CN111018994A

CN111018994A - VZV virus subunit fusion antigen

Info

Publication number: CN111018994A
Application number: CN201811180287.5A
Authority: CN
Inventors: 王建新; 顾云
Original assignee: Jiangsu Immuon Therapeutics Technology Co ltd
Current assignee: Jiangsu Immuon Therapeutics Technology Co ltd
Priority date: 2018-10-10
Filing date: 2018-10-10
Publication date: 2020-04-17

Abstract

The invention relates to a VZV virus subunit fusion antigen, which comprises a VZV virus subunit and a cytokine, wherein the VZV virus subunit is fused with the cytokine through a linker sequence. The VZV virus subunit fusion antigen can effectively treat herpes zoster and has low infectivity.

Description

VZV virus subunit fusion antigen

Technical Field

The invention relates to a method for producing an antigen, in particular to a VZV virus subunit fusion antigen.

Background

Varicella-zoster virus (VZV) is called varicella-zoster virus, which is a virus that causes varicella in children after primary infection, remains latent in the body after recovery, and recurs after adults in a small number of patients.

Herpes zoster is a common disease and frequently encountered disease affecting hundreds of millions of middle-aged and elderly people in China, and patients are very painful during the disease period, but no effective antiviral drug for controlling the disease is available at present. Herpes zoster is the result of the virus (varicella zoster virus) being latent in the body after a primary infection with VZV. When the immune function of the organism is reduced to a certain level or in a certain pathological state, the VZV virus reactivates in a nerve node, reversely prolongs sensory nerve transmission to the skin range innervated by the nerve endings after replication and is released, thereby generating the characteristic herpes zoster.

Attenuated virus-based prophylactic geriatric herpes zoster vaccine in msadont was marketed in 2008, but the attenuated virus itself has some infectivity. The concentration of attenuated virus in the vaccine for middle aged and elderly people is 14 times higher than the same virus concentration in the varicella vaccine for children, thereby increasing the possibility of infection. On the other hand, the middle-aged and the elderly, especially some chronic disease patients have the problem of reduced immunocompetence. The combination of these two issues makes the safety of attenuated virus vaccines a very demanding issue. It also therefore makes it impossible to use the vaccine in patients in whom immunoprophylaxis is highly desirable (e.g., HIV patients).

In response to the above problems, the international pharmaceutical company, puerarialan, has been studied for many years to develop recombinant subunit-based herpes zoster vaccines. The vaccine achieves 99.7% protection in clinical trials. In contrast, the clinical protection of attenuated viruses is only 69%. Meanwhile, clinical experiments prove that the product can generate protective antibodies for patients with low immune function (including cancer patients and HIV patients) without generating any immune side effect.

However, many elderly people develop tolerance to VZV virus in the case of chronic infection over a long period of time, where immune tolerance cannot be resolved by prophylactic vaccines alone. The clearance of chronic infections by therapeutic vaccines has become an important research direction.

In view of the above-mentioned drawbacks, the designer actively makes research and innovation to create a VZV virus subunit fusion antigen with a novel structure, so that the VZV virus subunit fusion antigen has industrial utility value.

Disclosure of Invention

In order to solve the above-mentioned problems, it is an object of the present invention to provide a VZV virus subunit fusion antigen which is effective in treating herpes zoster and has low infectivity.

The VZV virus subunit fusion antigen comprises a VZV virus subunit and a cytokine, wherein the VZV virus subunit is fused with the cytokine through a linker sequence.

Further, a VZV virus subunit fusion antigen of the invention, said VZV virus subunit comprising gE or gB or gI or gH or gK or gL or gC or gM.

Furthermore, the subunit fusion antigen of the VZV virus is gE, and the amino acid sequence of the gE is shown in SEQ ID NO. 1.

Further, the VZV virus subunit fusion antigen of the invention, the cytokine includes granulocyte colony-stimulating factor GM-CSF or interleukin or interferon or cell chemotactic factor.

Further, the VZV virus subunit fusion antigen of the invention, the interleukin is IL-1 or IL-2 or IL-4 or IL-5 or IL-6 or IL-10 or IL-12 or IL-15 or IL-18.

Further, the VZV virus subunit fusion antigen of the invention, the cell chemotactic factor is IL-8 or sdf-1 α or MCP1 or MCP2 or MCP4 or MCP5 or RANTES or MIP-5 or 3-MIP or MIP-1 α or MIP-1 β or LOGO or TARC or LARC or SLC.

Further, the VZV virus subunit fusion antigen of the invention, wherein the interferon is gamma-interferon.

Further, the VZV virus subunit fusion antigen is provided, the cytokine is granulocyte colony-stimulating factor GM-CSF, the amino acid sequence of the granulocyte colony-stimulating factor GM-CSF is shown by SEQ ID NO.2, and the nucleotide sequence of the VZV virus subunit fusion antigen is shown by SEQ ID NO. 3.

Further, the VZV virus subunit fusion antigen of the invention has a linker sequence (GGGGS)4 sequence.

Furthermore, the VZV subunit fusion antigen also comprises an expression vector, wherein the expression vector is a eukaryotic expression vector or a prokaryotic expression vector, or a recombinant adenovirus vector or a recombinant adeno-associated virus vector or a recombinant retrovirus vector or a recombinant lentivirus vector or a nanoparticle or a polymer or a liposome, the eukaryotic expression vector is pCMVp-NEO-BAN or pEGFP or pSV2, and the prokaryotic expression vector is a pET vector or pEGX vector.

By the scheme, the invention at least has the following advantages: the VZV virus subunit fusion antigen of the invention generates corresponding antibody after immunizing mice, and the antibody titer is obviously higher than the effect of pure gE immunization (p < 0.05). The results of in vitro T lymphocyte activation experiments show that the fusion protein expressing the gE antigen and GM-CSF can induce a stronger T cell immune response than the pure gE antigen. And the T cell immune response induced by the fusion protein of the gE antigen and GM-CSF is strongest. In addition, the expression of GM-CSF can significantly increase the T cell immune level of low dose groups and induce more specific T cells.

In conclusion, the VZV virus subunit fusion antigen of the invention can effectively treat herpes zoster and has low infectivity.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 shows Western blot for detecting the expression of recombinant fusion protein vector in E.coli cells under the conditions of transformation of the vector into E.coli, detection of foreign gene expression by anti-gE antibody, and no-load of pET-22b (+) as negative control;

FIG. 2 shows Western blot for detecting the expression of recombinant fusion protein vector in E.coli cells under the conditions that the vector transforms E.coli, an anti-GM-CSF antibody is used for detecting the expression of an exogenous gene, and pET-22b (+) no-load is used as a negative control;

FIG. 3: protein purification and recovery conditions, namely recovering the fusion protein by urea concentration gradient and then purifying by a nickel column, wherein a western blot detection shows that an obvious band is formed at a corresponding position and a miscellaneous band is not formed;

FIG. 4: elisa detects the generation condition of the antibody, corresponding gE antibody is generated after mice are immunized, but the antibody titer of the fusion protein gE-GM-CSF is obviously higher than that of the pure gE immunization (p is less than 0.05);

FIG. 5: one form of in vitro lymphocyte proliferation and specific T cell activation is the splenocyte proliferation fold of a corresponding gE or gE-GM-CSF immunized mouse under gE antigen stimulation, the ordinate is the relative proliferation fold, the o.d. value for gE-stimulated splenocytes is comparable to the o.d. value for splenocytes without gE stimulation, the asterisk indicates p < 0.05;

FIG. 6: another form of lymphocyte proliferation and specific T cell activation in vitro is the number of spleen cells secreting IFN γ from mice immunized with gE or gE-GM-CSF under stimulation by gE antigen, on the ordinate the number of spot-forming cells per 105 spleen cells, and asterisks indicate p < 0.05.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Referring to fig. 1-2, a VZV viral subunit fusion antigen according to a preferred embodiment of the present invention comprises a VZV viral subunit and a cytokine, the VZV viral subunit being fused to the cytokine via a linker sequence.

Preferably, the subunit of a VZV virus of the invention comprises a gE or gB or gI or gH or gK or gL or gC or gM fusion antigen.

Preferably, the subunit of the VZV virus of the present invention is gE, and the amino acid sequence of gE is shown in SEQ ID NO. 1.

Preferably, the VZV virus subunit fusion antigen of the invention, the cytokine comprises granulocyte-colony stimulating factor GM-CSF or interleukin or interferon or a cell chemotactic factor.

Preferably, the VZV virus subunit fusion antigen of the invention, the interleukin is IL-1 or IL-2 or IL-4 or IL-5 or IL-6 or IL-10 or IL-12 or IL-15 or IL-18.

Preferably, the VZV virus subunit fusion antigen of the invention, the cell chemotactic factor is IL-8 or sdf-1 α or MCP1 or MCP2 or MCP4 or MCP5 or RANTES or MIP-5 or 3-MIP or MIP-1 α or MIP-1 β or LOGO or TARC or LARC or SLC.

Preferably, the VZV virus subunit fusion antigen of the invention, the interferon is gamma-interferon.

Preferably, the VZV virus subunit fusion antigen of the invention, the cytokine is granulocyte colony-stimulating factor GM-CSF, the amino acid sequence of the granulocyte colony-stimulating factor GM-CSF is shown by SEQ ID NO.2, and the nucleotide sequence of the VZV virus subunit fusion antigen is shown by SEQ ID NO. 3.

Preferably, the VZV viral subunit fusion antigen of the invention has a linker sequence (GGGGS) 4.

Preferably, the VZV subunit fusion antigen further comprises an expression vector, wherein the expression vector is a eukaryotic expression vector or a prokaryotic expression vector, or a recombinant adenovirus vector or a recombinant adeno-associated virus vector or a recombinant retrovirus vector or a recombinant lentivirus vector or a nanoparticle or a polymer or a liposome, the eukaryotic expression vector is pCMVp-NEO-BAN or pEGFP or pSV2, and the prokaryotic expression vector is a pET vector or pEGX vector.

The following method for producing and verifying the VZV virus subunit fusion antigen comprises the following steps:

design and construction of fusion protein constructs

Subunits in such subunit fusion antigens include various subunits of the VZV virus, including but not limited to gE, gB, gI, gH, gK, gL, gC, gM, and the like. Among these proteins, glycoprotein E (gE) and glycoprotein B (gB) have strong humoral immunity and cellular immunity stimulating effects. Glycoprotein i (gi) may also enhance immunogenicity by forming a complex with gE. The combination of monovalent or polyvalent can be adopted depending on the purpose and result of the test. In the present method, the VZV viral subunit employed is the monovalent glycoprotein E (gE).

The subunit is linked to the cytokine in a linear manner by designing a suitable fusion protein Linker sequence (Linker) which should not be less than 3.5nm in length, since the distance between adjacent peptide bonds is 0.38nm, and thus the Linker peptide should comprise at least 10 amino acids. Most commonly, Huston designs a synthetic (GGGGS)3 sequence, and the fusion protein with the connecting peptide of (Gly4Ser)3 shows higher renaturation efficiency. It is probably that the (Gly4Ser)3 connecting peptide is longer and soft, and can reduce the steric hindrance between two components of the fusion protein during renaturation, thereby being more beneficial to the correct folding of each structural domain of the fusion protein. In this method, we used (GGGGS)4 sequence as fusion protein Linker sequence (Linker) on the original basis.

The cytokine to be linked is granulocyte colony stimulating factor GM-CSF, interleukin, interferon or a cytochemotactic factor, including but not limited to the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-18, GM-CSF, and gamma-interferon, the chemokines IL-8, sdf-1 α 1, MCP2 and MCP4 or MCP5, monocyte chemotactic protein 3, RANTES, MIP-5, 3-MIP, eotaxin, MIP-1 α -1 β, and SLC, and the stimulatory molecules CD80, CD86, ICAM-1, 3-LFA, C3d, CD40L and Flt 3L.

In the method, the amino acid sequences of the subunit gE of the human herpes zoster virus and the cell factor GM-CSF are shown as SEQ ID No.1 and No. 2.

Design and construction of fusion protein expression System

Further, the method provides an expression vector for expressing the recombinant gene containing the fusion antigen. The vector includes but is not limited to eukaryotic expression vectors (pCMVp-NEO-BAN, pEGFP, pSV2), prokaryotic expression vectors (pET vector series, pEGX vector series), recombinant adenovirus vectors (adenoviral vector), recombinant adeno-associated virus vectors (adeno-associated viral vector), recombinant retrovirus vectors (retroviraector), recombinant lentivirus vectors (lentiviral vector), nanoparticles, polymers or liposomes.

In a specific embodiment of the method, the fusion antigen carried by the expression vector has a DNA fragment with a nucleotide sequence shown in SEQ ID No. 3. The vector expresses herpes zoster virus subunit gE and granulocyte macrophage colony stimulating factor GM-CSF.

3, purification and recovery of expressed protein

The prokaryotic expression system expresses protein and has the defect that inclusion bodies are easy to form, and are high-density insoluble protein particles wrapped by membranes formed when exogenous genes are expressed in prokaryotic cells, particularly when the exogenous genes are efficiently expressed in escherichia coli. The proteins in inclusion bodies are aggregates in an unfolded state and have no biological activity, so that the recombinant proteins need to be recovered or renatured for use, namely, the target proteins are recovered from a denatured fully-extended state to a normal folded structure by slowly removing the denaturant, and disulfide bonds are normally formed by removing the reducing agent. In one embodiment of the method, protein resuscitation is performed using an automated protein resuscitation device that automatically adds water to the dialysate based on the turbidity of the solution containing the recombinant protein to change the concentration of urea in the dialysis tank to achieve control of the recombinant protein resuscitation process.

The recovered protein is purified by using a his tag designed on the fusion protein and a Ni + column.

4, animal immunization experiment and results

The fusion antigen provided by the method can generate corresponding gE antibody after a mouse is immunized, but the antibody titer of the fusion protein gE-GM-CSF is obviously higher than that of the pure gE immunization (p is less than 0.05). The results of in vitro T lymphocyte activation experiments show that the fusion protein expressing the gE antigen and GM-CSF can induce a stronger T cell immune response than the pure gE antigen. And the T cell immune response induced by the fusion protein of the gE antigen and GM-CSF is strongest. In addition, the expression of GM-CSF can significantly increase the T cell immune level of low dose groups and induce more specific T cells.

According to the existing experimental results in mice, the fusion protein can be predicted to activate the specific immune response to the antigen expressed by VZV virus in human body by the same mechanism, so as to stimulate and strengthen the strong killing effect of specific immune cells such as T cells on human herpes zoster virus infected cells. Meanwhile, the vaccine plays the roles of prevention and treatment, and lays a solid foundation for further developing preventive and therapeutic vaccines of the adult herpes zoster virus.

The following are specific examples of methods for producing and validating the VZV virus subunit fusion antigen:

s1 construction of fusion antigen plasmid

Taking the nucleic acid sequences of gE (NC-001348) and mGM-CSF (NC-000077) searched in Genebank as a model, carrying out codon optimization by using the expression adaptability of escherichia coli, removing a middle stop codon, adding a Linker sequence (Linker) to splice into a fusion gene open reading frame in sequence, and synthesizing the fusion gene open reading frame by a Jinzhi company complete sequence.

pET-22b (+) (from Novagen) prokaryotic expression vectors were used to construct pET-gE and pET-gE-GM-CSF, respectively, with pET-22b (+) empty as a control.

S2 recombinant fusion antigen expression

Transforming the recombinant fusion protein plasmid constructed in S1 into host bacteria BL21 (purchased in Biyun), plating a plate, picking a monoclonal colony, performing induced expression, taking a bacterial liquid for cracking, performing SDS electrophoresis on total cell protein after the total cell protein is denatured by boiling for 5 minutes, transferring the protein to a cellulose acetate membrane, and detecting by using a corresponding antibody: anti-gE protein antibodies (Santa Cru) and anti-GM-CSF antibodies (bosch biosciences).

The inclusion body is subjected to concentration gradient resuscitation by urea, then is purified by his-tag, and the foreign gene expression is detected by a Western-Blots method. The results show that expression bands were detected at the expected molecular weight band positions (FIG. 2). The results indicate that the VZV antigen-associated fusion protein is efficiently expressed with GM-CSF.

S3 production of antibodies

12 male BARB/c mice (purchased from the university of Nantong, laboratory animals center) of 3-4 weeks of age were selected and randomly divided into 4 groups, 1 group being a control group (saline group), 2 groups being immunized with gE protein, and 3 groups being immunized with gE-mGM-CSF. The injection is injected into abdomen at multiple points, and is administered 1 time each for 0, 2, and 4 weeks. And (4) after the immunization, collecting blood samples on the orbit, standing the collected blood samples at 4 ℃ overnight, centrifuging the blood samples at 12000rpm for 5min, and collecting supernatant. A coating solution containing gE antigen (5ug/ml) (purchased from Prospec) was added to a 96-well plate and coated overnight at 4 ℃; discard the liquid and wash 3 times with 0.05% PBST. 200ul of blocking solution was added to a 96-well plate and blocked at 37 ℃ for 2 hours. Discard the liquid and wash 3 times with 0.05% PBST. Mouse serum was added at 100ul37 ℃ and incubated for 1 h. Discard the liquid and wash 3 times with 0.05% PBST. 100ul HRP-labeled secondary antibody (1:20000 dilution) was added to a 96-well plate and incubated at 37 ℃ for 1 h. Discard the liquid and wash 3 times with 0.05% PBST. 100ul of TMB color developing solution (purchased in Byun day) was added to 96 wells, and incubated for 15min at room temperature in the dark. The color reaction was stopped by adding 50ul of 2M sulfuric acid solution, and the degree at 450nm was measured with a microplate reader.

Through detection, mice generate specific gE antibodies in groups 2 and 3 after immunization, but the antibody titer of group 3 is obviously higher than that of group 2, which indicates that the fusion antigen has stronger antibody generation effect than that of a simple subunit antigen.

S4, in vitro lymphocyte proliferation and specific T cell activation

4 weeks after immunization, mouse splenocytes (1X 105/well) were isolated and cultured, with or without co-stimulation with 10. mu.g/ml of gE protein for 5 days. CCK-8 was added on the fifth day. The results of the assay are expressed as relative fold proliferation, which is converted by comparison between the o.d. readings stimulated with gE protein and the o.d. readings not stimulated with gE protein.

T cell immune responses were assessed by counting interferon IFN γ -secreting T lymphocytes specific for the gE antigen using ELISPOT experiments. First, BARB/c mice were immunized with antigen as described above. After 2-4 weeks, the mouse splenocytes were isolated and cultured in 96-well plates at 1 × 105 cells/well. Cultured cells of each mouse were stimulated with gE antigen, and cells not stimulated with antigen were negative controls. After 24 hours incubation at 37 ℃, cells were discarded and washed 3 times with pbs (pbst) containing 0.5% tween 20. Then incubation with an enzyme-labeled antibody against IFN γ, and finally development by ACE substrate. After color development and drying termination, the IFN γ reaction spots were counted by an ELISPOT counter and analyzed.

The antigen-specific T cell immune response can be detected in vitro through the results of T cell proliferation experiments and ELISPOT experiments. In cell proliferation experiments, the relative cell number at day five can be determined by the number of cells measured by CCK-8. All immunized mice splenocytes proliferated significantly higher under stimulation by gE antigen than the saline-immunized mice splenocytes of the negative control. The number of splenocytes from mice immunized with gE-GM-CSF is higher than the proliferation fold of splenocytes from mice immunized with gE alone. The ELISPOT experiment result can show the antigen-specific killer T cell immune response intensity. The results show that all immunized mice acquire an antigen-specific killer T cell immune response under stimulation by gE antigen. Specifically, the number of IFN gamma secretory splenocytes of mice immunized with gE-GM-CSF is much higher than that of mice immunized with gE alone. In summary, the results of the T cell proliferation assay and the ELISPOT assay consistently reflect that the fusion protein antigen can enhance the gE antigen-specific T lymphocyte immune response.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description.

In addition, the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention. Also, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Jiangsu Jianan Biotechnology Ltd

<120> VZV virus subunit fusion antigen

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>546

<212>PRT

<213> herpes zoster virus subunit (gE)

<400>1

Met Gly Thr Val Ala Leu Pro Val Val Gly Val Leu Met Gly Pro Gly

1 5 10 15

Ile Ile Thr Gly Thr Leu Ala Ile Thr Ala Pro Val Ala Ala Ser Val

20 25 30

Leu Ala Thr Ala Ala Pro His Thr Ala Gly Ala Leu Leu Ala Thr Ala

35 40 45

Ser Val Thr Gly Pro Thr Thr His Ser Ala His Ala Gly Ser Ser Thr

50 55 60

Val Ala Ala Gly Gly Ser Ser Ala Leu Ala Thr Ala His Ala Ser Pro

65 70 75 80

Thr Ile Thr Pro Ala Ala Ala Thr Ala Gly Pro Leu Gly Ala Ala His

85 90 95

Gly His His Gly Val Thr Ala Gly Gly Ala Gly Ile Ala Ser Gly Gly

100 105 110

Ala Leu Met Gly Pro Thr Gly Met Ser Ala Gly Gly Ala Leu Gly Ala

115 120 125

Ala Thr Gly Ile His Val Ile Pro Thr Leu Ala Gly Ala Ala Ala His

130 135 140

Leu Ile Val Ala Val Ala Gly Ala Gly Thr Gly Ala Val Pro Leu Gly

145 150 155 160

Ala Leu Ala Pro Leu Pro Gly Gly Gly Ala Leu Ile Gly Val Ser Val

165 170 175

Gly Gly Ala His Pro Pro Thr Leu Ala Ala Pro Ile Gly Ala Ile Thr

180 185 190

Gly Val Ala Thr Thr Gly Thr Thr Ser Pro Leu Pro Ser Leu Thr Cys

195 200 205

Thr Gly Ala Ala Ala Pro Ala Ile Gly His Ile Cys Leu Leu His Thr

210 215 220

Thr Cys Pro Gly Ala Val Val Val Ala Val Ala Cys Ala Gly Ala Thr

225 230 235 240

Leu Gly Ala Gly Leu Ala Gly Ile Ser Thr Ala Pro Gly Gly Leu Leu

245 250 255

Gly Ala Ala Gly Pro Thr Ile Val Val Ala Thr Ser Thr Leu Pro Ala

260 265 270

Gly Leu Gly Leu Ala Pro Pro Gly Ile Gly Pro Gly Val Leu Leu Val

275 280 285

Leu Ala Thr Gly Leu Gly Thr Leu Gly Val Thr Ile Thr Ala Met Ala

290 295 300

Gly Ser Ala Gly Thr Ser Thr Thr Ala Thr Pro Leu Val Thr Thr Leu

305 310 315 320

Gly Ala Gly Leu Thr Ala Ala Pro Thr Pro Ala Val Thr Pro Gly Pro

325 330 335

Ala Gly Ala Gly Pro His Met Thr Ala Thr His Ser His Val Pro Ser

340 345 350

Val Gly Ala Thr Pro Ser Leu Ala Met His Leu Gly Thr Leu Ile His

355 360 365

Gly Ala Pro Pro Ala Leu Leu Leu Gly Thr Leu Thr Val Pro Ile Ala

370 375 380

Pro Thr Cys Gly Pro Met Ala Leu Thr Ser Thr Cys Leu Thr His Pro

385 390 395 400

Ala Ala Pro Gly Cys Leu Ser His Met Ala Ser Gly Cys Thr Pro Thr

405 410 415

Ser Pro His Leu Ala Gly Ala Val Ala Ser Thr Val Thr Gly Ala Cys

420 425 430

Gly His Ala Ala Ala Thr Thr Ala Thr Cys Leu Gly Ile Ser His Met

435 440 445

Gly Pro Ser Pro Gly Leu Ile Leu His Ala Gly Gly Thr Thr Leu Leu

450 455 460

Pro Val Ala Thr Pro Gly Ser Leu Ser Gly Leu Thr Val Pro Val Val

465 470 475 480

Thr Pro Ala Gly His Val Gly Ala Val Ala Thr Thr Val Val Ser Thr

485 490 495

Val Ala His Pro Val Ala Ala Ile Gly Gly Ala Gly Pro Pro Pro Thr

500 505 510

Ala Gly Gly Pro Pro Ala Thr Thr Leu Pro Leu Gly Ile Thr Pro Val

515 520 525

Ala Pro Gly Thr Ser Pro Leu Leu Ala Thr Ala Ala Thr Thr Gly Gly

530 535 540

Leu Ala

545

<210>2

<211>141

<212>PRT

<213> monocyte colony stimulating factor (GM-CSF)

<400>2

Met Thr Leu Gly Ala Leu Leu Pro Leu Gly Ile Val Val Thr Ser Leu

1 5 10 15

Ser Ala Pro Thr Ala Ser Pro Ile Thr Val Thr Ala Pro Thr Leu His

20 25 30

Val Gly Ala Ile Leu Gly Ala Leu Ala Leu Leu Ala Ala Met Pro Val

35 40 45

Thr Leu Ala Gly Gly Val Gly Val Val Ser Ala Gly Pro Ser Pro Leu

50 55 60

Leu Leu Thr Cys Val Gly Thr Ala Leu Leu Ile Pro Gly Gly Gly Leu

65 70 75 80

Ala Gly Ala Pro Thr Leu Leu Leu Gly Ala Leu Ala Met Thr Ala Ser

85 90 95

Thr Thr Gly Thr Thr Cys Pro Pro Thr Pro Gly Thr Ala Cys Gly Thr

100 105 110

Gly Val Thr Thr Thr Ala Ala Pro Ile Ala Ser Leu Leu Thr Pro Leu

115 120 125

Thr Ala Ile Pro Pro Gly Cys Leu Leu Pro Gly Gly Leu

130 135 140

<210>3

<211>2124

<212>DNA

<213> Artificial sequence (Artificial)

<400>3

atggggacag ttaataaacc tgtggtgggg gtattgatgg ggttcggaat tatcacggga 60

acgttgcgta taacgaatcc ggtcagagca tccgtcttgc gatacgatga ttttcacacc 120

gatgaagaca aactggatac aaactccgta tatgagcctt actaccattc agatcatgcg 180

gagtcttcat gggtaaatcg gggagagtct tcgcgaaaag cgtacgatca taactcacct 240

tatatatggc cacgtaatga ttatgatgga tttttagaga acgcacacga acaccatggg 300

gtgtataatc agggccgtgg tatcgatagc ggggaacggt taatgcaacc cacacaaatg 360

tctgcacagg aggatcttgg ggacgatacg ggcatccacg ttatccctac gttaaacggc 420

gatgacagac ataaaattgt aaatgtggac caacgtcaat acggtgacgt gtttaaagga 480

gatcttaatc caaaacccca aggccaaaga ctcattgagg tgtcagtgga agaaaatcac 540

ccgtttactt tacgcgcacc gattcagcgg atttatggag tccggtacac cgagacttgg 600

agctttttgc cgtcattaac ctgtacggga gacgcagcgc ccgccatcca gcatatatgt 660

ttaaaacata caacatgctt tcaagacgtg gtggtggatg tggattgcgc ggaaaatact 720

aaagaggatc agttggccga aatcagttac cgttttcaag gtaagaagga agcggaccaa 780

ccgtggattg ttgtaaacac gagcacactg tttgatgaac tcgaattaga cccccccgag 840

attgaaccgg gtgtcttgaa agtacttcgg acagaaaaac aatacttggg tgtgtacatt 900

tggaacatgc gcggctccga tggtacgtct acctacgcca cgtttttggt cacctggaaa 960

ggggatgaaa aaacaagaaa ccctacgccc gcagtaactc ctcaaccaag aggggctgag 1020

tttcatatgt ggaattacca ctcgcatgta ttttcagttg gtgatacgtt tagcttggca 1080

atgcatcttc agtataagat acatgaagcg ccatttgatt tgctgttaga gtggttgtat 1140

gtccccatcg atcctacatg tcaaccaatg cggttatatt caacgtgttt gtatcatccc 1200

aacgcacccc aatgcctctc tcatatgaat tccggttgta catttacctc gccacattta 1260

gcccagcgtg ttgcaagcac agtgtatcaa aattgtgaac atgcagataa ctacaccgca 1320

tattgtctgg gaatatctca tatggagcct agctttggtc taatcttaca cgacgggggc 1380

accacgttaa agtttgtaga tacacccgag agtttgtcgg gattatacgt ttttgtggtg 1440

tattttaacg ggcatgttga agccgtagca tacactgttg tatccacagt agatcatttt 1500

gtaaacgcaa ttgaagagcg tggatttccg ccaacggccg gtcagccacc ggcgactact 1560

aaacccaagg aaattacccc cgtaaacccc ggaacgtcac cacttctacg atatgccgca 1620

tggaccggag ggcttgcagg cggaggcgga tccggcggag gcggctccgg cggaggcggc 1680

tccggcggag gcggctccat gtggctgcag aatttacttt tcctgggcat tgtggtctac 1740

agcctctcag cacccacccg ctcacccatc actgtcaccc ggccttggaa gcatgtagag 1800

gccatcaaag aagccctgaa cctcctggat gacatgcctg tcacgttgaa tgaagaggta 1860

gaagtcgtct ctaacgagtt ctccttcaag aagctaacat gtgtgcagac ccgcctgaag 1920

atattcgagc agggtctacg gggcaatttc accaaactca agggcgcctt gaacatgaca 1980

gccagctact accagacata ctgcccccca actccggaaa cggactgtga aacacaagtt 2040

accacctatg cggatttcat agacagcctt aaaacctttc tgactgatat cccctttgaa 2100

tgcaaaaaac caggccaaaa atga 2124

Claims

1. A VZV virus subunit fusion antigen characterized by: comprising a subunit of VZV virus fused to a cytokine by a linker sequence.

2. The VZV virus subunit fusion antigen of claim 1, wherein: the subunit of VZV virus comprises gE or gB or gI or gH or gK or gL or gC or gM.

3. The VZV virus subunit fusion antigen of claim 2, wherein: the subunit of the VZV virus is gE, and the amino acid sequence of the gE is shown by SEQ ID NO. 1.

4. The VZV virus subunit fusion antigen of claim 1, wherein: the cell factor comprises granulocyte colony stimulating factor GM-CSF or interleukin or interferon or cell chemotactic factor.

5. The VZV virus subunit fusion antigen of claim 4, wherein: the interleukin is IL-1 or IL-2 or IL-4 or IL-5 or IL-6 or IL-10 or IL-12 or IL-15 or IL-18.

6. The VZV virus subunit fusion antigen of claim 4, wherein the cell chemotactic factor is IL-8 or sdf-1 α or MCP1 or MCP2 or MCP4 or MCP5 or RANTES or MIP-5 or 3-MIP or MIP-1 α or MIP-1 β or LOGO or TARC or LARC or SLC.

7. The VZV virus subunit fusion antigen of claim 4, wherein: the interferon is gamma-interferon.

8. The VZV virus subunit fusion antigen of claim 1, wherein: the cell factor is granulocyte colony-stimulating factor GM-CSF, the amino acid sequence of the granulocyte colony-stimulating factor GM-CSF is shown by SEQ ID NO.2, and the nucleotide sequence of the VZV virus subunit fusion antigen is shown by SEQ ID NO. 3.

9. The VZV virus subunit fusion antigen of claim 1, wherein: the linker sequence is (GGGGS)4 sequence.

10. The VZV virus subunit fusion antigen of claim 1, wherein: the expression vector is a eukaryotic expression vector or a prokaryotic expression vector, or a recombinant adenovirus vector or a recombinant adeno-associated virus vector or a recombinant retrovirus vector or a recombinant lentivirus vector or a nanoparticle or a polymer or a liposome, the eukaryotic expression vector is pCMVp-NEO-BAN or pEGFP or pSV2, and the prokaryotic expression vector is a pET vector or pEGX vector.