JP2001299353A - Adjuvant comprising rotavirus nsp4 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To create a practical and safe adjuvant for oral and nasal administrations, and simultaneously to provide a method for producing a rotavirus NSP4 protein, by which the protein capable of being used as the adjuvant can stably be provided in a large amount. SOLUTION: This adjuvant containing the rotavirus NSP4 is provided on the basis that the rotavirus NSP4 has the adjuvant activity. This method for producing the rotavirus NSP4 protein used as the adjuvant, comprising (1) a step of preparing a recombined Escherichia coli expression plasmid vector which contains a gene encoding the rotavirus NSP4 protein or its part and can produce the protein, (2) a step of transforming host Escherichia coli with the recombined Escherichia coli expression plasmid vector, (3) a step of culturing the transformed hoot Escherichia coli to produce the protein, and (4) a step of separating and recovering the produced protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the utility of a rotavirus nonstructural protein NSP4 as an adjuvant. More specifically, the present invention relates to a nasal / oral administration type adjuvant containing feline rotavirus NSP4, and the NSP4 protein. And a recombinant Escherichia coli expression plasmid vector incorporating a gene encoding the protein, which is prepared when the protein is produced, and a transformant transformed with the recombinant Escherichia coli expression plasmid vector. Things.

[0002]

2. Description of the Related Art At present, vaccines against various pathogenic microorganisms are clinically used, and the main administration methods of these vaccines include injection, oral and nasal administration. Nasal and oral vaccines are
Compared with an injection vaccine, there are advantages that there is no pain, administration is simple, and mucosal immunity can be induced.

[0003] On the other hand, on the other hand, adjuvants exhibiting remarkable immunostimulatory effects have been found and practically used in injection vaccines, whereas practical adjuvants have not been found in nasal and oral administration vaccines. Problems remain.

[0004] In recent years, there have been reports of studies showing that the B subunit of cholera toxin, which is known as a bacterial enterotoxin, exhibits an adjuvant effect when used in combination with a nasal or oral administration vaccine. As an example, when AgI / II, a surface protein of Streptococcus mutans, is mixed with cholera toxin B subunit and nasally administered to BALB / c mice, remarkable mucosal IgA compared to AgI / II alone administration
And the induction of blood IgG has been reported (H.-Y. Wu and MH Russell, 1998, Vaccine 16: 28
6-292).

[0005] As an example of oral administration, when a synthetic peptide vaccine corresponding to the main neutralizing region of human immunodeficiency virus is orally administered together with cholera toxin B subunit, a significantly higher immune response than administration of the synthetic peptide vaccine alone is obtained. There is a study report that was induced (H. Bukaw
a et al., 1995, Nature Med., 1: 681).

[0006] However, at present, cholera toxin B subunit has not been practically used as an adjuvant for nasal or oral administration vaccine.

[0007] Rotavirus infects warm-blooded animals including humans widely, and consists of eleven segments that cause severe acute diarrhea in infected animals, especially in infected animals when neonatal translocation antibodies have disappeared. It is a virus whose main strand is RNA. The feline rotavirus is no exception, with acute diarrhea infecting cats in childhood, but subclinical infections in mature cats.

[0008] Regarding the mechanism of the onset of acute diarrhea caused by rotavirus, one of the nonstructural proteins is one of a total of eleven viral proteins produced by the virus, namely six structural proteins and five nonstructural proteins. Enterotoxin of NSP4, a glycoprotein
Experimental results suggest that neonatal mice inoculated intraperitoneally or intestine with recombinant simian rotavirus NSP4 exhibited acute diarrhea (JM Ball
Et al., Science 1996, 272: 101-104). Further, Ball et al. (J.
M. Ball et al., Science 1996, 272: 101-104) discloses that amino acids constituting the NSP4 protein have SEQ ID NOS: 114 to 135.
It is reported that up to 22 amino acid residues are deeply involved in the action of NSP4 as an enterotoxin.

Regarding the enterotoxin activity of this rotavirus NSP4, Zhang et al. (M. Zhang et al., J. Virol. 1998, 72: 3666-3)
672) is a recombinant NSP4 derived from a pathogenic porcine rotavirus.
Shows positive experimental results that acute diarrhea was observed in mice inoculated intraperitoneally with. Meanwhile, Zhang
Et al., Recombinant NSP4 derived from non-pathogenic porcine rotavirus
Does not induce acute diarrhea in mice.

Further, Angel et al. Found no difference in the amino acid sequence of mouse rotavirus NSP4 regardless of pathogenicity (J. Angel et al., J. Infect. Diseases 199).
8,177: 455-458), and no definitive opinion is currently available on the relationship between NSP4 and enterotoxin activity.

[0011] Furthermore, it has not been studied at all whether such rotavirus NSP4 exhibits adjuvant activity by nasal and oral administration.

[0012]

Generally, vaccination is accompanied by side effects to some extent, which is a serious clinical problem. Therefore, application of an adjuvant capable of reducing the amount of antigen in a vaccine leads to a reduction in the amount of inoculated virus, particularly when the antigen is a virus,
It greatly contributes to the reduction and elimination of side effects. However, there is no practical and safe adjuvant for nasal and oral administration vaccines at present, and their development is strongly demanded.

In view of the above-mentioned situation, the present invention aims to create a practical and safe adjuvant for oral and nasal administration, and at the same time, provides a large and stable production of a protein that can be used as this adjuvant. The aim is to provide a method.

[0014]

[Means for Solving the Problems] Pathogenic simian rotavirus NSP4 (JMBal) reported as a viral enterotoxin
l et al., Science 1996, 272: 101-104) clearly induces acute diarrhea in mice and is therefore not suitable as an adjuvant for orally administered vaccines.

[0015] Further, it has not been known whether feline rotavirus NSP4 has an effect as an enterotoxin because its purification method and expression system of recombinant NSP4 have not been established so far.

However, in experiments using recombinant NSP4 derived from the feline rotavirus FRV-64 strain prepared by the production method established by the inventors, mice were intraperitoneally inoculated with NSP4,
Neither oral administration nor intranasal administration caused acute diarrhea, and unlike simian rotavirus NSP4 and pathogenic porcine rotavirus NSP4, it was revealed that they did not have enteric toxin properties. When the adjuvant activity of the protein obtained was examined, it was found that NSP4 of feline rotavirus also had adjuvant activity, and the present invention was completed.

That is, the present invention relates to the rotavirus NSP4
An adjuvant containing a protein. An adjuvant as used herein is a substance having an activity of regulating (for example, enhancing) an immune response when an antigen is administered, and administered an antigen such as a virus and NSP4 as a vaccine. Sometimes it can enhance antibody production induced by viral antigens. Dosage forms include viral fluid and NS
It is only necessary to prepare the mixture by mixing it with the P4 protein, and it is not necessary to perform a treatment such as emulsification as in a conventional adjuvant. Nasal, prepared in this way,
Administration is oral or by injection, but nasal or oral administration is preferred, and nasal administration is particularly preferred in that a remarkable adjuvant effect is observed in the administered animal.

As a method for intranasally or orally administering the NSP4 protein, a liposome or the like containing the protein can be used.

The rotavirus NSP4 used as an adjuvant in the present invention may be a rotavirus-derived NS that infects any species as long as it does not show pathogenicity to the administered animal.
P4 may be used (for example, NSP4 protein of non-pathogenic porcine rotavirus), and such an NSP4 protein may be the whole NSP4 protein or NS
It may be part of the P4 protein.

NSP4 having such adjuvant activity
Examples of a part of the protein include a recombinant NSP4 derived from the feline rotavirus FRV-64 strain. Specifically, it is the amino acid sequence shown in SEQ ID NO: 2 predicted from the partial gene sequence shown in SEQ ID NO: 1, which is a part of the full-length sequence of NSP4, or one or several amino acids in the amino acid sequence Has a deleted, substituted or added amino acid sequence.

Further, the protein may be a protein in which a partial amino acid sequence has been removed from the partial amino acid sequence of feline rotavirus NSP4. And the like are removed.

Such proteins can be prepared from new gene fragments produced by digesting / cleaving the genes encoding those proteins with one or more restriction enzymes, and joining the cut DNA fragments. . In addition, a DNA fragment encoding a target protein with a mutation or deletion is amplified by a gene amplification method (polymerase chain reaction, PCR) using primers into which a gene mutation or a deletion has been inserted, and the protein fragment is converted from the DNA fragment. It can be easily obtained by performing expression.

The NSP4 protein of the present invention can be obtained by disrupting rotavirus-infected cells and purifying the whole protein of the cells by various chromatography and the like. However, the present invention also aims to provide a large amount of the rotavirus NSP4, a method for producing the NSP4 protein having adjuvant activity,
(1) a step of preparing a recombinant Escherichia coli expression plasmid vector containing a gene encoding the feline rotavirus NSP4 protein or a part thereof and capable of producing the protein; Transforming host E. coli with the expression vector;
(3) culturing the transformed host Escherichia coli to produce a protein; and (4) separating and recovering the produced protein.

Further, the present invention provides the above-mentioned NSP4 protein,
A protein or peptide having a partial amino acid sequence of NSP4, or an NSP4 protein obtained according to the above-mentioned production method (1) to (4) is administered to an animal to activate immunity in the administered animal. It also includes an immunostimulation method characterized by the above. Animals to be administered are mammals including humans, preferably mammals excluding humans,
Even more preferred are felines.

In general, animals to be administered are
It is preferable that the rotavirus from which NSP4 is derived is the same as the species to be infected. When the feline rotavirus NSP4 protein is used, a feline animal is the most preferable as an immunostimulatory animal.

The gene encoding the feline rotavirus NSP4 protein or a part thereof is a feline rotavirus.
It may be the entire gene encoding NSP4 or a part of the gene encoding feline rotavirus NSP4 as follows. That is, some of the genes encoding NSP4 have the amino acid sequence represented by SEQ ID NO: 2, or have one or several amino acids deleted, substituted, or deleted in the amino acid sequence represented by SEQ ID NO: 2. Added amino acid sequence or SEQ ID NO: 2
For example, a gene encoding an amino acid sequence in which amino acids are removed from the N-terminal or C-terminal.

The present invention also includes a recombinant vector obtained when producing the rotavirus NSP4 and a transformant transformed with the recombinant vector.

The recombinant vector of the present invention comprises a gene encoding the whole or a part of the feline rotavirus NSP4 protein, for example, the amino acid sequence of SEQ ID NO: 2, or one or several amino acids in this amino acid sequence. To contain a gene encoding a deleted, substituted or added amino acid sequence or a gene encoding a partial amino acid sequence of the amino acid sequence of SEQ ID NO: 2 so that the gene can be expressed, An expression vector having a transcription initiation site, a promoter, an enhancer, and the like at the 5 'end and a terminator, a polyA addition signal, and the like at the 3' end, and various vectors such as a plasmid and a baculovirus vector.

The transformant of the present invention is obtained by transforming the above-mentioned recombinant vector and using a cell containing the recombinant vector,
As the host cell of the transformant, various cells such as microorganisms such as Escherichia coli, Bacillus subtilis, and yeast, as well as mammalian cells such as insect cells and cat-derived cell lines can be used. In addition,
As the transformant of the present invention, Escherichia coli and Bacillus subtilis are preferable in terms of mass production of the protein and highly and easily, and can be purified at low cost. Therefore, it is preferable to employ a plasmid as the most preferable vector system. .

E. coli, E-FRV64-NSP4 containing the above-mentioned recombinant vector and a transformant, that is, a plasmid vector into which a gene encoding the protein represented by SEQ ID NO: 2 was incorporated, was obtained on July 14, 1999. And deposited at the Patented Microorganisms Depositary Center of the Institute of Biotechnology, Ministry of International Trade and Industry under the accession number FERM-17466.

As described above, the production method of the present invention is characterized by using a plasmid as a recombinant vector and Escherichia coli as a host cell. A recombinant feline rotavirus NSP4 having adjuvant activity in E. coli transformed with an E. coli expression plasmid incorporating the gene fragment isolated as such a transformant.
After the production of, was separated and recovered for the first time according to the present invention. In addition, the present invention is the first to find that rotavirus NSP4 has adjuvant activity.

[0032]

BEST MODE FOR CARRYING OUT THE INVENTION The rotavirus NSP4 protein having adjuvant activity of the present invention can be produced as follows. Hereinafter, the feline rotavirus NSP4 protein will be described as an example, but other rotavirus-derived NS
P4 protein can be prepared in the same manner.

First, NSP4 of feline rotavirus FRV64 strain
The nucleotide sequence existing downstream of the 5 'end of the gene and the nucleotide sequence near the 3' end (gene data bank, accession nu
PCR using a cDNA fragment encoding NSP4 of feline rotavirus FRV64 strain (distributed by Dr. Osamu Nakagome, Faculty of Medicine, Akita University) using sense primers and antisense primers synthesized based on each of mberD88833).
Thus, a partial cDNA fragment of NSP4 of the feline rotavirus FRV64 strain was amplified.

Next, in order to produce the feline rotavirus partial NSP4 in Escherichia coli, the partial cDNA fragment of the NSP4 gene amplified by the above PCR was incorporated into a commercially available vector plasmid to obtain an Escherichia coli expression vector. Partial DN of feline rotavirus NSP4 gene prepared as described above
The expression plasmid containing the A fragment was named pGEX-NSP4.
Named FRV64-NSP4.

A cat having the adjuvant activity of the present invention
The rotavirus partial NSP4 is
It can be obtained by culturing E-FRV64-NSP4, separating the produced protein from Escherichia coli, and purifying it.

The above operation is carried out according to Sambrook et al.
ok et al., 1989, Molecular cloning: a laboratory manua
l. 2nd edition, Cold Spring Harbor, New York: Cold
Spring Harbor Press) and other known gene manipulation techniques and cell engineering techniques.

It has the adjuvant activity of the present invention,
Feline rotavirus partial NSP4 having the amino acid sequence of SEQ ID NO: 2 is a feline rotavirus NSP4
Is not necessarily transcribed / translated from the genome encoding, for example, may be produced by a chemical synthesis method, expression of a transformant, or the like. Also,
The base sequence encoding the amino acid of SEQ ID NO: 2 can also be obtained by any of a chemical synthesis method, a DNA replication method, a reverse transcription method, and a transcription method.

The present invention also relates to a recombinant vector capable of producing a protein comprising a gene fragment encoding feline rotavirus partial NSP4 having an adjuvant activity and having the amino acid sequence of SEQ ID NO: 2 as described above. The above-mentioned pGEX-NSP4 is exemplified as such a plasmid.

Further, the present invention relates to a synthetic plasmid capable of producing a feline rotavirus partial NSP4 having an adjuvant activity into which a gene fragment encoding a protein having the amino acid sequence of SEQ ID NO: 2 is incorporated. It is intended to provide a transformant obtained by transforming a host cell with a vector. Examples of such cells include those using Escherichia coli as a host cell.Examples of transformed Escherichia coli include Escherichia coli E-FRV64-NSP4 containing the above-mentioned plasmid pGEX-NSP4.
P-17466 has been deposited with the Ministry of International Trade and Industry at the National Institute of Advanced Industrial Science and Technology, Biotechnology Institute, Patent Microorganisms Depositary Center on July 14, 1999.

Such a cat / rotavirus partial NSP4
As a transformant which is a protein producing cell, not only Escherichia coli but also microbial cells such as Bacillus subtilis (JP-A-11-178574) and yeast can be used, but the most preferable in terms of the amount of expressed protein and the operation such as purification thereof. Is Escherichia coli or Bacillus subtilis.

The rotavirus NSP4 protein of the present invention can also be produced using various cells other than the above-mentioned microorganisms, and the case where the protein is expressed in insect cells using a baculovirus vector will be described.

When a recombinant virus vector such as a baculovirus is used, for example, the method of Smith et al. (GE Smith
Et al., 1983, Mol. Cell Biol. 3: 2156-2165) to produce a recombinant viral vector.
That is, a gene fragment encoding a feline rotavirus partial NSP4 having an adjuvant activity is ligated to a polyhedron promoter of a baculovirus transfer vector, and the transfer vector is baculovirus.
Introducing insect cells together with DNA, selecting a recombinant clone having adjuvant activity from the obtained recombinant baculovirus, infecting the recombinant clone with insect cells or insects, and producing a partial NSP4 protein Can be.

The present invention further relates to a method for producing the above-mentioned transformant by culturing the transformed microorganism, and separating and recovering the feline rotavirus partial NSP4 protein having adjuvant activity. It also provides a way to do that. For separation of the protein from the cultured cells, various types of chromatography can be used, but affinity chromatography to which a specific antibody or the like is bound is preferably used.

The obtained cat and rotavirus partially
The adjuvant activity of the NSP4 protein was determined by the nasal administration of only the antigenic protein in the mice of the negative control group, and the partial NS purified together with the antigenic protein.
About the mice in the test group to which P4 protein was intranasally administered,
Serum was prepared from each mouse, and IgG and IgA antibodies against the antigen protein in the serum were assayed by ELISA.
(enzyme-linked immunosorbent assay), which can be confirmed by determining whether significantly higher antibody production is observed in the serum of the test group than in the serum of the negative control group.

Examples of the protein having antigenicity as described above include antigens produced by various microorganisms that infect cats,
For example, the coat protein of feline leukemia virus, the VP2 protein of feline panleukemia reduction virus, the coat and core proteins of feline immunodeficiency virus, the capsid protein of feline calicivirus, the major outer membrane protein of chlamydia MOMP-1, and There are various viral proteins of feline herpes virus type 1 and the like, but ovalbumin (OVA) is preferred because it is easily and inexpensively available when used experimentally.

When a rotavirus NSP4 protein is used as an adjuvant in a vaccine, a mixture of an antigen protein solution and a rotavirus NSP4 protein solution is used as the vaccine. In this case, as the antigenic protein used as a vaccine, in addition to the protein having the above antigenicity, viruses and bacteria that infect various animals including cats and humans, and proteins such as their constituent proteins can be used. The NSP4 protein, when administered as a vaccine, acts to enhance the production of IgG and IgA antibodies to the antigen protein.

[0047]

The present invention will be described in more detail with reference to the following examples. FIG. 1 schematically shows the outline of the construction of the expression vector pGEX-NSP4.

1. Preparation of Transformant E-FRV64-NSP4 Expressing Feline Rotavirus Partial NSP4 Protein (1) Amplification of Feline Rotavirus NSP4 Partial cDNA Fragment by PCR Field Isolate of Feline Rotavirus N derived from FRV64 strain
Plasmid pCR-NSP4 DNA obtained by incorporating the full-length cDNA encoding SP4 into a commercially available plasmid vector pCR II (manufactured by Invitrogen) (distributed by Dr. Osamu Nakagome, Faculty of Medicine, Akita University, Y. Horie et al., 1997, J. Gen. . Virol. 78: 2341-23
46) was used as a template.

The above template DNA (100 ng) and a gene sequence encoding feline rotavirus NSP4 protein (accession number D8883) registered in advance in the Gene Data Bank were used.
0.5 µM of each of the sense primer and antisense primer synthesized based on 3) were added to PCR buffer (1.2
5 units of Taq DNA polymerase; Boehringer Mannheim, 200 M deoxynucleotide, 10 mM Tris-HCl, pH 7.4, 10 mM potassium chloride and 1.5 m
M magnesium chloride), paraffin oil was layered on the mixture, and the mixture was heated at 94 ° C for 60 seconds and at 55 ° C for 90 seconds.
For 30 cycles, PCR was performed at 72 ° C. for 90 seconds as one cycle. The sense primer and antisense primer used
The nucleotide sequences of the primers are as follows.

Sense primer: 5'-ATCGCCCGGGAACTA
AGGATGAGATTGAA (SEQ ID NO: 3, where CC near the 5 'end is
CGGG converts the NSP4 cDNA fragment amplified by PCR into a plasmid
This is a restriction enzyme SmaI cleavage site added for integration into the vector. ) Antisense primer: 5'-GTACCTCGAGCGCTTCCGTGG
ACAGACGAC (SEQ ID NO: 4, but CTCGAG near the 5 'end)
Is an XhoI cleavage site added for incorporating the NSP4 cDNA fragment amplified by PCR into a plasmid vector. (2) Construction of Feline Rotavirus Partial NSP4 Expression Plasmid The cDNA fragment amplified by the above PCR was separated by electrophoresis using 2% agarose gel, and the 314 base pair cDNA fragment was amplified. Was confirmed (FIG. 3). This is the N / F of the cat rotavirus FRV64 strain registered in the Genetic Data Bank.
Of the whole SP4 gene sequence (accession number D88833), it corresponded to the size derived from the partial NSP4 sequence of 297 base pairs from base number 310 to 603 and the restriction enzyme recognition site added to the primer. The resulting 314 base pair cDNA sequence is shown in FIG. Then the PCR product 5μ
l was cut at the SmaI cleavage site added to the nucleotide sequence of the sense primer and the XhoI cleavage site added to the nucleotide sequence of the antisense primer, and the resulting 298 base pair N
A commercially available plasmid vector pGEX that can express the target protein as a fusion protein with the partial cDNA fragment of SP4 and glutathione-S-transferase (GST)
-4T-3 (Amersham Pharmacia) was incorporated into the SmaI-XhoI site by binding using T4 ligase (Toyobo) at 16 ° C. for 16 hours. The plasmid constructed in this way was named pGEX-NSP4 (FIG. 1).

(3) Preparation of Feline Rotavirus Partially NSP4-Producing Escherichia coli A commercially available competent Escherichia coli BL from a DNA solution of pGEX-NSP4
21 (manufactured by Stratagene), and the plasmid was introduced into Escherichia coli according to the attached manual to transform Escherichia coli. The transformant was further transformed into an LB agar medium (50 L / ml) containing 50 μg / ml ampicillin. -Ampicillin resistance grown on an agar medium by culturing for 17 hours at 37 ° C using 10 g of tryptone, 5 g of Bacto yeast extract, 10 g of sodium chloride and 15 g of Bacto agar) Was obtained. Next, the plasmid DNA extracted from each transformant clone was digested with SmaI and XhoI and subjected to 2% agarose gel electrophoresis to combine the partial cDNA fragment of NSP4 with its associated restriction enzyme site (297 bases). A clone having a cDNA fragment corresponding to (pair) was selected, and the transformant E-
FRV64-NSP4 was obtained.

A transformed Escherichia coli containing the obtained expression plasmid pGEX-NSP4 was deposited as E-FRV64-NSP4 (Accession No. FERM P-17466).

2. Determination of base sequence of partial cDNA of feline rotavirus NSP4 DN of recombinant plasmid from E-FRV64-NSP4 obtained above
A is extracted and purified, and a commercially available automatic sequencer (Amersham Pharmacia), cycle sequence kit (Amersham Pharmacia) and pGEX-4T-3
The nucleotide sequence of the NSP4 partial cDNA derived from FRV64 strain integrated into pGEX-NSP4 was determined by the deoxy sequencing method using a forward primer and a reverse primer (Amersham Pharmacia) for vector. Were determined
The nucleotide sequence of the NSP4 partial cDNA is shown in SEQ ID NO: 1.

3. Purification and Identification of Feline Rotavirus Partial NSP4 Protein Next, a method for inducing expression of the partial NSP4 protein in which the E. coli prepared in the above 1 (3) was fused with GST at the N-terminal, The details of a method for separating and recovering an expressed protein and a method for identifying that the separated and recovered protein is an NSP4 protein will be described.

(1) Induction and Purification of Feline Rotavirus Partial NSP4 Protein The transformant, E-FRV64-NSP4, was cultured and GST-fused NSP4
Induces the expression of a protein (hereinafter abbreviated as GST-NSP4),
GST-NSP4 whose expression has been induced
(Amersham Pharmacia) according to the separation / purification manual attached to the kit.

That is, E-FRV64-NSP4 was added to 10 ml of LB liquid medium (medium obtained by removing agar from the LB agar medium) at 37 ° C.
For 15 hours, and 5 ml thereof was added to 500 ml of a new LB liquid medium, and further cultured until the turbidity at 600 nm of the liquid medium reached 0.6. Next, IPTG (isopropyl β-D-thiogalactoside) having a final concentration of 1.0 mM was added to the E. coli culture, and the mixture was cultured at 37 ° C. for 3 hours to induce the expression of GST-NSP4. FIG. 4 shows the expression results of the GST-NSP4 fusion protein induced by IPTG.

Next, centrifugation was performed at 3000 rpm for 5 minutes.
Escherichia coli that has induced the expression of GST-NSP4 is collected, and the Escherichia coli is collected in a phosphate buffer containing 1% Triton X-100 (137 mM sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium hydrogen phosphate). , And 1.47 mM potassium dihydrogen phosphate, pH 7.4), followed by sonication.

Subsequently, GST was performed by centrifugation at 5000 rpm for 5 minutes.
A soluble fraction containing -NSP4 was obtained, and the GST-NSP4 fusion protein was separated and purified from the soluble fraction using a column (manufactured by Amersham Pharmacia) containing glutathione sepharose alone according to the attached manual. Then, 10 units of thrombin (Amersham Pharmacia) was added to the solution containing the purified GST-NSP4 fusion protein.
Was added and reacted at room temperature for 16 hours to cleave the GST-NSP4 fusion protein into GST and NSP4 to obtain a mixture of GST and partial NSP4 protein. Finally, the solution containing this mixture is dialyzed in phosphate buffer to remove glutathione,
The dialysate was again layered on a glutathione-Sepharose column, and a partial NSP4 protein sample was obtained in a flow-through fraction.

(2) Identification of Feline Rotavirus Partial NSP4 Protein The partial NSP4 protein obtained by the above operation was subjected to sodium dodecyl sulfate (SDS) -polyacrylamide gel using a 15% polyacrylamide gel. Separated by electrophoresis. The acrylamide gel was then stained with a stain consisting of 0.25% Coomassie Brilliant Blue R250, 5% acetic acid and 50% methanol to visualize the separated proteins, and then the excess stain was stained with 7% acetic acid and It was removed with a destaining solution consisting of 5% methanol. The results are shown in FIG. As shown in FIG. 5, a protein of 11.4 kilodalton (kDa) corresponding to the size of the partial NSP4 protein was detected.

Next, the above 11.4 kDa protein was converted to N
In order to confirm that the protein was SP4 protein, ELISA using mouse anti-mouse rotavirus NSP4 serum as a primary antibody was performed. Hereinafter, the ELISA will be described in detail.

Purified partial NSP4 purified and recovered in 3 (1) above
(4 μg / ml solution) 100 μl was dispensed into each well of a 96-well ELISA plate (manufactured by Nunc), and then left at 4 ° C. overnight. Next, each well of the ELISA plate was washed three times with a phosphate buffer containing 0.05% Tween 20 (hereinafter abbreviated as PBST), and further diluted 4-fold with Block Ace (Dainippon Pharmaceutical) 200 μl. Was poured into each well and a blocking operation was performed at 37 ° C. for 60 minutes. Then, 58 g of sodium chloride, 8 g of potassium chloride, and 1.15 g of hydrogen phosphate in 1 L (liter) were added to each well. Mouse anti-mouse rotavirus diluted 10-fold, 20-fold, 40-fold, 80-fold, 80-fold, 160-fold and 320-fold with sodium, 0.2 g potassium dihydrogen phosphate, 2 g gelatin and 1 ml Tween 80) NSP4 serum (provided by Dr. Osamu Nakagome, Akita University, School of Medicine) was injected in 100 μl portions and incubated at 37 ° C. for 60 minutes. Subsequently, each well was washed three times with PBST, and each well was washed with PBST for 2000 times.
1: 2 dilution of alkaline phosphatase-labeled goat anti-mouse
After 100 μl of IgG serum (manufactured by DAKO) was added, and the mixture was incubated at 37 ° C. for 60 minutes, the wells were washed three times with PBST. Finally, 100 µl of the color developing solution (1 g of o-
Nitrophenyl phosphate, 97 ml diethanolamine,
A mixture containing 0.2 g of sodium azide and 100 mg of magnesium chloride, pH 9.8) was added to each well, and the mixture was added at 25 ° C.
After the color was developed by a reaction for 20 minutes, the color reaction was stopped by adding 50 μl of 0.25 N sodium hydroxide, and the absorbance at 405 nm was measured. As control mouse sera, non-antigen-sensitized mouse sera diluted 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, and 320-fold with a diluent (Akita University
(Provided by Dr. Osamu Nakagomi, School of Medicine).

(Results of Measurement by ELISA) Table 1 shows the measured values of ELISA by absorbance. As is clear from Table 1, 1: 1
ELISA using mouse anti-mouse rotavirus NSP4 serum diluted 1: 0, 1:20, 1:40, 1:80, 1: 160 and 1: 320
, The measured value of feline rotavirus partial NSP4 was 1.202, 0.848, 0.578, 0.430, 0.254 and 0.202, respectively.
0.155, 0.131, 0.129, 0.1
Obviously higher measured values were obtained compared to 28, 0.136 and 0.142. As a result, 11.4 k expressed and recovered in E. coli
The Da protein was shown to be a feline rotavirus partial NSP4 protein.

[0063]

[Table 1] 4. Measurement of adjuvant activity of partial NSP4 protein The adjuvant activity of partial NSP4 protein separated and recovered in (1) above is nasal administration of only ovalbumin (hereinafter abbreviated as OVA, manufactured by Sigma). The serum IgG and IgA levels of the treated mice (control group) or the mice to which the partial NSP4 protein was intranasally administered together with OVA (test group) were measured. The test group showed significantly higher IgG production than the control group. In this case, it was determined that the adjuvant activity for conferring systemic immunity was positive. In addition, when significantly higher IgA production was observed in the test group than in the control group, it was determined that the adjuvant activity for conferring mucosal immunity was positive. Hereinafter, the adjuvant activity of the partial NSP4 protein will be described in detail.

(1) Measurement of Serum Anti-OVA IgG and Anti-OVA IgA in Mice Administered with 50 μg of OVA Three 6-week-old CD1 mice were treated as a group, and 20 μl of a phosphate buffer containing 50 μg of OVA (hereinafter referred to as “20 μl”) was used. , Low concentration OVA),
And 2 containing 50 μg OVA and 50 μg partial NSP4
Mice were intranasally administered each of 0 μl of phosphate buffer. Nasal administration was performed twice, on the 14th day and 3rd after the 28th day, three times after the initial administration. Seven days after the third administration, blood was collected from the heart, the blood was allowed to stand at room temperature overnight, and then centrifuged at 3000 rpm for 5 minutes to obtain serum in the supernatant, and IgG against OVA in the serum.
And a sample for measuring IgA.

1) Test 1 (Measurement of Serum Anti-OVA IgG in Mice Administered at Low Concentration OVA) Serum anti-OVA antibody (IgG) was measured by the following ELISA. That is, 1 mg / ml OVA and 0.1% Triton X-100
TEN buffer (10 mM Tris, pH 7.54, 1 mM
Sodium ethylenediaminetetraacetate; EDTA, and 8
7.7 g of sodium chloride) in coating buffer (consisting of 35 mM sodium bicarbonate, 15 mM sodium carbonate, pH 9.6) to a final OVA concentration of 1
The volume was adjusted to 00 ng / 100 μl, and 100 μl was dispensed into each well of a 96-well ELISA plate (manufactured by Nunc), and then left at 4 ° C. overnight. Next, each well of the ELISA plate is washed three times with PBST, and a blocking solution (hereinafter abbreviated as blocking solution) comprising 0.05% Tween 20, 0.25% (V / V) Escherichia coli crushed product, and 1 mM EDTA. Pour 200 μl into each well
After performing a blocking operation at 37 ° C. for 60 minutes, 100 μl of each serum of the mice in the test group and control group diluted 100-fold with the diluent was injected into each well, and incubated at 37 ° C. for 60 minutes. Subsequently, each well was washed three times with PBST, and 100 μl of a peroxidase-labeled goat anti-mouse IgG antibody (manufactured by ICN) diluted 10000-fold with PBST was added to each well.
After incubation at 60 ° C for 60 minutes, the wells were washed three times with PBST. Finally, 100 μl of the coloring solution (110 μl of dimethyl sulfoxide solution containing 6 mg / ml of tetramethylbenzidine, 100 mM sodium acetate buffer (pH
5.5) A mixture of 11 ml and 3% hydrogen peroxide (11 μl) was injected into each well, and allowed to develop color at 25 ° C. for 20 minutes.
The color reaction was stopped by adding 50 μl of 2 M sulfuric acid, and the absorbance at 450 nm was measured.

(Serum anti-O in mice administered with low-concentration OVA)
VA IgG measurement results) Anti-OV in serum of mice administered with low concentration OVA
Table 2 shows the measurement results of A IgG by ELISA. As can be seen from the table, the absorbances measured in the test group receiving partial NSP4 with 50 μg OVA (0.346, 0.404 and 0.48
6) was significantly higher than the absorbance (0.226, 0.257, and 0.265) measured in the control group to which only OVA was administered.
As a result, it was revealed that partial NSP4 had adjuvant activity against systemic immunity.

[0067]

[Table 2] 2) Test 2 (Measurement of Serum Anti-OVA IgA in Mice Administered at Low Concentration OVA) Serum anti-OVA antibody (IgA) was measured by ELISA shown below. That is, 1 mg / ml OVA and 0.1% Triton X-10
The TEN buffer containing 0 is diluted with the coating buffer to a final OVA concentration of 500 ng / 100 μl, and 100 μl is
After dispensing into each well of a well ELISA plate, the plate was left overnight at 4 ° C. Then, each well of the ELISA plate was
After washing twice and blocking using a blocking solution, 100 μl of each serum of the test group and control group mice diluted 100-fold with the diluent was injected, and incubated at 37 ° C. for 60 minutes. Subsequently, each well was washed three times with PBST, 100 μl of a peroxidase-labeled goat anti-mouse IgA antibody (manufactured by OEM Concepts) diluted 1000-fold with PBST was added to each well, and incubated at 37 ° C. for 60 minutes. The wells were washed three times with. Finally, 100μl
Was added to each well, and the color was developed by reaction at 25 ° C. for 20 minutes. Then, 50 μl of 2 M sulfuric acid was added to stop the color reaction, and the absorbance at 450 nm was measured.

(Serum anti-O in mice administered with low-concentration OVA)
Measurement results of VA IgA) Table 3 shows the ELISA measurement values of anti-OVA IgA in serum of mice administered with low-concentration OVA. As can be seen in this table, the absorbances measured in the test group receiving partial NSP4 with 50 μg of OVA (0.360, 0.396 and 0.671)
Was significantly higher than the absorbance measured in the control group receiving only 50 μg of OVA (0.163, 0.195 and 0.246). This revealed that partial NSP4 had adjuvant activity against mucosal immunity.

[0069]

[Table 3] (2) Serum anti-OVA IgG in 500 μg OVA administered mice
And measurement of anti-OVA IgA Next, the dose of OVA to the mice in the test group and the control group in the above 4 was increased to 500 μg (hereinafter abbreviated as high concentration OVA), and the same operation as in 4 (1) was performed. The serum of the mouse was prepared, and the anti-OVA IgG and anti-OVA IgA in the serum were determined in 4) (1) and 2) above.
Each was measured by the same ELISA.

1) Test 3 (Results of Measurement of Serum Anti-OVA IgG in Mice Administered with High Concentration OVA) The absorbance measured by ELISA for anti-IgG in the blood of mice administered with high concentration OVA is shown in Table 2. As is evident from Table 2, the absorbance (0.295, 0.668 and 1.446) measured in the test group receiving partial NSP4 with 500 μg of OVA was 5
Absorbance (0.292, 0.312, and 0.314) measured in the control group to which only 00 μg of OVA was administered was significantly higher except for one case, and it was again confirmed that partial NSP4 had adjuvant activity against systemic immunity. .

2) Test 4 (Results of Measurement of Serum Anti-OVA IgA in Mice Administered with High Concentration OVA) ELIS of Anti-OVA IgA Serum in Mice Administered with High Concentration OVA
Table 3 shows the measurement results by A. As is evident from Table 3, the absorbance (0.405, 0.478 and 0.502) measured in the test group receiving partial NSP4 with 500 μg of OVA was 5
The absorbance was significantly higher than the absorbance (0.257, 0.279, and 0.385) measured in the control group to which only 00 μg of OVA was administered.
This again confirmed that partial NSP4 had adjuvant activity against mucosal immunity.

[0072]

Industrial Applicability According to the present invention, it is possible to produce a large amount of a feline rotavirus NSP4 protein having adjuvant activity by using a transformed microorganism. This protein can be used as an orally administered adjuvant.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing an outline of construction of an expression vector pGEX-NSP4.

FIG. 2 is a diagram showing the nucleotide sequence of a 314 base pair cDNA fragment amplified by PCR. The sequence shown in the figure is unrestricted enzyme treatment, the lower case part is the sequence of the restriction enzyme recognition site etc. added to the primer, the upper case part shows the partial NSP4 sequence, the underlined part Indicates a PCR primer or a sequence complementary to the primer.

FIG. 3 shows the results of electrophoresis of a cDNA fragment amplified by PCR on a 2% agarose gel. The obtained cDNA fragment was obtained by combining a partial cDNA fragment of NSP4 with a restriction enzyme site associated therewith. It is shown that this corresponds to a size of 314 base pairs. In the figure, φX174 / HaeIII is a marker.

FIG. 4 shows the results of SDS polyacrylamide gel (15%) electrophoresis showing the expression of GST-NSP4 fusion protein induced by IPTG. In the figure, IPTG (+) is the case where 1 mM of IPTG was added to induce the expression of the fusion protein, and IPTG (-)
Shows the electrophoresis pattern of the expressed protein when IPTG was not added and the expression of the fusion protein was not induced.

FIG. 5: GST-NSP4 fusion protein treated with thrombin and purified partial NSP4 SDS-polyacrylamide
It is a figure showing a result of gel electrophoresis. It can be seen that thrombin treatment generates GST and partial NSP4.

[Sequence List Free Text]

 SEQ ID NO: 3: <223> Sense primer for PCR SEQ ID NO: 4: <223> Antisense primer for PCR

[Sequence List] SEQUENCE LISTING <110> kyoritsu shoji <120> An adjuvant containing rotavirus NSP4s <130> krs5 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 292 <212> DNA <213> Feline rotavirus <220> <221> CDS <222> (1) .. (255) <400> 1 act aag gat gag att gaa aag caa atg gac aga gtt gtg aaa gaa atg 48 Thr Lys Asp Glu Ile Glu Lys Gln Met Asp Arg Val Val Lys Glu Met 1 5 10 15 aga cgt caa cta gaa atg att gat aaa tta acg act aga gaa ata gaa 96 Arg Arg Gln Leu Glu Met Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu 20 25 30 caa gta gaa ctt cta aag aga att tat gat atg cta ata gcg aca tca 144 Gln Val Glu Leu Leu Lys Arg Ile Tyr Asp Met Leu Ile Ala Thr Ser 35 40 45 gtt gac aaa ata gat acg aca caa gaa ttc aac caa aag cat ttt aaa 192 Val Asp Lys Ile Asp Thr Thr Gln Glu Phe Asn Gln Lys His Phe Lys 50 55 60 acg cta aac gaa tgg gca gaa ggt gaa aat cca tat aag cca aga gaa 240 Thr Leu Asn Glu Trp Ala Glu Gly Glu Asn Pro Tyr Lys Pro Arg Glu 65 70 75 80 gtg act gca tct ctg taagaggttg agctgccgtc gtctgtccac ggaagc g 292 Val Thr Ala Ser Leu 85 <210> 2 <211> 85 <212> PRT <213> Feline rotavirus <400> 2 Thr Lys Asp Glu Ile Glu Lys Gln Met Asp Arg Val Val Lys Glu Met 1 5 10 15 Arg Arg Gln Leu Glu Met Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu 20 25 30 Gln Val Glu Leu Leu Lys Arg Ile Tyr Asp Met Leu Ile Ala Thr Ser 35 40 45 Val Asp Lys Ile Asp Thr Thr Gln Glu Phe Asn Gln Lys His Phe Lys 50 55 60 Thr Leu Asn Glu Trp Ala Glu Gly Glu Asn Pro Tyr Lys Pro Arg Glu 65 70 75 80 Val Thr Ala Ser Leu 85 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence < 220> <223> Description of Artificial Sequence: Sence primer for PCR <400> 3 atcgcccggg caactaagga tgagattgaa 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Antisence primer for PCR <400> 4 gtacctcgag cgcttccgtg gacagacgac 30

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/02 (C12N 1/21 // (C12N 1/21 C12R 1:19) C12R 1:19) ( C12P 21/02 C (C12P 21/02 C12R 1:19) C12R 1:19) C12N 15/00 ZNAA (72) Inventor Masami Mochizuki 968-32 Obadai, Aoba-ku, Kanagawa F-term (reference) 4B024 AA01 BA32 CA04 DA06 EA04 GA11 HA01 HA14 4B064 AG32 CA02 CA19 CC24 CE02 CE06 CE12 DA01 4B065 AA26X AA95Y AB01 AC14 BA02 BB11 BD01 BD15 BD17 CA24 CA45 4C085 AA38 CC08 DD62 FF13 FF19 GG08 GG10 4H045 AA11 AA26

Claims (12)

[Claims] 1. An adjuvant comprising the rotavirus NSP4 protein. 2. The adjuvant according to claim 1, wherein the NSP4 protein is a feline rotavirus-derived NSP4 protein. 3. The NSP4 protein is a feline rotavirus FR
The adjuvant according to claim 1 or 2, which is an NSP4 protein derived from the V-64 strain. 4. The NSP4 protein is an amino acid sequence represented by SEQ ID NO: 2, or a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2. The adjuvant according to any one of claims 1 to 3, wherein 5. The adjuvant according to claim 1, which is for nasal administration. 6. A step of preparing a recombinant Escherichia coli expression plasmid vector containing a gene encoding a feline rotavirus NSP4 protein and capable of producing the protein, comprising the steps of: Producing a feline rotavirus NSP4 protein having adjuvant activity, comprising: a step of transforming Escherichia coli; a step of culturing the transformed host Escherichia coli to produce a protein; and a step of separating and recovering the produced protein. Method. 7. A recombinant Escherichia coli expression vector containing a gene encoding an amino acid of the feline rotavirus NSP4 protein and capable of expressing the protein. 8. The recombinant E. coli expression plasmid vector according to claim 7, wherein the recombinant E. coli expression vector is a synthetic plasmid. 9. Accession number FERM P-
The recombinant E. coli expression plasmid deposited at 17466
vector. 10. A transformant containing a recombinant Escherichia coli expression vector containing a gene encoding a feline rotavirus NSP4 protein. 11. The transformant is Escherichia coli.
The transformant as described above. 12. Accession number FERM P using Escherichia coli as a host.
The transformant deposited at -17466.