CN111925424A

CN111925424A - Japanese B encephalitis virus genetic engineering subunit vaccine, preparation method and application thereof

Info

Publication number: CN111925424A
Application number: CN202010971316.0A
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 张大鹤
Original assignee: Suzhou Midi Biotechnology Co ltd; Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2020-11-13
Anticipated expiration: 2040-09-16
Also published as: CN111925424B

Abstract

The invention discloses a Japanese B encephalitis virus genetic engineering subunit vaccine, a preparation method and application thereof. The vaccine comprises recombinant protein and a pharmaceutically acceptable carrier, wherein the recombinant protein has a sequence shown in SEQ ID NO. 2. The vaccine provided by the invention has high safety and good immunogenicity, can generate stronger humoral immunity in an animal body, the immunized animal can resist strong toxicity and attack toxicity, and the vaccine can be prepared by large-scale serum-free suspension culture of a bioreactor, and has the advantages of easy quality control, stable batch-to-batch, low production cost and the like.

Description

Japanese B encephalitis virus genetic engineering subunit vaccine, preparation method and application thereof

Technical Field

The invention relates to a genetic engineering vaccine, in particular to a Japanese B encephalitis virus genetic engineering subunit vaccine, a preparation method and application thereof, belonging to the technical field of animal immunity drugs.

Background

Japanese Encephalitis (JE), also called epidemic Encephalitis B (Japanese Encephalitis for short), is a mosquito-mediated infectious disease of zoonosis caused by Japanese Encephalitis Virus (JEV). JE can infect various organisms such as pigs, horses, cattle, sheep, mice, dogs, and humans, and is clinically characterized by neurological symptoms such as hyperpyrexia, disturbance of consciousness, convulsion or convulsion, respiratory failure, dyskinesia, and reflex disorder. The disease has obvious seasonality and certain regionality, is mostly generated in the season of mosquito breeding in summer and autumn, pigs are one of main storage hosts and diffusion hosts of JEV, and generally form circulating infection among mosquitoes, pigs and mosquitoes, and can also infect human beings through pig-mosquito-human approaches. Therefore, the prevention and control of the spread of the virus are of great importance not only to the breeding industry but also to human health. The world animal health group (OIE) classifies JE as a B-type infectious disease, and the Ministry of agriculture of China classifies JE as a B-type legal infectious disease and a second-type animal epidemic disease.

JEV belongs to the Flaviviridae (Flaviviridae) genus (Flavivirus) of the family Flaviviridae, has a cyst membrane, is spherical, and is icosahedral symmetric. Its genome is a single positive-stranded RNA, approximately 11kb in length, containing a long Open Reading Frame (ORF), and encoding a polyprotein. The polyprotein is cleaved by proteases into three structural proteins: nucleocapsid protein C protein, membrane protein precursor protein prM, fiber glycoprotein E protein, and seven non-structural proteins: NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS 5. The E protein is the main structural protein of the virus, is an important antigen protein of JEV, has the capacity of combining with a cell surface receptor, induces an organism to generate a neutralizing antibody, and is closely related to virus virulence, pathogenicity, membrane fusion, immune protection, hemagglutination reactivity, serum specificity and the like.

The propagation of Japanese encephalitis mainly takes mosquito bites as main measures, mosquitoes are rapidly bred in summer and autumn, the environment is difficult to control, and the mosquito preventing and killing effects are not ideal, so that large-scale vaccination becomes a main measure for preventing and controlling Japanese encephalitis. The currently widely used JEV vaccines mainly comprise inactivated vaccines and attenuated live vaccines, but the inactivated vaccines have the defects of more side reactions, complex production and purification processes, high requirements on production facilities, high production cost, low antibody maintenance level and the like in the application process; the SA14-14-2 attenuated strain in the attenuated live vaccine is obtained by screening and purifying primary hamster kidney cells (PHK) through repeated passage and repeated plaque, the source cell line is not approved by FDA, the application range is small, the risk of reversion is high, and the purification process is to be improved.

In view of the above, there is a need to develop a JEV genetic engineering subunit vaccine with high safety and strong immunogenicity.

Disclosure of Invention

The invention mainly aims to provide a Japanese B encephalitis virus genetic engineering subunit vaccine, a preparation method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a recombinant protein, which has a sequence shown in SEQ ID NO. 2 or a sequence which is 95% identical to the full-length sequence of the SEQ ID NO. 2. That is, the amino acid sequence of the recombinant protein provided by the embodiments of the present invention may be the original sequence, an added or truncated sequence.

The embodiment of the invention also provides a coding gene for coding the recombinant protein.

In some embodiments, the encoding gene has the sequence shown in SEQ ID NO. 1 or a sequence that is 95% or more identical to the full-length sequence of SEQ ID NO. 1.

The embodiment of the invention also provides a recombinant vector which comprises the coding gene of the recombinant protein.

In some embodiments, the recombinant vector includes, but is not limited to, pFastBac1, pVL1393, pFastBac dual, etc., and pFastBac1 is preferably used.

The embodiment of the invention also provides a host cell which comprises the coding gene of the recombinant protein.

In some embodiments, the host cell is selected from insect cells, such as the Sf9 cell line, preferably the Sf9 cell line includes but is not limited to Sf9, High Five or Sf21 cells, more preferably Sf9 cells.

The embodiment of the invention also provides an immune composition, which is characterized by comprising the following components: the recombinant protein; and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, any one or a combination of two or more of montainide ISA 206 VG, montainide ISA 201 VG, montainide ISA 15 VG, liquid paraffin, camphor oil, plant cell agglutinin, preferably, montainide ISA 201 VG.

The embodiment of the invention also provides a preparation method of the recombinant protein, which comprises the following steps:

cloning the encoding gene of the recombinant protein into a shuttle vector to obtain a recombinant shuttle vector containing a target gene;

transforming the recombinant shuttle vector into competent cells, and separating to obtain a recombinant baculovirus genome plasmid containing a target gene expression frame;

transfecting insect cells by using the recombinant baculovirus genome plasmid, and obtaining a recombinant baculovirus;

inoculating insect cells with the recombinant baculovirus, culturing, and separating to obtain the recombinant protein.

In some embodiments, the shuttle vector includes, but is not limited to, pFastBac1, pVL1393, pFastBac dual, etc., preferably pFastBac 1.

In some embodiments, the insect cells include, but are not limited to Sf9, High Five or Sf21 cells, and the like, preferably Sf9 cells.

The embodiment of the invention also provides a preparation method of the Japanese encephalitis virus genetic engineering subunit vaccine, which comprises the following steps: recombinant proteins are prepared using any of the methods described above and are admixed with a pharmaceutically acceptable carrier.

The embodiment of the invention also provides application of the recombinant protein or the immune composition in preparing a Japanese encephalitis virus detection reagent, in producing a medicament for inducing an immune response to a Japanese encephalitis virus antigen in a test animal or in producing a medicament for preventing the animal from being infected by the Japanese encephalitis virus.

The embodiment of the invention also provides application of the recombinant protein or the immune composition in preparing Japanese encephalitis virus genetic engineering subunit vaccine.

The embodiment of the invention provides a Japanese encephalitis virus genetic engineering subunit vaccine, which comprises any one of the immune compositions. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

The embodiment of the invention also provides application of the recombinant vector or the host cell containing the encoding gene of the recombinant protein in producing a reagent for detecting animal infection by Japanese encephalitis virus.

The embodiment of the invention also provides the application of the recombinant vector or the host cell containing the encoding gene of the recombinant protein in the production of a medicament for inducing an immune response against Japanese encephalitis virus antigen in a test animal.

The embodiment of the invention also provides application of a recombinant vector or a host cell containing the encoding gene of the recombinant protein in producing a medicament for preventing animals from being infected by Japanese encephalitis virus.

The embodiment of the invention also provides a method for inducing immune response against Japanese B encephalitis virus antigen, which comprises administering the Japanese B encephalitis virus genetic engineering subunit vaccine to a test animal.

The embodiment of the invention also provides a method for protecting a test animal from Japanese B encephalitis virus infection, which comprises the step of administering the Japanese B encephalitis virus genetic engineering subunit vaccine to the test animal.

Embodiments of the present invention also provide a vaccine suitable for generating an immune response against japanese b encephalitis virus infection in a test animal, comprising: recombinant proteins of the invention and adjuvant molecules.

An "adjuvant" as described in the present specification means any molecule added to the vaccine described in the present specification to enhance the immunogenicity of the antigen encoded by the encoding nucleic acid sequence described below. For example, the adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33, or a combination thereof; and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES. Further, the adjuvant may preferably be related adjuvants produced by Suzhou Shino biotechnology, Inc. to improve the effect of the vaccine.

Compared with the prior art, the embodiment of the invention optimizes the Japanese encephalitis B virus E protein, comprises deleting a membrane fusion region sequence in the sequence, uses a tandem repeat epitope in the sequence, and mutates a plurality of glycosylation sites in the sequence, thereby obtaining the recombinant protein (named as JEV E protein), obviously reducing the toxicity of the recombinant protein to cells, greatly enhancing the immunogenicity, well increasing the expression quantity, obviously reducing the glycosylation level, and reducing the irritability to animals, moreover, the recombinant protein can adopt a baculovirus insect cell expression system, uses suspension culture Sf9 cells and the like for expression and large-scale serum-free suspension culture preparation, greatly reduces the production cost of vaccines, simultaneously, the antigenicity and the function of the obtained product are similar to those of natural proteins, and the expression level is higher, the vaccine has strong immunogenicity, can provide good immune effect only by a small amount, has no pathogenicity to animals, and is suitable for being widely applied as a Japanese encephalitis virus genetic engineering subunit vaccine.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a gel electrophoresis photograph of the PCR-amplified product of the E gene after codon optimization in example 1, wherein the band of interest appeared at the 1.6kbp position.

FIG. 2 is a gel electrophoresis chart of the colony PCR amplification product in example 1, wherein the band of interest appears at the 1.6kbp position.

FIG. 3 is a schematic structural diagram of the transfer vector pF-JEV-E in example 1.

FIG. 4 is a SDS-PAGE detection profile of the cell culture obtained in example 3.

FIG. 5 is a graph showing the result of Western Blot detection of the product obtained in example 4 after SDS-PAGE electrophoresis.

FIG. 6 is a gray-scale scan of the purified recombinant protein obtained in example 7.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The embodiment of the invention mainly optimizes Japanese encephalitis virus E protein to obtain recombinant protein (JEV E protein), wherein the optimization mode and the function adopted by the method comprise:

the membrane fusion domain sequence in the E protein was removed (VMTVGSRSFLVHREWFHDLALPWT),

tandem repeat epitopes were used in the E protein,

two glycosylation sites (two N mutations to D) in the mutant E protein.

Through the optimization, the toxicity of the recombinant protein to cells can be obviously reduced, the expression quantity of the recombinant protein is greatly increased, the immunogenicity of the recombinant protein is obviously improved, the glycosylation level of the recombinant protein can be effectively reduced, and the animal allergy of the recombinant protein to animals is reduced.

Furthermore, the recombinant protein can be expressed by using Sf9 cells cultured in suspension based on a baculovirus insect cell expression system, and has high expression level and good protein immunogenicity.

Further, the recombinant protein can be used for preparing Japanese encephalitis virus genetic engineering subunit vaccines.

For example, in a specific embodiment of the present invention, a method for preparing a japanese encephalitis virus genetically engineered subunit vaccine specifically may comprise:

(1) preparing a nucleic acid molecule encoding the recombinant protein;

(2) cloning the nucleic acid molecule which is prepared in the step (1) and codes the recombinant protein into a shuttle vector to obtain a recombinant shuttle vector containing a target gene;

(3) transforming the recombinant shuttle vector obtained in the step (2) into DH10Bac bacteria, selecting recombinant bacteria, extracting genome to transfect Sf9 cells (or other insect cells) to obtain recombinant baculovirus;

(4) incubating said Sf9 cell (or other insect cell as described above) for recombinant expression to produce a recombinant protein;

(5) and mixing the recombinant protein and adding the recombinant protein into an adjuvant to obtain the vaccine.

In the specific embodiment of the invention, Sf9 cells are used for expressing recombinant proteins (JEV E proteins), the antigenicity, immunogenicity and functions of the products are similar to those of natural proteins, the expression level is higher, the immunogenicity is strong, no pathogenicity is caused to animals, and the vaccine can be prepared by using a bioreactor in large-scale serum-free suspension culture, so that the production cost of the vaccine is greatly reduced.

The Japanese B encephalitis virus genetic engineering subunit vaccine provided by the embodiment of the invention has high safety and good immunogenicity, can generate stronger humoral immunity in an animal body, has no pathogenicity to the animal, can resist strong toxicity attack by the immunized animal, and also has a series of advantages of large-scale batch production, easy quality control, stable batch-to-batch, low production cost and the like.

When the Japanese B encephalitis virus genetic engineering subunit vaccine provided by the embodiment of the invention is applied, only an effective amount of the Japanese B encephalitis virus genetic engineering subunit vaccine needs to be inoculated to animals such as pigs, horses, cattle, sheep, mice, dogs, humans and the like. As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein are performed using molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and techniques conventional in the art. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 construction and identification of transfer vector pF-JEV-E

E gene amplification and purification the codon optimized E gene (defined as JEV-E gene, SEQ ID NO: 1) was synthesized in Nanjing Kingsrey Biotech Co., Ltd and cloned into pUC17 vector to obtain pUC-JEV-E plasmid vector. PCR amplification was performed using pUC-E plasmid as template and JEV-E-F, JEV-E-R as upstream and downstream primers (the gene sequence of JEV-E-F, JEV-E-R is shown in SEQ ID NO:3, 4), and the amplification system is shown in Table 1.

TABLE 1 JEV-E Gene amplification System

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 45 seconds, annealing at 54 ℃ for 45 seconds, extension at 72 ℃ for 1 minute, 35 cycles; extension at 72 ℃ for 10 min.

The PCR product was subjected to gel electrophoresis to verify the size of the target gene, and as shown in FIG. 1, a band of interest appeared at a position of 1.6kbp, and the target gene was successfully amplified and recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion and purification the pFastBac1 plasmid and the PCR amplification product of the JEV-E gene were digested simultaneously for 3 hours at 37 ℃ with BamHI and Hind III, and the specific digestion reaction systems are shown in tables 2 and 3.

And (3) performing gel electrophoresis on the enzyme digestion product, and purifying the enzyme digestion pFastBac1 plasmid and the E gene fragment by using a gel recovery and purification kit respectively.

TABLE 2 JEV-E Gene restriction enzyme reaction System

TABLE 3 pFastBac1 plasmid digestion reaction System

3. Ligation the digested pFastBac1 plasmid and the product of the JEV-E gene digestion were ligated overnight using T4 DNA ligase in a water bath at 16 ℃ in the ligation system shown in Table 4.

TABLE 4 connection System of enzyme digestion product of JEV-E gene and pFastBac1 plasmid

4. Mu.l of the ligation product was added to 100. mu.l of DH 5. alpha. competent cells, mixed well, heat-shocked at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.l of LB medium without Amp, and incubated at 37 ℃ for 1 hour. 1.0 mL of the cell suspension was concentrated by centrifugation to 100. mu.l, and the concentrated solution was applied to LB solid medium containing Amp and cultured at 37 ℃ for 16 hours.

5. Colony PCR and sequencing identification single colonies on the selected plate are respectively inoculated to an LB liquid culture medium, cultured for 2 hours at 37 ℃, and colony PCR is carried out by taking a bacterial liquid as a template and taking JEV-E-F and JEV-E-R as primers. The size of the gene of interest was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 2, a sample showing a band of approximately 1.6kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. The schematic diagram of the constructed transfer vector pF-JEV-E containing the target gene is shown in FIG. 3.

Example 2 construction of recombinant baculovirus genome Bac-JEV-E

DH10Bac transformation mu.l pF-JEV-E plasmid from example 1 was added to 100. mu.l DH10Bac competent cells and mixed well, ice-bathed for 30 minutes, water-bathed heat shock at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.l LB liquid medium without Amp, and cultured at 37 ℃ for 5 hours. After 100. mu.l of the diluted bacterial solution was diluted 81 times, 100. mu.l of the diluted bacterial solution was applied to LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37 ℃ for 48 hours.

2. Selecting a single colony, selecting a large white colony by using an inoculating needle, then streaking on an LB solid culture medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, culturing for 48 hours at 37 ℃, selecting a single colony, inoculating an LB liquid culture medium containing gentamicin, kanamycin and tetracycline, culturing, preserving strains, and extracting plasmids. Obtaining the recombinant plasmid Bacmid-JEV-E.

Example 3 recombinant baculovirus transfection

Six well plates were seeded 0.8X 10 per well⁶The confluency of Sf9 cells is 50-70%. The following complexes were prepared for each well: diluting 4. mu.l of Cellffectin transfection reagent with 100. mu.l of transfection medium T1, and shaking briefly with vortex; mu.g of the recombinant Bacmid-JEV-E plasmid from example 2 was diluted with 100. mu.l of transfection culture T1 medium, and the diluted transfection reagent and plasmid were mixed and gently and evenly blown to prepare a transfection mixture. And adding the transfection compound after the cells adhere to the wall, incubating for 5 hours at 27 ℃, removing the supernatant, adding 2mLSF-SFM fresh culture medium, and culturing for 4-5 days at 27 ℃ to obtain the supernatant. Obtaining recombinant baculovirus rBac-JEV-E, detecting virus titer of the harvested P1 generation recombinant baculovirus by using a Reed-Muench method, wherein the titer of the rBac-JEV-E seed virus is 7.5 multiplied by 10⁷ TCID₅₀and/mL. And amplifying the recombinant baculovirus rBac-JEV-E for later use as a seed virus.

Recombinant baculoviruses expressing the following control groups were also constructed according to the above example method (Table 5).

TABLE 5

Example 4 SDS-PAGE detection

The cell cultures of rBac-JEV-E harvested in example 3 and each control group were subjected to SDS-PAGE while using Sf9 cells infected with empty baculovirus as a negative control. The specific operation is as follows: mu.l of the harvested cell culture was taken, 10. mu.l of 5 × loading buffer was added, the mixture was centrifuged in a boiling water bath for 5 minutes at 12000r/min for 1 minute, the supernatant was subjected to SDS-PAGE gel (12% strength gel) electrophoresis, and the gel was stained and decolored after electrophoresis to observe the band.

As shown in FIG. 4, the rBac-JEV-E cell culture showed a band of interest at a molecular weight of about 58kDa, and the protein expression was higher than that of each control group, and the negative control had no band at the corresponding position.

Example 5 Western Blot assay

The product of example 4 after SDS-PAGE electrophoresis was transferred to an NC (nitrocellulose) membrane, blocked with 5% skim milk for 2 hours, incubated with swine anti-JEV positive serum for 2 hours, rinsed, incubated with secondary goat anti-swine polyclonal antibody labeled with HRP for 2 hours, rinsed, and then added dropwise with an enhanced chemiluminescent fluorogenic substrate and photographed using a chemiluminescent imager. The results are shown in FIG. 5, where the recombinant baculovirus expression sample has a band of interest, and the negative control has no band of interest, indicating that the protein of interest was correctly expressed in Sf9 cells.

EXAMPLE 6 bioreactor serum-free suspension culture of insect cells

The Sf9 insect cells were aseptically cultured in 1000mL shake flasks for 3-4 days to a concentration of 3-5X 10⁶cell/mL, when the activity is more than 95%, inoculating the cells into a 5L bioreactor, wherein the inoculation concentration is 3-8 × 10⁵cell/mL. When the cell concentration reaches 3-55X 10⁶At cell/mL, cells were seeded into a 50L bioreactor until the cells grew to a concentration of 3-55X 10⁶cell/mL, inoculating into 500L bioreactor until cell concentration reaches 2-85 × 10⁶When the cell/mL is obtained, rBac-JEV-E is inoculated, and the culture conditions of the reactor are that the pH value is 6.0-6.5, the temperature is 25-27 ℃, the dissolved oxygen is 30-80 percent, and the stirring speed is 100-180 rpm. In view of the optimum conditions for cell culture, it is preferable to set pH6.2, the temperature at the stage of cell culture at 27 ℃, the dissolved oxygen at 50%, and the stirring speed at 100-180 rpm. Culturing for 5-9 days after infection, adding one-thousandth final concentration BEI, acting at 37 deg.C for 48 hr, adding two-thousandth final concentration Na₂S₂O₃The inactivation is terminated. Cell culture supernatant is harvested by centrifugation or hollow fiber filtration, and the JEV-E protein stock solution is stored at 2-8 ℃. Meanwhile, the same procedure was used to prepare protein stocks expressing the control groups 1 to 4 of example 3.

Example 7 protein purification

1. Purifying the harvested stock solution by cation exchange chromatography

Ion exchange chromatography was performed using a strong cation chromatography packing, POROS 50HS, sterilized using 0.5M NaOH before use. The vaccine stock was then equilibrated with microfiltration buffer at room temperature, and then loaded onto the column at a rate of 125 mL/min, followed by 8 column volumes eluted with rinse buffer a (0.05M MOPS (sodium salt), pH =7.0, 0.5M NaCl). Linear gradient elution was then performed with buffer a and buffer B from 0% buffer B (i.e. 100% buffer a) to 100% buffer B (0.05M MOPS (sodium salt), pH =7.0, 1.5M NaCl) (i.e. 0% buffer a), where a total of 10 column volumes were eluted by linear elution, and then the 10 column volumes were harvested on average. After linear elution, 2 column volumes were eluted with buffer B and collected separately. The collected sample was placed in a 2L sterile plastic bottle and placed at 4 ℃. The fractions collected under the last elution peak (A280) were then stored sterile filtered at 4 ℃.

2. Hydroxyapatite hydrophobic chromatography

Using a pre-packed Hydroxyapatite column (CHT;. Ceramic Hydroxyapatite Type II Media), first, 50 mM MOPS (sodium salt), pH =7.0, 1.25M NaCl was equilibrated, and then the above preliminary-purified sample was loaded at 90 cm/hour, and after loading, elution was carried out using 8 volumes of an equilibration solution until the UV value was zero. Then a gradient elution was performed using an eluent (0.2M phosphate, pH =7.0, 1.25M NaCl) with a concentration of eluent from 0% to 100%, the speed was still 90 cm/h and the elution volume was 4 column volumes. Purifying to obtain the target protein, namely the JEV-E protein.

The purified target protein is quantified by using BCA total protein, and then the purity of the target protein is determined by combining gray scanning, and the purified protein is shown in figure 6, wherein the concentration of the target protein is 1.2mg/mL, and the purity is 92%.

Example 8 agar amplification assay

Detecting the titer of the expressed JEV-E protein by using an agar-agar method, drilling a plum blossom hole on an agarose gel plate, adding JEV agar detection standard serum in the middle of the plum blossom hole, and respectively adding 2-diluted expression antigens of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9and 10 th power around the plum blossom hole. After incubation in an inverted position for 72h, the line of precipitation was observed. The maximum dilution at which a precipitate line appears is its agar titer. The agar titer detection results are as follows: the JEV-E protein agar titer is 1: 256.

example 9 vaccine preparation

The recombinant JEV-E protein stock solution harvested in example 7 is diluted and added into MONTANIDE ISA 201 VG adjuvant (volume ratio is 46: 54) so that the final protein concentration is 100 mug/ml, emulsified and stored at 4 ℃ after qualified quality inspection. Vaccines were prepared in the same manner for control group 1, control group 2, control group 3 and control group 4, respectively.

Example 10 immunization experiment

Test one: antibody detection

30 Balb/c mice with the age of 4-6 weeks are taken and randomly divided into 6 groups, the first five groups are respectively subjected to intraperitoneal injection of 0.2ml of vaccines of an immune group of the vaccine, a control group 1, a control group 2, a control group 3 and a control group 4, the sixth group is a negative control group, and 0.2ml of physiological saline is subjected to intraperitoneal injection, so that the immunity is strengthened once after 4 weeks of primary immunity. Blood was collected before immunization, 4 weeks after primary immunization, and 8 weeks after primary immunization (4 weeks after secondary immunization), and serum was isolated, and the antibody level of each group was measured by ELISA, and the results are shown in Table 6.

TABLE 6

And (2) test II: safety test

10 Balb/c mice of 4-6 weeks old are taken and randomly divided into 2 groups, 2ml of the vaccines in the immune group and the control group 1 in the example 9 are respectively injected into muscles, the cervical vertebrae are respectively removed after continuous observation for one week to be killed, and the conditions of the vaccine injection parts are dissected and observed. In the observation period, the mice in the immune group live normally without fever, and the mice in the control group 1 partially have fever, anorexia, diarrhea, weakness and other symptoms; the tissue of the vaccine injection part of the mice in the immune group is normal, and the inflammation reactions such as red swelling, hard lumps and the like basically appear at the vaccine injection part of the mice in the control group 1.

And (3) test III:

mice 7 days after the secondary immunization in trial one were sacrificed in 3 groups and splenocytes were taken for cytokine testing. The specific operation is as follows: prepared mouse spleen cell suspensions of each group are plated, 100 mu l of each well is inoculated to a 96-well cell culture plate, rMEP with the final concentration of 10 mu l/ml is added for stimulation, supernatant is collected after culture at 37 ℃ for 72h, and the content of Th1 cytokine (IFN-gamma) Th2 cytokine (IL-4) and Th2 cytokine (IL-4) in the supernatant is detected by a cytokine detection kit. The results show that IFN-gamma and IL-4 of the mice in the immune group are remarkably higher than those in other control groups, which indicates that the vaccine in the immune group can induce to generate higher levels of Th1 and Th2 cytokines and play an important role in resisting virus and immunity of the mice. The results are shown in Table 7.

TABLE 7

It is to be understood that the above-described embodiments are part of the present invention, and not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Sequence listing

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gaagtgggca tttttgtgca tggcaccacc accagcgaaa accatggcaa ctatagcgcg 540

caggtgggcg cgagccaggc ggcgaaattt accgtgaccc cggatgcgcc gagcattacc 600

ctgaaactgg gcgattatgg cgaagtgacc ctggattgcg aaccgcgcag cggcctgaac 660

accgaagcgt tttatccgcc gagcagcacc gcgtggcgcg atcgcgaact gctgatggaa 720

tttgaagaag cgcatgcgac caaacagagc gtggtggcgc tgggcagcca ggaaggcggc 780

ctgcatcagg cgctggcggg cgcgattgtg gtggaatata gcagcagcgt gaaactgacc 840

agcggccatc tgaaatgccg cctgaaaatg gataaactgg cgctgaaagg caccacctat 900

ggcatgtgca ccggcaaatt tagctttgcg aaaaacccgg cggataccgg ccatggcacc 960

gtggtgattg aactgagcta tagcggcagc gatggcccgt gcaaaattcc gattgtgagc 1020

gtggcgagcc tgaacgatat gaccccggcg ggccgcctgg tgaccgtgaa cccgtttgtg 1080

gcgaccagca gcgcgaacag caaagtgctg gtggaaatgg aaccgccgtt tggcgatagc 1140

tatattgtgg tgggccgcga agataaacag attaaccatc attggcataa agcgggcagc 1200

accctgggca aagcgtttct gaccaccctg aaaggcgcgc agcgcctggc ggcgctgggc 1260

gatatgtgca ccggcaaatt tagctttgcg aaaaacccgg cggataccgg ccatggcacc 1320

gtggtgattg aactgagcta tagcggcagc gatggcccgt gcaaaattcc gattgtgagc 1380

gtggcgagcc tgaacgatat gaccccggcg ggccgcctgg tgaccgtgaa cccgtttgtg 1440

gcgaccagca gcgcgaacag caaagtgctg gtggaaatgg aaccgccgtt tggcgatagc 1500

tatattgtgg tgggccgcga agataaacag attaaccatc attggcataa agcgggcagc 1560

accctgggca aagcgtttct gaccaccctg aaaggcgcgc agcgcctggc ggcgctgggc 1620

gattga 1626

<210> 2

<211> 541

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile

1 5 10 15

Ser Tyr Ile Tyr Ala Asp Arg Phe Asn Cys Leu Gly Met Gly Asn Arg

20 25 30

Asp Phe Ile Glu Gly Ala Ser Gly Ala Thr Trp Val Asp Leu Val Leu

35 40 45

Glu Gly Asp Ser Cys Leu Thr Ile Met Ala Asn Asp Lys Pro Thr Leu

50 55 60

Asp Val Arg Met Thr Asn Ile Glu Ala Ser Gln Leu Ala Glu Val Arg

65 70 75 80

Ser Tyr Cys Tyr His Ala Ser Val Thr Asp Ile Ser Thr Val Ala Arg

85 90 95

Cys Pro Met Thr Gly Glu Ala His Asn Glu Lys Arg Ala Asp Ser Ser

100 105 110

Tyr Val Cys Lys Gln Gly Phe Thr Asp Arg Gly Trp Gly Asn Gly Cys

115 120 125

Gly Leu Phe Gly Lys Gly Ser Ile Asp Thr Cys Ala Lys Phe Ser Cys

130 135 140

Thr Ser Lys Ala Ile Gly Arg Ala Ile Gln Pro Glu Asn Ile Lys Tyr

145 150 155 160

Glu Val Gly Ile Phe Val His Gly Thr Thr Thr Ser Glu Asn His Gly

165 170 175

Asn Tyr Ser Ala Gln Val Gly Ala Ser Gln Ala Ala Lys Phe Thr Val

180 185 190

Thr Pro Asp Ala Pro Ser Ile Thr Leu Lys Leu Gly Asp Tyr Gly Glu

195 200 205

Val Thr Leu Asp Cys Glu Pro Arg Ser Gly Leu Asn Thr Glu Ala Phe

210 215 220

Tyr Pro Pro Ser Ser Thr Ala Trp Arg Asp Arg Glu Leu Leu Met Glu

225 230 235 240

Phe Glu Glu Ala His Ala Thr Lys Gln Ser Val Val Ala Leu Gly Ser

245 250 255

Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile Val Val Glu

260 265 270

Tyr Ser Ser Ser Val Lys Leu Thr Ser Gly His Leu Lys Cys Arg Leu

275 280 285

Lys Met Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly Met Cys Thr

290 295 300

Gly Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly His Gly Thr

305 310 315 320

Val Val Ile Glu Leu Ser Tyr Ser Gly Ser Asp Gly Pro Cys Lys Ile

325 330 335

Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr Pro Ala Gly Arg

340 345 350

Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala Asn Ser Lys

355 360 365

Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val Val

370 375 380

Gly Arg Glu Asp Lys Gln Ile Asn His His Trp His Lys Ala Gly Ser

385 390 395 400

Thr Leu Gly Lys Ala Phe Leu Thr Thr Leu Lys Gly Ala Gln Arg Leu

405 410 415

Ala Ala Leu Gly Asp Met Cys Thr Gly Lys Phe Ser Phe Ala Lys Asn

420 425 430

Pro Ala Asp Thr Gly His Gly Thr Val Val Ile Glu Leu Ser Tyr Ser

435 440 445

Gly Ser Asp Gly Pro Cys Lys Ile Pro Ile Val Ser Val Ala Ser Leu

450 455 460

Asn Asp Met Thr Pro Ala Gly Arg Leu Val Thr Val Asn Pro Phe Val

465 470 475 480

Ala Thr Ser Ser Ala Asn Ser Lys Val Leu Val Glu Met Glu Pro Pro

485 490 495

Phe Gly Asp Ser Tyr Ile Val Val Gly Arg Glu Asp Lys Gln Ile Asn

500 505 510

His His Trp His Lys Ala Gly Ser Thr Leu Gly Lys Ala Phe Leu Thr

515 520 525

Thr Leu Lys Gly Ala Gln Arg Leu Ala Ala Leu Gly Asp

530 535 540

<210> 3

<211> 37

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ataggatcca tgaaatttct ggtgaacgtg gcgctgg 37

<210> 4

<211> 42

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ataaagcttt caatcgccca gcgccgccag gcgctgcgcg cc 42

<210> 5

<211> 1626

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

atgaaatttc tggtgaacgt ggcgctggtg tttatggtgg tgtatattag ctatatttat 60

gcggatcgct ttaactgcct gggcatgggc aaccgcgatt ttattgaagg cgcgagcggc 120

gcgacctggg tggatctggt gctggaaggc gatagctgcc tgaccattat ggcgaacgat 180

aaaccgaccc tggatgtgcg catgaccaac attgaagcga gccagctggc ggaagtgcgc 240

agctattgct atcatgcgag cgtgaccgat attagcaccg tggcgcgctg cccgatgacc 300

ggcgaagcgc ataacgaaaa acgcgcggat agcagctatg tgtgcaaaca gggctttacc 360

gatcgcggct ggggcaacgg ctgcggcctg tttggcaaag gcagcattga tacctgcgcg 420

aaatttagct gcaccagcaa agcgattggc cgcgcgattc agccggaaaa cattaaatat 480

gaagtgggca tttttgtgca tggcaccacc accagcgaaa accatggcaa ctatagcgcg 540

caggtgggcg cgagccaggc ggcgaaattt accgtgaccc cgaacgcgcc gagcattacc 600

ctgaaactgg gcgattatgg cgaagtgacc ctggattgcg aaccgcgcag cggcctgaac 660

accgaagcgt tttatccgcc gagcagcacc gcgtggcgca accgcgaact gctgatggaa 720

tttgaagaag cgcatgcgac caaacagagc gtggtggcgc tgggcagcca ggaaggcggc 780

ctgcatcagg cgctggcggg cgcgattgtg gtggaatata gcagcagcgt gaaactgacc 840

agcggccatc tgaaatgccg cctgaaaatg gataaactgg cgctgaaagg caccacctat 900

ggcatgtgca ccggcaaatt tagctttgcg aaaaacccgg cggataccgg ccatggcacc 960

gtggtgattg aactgagcta tagcggcagc gatggcccgt gcaaaattcc gattgtgagc 1020

gtggcgagcc tgaacgatat gaccccggcg ggccgcctgg tgaccgtgaa cccgtttgtg 1080

gcgaccagca gcgcgaacag caaagtgctg gtggaaatgg aaccgccgtt tggcgatagc 1140

tatattgtgg tgggccgcga agataaacag attaaccatc attggcataa agcgggcagc 1200

accctgggca aagcgtttct gaccaccctg aaaggcgcgc agcgcctggc ggcgctgggc 1260

gatatgtgca ccggcaaatt tagctttgcg aaaaacccgg cggataccgg ccatggcacc 1320

gtggtgattg aactgagcta tagcggcagc gatggcccgt gcaaaattcc gattgtgagc 1380

gtggcgagcc tgaacgatat gaccccggcg ggccgcctgg tgaccgtgaa cccgtttgtg 1440

gcgaccagca gcgcgaacag caaagtgctg gtggaaatgg aaccgccgtt tggcgatagc 1500

tatattgtgg tgggccgcga agataaacag attaaccatc attggcataa agcgggcagc 1560

accctgggca aagcgtttct gaccaccctg aaaggcgcgc agcgcctggc ggcgctgggc 1620

gattga 1626

<210> 6

<211> 1263

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

atgaaatttc tggtgaacgt ggcgctggtg tttatggtgg tgtatattag ctatatttat 60

gcggatcgct ttaactgcct gggcatgggc aaccgcgatt ttattgaagg cgcgagcggc 120

gcgacctggg tggatctggt gctggaaggc gatagctgcc tgaccattat ggcgaacgat 180

aaaccgaccc tggatgtgcg catgaccaac attgaagcga gccagctggc ggaagtgcgc 240

agctattgct atcatgcgag cgtgaccgat attagcaccg tggcgcgctg cccgatgacc 300

ggcgaagcgc ataacgaaaa acgcgcggat agcagctatg tgtgcaaaca gggctttacc 360

gatcgcggct ggggcaacgg ctgcggcctg tttggcaaag gcagcattga tacctgcgcg 420

aaatttagct gcaccagcaa agcgattggc cgcgcgattc agccggaaaa cattaaatat 480

gaagtgggca tttttgtgca tggcaccacc accagcgaaa accatggcaa ctatagcgcg 540

caggtgggcg cgagccaggc ggcgaaattt accgtgaccc cggatgcgcc gagcattacc 600

ctgaaactgg gcgattatgg cgaagtgacc ctggattgcg aaccgcgcag cggcctgaac 660

accgaagcgt tttatccgcc gagcagcacc gcgtggcgcg atcgcgaact gctgatggaa 720

tttgaagaag cgcatgcgac caaacagagc gtggtggcgc tgggcagcca ggaaggcggc 780

ctgcatcagg cgctggcggg cgcgattgtg gtggaatata gcagcagcgt gaaactgacc 840

agcggccatc tgaaatgccg cctgaaaatg gataaactgg cgctgaaagg caccacctat 900

ggcatgtgca ccggcaaatt tagctttgcg aaaaacccgg cggataccgg ccatggcacc 960

gtggtgattg aactgagcta tagcggcagc gatggcccgt gcaaaattcc gattgtgagc 1020

gtggcgagcc tgaacgatat gaccccggcg ggccgcctgg tgaccgtgaa cccgtttgtg 1080

gcgaccagca gcgcgaacag caaagtgctg gtggaaatgg aaccgccgtt tggcgatagc 1140

tatattgtgg tgggccgcga agataaacag attaaccatc attggcataa agcgggcagc 1200

accctgggca aagcgtttct gaccaccctg aaaggcgcgc agcgcctggc ggcgctgggc 1260

tga 1263

<210> 7

<211> 1698

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

atgaaatttc tggtgaacgt ggcgctggtg tttatggtgg tgtatattag ctatatttat 60

gcggatcgct ttaactgcct gggcatgggc aaccgcgatt ttattgaagg cgcgagcggc 120

gcgacctggg tggatctggt gctggaaggc gatagctgcc tgaccattat ggcgaacgat 180

aaaccgaccc tggatgtgcg catgaccaac attgaagcga gccagctggc ggaagtgcgc 240

agctattgct atcatgcgag cgtgaccgat attagcaccg tggcgcgctg cccgatgacc 300

ggcgaagcgc ataacgaaaa acgcgcggat agcagctatg tgtgcaaaca gggctttacc 360

gatcgcggct ggggcaacgg ctgcggcctg tttggcaaag gcagcattga tacctgcgcg 420

aaatttagct gcaccagcaa agcgattggc cgcgcgattc agccggaaaa cattaaatat 480

gaagtgggca tttttgtgca tggcaccacc accagcgaaa accatggcaa ctatagcgcg 540

caggtgggcg cgagccaggc ggcgaaattt accgtgaccc cggatgcgcc gagcattacc 600

ctgaaactgg gcgattatgg cgaagtgacc ctggattgcg aaccgcgcag cggcctgaac 660

accgaagcgt tttatgtgat gaccgtgggc agccgcagct ttctggtgca tcgcgaatgg 720

tttcatgatc tggcgctgcc gtggaccccg ccgagcagca ccgcgtggcg cgatcgcgaa 780

ctgctgatgg aatttgaaga agcgcatgcg accaaacaga gcgtggtggc gctgggcagc 840

caggaaggcg gcctgcatca ggcgctggcg ggcgcgattg tggtggaata tagcagcagc 900

gtgaaactga ccagcggcca tctgaaatgc cgcctgaaaa tggataaact ggcgctgaaa 960

ggcaccacct atggcatgtg caccggcaaa tttagctttg cgaaaaaccc ggcggatacc 1020

ggccatggca ccgtggtgat tgaactgagc tatagcggca gcgatggccc gtgcaaaatt 1080

ccgattgtga gcgtggcgag cctgaacgat atgaccccgg cgggccgcct ggtgaccgtg 1140

aacccgtttg tggcgaccag cagcgcgaac agcaaagtgc tggtggaaat ggaaccgccg 1200

tttggcgata gctatattgt ggtgggccgc gaagataaac agattaacca tcattggcat 1260

aaagcgggca gcaccctggg caaagcgttt ctgaccaccc tgaaaggcgc gcagcgcctg 1320

gcggcgctgg gcgatatgtg caccggcaaa tttagctttg cgaaaaaccc ggcggatacc 1380

ggccatggca ccgtggtgat tgaactgagc tatagcggca gcgatggccc gtgcaaaatt 1440

ccgattgtga gcgtggcgag cctgaacgat atgaccccgg cgggccgcct ggtgaccgtg 1500

aacccgtttg tggcgaccag cagcgcgaac agcaaagtgc tggtggaaat ggaaccgccg 1560

tttggcgata gctatattgt ggtgggccgc gaagataaac agattaacca tcattggcat 1620

aaagcgggca gcaccctggg caaagcgttt ctgaccaccc tgaaaggcgc gcagcgcctg 1680

gcggcgctgg gcgattga 1698

<210> 8

<211> 1338

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

atgaaatttc tggtgaacgt ggcgctggtg tttatggtgg tgtatattag ctatatttat 60

gcggatcgct ttaactgcct gggcatgggc aaccgcgatt ttattgaagg cgcgagcggc 120

gcgacctggg tggatctggt gctggaaggc gatagctgcc tgaccattat ggcgaacgat 180

aaaccgaccc tggatgtgcg catgaccaac attgaagcga gccagctggc ggaagtgcgc 240

agctattgct atcatgcgag cgtgaccgat attagcaccg tggcgcgctg cccgatgacc 300

ggcgaagcgc ataacgaaaa acgcgcggat agcagctatg tgtgcaaaca gggctttacc 360

gatcgcggct ggggcaacgg ctgcggcctg tttggcaaag gcagcattga tacctgcgcg 420

aaatttagct gcaccagcaa agcgattggc cgcgcgattc agccggaaaa cattaaatat 480

gaagtgggca tttttgtgca tggcaccacc accagcgaaa accatggcaa ctatagcgcg 540

caggtgggcg cgagccaggc ggcgaaattt accgtgaccc cgaacgcgcc gagcattacc 600

ctgaaactgg gcgattatgg cgaagtgacc ctggattgcg aaccgcgcag cggcctgaac 660

accgaagcgt tttatgtgat gaccgtgggc agccgcagct ttctggtgca tcgcgaatgg 720

tttcatgatc tggcgctgcc gtggaccccg ccgagcagca ccgcgtggcg caaccgcgaa 780

ctgctgatgg aatttgaaga agcgcatgcg accaaacaga gcgtggtggc gctgggcagc 840

caggaaggcg gcctgcatca ggcgctggcg ggcgcgattg tggtggaata tagcagcagc 900

gtgaaactga ccagcggcca tctgaaatgc cgcctgaaaa tggataaact ggcgctgaaa 960

ggcaccacct atggcatgtg caccggcaaa tttagctttg cgaaaaaccc ggcggatacc 1020

ggccatggca ccgtggtgat tgaactgagc tatagcggca gcgatggccc gtgcaaaatt 1080

ccgattgtga gcgtggcgag cctgaacgat atgaccccgg cgggccgcct ggtgaccgtg 1140

aacccgtttg tggcgaccag cagcgcgaac agcaaagtgc tggtggaaat ggaaccgccg 1200

tttggcgata gctatattgt ggtgggccgc gaagataaac agattaacca tcattggcat 1260

aaagcgggca gcaccctggg caaagcgttt ctgaccaccc tgaaaggcgc gcagcgcctg 1320

gcggcgctgg gcgattga 1338

Claims

1. The recombinant protein has a sequence shown in SEQ ID NO. 2.

2. A gene encoding the recombinant protein of claim 1.

3. The coding gene of claim 2, wherein: it has a sequence shown in SEQ ID NO. 1.

4. A recombinant vector comprising a gene encoding the recombinant protein of claim 1.

5. The recombinant vector of claim 4, wherein: the recombinant vector comprises pFastBac1, pVL1393 or pFastBac dual.

6. A host cell comprising a gene encoding the recombinant protein of claim 1.

7. The host cell of claim 6, wherein: the host cell is an insect cell, and the insect cell comprises Sf9, High Five or Sf21 cells.

8. An immunological composition characterized by comprising: the recombinant protein of claim 1; and a pharmaceutically acceptable carrier.

9. The immunogenic composition of claim 8, wherein: the pharmaceutically acceptable carrier comprises one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, MONTANIDE ISA 15 VG, liquid paraffin, camphor oil and plant cell agglutinin.

10. A method for producing a recombinant protein, comprising:

cloning a gene encoding the recombinant protein according to claim 1 into a shuttle vector to obtain a recombinant shuttle vector containing a target gene;

11. The method of claim 10, wherein: the shuttle vector comprises pFastBac1, pVL1393 or pFastBac dual; and/or, the insect cell comprises an Sf9, High Five or Sf21 cell.

12. Use of the recombinant protein of claim 1 or the immunological composition of claim 8 or 9 for the preparation of a japanese b encephalitis virus detection reagent, for the manufacture of a medicament for inducing an immune response against a japanese b encephalitis virus antigen in a subject animal, or for the manufacture of a medicament for preventing an animal from being infected with japanese b encephalitis virus.

13. Use of the recombinant protein according to claim 1 or the immunological composition according to claim 8 or 9 for preparing a japanese b-encephalitis virus genetically engineered subunit vaccine.