JP2003116385A - Transgenic plants containing the gene encoding the Japanese encephalitis vaccine - Google Patents

Abstract

(57) [Problem] To provide a transgenic plant containing a gene encoding Japanese encephalitis vaccine.
Still another object of the present invention is to provide a Japanese encephalitis vaccine expressed by the plant. The present invention relates to a mucosal immune carrier and a Japanese encephalitis virus outer protein (Eglycoprotei).
A transgenic plant into which a gene encoding the fusion protein of n) has been introduced. In particular, there is provided a transgenic plant wherein the mucosal immune carrier is cholera toxin B chain protein (CTB), and a transgenic plant wherein the plant is tobacco.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mucosal immunocarrier and a Japanese encephalitis virus outer coat protein (E glycopr).
The transgenic plant into which the gene encoding the fusion protein of

[0002]

BACKGROUND OF THE INVENTION Inactivated vaccines are the mainstream of conventional vaccines including Japanese encephalitis vaccines, and the method of inoculation is most widely used with a syringe. With the current Japanese encephalitis vaccine, the virus is inoculated into the brain of a mouse, and when the virus grows, the brain is removed and the virus is isolated, purified, and inactivated. These vaccines have the ethical problem that many animals are sacrificed, but the cost problem is more serious. Although the spread of conventional vaccines has no problem in developed countries, the epidemics of many infectious diseases such as malaria, dengue fever, and Japanese encephalitis are plagued by economic developing countries in Asia, Africa, and Latin America. Is the most.

[0003] It is extremely difficult to disseminate conventional vaccines in Southeast Asia and Africa for mass vaccination in economically developing countries because the dissemination of the vaccines is too expensive. The current situation is that it cannot be carried out smoothly.

The transmucosal immunization method such as oral and nasal administration of the recombinant protein vaccine produced by the gene recombination technology is an easy inoculation type vaccine that does not require a syringe, and is superior in cost to the conventional type vaccine. There is. It is also possible to obtain the same effect immunologically. Among them, especially, "eating vaccine" using recombinant plants
Is the ultimate vaccination method for transmucosal vaccines and is extremely cost effective. Considering the global situation that the spread of vaccines is extremely difficult due to economic problems, future vaccine development will focus on various transmucosal vaccines, including plant-derived recombinant vaccines such as edible vaccines. There is a need. In addition, it is an attractive field for developed countries as a new industry and from an international contribution standpoint.

Prior Art A substance that functions as a Japanese encephalitis virus vaccine is a protein. The Japanese encephalitis vaccine is an inactivated vaccine purified from infected mouse brain emulsion and is given by subcutaneous injection. In Japan's encephalitis, which is transmitted by the blood-sucking of mosquitoes, rather than the mucous membranes of the intestinal tract and respiratory system as in polio and influenza, there was no idea to inject the vaccine from the mucous membrane. However, if mucosal immunity is possible from the nasal cavity, it is expected that the syringe will become insoluble and the vaccination cost will be greatly reduced. Therefore, it was attempted to inject the Japanese encephalitis vaccine from the nasal mucosa. As a result, the intranasal inoculation method was able to induce the same virus infection protective effect as the conventional inoculation method. In order to specifically induce antibodies to Japanese encephalitis virus particles by mucosal administration, cholera toxin B chain (CTB) and Japanese encephalitis virus envelope protein (E g) having specific affinity for transmucosal immune system
The fusion protein (CTB-JE Egl) was prepared by producing a fusion gene with lycoprotein (JE) and introducing the gene into each species (bacteria, yeast).
Ycoprotein). CTB-JE that can be used as the Japanese encephalitis virus vaccine
The fusion protein of Eglycoprotein is produced in various gene expression systems such as bacteria, yeast, insect cells and the like. It was possible to induce systemic defense immunity by extracting the fusion protein from these expression systems and administering it by oral or nasal mucosa.

[0006] Conventionally, when inoculated by subcutaneous injection, the inclusion of contaminants may induce an immune response to the contaminants, but when administered orally, it is transmucosally administered. The immune response was not induced by the contaminants, but the immune response could be induced only by the protein that was absorbed and bound through the mucosa.

Particularly, in the case of a plant vaccine, it is possible to directly immunize through an intestinal mucosa by expressing a gene in an edible plant. The above-mentioned constitution and its mucosal immunization administration method can be applied to all vertebrates including humans and domestic animals, and is expected to be used as an "edible vaccine". Therefore, as a transmucosal Japanese encephalitis vaccine, the production of transgenic plants into which the above genes have been introduced has been desired.

[0008]

In order to solve such problems, the present invention provides a Japanese encephalitis vaccine (mucosal immune carrier and Japanese encephalitis virus outer protein (E glyc).
It is an object of the present invention to provide a transgenic plant into which a gene encoding a fusion protein (of protein) has been introduced.

[0009]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that the mucosal immune carrier (cholera toxin B chain protein (CTB)) and the Japanese encephalitis virus envelope protein (E). glycoprote
It was found that the fusion protein with (in) functions as a Japanese encephalitis vaccine, and succeeded in producing a transgenic plant expressing the protein, thus completing the present invention.

That is, the present invention relates to a mucosal immune carrier and a Japanese encephalitis virus outer coat protein (Eglycoprot).
ein) transgenic plants containing a gene encoding a fusion protein.

In the present invention, the mucosal immunocarrier is a cholera toxin B chain protein (CTB), and the Japanese encephalitis virus envelope protein is Eglycoprotein.
A transgenic plant according to claim 1 or 2 is provided.

[0012] More preferably, the present invention provides pBI121.
-Providing a transgenic plant into which CTB-JE has been introduced.

In the present invention, the plant is tobacco.
Provided is the transgenic plant.

The present invention further provides the transgenic plant for use as a Japanese encephalitis vaccine. ． The present invention will be described in detail below.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION A gene encoding a fusion protein of a mucosal immunocarrier and a Japanese encephalitis virus outer coat protein (JEV) is introduced into the transgenic plant of the present invention. The fusion protein is a protein in which the mucosal immunocarrier and the Japanese encephalitis virus outer coat protein are linked so as to be in frame.

The term "mucosal immunocarrier" as used herein means a protein having a specific affinity for mucosal tissues. As the mucosal immunocarrier, for example, a cholera toxin B chain protein, a toxinogenic Escherichia coli (LTB), a bacterium such as Salmonella or lactic acid bacterium, a virus, a pathogen, or a protein having an affinity for the mucosal cell surface is used. can do. In one aspect of the invention, the mucosal immunocarrier is preferably cholera toxin B chain protein (CTB).

In one embodiment of the present invention, the Japanese encephalitis virus outer coat protein is a protein of Japanese encephalitis virus, preferably Egly contained in the outer coat.
It is a coprotein.

The method for obtaining the gene encoding the fusion protein is not particularly limited, but the DNA can be prepared, for example, as follows.

Since the cDNA sequences encoding CTB and the Japanese encephalitis virus outer coat protein JEV (Eglycoprotein) are known, primers are designed based on the sequences and the primers are designed based on the sequences, using a usual PCR method, or by chemical synthesis. , Or DN having the base sequence
By hybridizing with the A fragment as a probe, the gene of the present invention can be obtained.

The primer may contain a sequence capable of fusing the E protein gene at the C'terminal side. Also,
Ko for increasing expression efficiency in eukaryotes on the N'-terminal side
A primer containing a zac sequence, a sequence enabling fusion of an exogenous gene in frame with the BamHI or SpeI site at the C ′ end may be designed.

Specifically, among the above fusion proteins,
DNA encoding cholera toxin B chain-derived protein
Can be amplified by performing PCR using the following reaction conditions, template and primer; template: plasmid pM2, N-terminal primer: 5′-GCCGCCATGGTTAAAATTAAAATT.
TGGTGTT-3 '(SEQ ID NO: 1) C-terminal primer: 5'-CGCGAGCTCTTAAAGTTCATCC
TTTTCGGATCTCTGGACTAGTAGGGG
TACGGGGCCCGGGTCCATTTGCCAT
ACTAATTGCGGCAATCGC-3 ′ (SEQ ID NO: 2) PCR conditions: 3 minutes at 94 ° C., then 45 seconds at 94 ° C., 1 minute at 55 ° C., 1 minute at 72 ° C. 3
0 cycles, then 10 minutes at 72 ° C.

Further, by the RT-PCR method using the RNA genome of Japanese encephalitis virus as a template, the Japanese encephalitis virus outer coat protein (Eglycoprotei) was detected.
n) may be amplified. The designed primer can be chemically synthesized according to its base sequence. PCR can be performed according to a conventional method. For example, PCR using the following reaction conditions, templates, and primers
N-terminal primer: 5'-GCGGGATCCCACTATGGCATGT
GCACA-3 ′ (SEQ ID NO: 3) C-terminal primer: (C1) 5′-GCGACATTAGTTCCGAAGGGGGGT
TCCAT-3 ′ (SEQ ID NO: 4) (C2) 5′-GCGACTAGTAGCTTTATGCCAA
TGGTG-3 '(SEQ ID NO: 5) (C3) 5'-GCGACTAGTCCCTTTGTGT
GATCCAAGA-3 ′ (SEQ ID NO: 6) After 3 minutes at 94 ° C., 1 minute at 94 ° C., 55 ° C.
1 minute at 30 ° C. for 1 minute at 72 ° C., then 10 minutes at 72 ° C.

The amplified sequence can be cloned into a commercially available plasmid after being cleaved with a restriction enzyme, for example. This plasmid was prepared by a known method (for example, J.
sambrook et al. , Molecula
r Cloning, 2nd Ed. , Cold
Spring Harbour Laboratory
Press, pp. Isolate and purify according to 1.21-1.52). Then, the base sequence can be determined using a known method such as the Sanger method or the Maxam-Gilbert method, or an automatic base sequence determination device (ABI DNA sequencer 310).

The fusion protein may have a mutation such as deletion, substitution, addition or the like in at least one amino acid in the amino acid sequence as long as it has an immune inducing activity.
The mutation as described above is, for example, at least one, preferably about 1 to 50, of the amino acid sequence of the fusion protein,
More preferably, 1 to 20 amino acids may be deleted, or at least 1, preferably about 1 to 50, more preferably 1 in the amino acid sequence of the fusion protein.
˜20 amino acids may be added, or at least one, preferably about 1 to 50, more preferably 1 to 20 amino acids in the amino acid sequence of the fusion protein may be replaced with another amino acid. Good. Shorter or longer peptide sequences may be encoded so long as they have immunogenic activity. To introduce a mutation into a gene,
It can be carried out by a known method such as the Kunkel method or the Gapped duplex method or a method analogous thereto, for example, using a mutation introduction kit utilizing the site-directed mutagenesis method.

The immunity-inducing activity means that an animal administered with the fusion protein recognizes the fusion protein as a foreign substance, produces an antibody against the fusion protein in the body, and the antibody produces the fusion protein. It is an activity that acts on the active portion and inhibits the activity of Japanese encephalitis virus.

Next, a method for producing a vector containing a gene encoding a fusion protein of a mucosal immunocarrier and a Japanese encephalitis virus outer coat protein (E glycoprotein) will be described.

Preparation of Recombinant Vector for Plant Introduction and Transformation of Agrobacterium In the recombinant vector for plant introduction, the DNA obtained as described above is directly or, if desired, digested with an appropriate restriction enzyme, or , Can be constructed by linking a suitable linker. As a vector for inserting DNA, pBI101, pBI1
21, binary vectors such as pGA482 and pLGV
Examples include intermediate vectors such as 23Neo, pNCAT, and pMON200, but are not limited to these vectors.

The gene encoding the above fusion protein needs to be incorporated into a vector so that the function of the gene is exerted. In particular, in order to express a foreign gene in a plant, the gene before and after the structural gene is used. It is necessary to arrange a promoter and a terminator for the plant respectively. Examples of promoters that can be used in the present invention include cauliflower mosaic virus (CaM
V) -derived 35S transcript [Jefferson, R. et al.
A. et al. : The EMBO J 6: 3
901-3907 (1987)], ubiquitin of corn [Christensen, A .; H. et
al. : Plant Mol. Biol. 18:67
5-689 (1992)], nopaline synthase (NO
S) gene, octopine (OCT) synthase gene, rice actin (Act1) gene, and other promoters, and examples of the terminator sequence include cauliflower mosaic virus-derived and nopaline synthase gene-derived terminators. However, the promoter and terminator are not limited to these as long as they are known to function in a plant.

In addition, if necessary, an intron sequence having a function of enhancing the gene expression, such as a maize alcohol dehydrogenase (Adh1) intron [Genes], may be present between the promoter sequence and the DNA.
& Development1: 1183-1200
(1987)] can be introduced. Furthermore, an effective selectable marker gene may be used in combination in order to efficiently select a target transformed cell. As a selectable marker used at that time, bialaphos (bia), a hygromycin phosphotransferase (http) gene that imparts resistance to the antibiotic hygromycin to plants
a phosphinothricin acetyltransferase (bar) gene that confers resistance to laphos),
Examples include the blasticidin S deaminase (BSD) gene that imparts resistance to blasticidin S.

A hinge region may be inserted between CTB and the fusion gene in order to minimize intermolecular friction between CTB and the fusion gene. The hinge area is, for example,
With GPGP as one unit, three types can be constructed in single, double, triple and tandem. At that time, codons in the hinge region may be used with relatively low frequencies in plant species so that the ribosome is temporarily stopped or slowed down during protein translation.

Furthermore, in order to facilitate the efficient accumulation of the recombinant protein in the endoplasmic reticulum of plant cells and the fusion protein to form a pentamer, the endoplasmic reticulum retention signal (SEKD
EL) may be inserted at the C ′ end of the fusion protein.
At this time, SEKDEL should be the one with the highest codon frequency, and the stop codon TGA should be inserted after that.

If desired, those containing a cis element such as an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence) and the like can be ligated.

To insert the gene encoding the fusion protein into the binary vector, first, purified DN
A vector is cleaved with an appropriate restriction enzyme to obtain an appropriate vector DNA.
The method of inserting into the restriction enzyme site or the multi-cloning site and ligating to the vector is adopted.

As an example of the above-mentioned recombinant vector, for example, a gene encoding the above fusion protein is added to pBI1.
21 vector.

First, an expression vector containing a gene encoding the CTB region is constructed downstream of the pBI plant expression vector (FIGS. 3 and 4). In such a vector, JE
Insert the E protein region of VcDNA and use JEV / C
A vector capable of expressing the TB fusion protein is constructed (Fig. 5).

For example, a CTB constructed as described above
The fusion gene containing the gene is CTBh _1-3SEKDEL
The plasmid vector pIBT210.1.
Downstream of the reflower mosaic virus 35S promoter
Inserted into pIBT210.1-CTBh_1-3SE
Make KDEL (FIG. 3).

Next, from this plasmid vector, CTBh
_{1-3 The} entire T-DNA region having the SEKDEL expression cassette was excised with HindIII-EcoRI, and HindIII-EcoRI of the plant expression vector pBI121 was excised.
Replace with fragment pBI121-CTBh
_1-3 SEKDEL is prepared (FIG. 4). This plant expression vector contains BamHI and SpeI downstream of CTB.
When the site has a site and a foreign gene is inserted into these restriction enzyme sites, it becomes in-frame with CTB, and a CTB fusion protein can be produced.

The above-mentioned pBI121-CTBh _1-3 SEK
JEVEgly on the BamHI-SpeI site of DEL
CTB-JEV by inserting the full length of coprotein
Construct E fusion gene (pBI121-CTB-J
E) (Fig. 5). As described above, DNA sequence
Confirm the pre-base sequence of the fusion gene using r
Confirm that the B and JEVE genes are in frame.

Next, a method for producing a transgenic plant into which a gene encoding the above fusion protein has been introduced will be described.

In the present invention, a plant means a cultured cell of a plant, a plant of a cultivated plant, an organ of a plant (for example, leaves, petals,
Stalk, root, rhizome, seed, etc.) or tissue (eg, epidermis, phloem, parenchyma, xylem, vascular bundle, etc.). The plant species used here is not particularly limited, but various plants such as rice, wheat, tobacco, tomato, and Arabidopsis can be mentioned. Preferred are solanaceous plants such as tomato, eggplant, peppers and tobacco. More preferably, tobacco is used.

The morphology of the plant into which the gene is introduced in the present invention includes all kinds of plant cells that can be regenerated into a plant. Examples include, but are not limited to, cultured cells, protoplasts, shoot primordia, multiple shoots, hairy roots, and callus. The plant cells in the present invention also include cells in plants.

The transformation of plants with the gene encoding the above fusion protein can be introduced using various methods known to those skilled in the art. It can be carried out by introducing a vector containing the gene into a plant host by the Agrobacterium binary vector method, particle gun method, polyethylene glycol method, or the like. Alternatively, it can be transformed by introducing it into protoplasts by electroporation.

Direct gene transfer by the particle gun method is a co-transformation in which a vector containing a selectable marker gene and a vector containing the gene are mixed and simultaneously shot into plant cells.
Ration) method. The shoots, hairy roots, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture, and using conventionally known plant tissue culture methods, The plant can be regenerated by administration or the like.

When the Agrobacterium binary vector method is used, the gene of interest is inserted between the boundary sequences (LB, RB) of the above binary vector, and this recombinant vector is amplified in E. coli. Then, the amplified recombinant vector is introduced into Agrobacterium tumefaciens LBA4404, EHA101, C58C1RifR and the like, which is used for transduction into plants.

For example, when introduced into tobacco, the following
Transduction may be performed in this manner. Aseptic culture non-recombinant
Cut leaves of tobacco (N. tabacum) 1.
Cut into 5-2 cm squares. The cut leaves are infected
To prevent this from happening, scratch it in places with a knife and
Culture solution of recombinant Agrobacterium (OD
₆₀₀= 1 x 10) for 3 minutes. Then on the leaf
Remove Agrobacterium solution as much as possible, MS agar culture
Be sure to turn it over on the ground (with the pore side up) and lay it without gaps.
Close up.

The petri dish was closed with parafilm, air holes were made at two places, and the dish was incubated in a dark room for 3 days at room temperature to promote infection.

After 3 days, leaves placed in a dark room were treated with MS sho containing an antibiotic (kanamycin) (50 μg / ml) and plant hormones (auxin and cytokinin).
Transfer to the otgrowth medium to induce callus. Since shoots start to emerge from the callus within 2-3 weeks, MS root containing only the antibiotic (kanamycin) (50 μg / ml) is cut off from the callus that emerged.
Magneta with growth medium
Recombinant tobacco can be obtained by transferring to Box and producing sterile culture of plants.

In the plant into which the gene of the present invention has been introduced, it is possible to select transformed cells carrying the gene by screening with a selectable marker or by analyzing the gene or its expression product. For example, to confirm that the target gene has been incorporated into the obtained transgenic plant and its next generation, DNA is extracted from these cells and tissues according to a conventional method, and known PCR is performed.
It can be performed by detecting the introduced gene using the method or Southern blotting. The obtained transgenic plants are cultivated in a pot filled with soil or vermiculite, and are divided into strains. The transgenic plant thus obtained is also included in the scope of the present invention.
The expression site of the gene of the present invention in plant tissues can be confirmed by, for example, analyzing mRNA expression or protein expression in each tissue. Specifically, examples of the method for confirming the expression by the transgenic plant of the present invention include RT-PCR method, Northern blotting method and the like, and Western analysis method using an antibody and the like, but are not limited thereto.

[0049]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the technical scope of the present invention is not limited to these examples.

1. CTB-J by Agrobacterium
Expression of E.

Method Construction of CTB-JEV E Protein Fusion Gene Cholera toxin B chain protein and Japanese encephalitis virus envelope protein (Eglycoprote) were constructed as follows.
in) was prepared (FIG. 3).

As a mucosal immunoadjuvant, the cholera toxin B chain gene was PCR-amplified from the plasmid pM2 so that the E protein gene could be fused to its C'terminal side. When amplifying the CTB gene, the N'-terminal side is the Kozac sequence (GCCA) for enhancing the expression efficiency in eukaryotes.
(TGG: ATG is the initiation codon) was introduced so that the foreign gene could be fused in-frame to the BamHI or SpeI site on the C′-terminal side. PCR was performed using the following reaction conditions, template, and primers.

Template: plasmid pM2, N-terminal primer: 5'-GCGCCATGGTTAAATTAAAAATT
TGGTGTT-3 ′ (SEQ ID NO: 7) C-terminal primer: 5′-CGCGAGCTCTTAAAGTTCATCC
TTTTCGGATCTCTGGACTAGTAGGGG
TACGGGGCCCGGGTCCATTTGCCAT
ACTAATTGCGGCAATCGC-3 ′ (SEQ ID NO: 8) PCR conditions: 3 minutes at 94 ° C., then 45 seconds at 94 ° C., 1 minute at 55 ° C., 1 minute at 72 ° C. 3
0 cycles, then 10 minutes at 72 ° C.

To minimize intermolecular friction between CTB and the fusion gene, a hinge region (GPGP) was inserted between the CTB and the fusion gene. The hinge region is GPGP 1
Single, double, triple and tandem 3 as units
Seed constructed. At that time, codons in the hinge region were used with relatively low frequency in plant species, and the ribosome was temporarily stopped or slowed down during protein translation. In addition, an endoplasmic reticulum retention signal (SEKDEL) was inserted at the C′-end of the fusion protein in order to efficiently accumulate the recombinant protein in the endoplasmic reticulum of plant cells and facilitate the fusion protein to form a pentamer. . Specifically, SEKDE
For L, the one with the highest codon frequency was used, and the stop codon TAA was inserted after that (TCC GAA AA
G GAT GAA CTT TAA: S
Corresponding to the E K E D E L stop codon).

The fusion gene constructed in this manner is ligated to CTB
plasmid vector pI as h _1-3 SEKDEL
Cauliflower mosaic virus 35 in BT210.1
Inserted downstream of the S promoter, pIBT210.1
-CTBh _1-3 SEKDEL was prepared (Fig. 3).

The insertion into pIBT210.1 was SacI
After digestion with (the S-terminal recognition site (GAGCTC) was incorporated in the C-terminal primer), ligation was performed by a usual method.

The total length of CTBh _1-3 SEKDEL is determined by an automatic nucleotide sequencer (ABI DNA sequence).
er 310). Next, the entire T-DNA region having the CTBh _1-3 SEKDEL expression cassette was excised with HindIII-EcoRI from this plasmid vector, and HindII of the plant expression vector pBI121 was excised.
PBI121 by replacing with I-EcoRI fragment
-CTBh _1-3 SEKDEL was prepared (Fig. 4). This plant expression vector has BamHI and SpeI sites downstream of CTB, and when a foreign gene is inserted into these restriction enzyme sites, it becomes in-frame with CTB and C
A TB fusion protein can be produced.

Next, Japanese encephalitis virus E glycopr
The full-length otein gene was amplified from the viral RNA genome by the RT-PCR method. Based on the cDNA, about 30% of the 3'side of the gene (E glycopr
amino acid residue 300 to amino acid residue 377 (C1), amino acid residue 399 to amino acid residue 399 (C
2), BamH up to amino acid residue 426 (C3))
Inserted at the I-SpeI site to construct a CTB-JEVE fusion gene (pBI121-CTB-JE) (FIG. 5), and confirm the pre-base sequence of the fusion gene using the DNA sequencer as described above, CTB and JE
It was confirmed that the VE gene was in frame.

Here, with reference to FIGS.
BI121-CTB-JE will be described. pBI1
The T-DNA region of 21-CTB-JE is a region sandwiched between RB and LB and contains the following genes (RB and LB are recognized by Agrobacterium, and T-D in the plant nucleus).
It is an essential nucleotide sequence region when inserting a copy of NA).

1. It contains a phosphotransferase gene (NPT-II) expression cassette (containing the NOS promoter and NOS terminator regions) that confers resistance to the antibiotic kanamycin in transgenic plants. This is a gene required for recombinant selection.

2. CTB-JE expression cassette (eCa
MV35S promoter *, TEV5 ′ translation enhancer region **, and VSP3 ′ terminator).
Japanese encephalitis virus E-glycoprotein is located behind the hinge region and in front of the SEKDEL endoplasmic reticulum retention signal.
Was inserted so as to be in frame with the CTB-hinge region, whereby the CTB protein and the E protein are separated by the hinge site. In addition, E-glycop is used so that the recombinant protein may be accumulated in the endoplasmic reticulum of plants.
The SEKDEL endoplasmic reticulum retention signal was bound immediately behind the protein. The leader peptide at the N'terminus of CTB is the original CTB gene and functions when translocating into the periplasm of Gram-negative bacteria, but in the plant expression construct, it translocates to endoplasmic reticulum in plant cells. Fulfill the function of

* ECaMV35S promoter: The 5'side of the promoter is doubled (tandem), and the transcription efficiency is several times higher than the original CaMV35S.

** TEV 5'translation enhancer: A sequence derived from a virus that infects tobacco and is a nucleotide sequence that improves translation efficiency.

PBI121-C to Agrobacterium
Introduction of TB-JE Gene A binary vector (pBI121-CTB-JE) having a CTB-JEVE fusion gene was introduced into Agrobact.
erium tumefaciens LBA4404
Was introduced by electroporation. At the time of gene transfer, the same protocol as the gene transfer to Escherichia coli was used using Gene Pulser of Bio Rad. Specifically, at 2.5 kV, 25 μF, 200 Ω, about 3
1 μl of 0 ng / μl DNA (about 30 ng DNA)
Was done using.

The gene-introduced Agrobacterium was selected with kanamycin (50 μg / ml), and the binary vector introduced by the plasmid miniprep method was taken out from Agrobacterium. It was confirmed by cutting with several restriction enzymes whether or not there was a change in its size compared to before insertion.

Detection of Fusion Protein Derived from Transformed Agrobacterium Agrobacterium was transformed into YEB solution (rifampicin 50
μg / ml), streptomycin 100 μg / ml,
Cultivation with kanamycin 50 μg / ml), bacterial protein extract (50 mM Tris-Cl, 1 mM E)
Total water-soluble protein was extracted as follows using DTA, 100 mM NaCl).

1. 8 μl of phenylmethylsulfol per gram of transformed Agrobacterium
Nylfluoride (PMSF) and 80 μl lysozyme (10 mg / ml) were added to give 20
Incubate at room temperature for minutes.

2. Add 4 mg of deoxychoic acid per gram of Agrobacterium,
Incubate at 37 ° C.

3. When the lysate became muddy, DNaceI was added for each gram of Agrobacterium.
Add (1 mg / ml) and incubate for about 30 minutes at room temperature.

4. Precipitate the lysate by high-speed centrifugation (40,000 g) (30 minutes centrifugation) and separate the supernatant.

Next, C derived from transformed Agrobacterium
GM in which TB-JE fusion protein is a receptor for CTB
In order to confirm that the specific affinity for 1 ganglioside was maintained, GM1-ELISA was performed as follows.

1. Microtiter plate mon
sialoganglioside-GM1 (Sig
ma G-7641), 100 μl / well (3.0 μm
g / ml bicarbonate buffer (15 mM Na2CO
3, 35 mM NaHCO 3), PH 9.6) 4
Coating overnight at ° C.

2. Plate the PBST [phosph
ate buffered saline (PBS),
0.05% Tween-20], blocked with 1% bovine serum albumin (BSA) at 37 ° C for 2 hours, and washed again.

3. Transformed Agrobacterium extract was diluted in PBS in several steps and incubated at 100 μl / well overnight at 4 ° C. Then wash.

4. Primary antibody recognizing CTB or JEV E protein (1000 to 5000-fold dilution) was added and incubated at 37 ° C. for 2 hours. Then wash.

5. Add a secondary antibody of alkaline phosphatase conjugate (100 μl / well),
Incubate at 37 ° C for 1 hour. Then wash.

6. Alkaline phosphatase substrate (10
(0 μl / well), and color was developed for 20 minutes at room temperature, and then OD was added. D. Measured at 415 nm.

7. CTB fusion protein derived from plant was measured using CTB protein (Sigma) as a control.

Results It was confirmed that the transformed Agrobacterium produced the CTB-JEVE fusion protein existing downstream of the cauliflower mosaic virus 35S promoter.

2. Mouse mucosal immunization experiment using fusion protein derived from Agrobacterium.

Method A mouse immunization experiment was carried out using a crude extract of bacterial cells. As an immunization route, three routes of oral, nasal and abdominal cavity were given, and immunization was performed once a week for a total of 4 times. One week after the final immunization, the antibody titer was measured using the serum collected from the tail.
In the neutralization test, using a Beijing strain of Japanese encephalitis virus, a case where the plaque number was halved at a serum dilution of 10 times or more, as in the Japanese encephalitis vaccine test, was defined as neutralizing antibody positive.

As a result, the induction of the antibody specifically recognizing only the expressed protein was confirmed without the rise of the antibody against the bacterial component only in the oral and nasal routes. 4 out of 5 animals in the oral administration group
It was found that the animal produced virus neutralizing antibody.

Although the fusion protein is mucosally administered and the conventional Japanese encephalitis vaccine administration method induces the antibody by injection vaccination, an equivalent effect can be obtained. That is,
By producing a fusion protein with the cholera toxin B chain protein, it is possible to efficiently transport the virus outer coat protein to the intestinal mucosal tissue and induce a systemic immune response (Table 1).

[0084]

[Table 1] 3. Induction experiment of virus neutralizing antibody by nasal vaccination with Japanese encephalitis vaccine.

Method To examine the antibody-inducing ability of the current Japanese encephalitis virus vaccine by mucosal immunization, a nasal immunization experiment was performed using mice. Inoculation was performed once a week for a total of 4 times, and the antibody titer was measured one week after the final immunization. The neutralization test was performed in the same manner as the above-mentioned CTB-JE fusion protein intake.

To examine the efficiency of the mucosal immunization route, the current Japanese encephalitis vaccine was diluted and nasally immunized, and then ELI was used.
The antibody titer and neutralizing antibody titer were examined by SA.

Results The results are shown in Table 2. An increase in antibody titer was observed 3 weeks after the start of immunization. Although the antibody titer was increased both by the vaccine alone and by mixing with the adjuvant, a marked increase in the ELISA antibody titer and the neutralizing antibody titer was confirmed by the admixture of the adjuvant (Table 2).

[0088]

[Table 2]

It was revealed that the efficiency of the mucosal immunization route required about 3 times the amount of vaccine in the case of the combined vaccine, as compared with the amount of vaccine used for intraperitoneal administration (134 ng). When no adjuvant was used, an increase in antibody titer was not confirmed even when the 10-fold amount thereof was administered (Table 3).

[0090]

[Table 3] 4. Expression of fusion proteins by transgenic tobacco.

Method Transformation of tobacco by Agrobacterium infection Constructed binary vector pBI121-CTB-J
One of the soil bacteria having the ability to introduce E into the plant nuclear chromosome
Agrobacterium (Agrobacterium)
um tumefaciens). pBI1
21-CTBh _1-3Ag with SEKDEL inside the cell
Rofampicin (50ng / ml),
Streptomycin (100 μg / ml), kanamai
YEB medium (1 liter: 50 μg / ml):
Beef Extract 5g, Bacto yea
st extract 1g, Sucrose 5g,
Bacto Ager 9g, pH 7.3) for 3 days
Nourished and sterilized with acetosyringone (370 μM)
MSO culture solution (1 pack / l MS salts,
3.0% sucrose, 1 × B5 vitamin
s, pH 5.8) 109-1010 bacteria
The suspension was made to be about / ml. The taste of sterilized cigarettes
Germination in MS medium until about 7-8 leaves appear
After culturing, remove the leaves and use a scalpel to make a grid (2 cm square).
I cut it out. These cut leaf disks are
To make it easier to get infected, use a knife to scratch it in places,
Immerse in a suspension of transformed Agrobacterium for 1-2 minutes
Then, remove the excess liquid after taking it out, and then use MS agar.
Make sure the back side is on top of the medium (pore side up.
I laid it without any gaps. Petri dish with parafilm
Close it and open air holes in two places, and let it reach room temperature in the dark for 3 days.
Incubate at room temperature to promote infection. 3 days later
Remove the soft disk and load kanamycin (50 μg / m
l), NAA (100 μg / ml), Claforan (3
Transferred to MS selection medium containing 100 μg / ml). Or
After about 2 weeks, callus progressed and shoots formed
And cut from the callus when the leaves are about 2-3 leaves
Then, the cells were individually cultured in MS selection medium. Shoot
After the roots are formed and the plant begins to grow, it is transplanted to soil.
Then grown further.

Analysis of Transformed Tobacco Primer set for extracting genomic DNA from tobacco showing kanamycin resistance and specifically amplifying CTB region,
PCR amplification (first at 94 ° C for 3 minutes, then 94 ° C for 45 seconds, 55 ° C for 1 minute, 72 ° C for 1 minute for 30 cycles, finally 72 ° C for 10 minutes) using the sequence, It was confirmed that the tobacco was transformed. The transformed tobacco can be transformed into the plant protein extraction buffer (2
00 mM Tris-Cl, pH 8.0, 100 mM
NaCl, 400 mM Sucrose, 10 mM E
DTA, 14 mM 2-mercaptoethano
l, 1 mM phenylmethylsulfony
l fluoride, 0.05% Tween-2
0) was used to extract the water-soluble total protein.

1. Use 1 ml of extraction buffer per 1 g of plant tissue (for tobacco, rhizome of potato, etc.) and make a mortar on ice.

2. 17,000 xg homogenate,
Centrifuge for 15 minutes at 4 ° C.

3. Collect the supernatant and centrifuge again.

4. The supernatant is used for protein assay. At this time, protein is about 50 to about 20 to 20 μl.
It becomes 100 μg.

Using the protein extract, CTB-JE
In order to confirm that the fusion protein retains a specific affinity for GM1 ganglioside, which is a receptor for CTB, GM1-ELISA was performed as follows.

1. Microtiter plate mon
sialoganglioside-GM1 (Sig
ma G-7641), 100 μl / well (3.0 μm
g / ml bicarbonate buffer (15 mM Na2CO
3, 35 mM NaHCO3), PH 9.6)
Coating overnight at 4 ° C.

2. Plate the PBST [phosph
ate buffered saline (PBS),
0.05% Tween-20], blocked with 1% bovine serum albumin (BSA) at 37 ° C for 2 hours, and washed again.

3. Transformed Agrobacterium extract was diluted in PBS in several steps and incubated at 100 μl / well overnight at 4 ° C. Then wash.

4. Primary antibody recognizing CTB or JEV E protein (1000 to 5000-fold dilution) was added and incubated at 37 ° C. for 2 hours. Then wash.

5. Add a secondary antibody of alkaline phosphatase conjugate (100 μl / well),
Incubate at 37 ° C for 1 hour. Then wash.

6. Alkaline phosphatase substrate (10
(0 μl / well), and color was developed for 20 minutes at room temperature, and then OD was added. D. Measured at 415 nm.

7. CTB fusion protein derived from plant was measured using CTB protein (Sigma) as a control.

Results As a result of several solid analysis of transgenic tobacco, 2 to 1 g of total water-soluble protein derived from tobacco leaves was analyzed.
It was confirmed that 12 μg of recombinant CTB-JE fusion protein was produced (FIG. 6).

[0106]

In the conventional Japanese encephalitis vaccine administration method, antibody induction is carried out by injection inoculation, but in the present invention, the same effect can be obtained by mucosal administration of the fusion protein. That is, by producing a fusion protein with the cholera toxin B chain protein, it is possible to efficiently deliver the virus envelope protein to the intestinal mucosal tissue and induce a systemic immune response. Because it is possible to create a transgenic plant into which this fusion protein has been introduced. By gene expression in edible plants,
It is possible to immunize directly via the intestinal mucosa. The above-mentioned constitution and its mucosal immunization administration method can be applied to all vertebrates including humans and domestic animals, and is expected to be used as an "edible vaccine".

[0107]

[Sequence list]                          SEQUENCE LISTING <110> The transgenic plant that contain the gene coding a japanese ence phalitis virus vaccines. <120> The president of Uneversity of Ryukyu <130> A0001001463 <160> 8 <170> PatentIn version 3.1 <210> 1 <211> 29 <212> DNA <213> Primer <400> 1 gcgccatggt taaattaaaa tttggtgtt 29 <210> 2 <211> 90 <212> DNA <213> Primer <400> 2 cgcgagctct taaagttcat ccttttcgga tcctggacta gtaggggtac cgggcccggg 60 tccatttgcc atactaattg cggcaatcgc 90 <210> 3 <211> 27 <212> DNA <213> Primer <400> 3 gcgggatcca cctatggcat gtgcaca 27 <210> 4 <211> 27 <212> DNA <213> Primer <400> 4 gcgactagtt ccgaaggggg gttccat 27 <210> 5 <211> 27 <212> DNA <213> Primer <400> 5 gcgactagta gctttatgcc aatggtg 27 <210> 6 <211> 27 <212> DNA <213> Primer <400> 6 gcgactagtc ccttgtgtga tccaaga 27 <210> 7 <211> 29 <212> DNA <213> Primer <400> 7 gcgccatggt taaattaaaa tttggtgtt 29 <210> 8 <211> 90 <212> DNA <213> Primer <400> 8 cgcgagctct taaagttcat ccttttcgga tcctggacta gtaggggtac cgggcccggg 60 tccatttgcc atactaattg cggcaatcgc 90

[Brief description of drawings]

FIG. 1 is a diagram showing an application example of a CTB-JE fusion protein.

FIG. 2 Japanese encephalitis virus Eglycoprotein
Schematic diagram showing the amplification site of

FIG. 3. Expression vector containing fusion protein with cholera toxin B chain protein (CTB), pIBT210.
The schematic diagram which shows 1-CTBh _1-3 SEKDEL.

FIG. 4 Cholera toxin B chain protein (CTB) Japanese encephalitis virus envelope protein (E glycoprot)
Ein) (JEV) plant expression vector containing fusion protein, pBI121-CTBh _1-3 SEKDEL
FIG.

FIG. 5: Cholera toxin B chain protein (CTB) Japanese encephalitis virus envelope protein (E glycoprot)
A schematic diagram showing pBI121-CTB-JE, a binary vector containing a fusion protein with ein) (JEV).

FIG. 6 shows the expression level of CTB-JE fusion protein in tobacco.

Continued front page    (72) Inventor Naoshi Tsuji             4-415-1, Matsushiro, Tsukuba City, Ibaraki Prefecture (72) Inventor Yoshiya Sato             693-1 Yasaya, Kitanakagusuku Village, Okinawa Prefecture (72) Inventor Toshihiko Fukunaga             4-58 Shuriishimine Town, Naha City, Okinawa Prefecture (72) Inventor Shigetoshi Sato             Okinawa Prefecture Nakahara-gun Nishihara-cho character Chihara 1 Ryukyu University             Faculty of Agriculture (72) Inventor Nagamine Masaru             1-180-9 Sueyoshi-cho, Shuri, Naha City, Okinawa Prefecture (72) Inventor Ichiro Kurane             17-7-601 Ichibancho, Chiyoda-ku, Tokyo F-term (reference) 2B030 CA15 CA17 CA19                 4B024 AA01 AA07 BA32 BA38 CA04                       CA07 DA01 EA02 EA04 GA11

Claims (6)

[Claims] 1. A transgenic plant into which a gene encoding a fusion protein of a mucosal immunocarrier and a Japanese encephalitis virus outer coat protein has been introduced. 2. The mucosal immunocarrier is cholera toxin B.
The transgenic plant according to claim 1, which is a chain protein (CTB). 3. The transgenic plant according to claim 1, wherein the Japanese encephalitis virus outer coat protein is Eglycoprotein. 4. A transgenic plant into which pBI121-CTB-JE has been introduced. 5. The method according to claim 1, wherein the plant is tobacco.
The transgenic plant according to any one of 1. 6. The transgenic plant according to any one of claims 1 to 5, for use as a Japanese encephalitis virus vaccine. ．