CN117285652A

CN117285652A - Fusion protein comprising varicella zoster virus gE, preparation method and application thereof

Info

Publication number: CN117285652A
Application number: CN202311261716.2A
Authority: CN
Inventors: 李海; 张燕; 李海鑫; 曹蕾; 郭晋源
Original assignee: National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
Current assignee: National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
Priority date: 2023-09-27
Filing date: 2023-09-27
Publication date: 2023-12-26

Abstract

The invention relates to a fusion protein comprising varicella zoster virus gE, a preparation method and application thereof. The fusion proteins of the present invention include varicella zoster virus gE protein and multimeric proteins, including Foldon, such that the fusion proteins form multimers, thereby improving the stability of the protein. Vaccines comprising the fusion proteins can induce a cellular and/or humoral immune response in an individual, such as increasing IgG antibody titer, igG1 antibody titer, igG2a/IgG1 antibody ratio, neutralizing antibody titer, and/or the number of lymphocytes secreting IFN- γ, IL-2, and/or IL-4, as well as inducing a Th1 and/or Th2 immune response.

Description

Fusion protein comprising varicella zoster virus gE, preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a fusion protein comprising varicella zoster virus gE, a preparation method and application thereof.

Background

Herpes Zoster (HZ) is an infectious skin disease caused by recurrent infections of latent Varicella-zoster virus (VZV) in humans. VZV is a neurotropic human herpesvirus belonging to the family herpesvirus alpha and of the human herpesvirus type 3, which causes varicella and/or shingles, and humans are the only hosts for this virus. VZV can spread via droplets or contact, primary infection mainly causes varicella, and subsequently VZV can establish latency within the spinal posterior root ganglion or cranial ganglion. When the body's resistance is reduced, latent viruses, after reactivation, cause inflammation and HZ. Age is the most important risk factor for HZ development, and the incidence rate appears to increase year by year with age. Except for age, the immune status of the organism is low (HIV, malignant tumor, etc.), diabetes, chronic kidney disease, depression, asthma, etc. are all dangerous factors for HZ occurrence, and the risk of HZ morbidity and recurrence is greatly increased. The most common chronic sequelae of HZ is postherpetic neuralgia, which can lead to physical disability, emotional distress, and disturbance of daily activities and sleep.

The VZV genome comprises 70 open reading frames (Open Reading Frame, ORF) encoding a variety of glycoproteins, of which the gE protein is the most abundant viral glycoprotein, encoded by ORF68, is an essential glycoprotein for the production of infectious VZV particles, comprises 623 amino acids, and the extracellular region contains 544 amino acids, forming a heterodimer with gl. Is the most antigenic and abundant glycoprotein on the envelope and infected cell membrane of the virus, and is widely present on the surface of the VZV particles and in the cell membrane and cytoplasm of host cells, and is abundantly expressed in skin lesions and ganglia at the onset of HZ. As targets for neutralizing antibodies and T cells during VZV infection, cellular and humoral immunity can be induced. Thus, gE glycoproteins are considered as the most suitable candidate antigens for vaccine development. Currently, three herpes zoster vaccines are marketed worldwide, comprising two attenuated live vaccines Zostavax and Gansv, and developed by applying the Oka attenuated strain VZV. The recombinant protein vaccine shintrix is developed by GSK company, and in 2017, FDA is formally marketed in China through the application of shintrix, 2020, and is used for inoculation of people over 50 years old to prevent herpes zoster and complications thereof. The application of Smingrix is adjuvant AS01B, which consists of monophosphoryl lipid A (MPL) and QS-21, wherein MPL is an immunopotentiator isolated from bacterial surface, and QS-21 is a natural compound extracted from Quillaja saponaria (Quillaja saponaria Molina).

The Foldon structure is a small polypeptide molecule, originally found in the fibrin of T4 phage, consisting of 27 amino acids, that can form an alpha helix, and 3 identical alpha helices can form a stable trimer by virtue of intermolecular hydrogen bonding and hydrophobic forces. However, it is not known whether the immunogenicity of the gE can be improved in the preparation of the fusion protein.

Disclosure of Invention

In order to solve the technical problems, the invention provides a fusion protein containing varicella zoster virus gE protein, a preparation method and application thereof, in particular,

in a first aspect of the invention, there is provided a fusion protein comprising varicella zoster virus gE and a multimeric protein comprising Foldon.

Preferably, the multimeric protein is such that the fusion protein forms a multimer. More preferably, the multimeric protein is such that the fusion protein forms dimers, trimers, tetramers or other numbers of multimers.

Preferably, the gE protein comprises any of the following groups:

a1 An extracellular region of the gE protein, or an extracellular region and a signal peptide;

a2 24-537 of the gE protein;

a3 Positions 1-537 of the gE protein;

a4 Any one of A1) to A3) is substituted and/or deleted and/or added by one or more amino acid residues to obtain protein with the same function; or alternatively, the first and second heat exchangers may be,

a5 A protein which has 80% or more identity with any one of the sequences A1) to A4) and has the same function.

Preferably, the Foldon comprises any of the following groups:

B1)SEQ ID No：2；

b2 1) a protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues; or alternatively, the first and second heat exchangers may be,

b3 A protein which has 80% or more identity with any one of the sequences B1) to B2) and has the same function.

It will be appreciated that the amino acids of proteins of different subtypes, different strains of viruses or different transcripts are not necessarily identical, and that there may be substitution, insertion or deletion mutations, the gE proteins and Foldon reference sequences described above being shown in SEQ ID Nos. 1-2, but that other structurally different wild-type sequences are also possible, the mutation sites corresponding to the above-described sites of SEQ ID Nos. 1-2, the correspondence being understood as being based on the correspondence after analysis of the amino acid structure and/or function.

The 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

Preferably, the gE protein and multimeric protein are linked directly or via a linking peptide.

Preferably, the multimeric protein is linked at the N-terminus or C-terminus of the gE protein.

Preferably, the N-terminal or/and the C-terminal of the fusion protein is/are linked to a tag.

The tag refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. More preferably, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

In a specific embodiment, the fusion protein comprises varicella zoster virus gE protein and Foldon.

Preferably, the fusion protein forms a trimeric protein.

Preferably, the fusion protein comprises glycosylation modification.

In a second aspect of the present invention, there is provided a recombinant gene encoding the above fusion protein.

Preferably, the recombinant gene comprises DNA or mRNA.

In a third aspect of the present invention, there is provided a vector comprising the recombinant gene described above.

Preferably, the vector includes a plasmid or the like.

In a fourth aspect of the invention, there is provided a host cell comprising a gene or vector as described above.

Preferably, the host cells include, but are not limited to CHO cells and the like.

In a fifth aspect of the present invention, there is provided a method for producing the above fusion protein, comprising culturing the above host cell and expressing the above fusion protein.

Preferably, the method of preparation further comprises the step of purifying the fusion protein.

In a sixth aspect, the present invention provides the use of the fusion protein, recombinant gene, vector and/or host cell described above for the preparation of a pharmaceutical composition for the prevention and/or treatment of Varicella-zoster virus (VZV) -mediated infectious disease.

Preferably, the pharmaceutical composition is a vaccine.

Preferably, the disease is varicella and/or shingles.

Preferably, the vaccine induces a cellular immune response and/or a humoral immune response in the individual. The humoral immune response includes secretion of antibodies by B cells, which may include T cell immune responses.

Such as increasing IgG antibody titer, igG1 antibody titer, igG2a/IgG1 antibody ratio, neutralizing antibody titer, and/or the number of lymphocytes secreting IFN-gamma, IL-2, and/or IL-4, and inducing a Th1 and/or Th2 immune response.

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising the fusion protein described above.

The pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient may be a carrier, diluent, adjuvant or nucleotide sequence encoding an adjuvant, solubilising agent, binder, lubricant, suspending agent, transfection facilitating agent and the like.

Preferably, the pharmaceutical composition is a vaccine.

The term "vaccine" in accordance with the present invention refers to a composition suitable for application to animals (including humans) that induces an immune response after administration that is sufficiently strong to minimally aid in the prevention, amelioration or cure of clinical disease resulting from infection by a microorganism.

Preferably, the vaccine contains gE3-100 μg in gE protein per unit dose. The gE protein may be any number or range within the above range, for example 3, 5, 8, 10, 15, 20, 25, 28, 30, 32, 35, 38, 40, 45, 50, 58, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg.

More preferably, the vaccine further comprises an adjuvant.

Further preferred, the adjuvant comprises an aluminium adjuvant and/or CpG.

Preferably, the adjuvant comprises an aluminum adjuvant and CpG, more preferably, the aluminum adjuvant: the mass ratio of CpG is 1: (1-5), specifically 1:1, 1:2, 1:3, 1:4, 1:5, etc.

In a specific embodiment, the aluminum adjuvant is an AlOH (aluminum hydroxide) adjuvant.

More preferably, the mass ratio of the fusion protein to the adjuvant is 1: (10-20), specifically 1:10, 1:12, 1:14, 1:15, 1:17, 1:18, 1:20, etc.

Preferably, the vaccine comprises 30-200 μg of adjuvant per unit dose.

The vaccine may be suitable for intramuscular liquid injection, intravenous liquid injection, intranasal liquid injection, intradermal liquid injection, subcutaneous liquid injection, mucosal administration, nebulization, oral administration, and the like.

In an eighth aspect of the invention, there is provided a method of preventing and treating a disease, including Varicella-zoster virus (VZV) -mediated infectious disease, comprising administering to a subject a pharmaceutical composition as defined in any of the above.

Preferably, the disease is varicella and/or shingles.

In the above methods, an immune response to VZV may be elicited in the subject following administration of the pharmaceutical composition to the subject.

In any of the above subjects, the subject described herein can be a human or a non-human animal.

Further, the non-human animal may be a non-human mammal.

The non-human mammal may be any one of, but not limited to, a mouse, a rat, a guinea pig, a hamster, a pig, a dog, a sheep, a monkey, a rabbit, a cat, a cow, and a horse.

The subject described herein includes, but is not limited to, healthy subjects, symptomatically infected subjects, asymptomatic infected subjects, or recovered subjects (subjects recovered after infection).

Administration as described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral nasal spray, or aerosol inhalation.

In summary, compared with the prior art, the invention has the following beneficial effects:

1. the fusion protein of the invention forms a polymer under the action of a multimeric protein, particularly forms a trimer after being connected with varicella zoster virus gE and Foldon, thereby improving the stability of the protein and the immunogenicity of the antigen.

2. Vaccines comprising the fusion proteins of the invention can induce a cellular and/or humoral immune response in an individual, such as increasing IgG antibody titer, igG1 antibody titer, igG2a/IgG1 antibody ratio, neutralizing antibody titer, and/or the number of lymphocytes secreting IFN- γ, IL-2, and/or IL-4, as well as inducing a Th1 and/or Th2 immune response. In particular vaccines comprising gE-Foldon, various immunoprotection efficacy was optimal.

3. The adjuvant in the vaccine has low cost, but can assist the antigen to generate good and stable cellular immune response and/or humoral immune response.

Drawings

FIG. 1 shows the strategy for the construction of fusion proteins.

FIG. 2 shows the detection of protein expression by SDS-PAGE gel electrophoresis and Western blot Western immunoblotting of three fusion proteins, wherein A, B, C corresponds to gE, gE-Foldon and gE-Fc proteins respectively, and the left side shows the result of the SDS-PAGE gel electrophoresis and the right side shows the result of the Western blot Western immunoblotting.

FIG. 3 is an electron microscope image of electron microscope negative staining of fusion proteins, wherein A, B, C corresponds to gE, gE-Foldon, gE-Fc proteins, respectively.

FIG. 4 shows the humoral immune response of the vaccine in mice, wherein A is the IgG antibody titer produced by each group, B is the IgG1 antibody titer produced by each group, C is the IgG2a antibody titer produced by each group, and D is the IgG2a/IgG1 antibody ratio produced by each group.

FIG. 5 shows the neutralizing antibody titers generated for each group.

FIG. 6 shows the number of lymphocytes induced to secrete IFN-gamma, IL-2, IL-4 for each group, corresponding to A, B, C, respectively.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The main materials and instruments in the examples:

1. cell, experimental animal

CHO cells were maintained in the measles laboratory at the chinese disease prevention and control center for viral disease;

the experimental animals adopt 30 SPF-class female BALB/c mice with the age of 6-8 weeks, and are purchased from Beijing Veantoni experimental animal technology limited company;

the animal experiment related to the research has been approved by the ethical committee for animal experiments (number: 20230309024) of the prevention and control center for viral diseases of Chinese disease prevention and control center, and the experimental operation meets the ethical requirements of experimental animals.

2. Main instrument and reagent

Plasmid extraction kit QIAGENplasmidMaxi Kit (25) was purchased from qiagen;

CHO cell transfection kit gibco ExpiFectamine CHO Transfection Kit, countess3, micro uv-vis spectrophotometer PierceTM BCAProteinAssay Kit from Invitrogen company;

protein purification instrument AKTApure, CTL fluorescence (enzyme linked) immune spot analyzer (S6 Ultra M2), recombinant herpes zoster vaccine (CHO cell) euphoria An Lishi is provided by GSK company;

PW-960 full-automatic enzyme-labeled plate washer is purchased from Shenzhen Pinaceae technology development Co., ltd;

goat anti-mouse IgG and goat anti-human antibodies were purchased from sequoyitol biotechnology limited;

IgG1 and IgG2a were purchased from abcam corporation;

AlOH adjuvants are offered by Heda Biotechnology Co;

type B CpG adjuvants are offered by wampe biotechnology limited;

mouse IFN-. Gamma. precoated ELISPOT kit, mouse IL-2precoated ELISPOT kit, mouse IL-4precoated ELISPOT kit, PMA+Ionomycin lymphocyte stimulator, mouse lymphocyte isolate and Nylon Net were purchased from Daidae Biotechnology Co., ltd;

phytohemagglutinin (PHA) was purchased from beijing soeba technologies limited.

Example 1 plasmid construction and protein purification characterization

Three recombinant varicella zoster multimeric vaccines gE, gE-Foldon, gE-Fc were prepared in total.

The extracellular varicella zoster virus E protein (VZV gE) of the gE protein has 537 amino acids, including positions 1-537 of the gE protein, and positions 1-23 are a signal peptide (SS) involved in secretion. The full-length sequence of the gE protein is shown in SEQ ID No:1, reference is made to the gene bank PDB ABE03086.1.

Foldon is a C-terminal fragment derived from the fibrin of the T4 bacteriophage, which is 27 amino acids in total. The structure is shown as SEQ ID No:2 (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID No: 2)

The Fc fragment was human IgG1 Fc, with a total of 227 amino acids. The full length sequence of human IgG1 Fc was referenced to gene pool PDB 3S7G_A.

And (3) connecting the gE and the gE-Foldon directly, then connecting the gE and the gE-Foldon in series with the His tag, and entrusting a general biotechnology company to synthesize the gene sequence of the gE, gE-Foldon and gE-Fc fusion protein, so as to construct the recombinant plasmid.

The strategy for constructing a specific fusion protein can be seen in FIG. 1.

The three target genes obtained by synthesis are transferred into an ExpiCHO eukaryotic cell to express proteins. And (3) carrying out identification of the expression condition of the target protein by an SDS-PAGE gel electrophoresis test and a Westernblot Western immunoblotting test, and verifying the conformation and the morphology of the protein by a negative dye electron microscope.

CHO fineAfter 3 days of shake flask culture, collecting culture supernatant, and purifying the culture supernatant to obtain target protein. Protein expression was detected by SDS-PAGE gel electrophoresis (left) and Westernblot Western immunoblotting (right), as shown by A in FIG. 2, the relative molecular mass (Mr) of gE protein bands was 70X 10 ³ About, slightly greater than the theoretical Mr60×10 ³ Indicating that the gE protein is glycosylated. B and C in FIG. 2 are bands of gE-Foldon and gE-Fc proteins, respectively, with Mr at 90X 10 ³ 、110×10 ³ About, like the gE proteins, gE-Foldon and gE-Fc also undergo some degree of glycosylation. The size of the catalyst is 62 multiplied by 10 slightly larger than the theoretical Mr ³ 、84×10 ³ 。

The electron microscope image taken by electron microscope negative staining technique shows that the gE-Foldon protein has a specific conformation length of about 10nm, and forms a trimer structure (see arrow B in FIG. 3).

Example 2 protective efficacy of vaccine

1. Immunization strategies

30 female BALB/c mice of 6-8 weeks are randomly divided into 5 groups (6 in each group), intramuscular injection immunization is carried out on days 0 and 21 according to the immunization strategy of the table 1, blood is taken after the first immunization on day 14, gE-Foldon and gE-Fc immunization doses are 5 mug/g total protein, alOH+CpG groups are taken as adjuvant control groups, eyeballs are taken after the second immunization for 14 days, and the mice are euthanized after dislocation, and spleen is collected for standby.

TABLE 1 grouping and immunization strategy for mice

Group of

Grouping

Quantity of

Antigens

Adjuvant

(Mode)

Immunization time

1

gE+AlOH+CpG

6

5μggE

25μg CpG+50μg AlOH

Intramuscular injection

0，21

2

gE-Foldon+AlOH+CpG

6

5μg gE-Foldon

25μg CpG+50μg AlOH

Intramuscular injection

0，21

3

gE-Fc+AlOH+CpG

6

5μg gE-Fc

25μg CpG+50μg AlOH

Intramuscular injection

0，21

4

Shingrix

6

5μg gE

AS01 _B

Intramuscular injection

0，21

5

AlOH+CpG

6

-

25μg CpG+50μg AlOH

Intramuscular injection

0，21

2. Statistical analysis

Data conditioning, analysis and mapping were performed using Grahpadprism 9.0. Count data is expressed in absolute numbers and percentages (%), and metering data is expressed in median (M) and quartile spacing (P25-P75). Significant differences between experimental groups were analyzed with ONE-WAY analysis of variance ONE WAY-ANOVA, followed by a multiple comparison test of Tukey, comparing the mean of each group with the mean of each other group. Asterisks indicate P value classification, and P < 0.05; * Represents P < 0.01; * Represents P < 0.001; * P < 0.0001.

3. ELISA detection of specific IgG, igG1 and IgG2a antibody levels in mouse serum

Specific IgG, igG1 and IgG2a antibodies in mouse serum are measured by an ELISA method, each well of a 96-well ELISA plate is respectively coated with 50nggE protein, gE-Foldon protein and gE-Fc protein, the mixture is placed in a refrigerator at 4 ℃ for standing overnight, PBS containing 10% FBS is used for sealing for 2 hours in the next day, a full-automatic ELISA plate washer is used for washing the plates, the plates are then patted dry, the collected mouse serum is taken for continuous 4-fold ratio dilution (dilution is started from 1:200), the mixture is placed in a constant temperature incubator at 37 ℃ for incubation for 1 hour, and horseradish peroxidase (HRP) -marked goat anti-mouse IgG and 1 are respectively added with 1:5000 dilution: goat anti-mouse IgG1 and IgG2a marked by 20000 diluted horseradish peroxidase (HRP) are incubated at 37 ℃ for 30min, developed by using TMB reagent, incubated at 37 ℃ in the dark for 15min, and then stop solution is added, and absorbance (OD value) is measured by a microplate reader (wavelength of 450 nm). EC50 values were calculated by 4 parameter fitting using GraphPad Prism software.

Results:

as shown in A in FIG. 4, the mice in the experimental group gE+AlOH+CpG (19306), gE-Foldon+AlOH+CpG (40492), gE-Fc+A1OH+CpG (17550) and the positive control group Sringrix (21737) all produced higher IgG antibodies than the adjuvant group A1OH+CpG (25). The IgG antibody levels generated after immunization of mice with the gE-Fc+A1OH+ CpG group were minimal (1.755X10) ⁴ ) Significantly lower than the gE-Foldon+AlOH+CpG group (P < 0.001). The highest IgG antibody level (4.0492 ×10) was obtained after immunization of mice with gE-Foldon+AlOH+CpG groups ⁴ ) Is significantly higher than the other three groups (P<0.01)。

To further verify the bias of humoral immune responses induced by the three proteins, the serum IgG1 and IgG2a antibody titers were measured by ELISA, respectively, and as shown in fig. 4B and C, the ge+aloh+cpg group, gE-fc+aloh+cpg group IgG1 antibody titers were higher than IgG2a, but the gE-foldon+aloh+cpg group IgG2a antibody titers were higher than IgG1. Comparing the ratios of IgG1 and IgG2a antibodies, as shown by D in FIG. 4, the ratios of gE+AlOH+CpG and gE-Fc+AlOH+CpG groups IgG2a/IgG1 were 0.52 and 0.48, respectively, each less than 1, and were lower than the Shangrix group. The ratio of gE-Foldon+AlOH+CpG group IgG2a/IgG1 was 1.59, higher than that of the Shangrix group. The gE-Foldon+AlOH+CpG group induced the mice to develop a Th1 biased immune response relative to the Sringrix immune group. The gE+AlOH+CpG groups and gE-Fc+AlOH+CpG groups induced Th 2-biased immune responses in mice. Because HZ patients act primarily by means of VZV-specific T cell mediated immune responses, TH1 mediates primarily cellular immune responses, biasing to TH1 may produce better cellular immune responses, meaning that the gE-foldon+aloh+cpg groups produce better cellular immune responses and the immunoprotection efficacy is more durable.

4. Plaque reduction neutralization assay to detect neutralizing antibody titres

MRC-5 cell density is regulated, inoculated into a 96-well plate, placed in a cell culture box (37 ℃ C., 5% CO 2) for culture overnight, and the experiment can be started after ensuring that the cell confluence in the next day is about 90%. Inactivating in water bath at 56 ℃ for 30 minutes.

The samples were diluted in a gradient, 30-fold in the first well, 3-fold in the gradient, and 8 dilutions were set. The sample wells and virus control wells were each filled with diluted virus (initial final initial dilution of 30-fold) and the drop-back wells were sequentially diluted down a 2-fold gradient for a total of 3 dilutions in a 5% CO2 incubator at 37℃for about 1 hour. The virus, the serum neutralization product and the positive and back drop hole viruses are added into the cells prepared in advance, 50 mu l of each hole is cultured in a 5% CO2 incubator at 37 ℃ for about 2 hours, the liquid is changed, 100 mu l of the culture medium is added into each hole for further culture for about 48 hours, the supernatant is discarded, the cells are fixed, and then fluorescent marked detection antibodies are added, and a CTL instrument is used for reading the plate.

Results:

neutralizing antibodies are one of the important indicators of vaccine and therapeutic drug development. The invention adopts plaque reduction neutralization test to evaluate the level of neutralizing antibodies generated by mice induced by three proteins combined with AlOH+CpG adjuvant. As shown in fig. 5, mice were induced to produce high titers of neutralizing antibodies in all of the experimental groups (ge+aloh+cpg, gE-foldon+aloh+cpg, gE-fc+aloh+cpg), and the differences in neutralizing antibody titers were similar to IgG antibodies in the 3 groups of experimental groups, with the lowest neutralizing antibody titers (719.25) in the gE-fc+aloh+cpg immunized group mice being significantly lower than in the gE-foldon+aloh+cpg group (P < 0.0001), and the highest neutralizing antibody titers (1190.5) in the gE-foldon+aloh+cpg immunized group, comparable to the shintrix group (p= 0.9225).

5. ELISPOT detection of IFN-gamma, IL-2, IL-4 secreting lymphocyte numbers

Taking out spleen tissue of a mouse from RPMI 1640 complete culture medium, putting the mouse spleen tissue into a 70 mu m cell screen containing 4.5mL of mouse lymphocyte separation liquid, cutting the mouse spleen by scissors, gently grinding the mouse spleen by using a syringe inner core until only one layer of white tissue remains in the screen, fully grinding, transferring the lymphocyte separation liquid into a 15mL centrifuge tube, covering 1mL of 1640 culture medium, centrifuging 800g for 30min, sucking out the lymphocyte layer, adding 10mL of 1640 culture medium, uniformly mixing, centrifuging for 10min 250g, collecting spleen cells of the mouse, and diluting the cell concentration with the culture liquid to 3x10 ⁶ And each mL. Each well was added with 10. Mu. LgE protein (10. Mu.g/ml), positiveControl was added with 10. Mu.L of positive stimulus PHA (25. Mu.g/ml), negative control was added with 10. Mu.L of medium, and ELISPOT assay was performed every 3X10 ⁵ The number of IFN-gamma, IL-2, IL-4 secreting lymphocytes in the cells, 6 samples, and the mean value was taken and other procedures were performed with reference to the ELISPOT kit instructions.

The results are shown in table 2 and fig. 6:

table 2:3 proteins induce cellular immune responses in mice

Group of	Grouping	IFN-Y	IL-2	IL-4
					1	gE+AlOH+CpG	17	51	24
2	gE-Foldon+AlOH+CpG	90	192	115
					3	gE-Fc+AlOH+CpG	14	26	26
4	Shingrix	75	85	89
					5	AIOH+CpG	21	24	42

The intensity of cellular immune response induced by the 3 proteins was measured using ELISPOT, and the results are shown in fig. 6, a, in which the mice secrete IFN- γ in the gE-Foldon + AlOH + CpG group and the positive control group shintrix with a higher number of lymphocytes than in the adjuvant group AlOH + CpG. As shown in B in fig. 6, the mice in experimental group ge+aloh+cpg, gE-foldon+aloh+cpg, gE-fc+aloh+cpg, and positive control group Shingrix secreted IL-2 in a higher number of lymphocytes than the adjuvant group aloh+cpg, in which gE-foldon+aloh+cpg group was significantly higher than ge+aloh+cpg group (P < 0.0001), gE-fc+aloh+cpg (P < 0.0001), positive control group Shingrix (P < 0.01), and adjuvant group aloh+cpg (P < 0.0001). As shown by C in FIG. 6, the number of lymphocytes secreting IL-4 in mice in the gE-Foldon+AlOH+CpG group and the positive control group Shangrix was higher than that in the adjuvant group AlOH+CpG, where the gE-Foldon+AlOH+CpG group was significantly higher than that in the gE+AlOH+CpG group (P < 0.05), and the gE-Fc+AlOH+CpG group (P < 0.05). The maximum number of lymphocytes secreting IFN-gamma, IL-2 and IL-4 in the gE-Folden+AlOH+CpG group in the 5 groups shows that gE-Folden+AlOH+CpG induces mice to generate a stronger cellular immune response.

In general, gE+AlOH+CpG, gE-Folden+AlOH+CpG, gE-Fc+AlOH+CpG can stimulate the generation of certain humoral immunity and cellular immunity, so that gE-Folden+AlOH+CpG has better effect, and compared with other experimental groups and positive control groups, the generated IgG antibody level is highest, the ratio of IgG2a/IgG1 is highest, the quantity of lymphocytes for inducing secretion of IFN-gamma, IL-2 and IL-4 is the greatest, and the recombinant subunit vaccine composition has obvious effect in both humoral immunity and cellular immunity and is in the optimal form.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

SEQ ID No：1

MG TVNKPVVGVL MGFGIITGTLRITNPVRASV LRYDDFHIDE DKLDTNSVYE PYYHSDHAES SWVNRGESSR KAYDHNSPYIWPRNDYDGFL ENAHEHHGVYNQGRGIDSGE RLMQPTQMSA QEDLGDDTGI HVIPTLNGDDRHKIVNVDQR QYGDVFKGDLNPKPQGQRLI EVSVEENHPF TLRAPIQRIY GVRYTETWSFLPSLTCTGDAAPAIQHICLK HTTCFQDVVV DVDCAENTKE DQLAEISYRF QGKKEADQPWIVVNTSTLFD ELELDPPEIE PGVLKVLRTE KQYLGVYIWN MRGSDGTSTY ATFLVTWKGDEKTRNPTPAV TPQPRGAEFH MWNYHSHVFS VGDTFSLAMH LQYKIHEAPF DLLLEWLYVPIDPTCQPMRL YSTCLYHPNAPQCLSHMNSG CTFTSPHLAQ RVASTVYQNC EHADNYTAYCLGISHMEPSF GLILHDGGTT LKFVDTPESL SGLYVFVVYF NGHVEAVAYT VVSTVDHFVNAIEERGFPPT AGQPPATTKP KEITPVNPGT SPLLRYAAWT GGLAAVVLLC LVIFLICTAKRMRVKAYRVD KSPYNQSMYY AGLPVDDFED SESTDTEEEF GNAIGGSHGG SSYTVYIDKTR

Claims

1. A fusion protein comprising varicella zoster virus gE protein and a multimeric protein comprising Foldon.

2. The fusion protein of claim 1, wherein the gE protein comprises any of the following groups:

a2 24-537 of the gE protein;

a3 Positions 1-537 of the gE protein;

3. The fusion protein of claim 2, wherein the Foldon comprises any of the following groups:

B1)SEQ ID No：2；

4. A fusion protein according to claim 3, wherein the fusion protein forms a trimer.

5. A recombinant gene encoding the fusion protein of any one of claims 1-4.

6. A vector comprising the recombinant gene of claim 5.

7. A host cell comprising the recombinant gene of claim 5 or the vector of claim 6.

8. A method of producing a fusion protein according to any one of claims 1 to 4, comprising culturing the host cell of claim 7 to express the fusion protein.

9. Use of the fusion protein of any one of claims 1-4, the recombinant gene of claim 5, the vector of claim 6 and/or the host cell of claim 7 for the preparation of a pharmaceutical composition for the prevention and/or treatment of Varicella-zoster virus (VZV) -mediated infectious disease.

10. A pharmaceutical composition comprising the fusion protein of any one of claims 1-4.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is a vaccine, the vaccine further comprising an adjuvant comprising an aluminum adjuvant and/or CpG.

12. The pharmaceutical composition according to claim 11, wherein the vaccine contains gE3-100 μg in gE protein per unit dose, comprising an adjuvant 30-200 μg per unit dose, or the aluminum adjuvant: the mass ratio of CpG is 1: (1-5).