Folden sequence as shown in SEQ ID NO: 1; or, The gE fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 1, a signal peptide and a ferritin sequence.

3. The varicella-zoster virus gE fusion protein according to claim 1, characterized in that: The sequence of the signal peptide is shown in SEQ ID NO:2; the sequence of Foldon is shown in SEQ ID NO:3; and the sequence of Ferritin is shown in SEQ ID NO:

4.

4. The varicella-zoster virus gE fusion protein according to any one of claims 1 to 3, characterized in that: The gE fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5-7.

5. A recombinant nucleic acid comprising a nucleotide sequence encoding the varicella-zoster virus gE fusion protein according to any one of claims 1 to 4. An expression vector comprising the recombinant nucleic acid according to claim 5 .

7. A host cell comprising the recombinant nucleic acid according to claim 5 or the expression vector according to claim 6.

8. The host cell according to claim 7, characterized in that The host cell is selected from Escherichia coli, yeast cells, insect cells or mammalian cells; preferably, the host cell is a CHO cell, and the recombinant nucleic acid is a nucleic acid that has been codon-optimized according to the CHO expression system.

9. The method for preparing the varicella-zoster virus gE fusion protein according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Step 1: Cultivating the host cell according to claim 7 or 8 under conditions suitable for the expression of the gE fusion protein; Step 2: Collect the cell supernatant by centrifugation; Step 3: Purify and obtain the gE fusion protein.

10. Use of the varicella-zoster virus gE fusion protein according to any one of claims 1 to 4, or the varicella-zoster virus gE fusion protein prepared by the preparation method according to claim 9 in preparing a vaccine for preventing varicella-zoster virus infection.

11. A vaccine for preventing varicella-zoster virus infection, comprising the varicella-zoster virus gE fusion protein according to any one of claims 1 to 4, or the varicella-zoster virus gE fusion protein prepared by the preparation method according to claim 10, and a pharmaceutically acceptable carrier or excipient.

12. The vaccine according to claim 11, characterized in that The vaccine further comprises an adjuvant, and the adjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, CpG, cytokine, liposome and QS-21.

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