JP2002253262A - Antibody-forming transgenic plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensitive means for human calicivirus detection, which can be easily carried out at a clinical examination site, or a food processing/ examination site. SOLUTION: A plant body cell having a gene encoding at least a variable region among genes encoding anti-human calicivirus monoclonal antibody in a self cell is prepared and the gene is expressed in a recombinant plant body cell to give an anti-human calicivirus monoclonal antibody, which is used and a human calicivirus detection system is constructed to solve the problem.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a recombinant plant into which a virus gene has been introduced, and more particularly to a recombinant plant into which a gene encoding a monoclonal antibody against a virus has been introduced.

[0002]

2. Description of the Related Art Human calicivirus is one of the viruses that cause viral food poisoning that frequently occurs in winter and viral gastroenteritis in infants and infants, and has been called a small spherical virus in Japan. Food poisoning caused by this virus, mainly caused by marine products (cultured oysters, etc.), is increasing in recent years. In 1997, the Food Sanitation Law was amended, and a report on a food poisoning case caused by human calicivirus was required. And
It was counted in food poisoning statistics. By the way, 20
According to the Ministry of Health, Labor and Welfare statistics from January to November 2000, there were 197 cases of poisoning identified as the cause of this virus and 6392 patients, about 1/10 of the total number of food poisoning cases and 1 of the number of food poisoning cases. / 6.

[0003] As described above, the human calicivirus infection route is mainly via marine products. Specifically, the human calicivirus infects human calicivirus by ingesting marine products contaminated with the virus. There are many.
It is also known that the virus is transmitted through the stool of a human calicivirus-poisoned patient through the hand or finger. Human calicivirus is so infectious that infection can be established by invasion of only 1 to 10 virus particles.

[0004] Despite this situation, the in vitro culture method of human calicivirus and the experimental animal model infection system have not been established yet, and the tissue culture method generally used for virus detection, mouse and the like have not been established. The inability to detect and identify the virus by inoculation into humans also makes it difficult to detect human calicivirus.

[0005] Even in the current situation, the diagnosis of a patient with human calicivirus poisoning is carried out by detecting virus particles using an electron microscope ("Testing Methods for Human Calicivirus", February 10, 1999, Ministry of Health and Welfare No. 20; However, this detection method requires a virus concentration of 10 ⁶ cells / mL or more, and the detection sensitivity cannot be said to be high. Further, even if the virus particles are detected morphologically, they cannot be immediately identified as human calicivirus.

Therefore, human calicivirus has been detected by identifying a viral gene by a PCR reaction which is a gene amplification reaction (“Testing method for human calicivirus”, February 10, 1999). Ministry of Health and Welfare No. 20, No. 28). At present, the detection method by the PCR reaction is the only sensitive method for detecting calicivirus because the virus in the food causing food poisoning is generally extremely low in concentration.

[0007]

However, first of all,
In the method using an electron microscope, in addition to the above-mentioned problems, problems such as an expensive electron microscope and the need for a skilled technician to identify virus particles are recognized.

Further, in the method using the PCR reaction, there are problems that the gene amplification detection device is expensive, it takes a long time to obtain a judgment result, and the test method is complicated and requires a skilled technician. .

[0009] Therefore, it is desired that a clinical test site or a food processing / test site provide a simpler and more sensitive means for detecting human calicivirus. The problem to be solved by the present invention lies exactly in the provision of this detection means.

[0010]

Means for Solving the Problems To solve this problem, the present inventor aimed to produce an antibody against human calicivirus and provide a virus detection means using the antibody.

However, in the case of polyclonal antibodies, the titer and reactivity of the antibodies vary depending on the individual differences between animals and the immunization method, so that it is difficult to always obtain antibodies of a constant quality. Also, when producing monoclonal antibodies, the immunogen is inoculated into pure mice,
Since the ascites is collected and produced or produced by culturing a hybridoma, it is generally difficult to produce it in large quantities at low cost. Particularly, production of polyclonal antibodies and production of monoclonal antibodies from ascites using mice at the stage of producing antibodies has become extremely difficult from the viewpoint of animal welfare.

[0012] The present inventor has disclosed that a gene encoding a monoclonal antibody against human calicivirus (at least a gene encoding a variable region of a monoclonal antibody characterized as being a monoclonal antibody against human calicivirus) is added to a plant cell. To solve the above-mentioned problems related to the production of antibodies by mass-cultivating the plant cells, that is, the technique can be used in a wide range of uses, and in a simple and convenient manner. We thought that it would be possible to provide an anti-human calicivirus monoclonal antibody that is easy to apply and has extremely high cost performance.

That is, the present invention relates to an anti-human calicivirus monoclonal antibody (hereinafter, also referred to as an anti-calicis antibody).
Of the present invention is to provide a plant somatic cell (hereinafter, also referred to as the present plant somatic cell) which has at least a gene encoding the variable region in its own cell among the genes encoding the above.

In general, an antibody molecule becomes a completed molecule when two molecules called an H chain and an L chain are covalently bonded by a disulfide bond, and an antigen binding region present at each of three positions in the H chain and the L chain is a space. By being correctly arranged, specific binding to the antigen is exhibited. Therefore,
In the cells of animals and plants into which the gene encoding the antibody has been introduced,
In order to express an antibody having the desired antigen-binding activity as a recombinant protein, it is necessary that H chains and L chains associate at a molar ratio of 1: 1 and that the three-dimensional structure of the antigen-binding site is correctly reproduced. Will be needed.

In order to correctly associate the H chain and the L chain and correctly reproduce the three-dimensional structure of the antibody molecule, a construct gene synthesized as a single-chain gene in which genes encoding both chains are linked via a spacer sequence is used. It has been known that a technique of introducing an H chain and an L chain as a fusion protein (single-chain antibody) into various gene expression systems is effective for producing an antibody molecule having an antigen-binding activity.

In view of the above, the present invention provides, as an embodiment of the present plant somatic cell, at least a gene encoding a variable region of a gene encoding an anti-caliche antibody,
Genes encoding the H chain and L chain of the anti-kalisi antibody
It is a construction gene linked via a spacer sequence,
The present plant somatic cells are also provided.

Human calicivirus (hereinafter, unless otherwise specified, "calicis virus" means human calicivirus) is mainly based on the nucleotide sequence of the polymerase gene, Genogroup I (Norwalk-like virus), Geno
group II (Snow Mountain-like virus), Genogroup III
(Sapporo-like virus). Classifications based on structural protein genes that define serotypes are also classified into these three groups.
Serotype, 7 serotypes for Genogroup II, 3 for Genogroup III
It is likely that serotypes are present.

[0018] Monoclonal antibodies have already been produced in caliciviruses and these have been reported to be effective in determining the serotype of the virus.
However, there has been no report of producing a monoclonal antibody against calicivirus based on plant cells into which a gene encoding the monoclonal antibody has been introduced, as in the present invention.

[0019]

Embodiments of the present invention will be described below. In the present invention, an anti-calicidal antibody-producing cell serving as a source of a gene for transforming a plant is generally obtained by fusing myeloma cells with immune cells obtained by immunizing an animal with a calicivirus antigen. Hybridoma cells, and the antibodies produced in such hybridoma cells are monoclonal antibodies that have binding properties to the calicivirus capsid antigen. This antibody is widely cross-reactive with currently known different serotypes of calicivirus.

The anti-calicivirus monoclonal antibody gene is composed of heavy chain (H chain), Light
Each of the two genes encodes an “antibody variable region” that defines at least the binding specificity to an antigen (calicis virus capsid antigen). Genes must be included. In addition, it is preferable that this gene (the gene encoding the H chain and the L chain of the anti-calcic antibody) be a construction gene linked via a spacer sequence.

First, the above-mentioned hybridoma cells are prepared by preparing spleen cells from an immunized animal such as a mouse immunized with a calicivirus as an immunogen, and fusing the cells with myeloma cells (myeloma) of the immunized animal in a test tube. Can be done. The monoclonal antibodies produced by the hybridoma cells bind to calicivirus specifically and show wide cross-reactivity with caliciviruses of different serotypes, so that it can recognize many kinds of caliciviruses. Yes, it can be used to efficiently diagnose a calicivirus infection.

The cloning of the gene encoding the anti-kalisi antibody can be performed by a generally known method. For example, RNA is isolated from a hybridoma cell (anti-calicidal antibody-producing cell) that produces the above-mentioned anti-calicin antibody, and cDNA is prepared using the gene RNA as a template and reverse transcriptase.
Can be used to clone a gene encoding an anti-caliche antibody using the cDNA.

The isolation of the RNA of the anti-kalici antibody-producing cells comprises
Usually, it can be performed by a known method. For example, a hybridoma cell producing an anti-kalisi antibody is cultured in a test tube, and a gene RNA is obtained from the hybridoma cell.
By extracting using a generally known RNA extraction method such as a phenol method, a guanidine hot phenol method, etc.,
RNA of the anti-kalici antibody-producing cells can be isolated.

[0024] Synthesis of cDNA using the gene RNA as a template can also be performed by a generally known method. For example, a single-stranded cDNA can be synthesized and prepared by using reverse transcriptase and the resulting gene RNA of an anti-caliche antibody-producing cell as a template and oligo dT primers.

Next, primers for specifically amplifying the H chain and L chain of the antibody gene from the cDNA obtained in this manner (ie, a base sequence complementary to a gene in the vicinity of these gene regions). Antibody gene fragment group is prepared by a gene amplification method such as a PCR method using the primers having the above-mentioned primers, incorporated into an amplification vector using Escherichia coli as a host, and further amplified in an appropriate host to obtain a large amount. To amplify the antibody gene. The amplification vector that can be used here is not particularly limited.
Amplification vectors that are commonly used for cloning, such as the pUC vector series, pGEM vector series, pBR vector, PBluescript vector, and λ phage vector, can be used.

The nucleotide sequence of the DNA fragment incorporated in the thus obtained cDNA clone can be confirmed by, for example, the Maxam-Gilbert method (Maxam, A. and Gilbert,
USA. 74,560 (1977)), Genomica sequencing method (Church.GMand Gilbert, W .: Genomi
c sequencing., Proc. Natl. Acad. Sci. USA 81, 1991-1995 (1
984)), multiplex method [Church, GM, Science
240, 185-188 (1988)), cycle sequence method (Natur
e, 321, 674 (1988)), the Sanga method (Sanger, F., Nicklen,
S. and Coulson, AR, Proc. Natl. Acad. Sci. USA 74,5463
(1977)]. It should be noted that a base sequence automatic analyzer (for example, ABI PRISM 310 Genetic Analyzer, ABI Model 37
3A (both from PERKIN ELMER), ALF DNA SequencerI
I (Pharmacia) etc.] can be used to confirm the nucleotide sequence.

The antibody H chain and L chain gene cDNA clones obtained by confirming the nucleotide sequences in this manner are ligated with a gene encoding a spacer peptide, for example, (Glycine-Glycine-Glycine-Gly
cine-Serine) × 4, artificially ligated into the vector for amplification as a single-chain antibody gene, and further amplified in an appropriate host to produce a large amount of the single-chain antibody gene. Can be amplified.

Based on the base sequence of a part or all of the single-chain anti-caliche antibody gene obtained by the above-mentioned method, a means for changing the base sequence of a generally known gene, such as a so-called site-directed mutagenesis method, is used. Thus, the amino acid sequence of a protein that can be produced based on the gene can be changed by partially modifying the base sequence of the single-chain anti-caliche antibody gene prepared in the above step. The present inventor has recognized that the use of such a single-chain anti-caliche antibody gene artificially modified gene as a transgene into a plant described below is within the technical scope of the present invention. I do.

Next, as a prerequisite for introducing the anti-caliche antibody gene obtained as described above into a plant, an expression vector capable of introducing and expressing the anti-caliche antibody gene and the like into a plant ( Hereinafter, this expression vector is also referred to).

As described above, the present expression vector is a plant expression vector containing the gene coding for the anti-Calici antibody, and the gene expression vector containing the gene coding for the anti-Calici antibody as an essential element. It is. In addition to the anti-calicivirus monoclonal antibody gene, if necessary, for example, a promoter sequence that functions in plants such as the cauliflower mosaic virus 35S promoter, rice actin promoter, rice waxy promoter;
/ Ds transposons, sequences related to the regulation of gene expression in plants, such as intron sequences of castor catalase; terminator sequences such as nopaline terminator and 35S terminator; necessary for self-replication in E. coli and Agrobacterium It can contain a unique replication origin sequence and the like. Further, if necessary, it may contain a gene obtained by fusing a gene such as a cell localization signal peptide.

Specific embodiments of the expression vector will be described in Examples described later. Using this expression vector,
According to the present invention, it is possible to introduce the above-mentioned anti-caliche antibody gene product into a plant and transform the plant to express the integrated anti-caliche antibody gene product, that is, the anti-caliche antibody, in the plant cell or the plant body. Is one of the main purposes.

First, the plant into which the above-mentioned anti-caliche antibody gene is to be introduced is not particularly limited. For example, rice (rice, upland rice), Italian ryegrass, sorghum, etc .; , Red clover,
White chopper, adzuki bean, soybean, alfalfa and the like; in the Solanaceous plant, potato, tobacco, tomato and the like can be widely exemplified.

The method for introducing the present expression vector into a plant can be carried out by a generally known method. For example, the introduction method using Agrobacterium [Horsch, RBe
t al., Science 227, 1229-1231 (1985), Hiei, Y. et al.,
Plant J. 6.271-282 (1994)], a protoplast is prepared from the target plant using a generally known method, and the protoplast is subjected to electroporation (Fromm, M. et a.
Acad. Sci. USA 82, 5824-5828), polyethylene glycol method (Zhang, W. and Wu, R. Theor. Appl. Gen.
et 76, 835-840 (1988)], for example, a particle gun method [Gordon-kamm, WJ. et al., Plant Cell
2,603 (1990)], a method of immersing a tissue or embryo in a solution of the present expression vector [Topfer.R. Et al., Plant Cell 1,133 (198
9)].

In a plant cell group into which the expression vector has been introduced, selection of plant cells into which the anti-caliche antibody gene has been incorporated can be carried out by simultaneously introducing an appropriate marker gene or a vector containing the same into a plant, It is preferable to first select a plant cell group into which the marker gene or the like has been introduced, and to take auxiliary measures such as increasing the probability of the presence of the plant cell into which the anti-caliche antibody gene has been introduced. Such marker genes include kanamycin resistance gene, hygromycin resistance gene, herbicide vasta resistance gene (ba
r gene).

In this manner, the present plant somatic cells into which the anti-caliche antibody gene has been incorporated can be produced. Next, a method of redifferentiating the present plant somatic cells into a plant and a method of growing the same will be described.

The regeneration of the plant from the present plant somatic cell (group) into which the above-described anti-caliche antibody gene produced as described above has been introduced can be carried out by a conventional method. For example, a method of isolating and culturing protoplasts from suspension culture cells having a strong regenerative and dividing ability [Yamada, Y. et al., Rice genetic
s newsletter 2,94 (1985)), agarose bead method and nurse culture method (Kyozuka, J. et al., Mol. Gen Genet, 206, 40
8 (1987)), leaf disk method (De Block, M. et al., The
ro.appl.Genet. 76, 767-774 (1988)), Tuber disk method (Sheerman, S. et al., Plant Cell Reports 7, 13-
16 (1988)] can be used, but the method is not limited thereto.

When the seedlings are obtained by these methods, the desired plant (the present plant) is obtained by, for example, picking up the seedlings on a cultivation soil for acclimation and acclimating the seedlings. Can be.

In the present invention, the term "plant" is a general term for roots, stems, leaves, flowers, fruits (seeds) and tubers of plants. Therefore, the technical scope of the plant of the present invention extends not only to seeds and seedlings produced by introducing the anti-kalisi antibody gene, but also to harvests thereof.

In the present invention, plant somatic cells are
It contains both protoplasts and calli. Therefore, the technical scope of the present plant somatic cells includes both protoplasts and calli of the plant.

Specific embodiments of the above-described series of steps for producing the present somatic cells or the present plant will be described in Examples described later. When the present plant body produced through the above steps is capable of self-breeding and cross-breeding, it is possible to produce an F1 plant body using a generally known crossing technique, and the characteristics of the present plant body are as follows: It is inherited stably in progeny.

Next, a method for producing the anti-calcic antibody of the present invention (hereinafter, also referred to as the present anti-calcic antibody) from the present plant or the present plant cell obtained as described above will be described.

As described above, in the present plant (including the present plant somatic cells), the anti-caliche antibody incorporated as a gene is expressed in the plant. In the present plant body (including the present plant somatic cells), the present anti-kalici antibody may be a root, if it is a site where the incorporated anti-kalici antibody gene is expressed.
It can be used as a raw material for any part, at any stage of differentiation, such as stems, leaves, flowers, fruits (seeds), tubers, calli, and the like. In addition, callus is a preferable material as an extraction material of the present anti-caliche antibody because it can be cultured in a tank throughout the year.

From these raw materials, the present anti-kalisi antibody
It can be extracted by a conventional method. For example, by grinding these raw materials together with a suitable buffer, a crude extract of the present anti-caliche antibody can be obtained. For example, milling these raw materials, subjecting the milled liquid to quick-centrifugation,
The centrifuged supernatant is obtained and can be used as the above crude extract. Next, a protein precipitant such as ammonium sulfate is added to the crude extract to obtain the target anti-caliche antibody as a precipitate.

Depending on the use of the present anti-kalisi antibody, the purpose can be achieved even at this stage of crude extraction, but if higher purity is desired, dialysis, ultrafiltration, molecular sieve chromatography, ion exchange chromatography E, affinity chromatography, electrophoresis or a combination of these methods can be used for purification.

For example, the obtained precipitate of the present anti-kalisi antibody is dissolved in a specific buffer, dialyzed, the dialyzed sample is centrifuged, and the supernatant is subjected to anion column chromatography. The desired purified anti-kalici antibody can be obtained by fractionation.

Further, it is possible to improve the purity of the anti-caliche antibody purified by the anion exchange by affinity column chromatography using c-mycTag.

Surprisingly, in the present plant (including the present plant somatic cells), a monoclonal antibody against calicivirus is a non-host that cannot be found in nature, despite being a molecule originally produced in animals. It is expressed as a gene product in a certain plant.

For example, a gene encoding a calicivirus protein is inserted into a baculovirus vector using a conventional method, and the gene is introduced into insect cells and expressed to collect virus particles. Thus, the virus can be recovered as a baculovirus-expressed calicivirus protein, and such a virus protein can be used as a standard product in various tests and the like related to the present invention.

Also, by inoculating the calicivirus virus protein expressed in the baculovirus into an immunized animal by a generally known method,
It is possible to obtain an antibody (polyclonal antibody or monoclonal antibody) against the calicivirus protein, and such an antibody can be used as a standard similarly.

[0050] The present anti-caliche antibody can be appropriately labeled as necessary according to a conventional method. For example, the anti-calicin antibody may be an enzyme (an enzyme labeling method such as a maleimide method, a glutaraldehyde method, a periodate method, etc.), a fluorescent dye (fluorescein isothiocyanate, rhodamine, Texas Red, etc.) or colloidal gold may be used. Can be labeled. These labeled anti-caliche antibodies are also included in the present calici antibodies.

Detecting a human calicivirus in a test article by associating an antigen-antibody reaction to a calicivirus in the present anti-caliche antibody produced as described above with the presence of the calicivirus in the test article. Is possible (this detection method).

Specifically, it is possible to construct the present detection method in various modes by making full use of immobilized or non-immobilized unlabeled or labeled present calici antibodies, standard virus antigens and the like. For example, enzyme immunoassays such as ELISA and EIA (non-competitive solid-phase enzyme immunoassay, non-competitive homogeneous enzyme immunoassay, competitive homogeneous enzyme immunoassay, competitive solid-phase enzyme immunoassay, etc.), RIA, flow cytometry, colloidal gold method In a detection method using an antigen-antibody reaction, such as an immunoturbidimetric method, an octalony method, or a Western blotting method, elements necessary for these detection methods are replaced with elements relating to the calicivirus in the present invention (standards of the calicivirus and calicivirus). Antigen).

[0053]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention should not be limited by these examples. A. Obtaining antibody genes from monoclonal antibody-producing hybridomas Culture of Hybridoma and Purification of Antibodies In 1996, monoclonal antibody-producing cells (NSFG22, NSFG2) were prepared by Tomoyuki Tanaka and others at the Sakai Animal Health Laboratory.
8, NSFG8, NSFG941: Virology vol.207 (1), p252-261,199
6) is RPMI164 supplemented with 10% (v / v) fetal bovine serum.
The cells were cultured in 0 medium (GIBCOBL) at 37 ° C. in the presence of 5% CO _{2 for} 3 to 4 days. Hybridoma typing is performed using the Mouse monoclonal antibody isotyping kit code R
This was performed using PN29 (Amersham Pharmacia biotech, UK).

First, 0.5 mL of the culture supernatant containing the antibody contained in the typing stick and TBS-Tween buffer (TBS-T / 20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% (v / v))
Tween 20) was added and reacted at room temperature for 15 minutes. Typing stick is 5mL TBS-Tween
Wash 2x / 5 min with buffer, 10 μL peroxidase lab
TBS- containing elled anti-mouse antibody (containing Kit)
The reaction was carried out at room temperature for 15 minutes in 5 mL of Tween buffer. In addition, 2 times / 5 times with 5 mL of TBS-Tween buffer.
Wash for 4 minutes and color reaction solution (4-chloro-1-naphthol (Kit
1 drop of H2O2, 50 mL of TBS (not containing Tween 20)
Solution in 5 mL) at room temperature for 15 minutes,
The reaction was stopped with distilled water. It was revealed that the antibodies of all hybridomas encode κ-chain in IgG1.

The antibody was purified from the monoclonal antibody-producing cells expanded in the abdominal cavity of mice using Affi-Gel Protein A
This was performed using the MAPS II Kit (BIO-RAD, CA). First, the monoclonal antibody-producing cells were subcultured several times, and then passed about 1 ×
Prepare to 10 ⁷ cells / mL and prepare in advance 2,4,10,14-tetramethylpentadecane (usually pristane, Wako Pure Chemical)
BAL pretreated with 0.5 mL, 7 to 14 days old
B / c mice were inoculated in 0.5 mL portions. Seven to ten days after the inoculation, the ascites of the mouse was collected, ammonium sulfate was added thereto with stirring to a final concentration of 33% (v / v), and the mixture was centrifuged at 10,000 rpm for 15 minutes (TOMY SRX).
-200, using a TA-8 rotor). The precipitate was dissolved in phosphate buffered saline (PBS) and allowed to stand at 4 ° C for 1 hour.
It was placed in an alysis membrane (size 8, Wako Pure Chemical Industries) and dialyzed in phosphate buffered saline (PBS) at 4 ° C for 2 days. 3mL
1 × 10cm E containing Affi-Gel Protein A agarose
After flowing 20 mL of a binding buffer (pH 9) through the cono-Column chromatography column, 15 mL of a solution obtained by diluting the crude antibody extract with PBS was flowed. After passing 50 mL of the binding buffer and washing the column, 20 mL of the elution buffer was poured, and I
The gG protein was eluted. Purified IgG was used as a positive control.

2. Extraction of mRNA and synthesis of cDNA Monoclonal antibody-producing cells obtained by the above operation
To (5 × 10 ⁶ ), 1 mL of TriZol (GIBCO) was added, stirred vigorously, allowed to stand at room temperature for 5 minutes, added with 0.2 mL of chloroform, stirred, and reacted at room temperature for 3 minutes.

Centrifugation operation (10000 rpm / 15 minutes, TOMY M
C-150) and the aqueous phase was transferred to a new tube. To this, 0.5 mL of isoamyl alcohol was added, and after stirring,
It was left at room temperature for 10 minutes. After collecting the precipitate by centrifugation at 10,000 rpm for 10 minutes, wash with 70% ethanol,
It was air-dried and dissolved in a small amount of DEPC-treated water to obtain a monoclonal antibody-producing cell RNA solution containing the antibody gene.

Using the total gene RNA of the monoclonal antibody-producing cells obtained by the above operation as a template, cDNA
The fragment was amplified. That is, 1 μg of monoclonal antibody-producing cell RNA was added to reverse transcription reaction buffer (50 mM Tris-HCl pH 8.3, 75 mM potassium chloride, 8 mM magnesium chloride, 10 mM thiothreitol), 10 mM dNTP, 50 pmolOl
igo (dT) 18primer, RNase inhibitor, RAV-2 Re
Reverse Transcriptase 10 U (Takara, Japan) was added and reacted at room temperature for 10 minutes and at 42 ° C. for 60 minutes. A part of this reaction product was used in a PCR reaction.

3. PCR detection of antibody gene Immunogene M
(NISSINBO, Japan) H chain 5 'Fab / Fv pri
mer (SEQ ID NOS: 33-39) and pri for H chain 3 ′ Fv
mer (SEQ ID NOs: 40 to 43) or κ chain 5 ′ Fa
Using the combination of the b · Fv primer (SEQ ID NOs: 44 to 53) and the κ chain 3 ′ Fv primer (SEQ ID NO: 54), the respective H chain and κ chain genes were detected from each hybridoma RNA. Thermostable DNA polymerase [PLATINUM Taq DNA Polymerase High Fidelity (GIB
CO BRL)], and using the buffer and reaction reagents attached thereto, and the method described in the attached instruction manual (1 × High
Fidelity PCR buffer, 200μM each dNTP mixture, 2m
M MgSO ₄ , 0.2 μM each primer, cDNA 500ng, 1 unit
PLATINUMTaq High Fidelity). After heating at 94 ° C for 2 minutes, the thermal reaction was performed at 94 ° C for 30 seconds at 65 ° C.
The thermal cycling reaction at ５５55 ° C./1 minute (decrease by 1 ° C. per cycle) and 68 ° C./2 minutes was performed for 10 cycles. Subsequently, 94 ° C./30 seconds, 55 ° C./1 minute and 68 ° C.
Twenty-five cycles of thermocycling at 2 ° C. for 2 minutes were performed.
Finally, an extension reaction was performed at 68 ° C. for 5 minutes to terminate the PCR reaction.

The cDNA fragment amplified by the above PCR reaction is electrophoresed on a 2% agarose gel (100 Volt / 30 min).
Then, an amplified band having a size of about 350 to 400 bp was confirmed. Cut out the amplified product and use GENECLEAN II Kit (B
Purification was performed using IO101 (CA).

First, add 1 mL of sodium iodide solution,
After treating at 56 ° C. for 5 minutes to dissolve the gel, 10 μL of gl
The assmilk was added and allowed to bind to the DNA on ice. Twice N
After washing with 500 μL of ewWash solution, it is dried,
It was dissolved in 30 μL of TE buffer and left standing at 56 ° C. for 10 minutes. Centrifuge at 12,000 rpm for 5 minutes at room temperature (TOMY M
C-150) to remove impurities and purify. The purified amplification product was subcloned according to the method using pGEM-T Easy vector system (Promega). T
4 DNA ligase (1 μL), pGEM-T Easy
Vector (1 μL), amplification product (200 ng), ligation reaction buffer (300 mM Tris-HCl, pH 7.8, 100 mM MgCl ₂ ,
A ligation reaction was performed at 16 ° C. overnight with 10 μL of a reaction solution containing 1 μL of (100 mM DTT, 5 mM ATP).

<Competent E. Preparation of E. coli> E. coli DH5a, A stored at ultra-low temperature (-80 ° C)
D494) was streaked on an LB agar plate (GIBCO BRL), cultured overnight at 37 ° C., a single colony formed was scraped off, and 250 mL of SOB medium (Bacto Tryptone) was removed.
2%, BactoYeast extract 0.5%, 10mM NaCl, 2.5mM KCl
). Rotary shaker adjusted to 18 ° C (EYELA UNI ACE SHAKER NCS-1300, TOKYO RIKAKIKAI
After cooling for 10 minutes and centrifugation at 4 ° C and 3000 rpm for 15 minutes (GS-6R Centri
fuge, BECKMAN) and the cells were collected. Remove the supernatant and add 8
The suspension was suspended in 0 mL of TB (10 mM PIPES, 15 mM CaCl ₂ , 250 mM KCl, Nacalai) and cooled for 10 minutes. 4 ° C
Centrifuge at 3000 rpm for 15 minutes (GS-6R Centrifuge, B
(Eckman) and the cells were collected. Remove the supernatant, suspend in 20 mL of TB, and add DMSO to a final concentration of 7%.
(Dimethyl Sulfoxide, Nacalai) was added, cooled for 10 minutes, dispensed, and stored in a liquid nitrogen solution.

After ligation, the plasmid competent E. E. coli (DH5α strain) was transformed as follows. Competent E. coli was added to 10 μL of the ligation reaction. After suspending 50 μL of E. coli and reacting on ice for 20 minutes, the mixture was treated at 42 ° C. for 2 minutes. Immediately, L
1 mL of B liquid medium (GIBCO BRL) was added, and the cells were cultured at 37 ° C for 1 hour. After the culture, the cells were seeded on an LB agar plate (containing ampicillin), cultured at 37 ° C overnight, and the transformed cells were selected and added to 5 mL of LB liquid medium (GIBCO BL).
Overnight.

Purification of the cloning plasmid was performed using QIA pr
The ep Spin Miniprep Kit (QIAGEN) was used. The cultured cells were centrifuged at 12,000 rpm for 2 minutes at room temperature (TO
MY MC-150), remove the supernatant, and add 250 μL of P1 buffer,
After adding and suspending 250 µL of P2 buffer and 350 µL of N3 buffer, the mixture was centrifuged at 14000 rpm for 10 minutes at room temperature (TOMY MC-150). The supernatant is put into a QIAprep column and centrifuged at 12,000 rpm for 1 minute (TOMY MC-150).
After washing the column with 750 μL of PE buffer, add 50 μL of TE buffer, and centrifuge at 12000 rpm for 1 minute (TO
MY MC-150) and the plasmid was purified.

4. Analysis of nucleotide sequence At least 24 clones / maximum 90 clones were selected from the obtained plasmids and used for the analysis of the nucleotide sequence of the antibody gene.

First, 200 ng of plasmid DNA, T7 primer (TAA TAC GAC TCA CTA TAG GG, PROMEGA)
Or SP6 primer (TAT TTA GGT GAC ACT ATA G,
PROMEGA) 1μL, ABI PRISM Dye Terminator Cycle
Sequencing Ready Reaction Kit (PERKIN ELMER) B
ig Dye Terminator RR Mix 4μL Reaction solution 10μL)
By PCR (96 ° C./15 seconds, 50 ° C./15 seconds and 60 ° C.)
C./4 minutes of thermal cycle reaction × 25 cycles). Add 50 μL of ethanol to the reaction solution, and add
After standing for 0 minutes, centrifugation (KUBOTA1720) at 14000 rpm for 20 minutes at 4 ° C., removing the supernatant, adding 200 μL of 70% ethanol, and centrifuging (KUBOTA1720) at 14000 rpm for 20 minutes at 4 ° C. to remove impurities And dried. Te
mplate Suppression Reagent (ABI PRISM, Applied Bio
13 μL was added to dissolve the sample, boiled for 3 minutes, and quenched on ice. The nucleotide sequence of this cDNA fragment portion was determined by cycle sequencing (Nature 321,674 (1
986), 310 Genetic Analyzer, ABI PRISM, PERKIN ELME
R), and the inserted cDNA fragments were isolated from mouse H chains (NSFG22 (SEQ ID NOs: 1 and 2),
NSFG28 (SEQ ID NOS: 5, 6), NSFG8 (SEQ ID NOs: 9, 1)
0), NSFG941 (SEQ ID NOs: 13 and 14)) and κ chain (NSFG
22 (SEQ ID NOs: 3 and 4), NSFG28 (SEQ ID NOs: 7 and 8), NS
FG8 (SEQ ID NOS: 11 and 12), NSFG941 (SEQ ID NO: 1)
5, 16)) It was confirmed that it was an antibody gene.

5. Preparation of single-chain antibody gene To connect each H chain and κ chain antibody gene with a linker [spacer (SEQ ID NO: 19, 20)]
On the 5 'side, a SmaI restriction enzyme recognition site (EcoRI in the case of expression in baculovirus and Escherichia coli) and a secretory signal sequence (SEQ ID NOs: 17 and 18), and on the 3' side, a c-myc antibody recognition site (tag, SEQ ID NOS: 21 and 22) and KDEL signal (SEQ ID NOS: 23 and 24) and restriction enzyme SacI (SmaI for expression in baculovirus, HindII for expression in E. coli)
In order to add the recognition site of I), a PCR reaction was performed using the designed primers.

That is, a primer designed to encode a secretory signal sequence at the 5 ′ end of each H chain and a linker (so that 35 bp overlaps the 5 ′ end of the κ chain) at the 3 ′ end [ VH-l for NSFG22 and NSFG28
eader (22,28) (SEQ ID NO: 55), VH-le for NSFG8
ader (8) (SEQ ID NO: 60), VH-leade for NSFG941
5'primer of r (941) (SEQ ID NO: 61) and VH-linker (22,
28) Combination of 3'primer of (SEQ ID NO: 57)],
At each 5 ′ end of the κ chain, a linker (so that 35 bp overlaps with the 3 ′ end of the H chain) is attached at the 3 ′ end.
Primers designed to encode the c-myc antibody recognition site (tag, EQKLISEEDL) and KDEL signal [NSFG22, NS
In the case of FG8, VL-linker (22) (SEQ ID NO: 58), NSFG28
5'primer of VL-linker (941) (SEQ ID NO: 62) for NSFG941 for VL-linker (28) (SEQ ID NO: 59) for NSFG941
And VL-Tag (22,28) for NSFG22, NSFG28, NSFG8
(SEQ ID NO: 56), in the case of NSFG941, 3'primer combination of VL-Tag (941) (SEQ ID NO: 63)] and PLATINUM
Taq DNA Polymerase High Fidelity (GIBCO BRL)
And a buffer and a reaction reagent attached thereto, and the reaction was carried out according to the procedure of the PCR reaction described above.

The thermal reaction was carried out after heating at 94 ° C./2 minutes,
A thermocycling reaction at 4 ° C./30 seconds, 45 ° C./1 minute and 68 ° C./2 minutes was performed 30 times. The DNA fragment amplified by the PCR reaction was subjected to electrophoresis (100 Volt / 30 min) on a 2% agarose gel. After confirming the amplified band of about 400 bp in size, the DNA fragment was cut out, and the above-mentioned GENECLEAN II Kit (B
Purification was performed using io101 (CA). The recovered H chain and κ chain DNA fragments were made into single strands by the PCR method.

That is, a primer having a restriction enzyme recognition site at the 5 'end of the H chain [SmaI in the case of a plant expression system]
(SEQ ID NO: 64), EcoRI (SEQ ID NO: 67) in the case of a baculovirus and Escherichia coli expression system] and a primer having a restriction enzyme recognition site at the 3 ′ end of the κ chain [SacI (SEQ ID NO: 65, 66), in the case of the baculovirus expression system, SmaI (SEQ ID NO: 69), in the case of the E. coli expression system, HindIII (SEQ ID NO: 68)] and a linker overlap sequence, and PLATINUM Taq DNA Polymer was used.
The reaction was carried out using ase High Fidelity (GIBCO BRL) and the buffer and reaction reagent attached thereto according to the above-described PCR reaction procedure.

The thermal reaction was performed after heating at 94 ° C. for 2 minutes,
Five thermal cycling reactions were performed at 4 ° C./30 seconds, 45 ° C./1 minute, and 68 ° C./2 minutes. Subsequently, at 94 ° C /
The thermal cycling reaction for 30 seconds, 55 ° C./1 minute, and 68 ° C./2 minutes was performed for 15 cycles. Finally, at 68 ° C, 5
The extension reaction was performed for 1 minute, and the PCR reaction was completed. The DNA fragment amplified by the PCR reaction was subjected to electrophoresis (100 Volt / 30 min) on a 2% agarose gel, and after confirming the amplified band of about 400 bp in size, cut out, and excised from the above-mentioned GENECLEAN II.
Purification was performed using Kit (Bio101, CA). The purified amplification product is pGEM-T Eazy vector system (Promega)
And cloning and competent E. coli according to the method described above. After transformation into E. coli (DH5α strain), the cloning plasmid was purified using QIAGEN Spin Miniprep Kit (QIAGEN).

The nucleotide sequence of the antibody gene in at least 24 obtained plasmids was analyzed by the above method. Then, it was confirmed that the determined base sequence was such that the H chain and κ chain of each hybridoma were correctly connected by a linker and that there were no mutations, and that 22 + 15 (SEQ ID NOS: 25 and 26) and 28 + 19 (sequence) No. 27, 28), 8
+6 (SEQ ID NO: 29, 30), 941 + 3 (SEQ ID NO: 3
1, 32).

6. Expression in Baculovirus System Baculovirus vector pVL1392 (Pharmingen
E.). After transforming this E. coli strain into E. coli DH5α by the method described above, it was cultured overnight at 37 ° C. in 5 mL of LB medium containing 100 μg / mL of ampicillin, and the QIAGEN Spin Miniprep K
Plasmid DNA was prepared using it (QIAGEN). To 50 μL of this pVL1392 plasmid DNA,
Add 4 μL of EcoRI and 6 μL of the attached buffer, and add
After reacting for 1 hour and cutting the plasmid strand, 1%
Separated by agarose electrophoresis, cut out the gel, and
It was purified by lean and dissolved in 60 μL of ultrapure water. This DN
Alkaline phosphatase (10 units) 2 in solution A
After addition of 10 μL of the attached buffer and 28 μL of ultrapure water, the mixture was reacted at 37 ° C. for 30 minutes, extracted with an equal volume of phenol and chloroform, and precipitated with ethanol at 4 ° C. overnight to precipitate DNA. After washing with ethanol, it was dried and dissolved in 20 μL of ultrapure water. pVL139
22 + 15, 28 + 19, 8+ to be inserted into 2 vectors
6, 941 + 3 single-chain antibody gene fragment DNA was obtained from the plasmid DNA obtained using the pGEM-T vector described above.
After 15 μL was cut by reacting with 2 μL of EcoRI at 37 ° C. for 4 hours, the mixture was separated by 1% agarose gel electrophoresis to obtain about 8 μL.
A 50 bp band was cut out, purified by Gene Clean, and dissolved in 20 μL of ultrapure water. Vector DN obtained above
A mixture of 7 μL of the A solution, 1 μL of the DNA solution to be inserted, 1 μL of T4 ligase, and 1 μL of the attached buffer were mixed to obtain
The reaction was carried out at 0 ° C overnight. Using this ligation solution,
E. FIG. E. coli DH5α competent cells were transformed, and about 20 obtained strains were examined for insertion of single-chain antibody gene fragment DNA by PCR. For the six positive strains, plasmid DNA was prepared and the nucleotide sequence was examined, and those having the inserted gene in the positive direction and having no mutation were selected.

[0074] Gene of Single Chain Antibody Gene to Baculovirus
Recombination is performed using the Baculo Gold Transfection Kit (Pharming
en) to transform insect-derived Sf9 cells.
Was. First, the plasmid DNA obtained above was placed in 0.22
Sterilize with a μm filter, and use 25 μL (2-5 μg
DNA), Baculo Gold Transfection Kit 5μ
Mix with L and leave at room temperature for 5 minutes.
 Add 1 mL of buffer B and add recombinant baculovirus
Was created. Sf9 cells contain 10% fetal bovine serum
Using Grace's medium (Gibco BRL) at 28 ° C
After subculture several times, 2 × 10⁶5mL of cells
And suspended in a 60% tissue culture medium (CELLSTA
R, Greiner) and cultured at 28 ° C overnight
Was used as a host cell. Sf9 cells use old media
Remove and add 1 mL of Transfection buffer A.
Add the replacement baculovirus solution slowly and drop by drop.
Was. After culturing at 28 ° C for 4 hours, replace with a new medium,
The cells were further cultured at 28 ° C. for 5 days for infection. Culture supernatant
Was centrifuged at 3000 rpm for 10 minutes to remove cells,
A recombinant baculovirus solution was obtained. Furthermore, Sf9 fine
Vesicle (3.2 × 10⁶ Pcs / 5mL) with the above low concentration combination
Add 300 μL of the replacement baculovirus solution, and for 3 days,
Cultured. Repeat this operation several times to obtain a high concentration
A baculovirus solution was obtained.

Whether the single-chain antibody gene has been recombined with the baculovirus was determined by determining whether the infected cultured cells were at 3000 rp.
m, collected by centrifugation for 10 minutes, DNA was extracted according to the package insert of Sepa Gene (Sanko Junyaku) and confirmed by PCR.

The recombinant baculovirus propagated in Sf9 cells was used to infect 100 mL of insect-derived TN5 cells (5 × 10 ⁵ cells / mL) having good protein expression. Four days later, the cells were collected, and a surfactant (1% Triton X-305) and a protease inhibitor (Protease-Inhibitor-Cocktail,
The mixture was suspended in PBS containing Roche), sonicated for 10 seconds (Astorason, MINOX) six times under ice-cooling, and then centrifuged at 4 ° C and 12,000 rpm for 2 minutes. Was used as a crude antibody extract.

7. Analysis of baculovirus-expressed proteins To express single-chain antibody proteins in solution, a portion of the infected cells was expressed in a small amount of sample buffer (0.5M Tris-HCl pH6.8).
1 mL, 10% SDS 2 mL, β-mercaptoethanol 0.6 mL, glycerol 1 mL, distilled water 1 mL, 1% BPB several drops)
The solution after heating at 0 ° C. for 5 minutes was analyzed by SDS-PAGE and Western blotting. SDS-PAGE (1
2%), the gel was fixed for 30 minutes after electrophoresis (50% methanol, 10% acetic acid) and Quick
After staining with -CBB (Wako Pure Chemical) solution for 1 hour, excess dye was shaken overnight in distilled water to decolorize, and the appearance of a protein of about 30 kilodalton was used as an index. In the western blot method, after SDS-PAGE (12%), the protein was transferred from the gel to a PVDF membrane at a constant current of ² mA / cm ² for 1 hour in a blotting device (horizon blot, ATTO). The membrane was washed with PBST containing 3% skim milk (SM) (PBS containing 0.5% Tween-20).
After blocking overnight at 4 ° C. in 1% SM-PBST
Anti-c-myc mouse serum (Resaerch
Diagnostics) for 1 hour at room temperature, PBS
After washing 3 times with T for 10 minutes, 10% with 1% SM-PBST.
In HRP-labeled anti-mouse IgG serum diluted 1:00,
The reaction was performed at room temperature for 1 hour. After washing three times, the substrate solution (PB
In 10 mL of ST, contains 25 μg of diaminobenzidine, 15 μg of cobalt chloride hexahydrate, and 10 μL of hydrogen peroxide solution)
The color was developed. As a result, expression of the anti-calicivirus single chain antibody in the baculovirus system was confirmed. For purification of single-chain antibody molecules, use Sepharose conjugated Mouse anti-cmyc
This was performed using clone (ResearchDiagnostics).

8. Antibody activity measurement The antigen used for the measurement of the expressed protein activity was baculovirus incorporated with the calicivirus (Chiba strain) particle protein gene distributed by the National Institute of Infectious Diseases.
It was prepared from a culture supernatant infected with N5 cells.

First, TN5 cells (5 × 10 ⁵ cells / m 2)
L) 1 mL of the recombinant virus solution was added per 100 mL, and 750 mL of the supernatant cultured at 28 ° C. for 7 days was added to 1450 mL.
The cells were removed by centrifugation (TOMY SRX-200, TA-8 rotor) at 0 rpm for 30 minutes, and the precipitate obtained by ultracentrifugation (Hitachi CP80α) at 100,000 g for 2 hours was further added to ultrapure water. Dissolved. This crude calicivirus protein solution was mixed with a discontinuous sucrose density gradient solution (10%, 20%, 30%).
%, 40%, 50%), and 113000 g is 1
Centrifuge for 0.5 hours, fractionate 0.5 mL each, and use BCA Protei
n The amount of protein was measured using Assay Reagent Kit (PIERCE). The protein-rich fractions at the 20% and 30% interface were collected and centrifuged at 52,000 rpm for 3 hours (Hi
The calicivirus-like particles were collected using mac CS100, RP80AT, Hitachi koki) and dissolved in a small amount of ultrapure water. The protein content of the purified calicivirus-like particles was measured and analyzed by SDS-PAGE (12%) and Western blotting. In the Western blotting method, a 1000-fold dilution of the monoclonal antibody NSFG22 (mouse ascites) was used as the primary antibody, and a 1000-fold dilution of the HRP-labeled anti-mouse IgG serum was used as the secondary antibody.

The expressed single-chain anti-calicivirus antibody activity was
The binding to the antigen was measured by ELISA. First, 0.
05M sodium carbonate buffer (1.59 g disodium carbonate, 2.93 g sodium bicarbonate per liter)
, Sodium azide 0.2 g, pH 9.6) at 1 μg /
The purified antigen particles diluted to a concentration of mL were dispensed in 100 μl aliquots into 96-well microplates for ELISA (Corning) and allowed to stand overnight at 4 ° C. for coating. After washing the coated plate twice with PBST, 3% SM-P
Blocking was performed with BST at 37 ° C for 2 to 3 hours. PBS
After washing twice with T, 100 μL of a 2-fold dilution series of baculovirus-expressed crude antibody extract was dispensed into each well, and
The reaction was performed at 7 ° C. for 2 hours. Further, after washing three times with PBST, a c-myc antibody diluted 1000-fold with 1% SM-PBST was dispensed to the plate and reacted at 37 ° C. for 1 hour. P
After washing three times with BST, the mixture was reacted for 1 hour at room temperature in HRP-labeled anti-mouse IgG serum diluted 1000-fold with 1% SM-PBST. After washing the plate three times with PBST, a substrate solution (citrate monohydrate 6.45 in 1 liter) was used.
g, disodium hydrogen phosphate 12 water 13.8 g, ABTS 11 mg
, Hydrogen peroxide solution (13 μL) and 100 μL were added to the plate and reacted at 37 ° C. After 30 minutes, the absorbance at 405 nm was measured using a microplate reader (Bio-Rad model 550).
Microplate reader). As a result, it was revealed that the single-chain calicivirus antibody had a calicivirus antigen-binding activity (FIG. 1: Reactivity of antibodies produced by baculovirus to calicivirus was confirmed. 405 corresponding to antibody dilution
The absorbance at nm is shown).

<Expression system in Escherichia coli> 22 + 15, 28+
19, 8 + 6, 941 + 3 single chain antibody gene fragments
After integration into an E. coli expression vector (Escherichia coli AD494 strain) and culturing at 37 ° C. until the logarithmic growth phase (OD600 = 0.6), IPTG (isopropyl-β) was adjusted to a final concentration of 1 mM.
-D-thiogalactopyranoside), further culture for 7 hours, and centrifuge the cells at 10,000 rpm for 15 minutes (TOMY)
SRX-200, TA-8 rotor). The precipitate is suspended in a PBS (0.05% PBST) containing a surfactant (0.05% Triton X-305), and sonicated (10 seconds, 6 times on ice) using an ultrasonic crusher (Astorason, MINOX). And centrifugation (4 ° C., 12000 rpm
After 30 minutes), the supernatant was used as the E. coli soluble fraction, and the precipitate was used as the insoluble fraction. The expression of each single-chain antibody molecule in each fraction was confirmed by the above-described SDS-PAGE method and Western blot method. In the Western blot method, anti-c-myc
A mouse monoclonal antibody diluted 1000-fold with 0.05% PBST was reacted at room temperature for 1 hour, and a secondary antibody peroxidase-labeled anti-mouse IgG diluted 1000-fold with 0.05% PBST was reacted at room temperature for 1 hour to detect. did.

B. Anti-calicivirus monoclonal inheritance
Introduction and expression of offspring into tobacco Introduction of anti-calicis virus monoclonal antibody gene into Agrobacterium The above anti-caliris virus single-chain monoclonal antibody genes 22-5, 22 + 15 (SEQ ID NOS: 25 and 26), 22
-8, and 28 + 19 (SEQ ID NOs: 27 and 28) were constructed using restriction enzymes SmaI and SacI.
To prepare a single-chain antibody cDNA fragment.

Next, this cDNA fragment was similarly digested with the restriction enzymes SmaI and SacI to obtain a plant expression vector pBE2113 (plant cell physiology vol. 37 49).
-59,1996), and Escherichia coli HB1 was ligated with this vector.
Strain 01 was transformed.

Plasmids were extracted from the obtained transformant in accordance with a conventional method, and pBECAB22 + 15
(Plasmid containing the cDNA fragments of SEQ ID NOS: 25 and 26), pBECAB22-5 (pBECAB22 + 15
Deleted from the cDNA fragment of SEQ ID NO: 4), pBECAB28 + 19 (including the cDNAs of SEQ ID NOs: 27 and 28), and pBECAB
28-8 (deletion of the portion encoding the 4 amino acid residues at the C-terminal side of the cDNA fragment of pBECAB28 + 19)
4 types of plant expression genes were constructed.

The four types of the anti-caliche antibody genes thus obtained were directly introduced into the Agrobacterium tumefaciens LBA 4404 (Clonte
ch company).

Specifically, Agrobacterium tumefaciens
LBA 4404 in 50 mL of LB broth (1% Bacto
Tryptone, 0.5% Bacto Yeast Extracts, 1% sodium chloride) was used for shaking culture at 28 ° C. until the absorbance of A600 reached about 1.0. And after cooling on ice,
After centrifugation at 3,000 g (using Kubota RA-6) at 4 ° C., the cells were collected, suspended in 1 mL of ice-cooled 20 mM calcium chloride solution, and transferred to an Ebendorf tube every 0.1 mL. Noted.

The recombinant plasmid pBECAB2
2 + 15, pBECAB22-5, pBECAB28 +
19 and pBECAB28-8, each in 1 μg, were snap frozen in liquid nitrogen. Next, after the obtained frozen cells were thawed at 37 ° C., they were allowed to stand for 5 minutes. To this, 1 mL of LB medium was added, followed by shaking culture at 28 ° C. for 2 to 4 hours. Centrifuge at 10,000g for 1 minute (Kubota KM-15200
And suspended in 0.1 mL of LB medium, then rifampicin (100 µg / mL), kanamycin (25 µg / mL) and streptomycin (300 µg / mL).
g / mL) after spreading on LB solid medium containing
Culture at 28 ° C, pBECAB22 + 15, pBEC
AB22-5, pBECAB28 + 19 or pBEC
A transformant in which AB28-8 was integrated was obtained.

The transformed cells thus obtained
Agrobacterium tumefaciens LBA 4404 is cultured in a LB liquid medium with shaking at 28 ° C., followed by centrifugation at 3,000 g (using Kubota RA-6) at 4 ° C., cell collection, MS liquid medium [Physiol, Plant. : 473 (1962)], which was used for plant transformation.

2. Transfection into Tobacco by the Agrobacterium Method The anti-calicivirus monoclonal antibody gene was introduced into tobacco using the Agrobacterium produced above by the leaf tisque method.

First, tobacco leaves were sterilized with a 1% sodium hypochlorite solution for 15 minutes and washed six times with sterile distilled water. From this leaf, about 1cm in diameter with a sterilized cork borer
And a leaf disk was cut out. Insert this disk in 1. Ag with pBECAB28 + 19 created in
The cells were immersed in a suspension of robacterium tumefaciens LBA 4404 in an MS liquid medium for 15 minutes. Thereafter, the MS solid medium [3%
Sucrose, 0.1 μg / mL 6-benzylaminopurine, 0.
1μg / mL naphthalene acetic acid, B5 vitamin, 0.8%
After containing the agar (pH 5.7)] at 28 ° C. for 3 days, the disc was discarded with the antibiotic kanamycin 100 μg / mL.
And 500 μg / mL of carbenicillin (both manufactured by Sigma).

After washing, the disc was placed on an MS solid medium (containing 3% sucrose) containing the above antibiotic at 25 ° C. for 2 hours.
The cells were subcultured every week (16 hours illumination, 8 hours dark).
After 4 to 8 weeks of culture, calli were formed on the disk surface, and further subcultured to induce shoots.

The shoot was cut off from the root and a hormone-free MS solid medium [3% sucrose, kanamycin 5
0 μg / mL and 500 μg / mL of carbenicillin (PH
5.7)] and cultured. Plants that have rooted after 2 to 4 weeks are placed in pots (diameter 10 cm) containing culture soil.
And cultivated in an artificial weather vessel.

3. Confirmation of introduction of the anti-calicivirus monoclonal antibody gene and expression of the gene in the regenerated plant (1) Confirmation of expression of the anti-calicivirus monoclonal antibody gene by ELISA The regenerated tobacco leaves were washed with 5 times the fresh weight of PBS- Tw
een buffer (135 mM sodium chloride, 1.5 mM potassium dihydrogen phosphate, 2.7 mM sodium chloride, 0.5%
(V / v) After grinding with Tween20, pH 7.2), 3,000 g
The supernatant obtained by centrifugation for 5 minutes (using Kubota KS-5000) was used as a crude juice. The juice was dispensed into an immunoplate (Maxisope, manufactured by Nunc) and coated at 4 ° C. overnight.

The coated plate was washed with PBS-Tw
Washed 3 times with eeen buffer. Thereafter, the mixture was diluted to a concentration of 5 μg / mL with 0.05 M sodium carbonate buffer (1.59 g of disodium carbonate, 2.93 g of sodium hydrogen carbonate, 0.2 g of sodium azide, pH 9.6 in 1 liter). Anti-c-myc monoclonal antibody (Reserch Diagro
stic.INC: mouse-derived) and immunoplate (Nunc
(Maxisope), and allowed to stand at room temperature for several hours to react.

The reacted plate was washed with PBS-Tween.
After washing three times with a buffer solution, H was diluted 400-fold with the same buffer solution.
RPO-labeled anti-goat mouse IgGF (ab ') 2 antibody (CAPE
L (manufactured by L Company) was dispensed to the plate and reacted at 37 ° C. for 3 hours. After washing the plate three times with PBS-Tween buffer, the substrate solution (7 g of citric acid monohydrate in 1 liter) was used.
, 23.9 g of disodium hydrogen phosphate, 12 water, O
0.5 g of phenylenediamine and 1 mL of aqueous hydrogen peroxide) were added to the plate and reacted at room temperature. The reaction is
After stopping with a stop solution (5% sulfuric acid aqueous solution), the absorbance at 492 nm was measured using a microplate reader (Bio-Rad model 25).
50 EIA reader). As a result, it was revealed that several regenerated tobacco individuals expressed the protein of the anti-calicivirus monoclonal antibody [FIG. 2: the vertical axis represents the absorbance at 492 nm, and the horizontal axis represents, for example, 22-. The box indicated by 5 is 22-5.
This indicates that the data is for antibodies expressed from the genes. Is an individual No. of the recombinant tobacco. (Other frames are provided with the same intention). P is
Data on a single-chain antibody expressed in Escherichia coli used as a positive control, Cont. Is
Data from non-recombinant tobacco used as a negative control, PBS shows data for PBS].

(2) Confirmation of Transcription of the Introduced Anti-Calicivirus Monoclonal Antibody Gene Product in the Tobacco Body Some of the leaves which had a reaction by ELISA were powdered using a mortar in the presence of liquid nitrogen. Ground. Add reagent ISOPLANT (Nippon Gene)
Extraction of total RNA from the above potato leaves was performed using the description in the instructions. Using 4 μg of the extracted RNA as a template, primers (5'-CCCGGGATGGGTTGGTCTTGGATT-
3 ′: SEQ ID NO: 64) and primer (5′-GAGCTCTTAC)
AAATCTTCTTCAGA-3 ′: SEQ ID NO: 66)
PCR was performed. This RT-PCR includes One-step RT
-Using PCR kit (TAKARA), 30 seconds / 94 ° C,
A thermal cycle reaction of 30 seconds / 55 ° C. and 1 minute / 72 ° C. was performed for 35 cycles. A thermal cycler (TAKARA MP-30) was used for this heat cycle reaction. A part of the sample after the reaction was subjected to 2% agarose gel electrophoresis. As a result, bands were confirmed at positions amplified from the transcript of the anti-calicivirus monoclonal antibody, and the anti-calicis virus monoclonal antibody gene was transcribed in these tobacco bodies. [FIG. 3: shows an electrophoresis image of the above transcript (28 + 19) in a Western assay. In the figure, the arrow indicates the migration position of the single-chain antibody. As a positive control, a single-chain antibody expressed in Escherichia coli was used and is indicated by (Posi) in the figure. Non-recombinant tobacco is used as a negative control, and is indicated by (Cont) in the figure.
As the primary antibody, an anti-c-myc (9E10) mouse antibody was used.
HRPO-labeled anti-mouse IgG goat antibody was used as the secondary antibody].

(3) Purification of Antibody Molecule As described above, purification of the antibody molecule obtained by introducing the antibody molecule-purified anti-calicivirus mouse monoclonal antibody variable region gene from the plant into which the antibody gene had been introduced into tobacco leaves and expressing it. went.

First, as described above, tobacco leaves in which transcription of the anti-caliche antibody gene was recognized were added to a Tris buffer solution (pH 8.
0) was added, and the mixture was ground on ice using a homogenizer.
For this ground leaf extract, 15000rpm, 30min, 4 ℃
Was performed. Ammonium sulfate was added to the supernatant obtained by this centrifugation operation so as to be 50% saturated. The mixture was stirred at 4 ° C. for several hours to insolubilize the antibody molecules by salting out, and the resulting salted-out precipitate fraction was collected by centrifugation. Next, the obtained precipitate fraction was suspended in Tris buffer, dialyzed against the same, and solvent replacement was performed.

The solution containing the substituted antibody molecule was subjected to anion exchange column chromatography,
A purified antibody molecule elution fraction was obtained by a salt concentration gradient (0.1 to 1.0 M), and this fraction was pooled.

Further, in order to increase the purity of the purified fraction containing the antibody molecule, the gene sequence of the antibody variable region
Affinity column chromatography using the c-myc tag sequence added to the 3 'side (Clontech)
To obtain an antibody molecule elution fraction purified by a pH gradient and pooled.

As described above, the purification purity of the anti-kalisi antibody recovered from tobacco leaves was increased, and the desired purified anti-karisi antibody could be obtained effectively from the antibody-transfected plant [FIG. : The purification result of the plant-expressed antibody was
It is the drawing shown by silver staining after S-PAGE. In the figure, M
Is a molecular weight marker, 1 is a DEAE ion exchange step
wise purified fraction (0.1 M NaCl)
2 is the same (0.2M NaCl) and 3 is the same (0.3M NaCl).
M NaCl), and 4 is the same (0.4 M NaCl)
5 is a pool of 1-4, c-myc Tag Affi
6 is the purified fraction (Fraction 2), and 7 is a suspension of tobacco leaves and an ammonium sulfate precipitate of IgG in a Tris buffer.]

(4) Labeling of Antibody Molecule and Detection of Calicivirus Using the Present Anti-Calici Antibody Production of HRP-Labeled Anti-Calici Antibody In the above (3), HRP was added to the purified anti-kalici antibody by the periodate method. Was labeled. That is, an aldehyde group formed by oxidizing a sugar chain of the enzyme with sodium periodate and an amino group of the antibody were reacted to obtain an HRP-labeled anti-caliche antibody.

ELISA using HRP-labeled anti-kalisi antibody
Simple Detection of Calicivirus by Method A The baculovirus-expressed calicivirus described above was bound by applying a predetermined amount to a 96-well microplate. Next, after the blocking operation by BSA,
The HRP-labeled anti-kalisi antibody was diluted and reacted. The substrate for detection is tetramethylbenzidine (hereinafter referred to as TMB).
) At 450 nm. As a result, color development by TMB was clearly observed in the plate hole to which the baculovirus-expressed calicivirus was bound.

Using the HRP-labeled anti-kalisi antibody obtained in the construction of a sandwich ELISA measurement system, a sandwich ELISA was examined.

First, the unlabeled anti-caliche antibody used for the enzyme labeling was diluted to 1 μg / mL, and allowed to bind to the surface of the microplate. After blocking with BSA and washing with PBS-0.02% Tween 20, calicivirus (described above) by baculovirus expression was serially diluted and added to the wells of each microplate. After the reaction at 37 ° C., the HRP
The labeled anti-caliche antibody was diluted to a concentration of 10 μg / mL and reacted. The chromogenic substrate used for detection was TMB, 4
It was measured at an absorbance of 50 nm.

As a result, it was shown that the anti-kalici antibody reacts in an antigen concentration-dependent manner. [FIG. 5: The calibration curve obtained by the above ELISA is shown. The abscissa indicates the concentration of the antigen (calicivirus) (lighter toward the right), and the ordinate indicates 450
nm absorbance]. The microplate used in such a sandwich ELISA is, for example,
By selecting a substance capable of covalent bonding, more sensitive detection can be performed.

Production of Colloidal Gold-Labeled Anti-Calici Antibodies By labeling the antibodies with colloidal gold, simpler and more visible detection can be performed.

First, chloroauric acid was suspended in Milli Q water and heated at 100 ° C. A sodium citrate solution was added thereto, and heating was further performed at 100 ° C. Finally, add potassium carbonate to 200 mM, and add pH.
A gold colloid solution of 9.0 was used. In this gold colloid solution,
The same unlabeled anti-caliche antibody as used in the above was added, and the reaction was carried out at room temperature for 10 to 15 minutes. After the reaction, centrifugation is performed, and 20 mM Tris-1 is used to remove unreacted antibodies.
The gold colloid to which the antibody was bound was washed using a% BSA solution (pH 8.2). Finally, this 20 mM Tris-1%
The suspension was suspended in a BSA solution (pH 8.2) to give a colloidal gold-labeled antibody.

Preparation of immunostrip A test strip for immunochromatography was prepared using the colloidal gold-labeled anti-kalisi antibody obtained in the detection.

First, the nitrocellulose membrane was cut into strips of a predetermined length, and a backing laminate was attached.
On this membrane, a predetermined concentration of anti-calicis virus antibody solution for trapping and an anti-mouse antibody as a positive control were added to each membrane.
0.5 μL was spotted. In addition, in order to apply a colloidal gold-labeled anti-calicidal antibody, a glass filter was cut along the membrane, and a colloidal gold-labeled anti-calicitic antibody solution was permeated and rapidly dried with cold air. The glass filter coated with the gold colloid-labeled anti-kalisi antibody was affixed 0.5 cm from the lower end of the membrane. Further, a filter paper for absorbing the sample was attached so as to be in contact with a glass filter coated with a colloidal gold-labeled anti-kalisi antibody.

The baculovirus-expressed calicivirus diluted with a 0.01% SDS-PBS solution was permeated through a filter paper for sample absorption and developed. At room temperature, about 10
After standing for about 15 minutes, spots exhibiting reddish purple were detected [FIG. 6: This is a drawing showing a color image showing the detection results by immunochromatography using a gold colloid-labeled anti-kalisi antibody. In the figure, the solid arrow indicates the location where anti-mouse IgG (positive control) was applied, and the dotted arrow indicates the location where anti-caliche antibody (1) or 0.01SDS-PBS (2) was applied. ].

As described above, a detection system by immunochromatography using a gold colloid-labeled anti-kalisi antibody was constructed.

[0113]

According to the present invention, a means for producing an anti-calicis virus monoclonal antibody using plant cells is provided, and an excellent means for detecting calicivirus using such an anti-calicis virus monoclonal antibody is provided.

[0114]

[Sequence List] SEQUENCE LISTING <110> Hokkaido Green-Bio Institute <120> Antibody-Forming Transgenic Plant <130> PHO6 <160> 69 <170> PatentIn Ver. 2.1 <210> 1 <211> 119 <212> PRT < 213> Calisivirus <400> 1 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Gly Tyr Tyr Glu Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 2 <211> 357 <212> DNA <213> Calisivirus <400> 2 gaggtgcagc ttcaggagtc gggacctggc ctggtggcgc cctcacagag cctgtccatt 60 acctgcactg tctctggggtt ctcattaacc agctatgata taagctggat tcgccaggat tcgcggagt 120g gcac aaattataat 180 tcagctttca tgtccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccatatatt actgtgtaag aggttactac 300 gagggttact <g> attg <tt> atgctatgca ctactggtca tcagt3 gtactac 300gagttc <acc> Ala Phe Ser Asn Pro Val Thr Leu Gly Thr 1 5 10 15 Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn 20 25 30 Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu 85 90 95 Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala 115 <210> 4 <211> 345 <212> DNA <213> Calisivirus < 400> 4 gatattgtga cgcaggctgc attctccaat ccagtcactc ttggaacatc agcttccatc 60 tcctgcaggt ctagtaagag tctcctgcat agtaatggca tcacttattt gtattg gtat 120 ctgcagaagc caggccagtc tcctcagctc ctgatttatc agatgtccaa ccttgcctca 180 ggagtcccag acaggttcag tagcagtggg tcaggaactg atttcacact gagaatcagc 240 agagtggagg ctgaggatgt gggtgtttat tactgtgctc aaaatctaga acttcctcgg 300 acgttcggtg gaggcaccaa gctggaaata aaacgggctg atgct 345 <210> 5 <211> 125 <212> PRT <213> Calisivirus <400> 5 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Leu Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Ala Ser Ser Asn Lys Val Asn Asp Tyr Thr Glu Tyr Ser Val 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Gly Pro Tyr Tyr Tyr Gly Thr Ser Gly Ala Met 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 375 <212> DNA <213> Calisivirus <400> 6 gaggtgcagc ttcaggagtc gggaggaggc ttgg tacagt ctgggggttc tctgagactc 60 tcctgtgcaa cttctgggtt caccctcagt gatttctaca tggagtgggt ccgccagcct 120 ccagggaagg gactggagtg gattgctgca agcagtaata aagttaatga ttatacaaca 180 gagtacagcg tctctgtgaa gggtcggttc atcgtctcca gagacacttc ccaaagcatc 240 ctctacctgc agatgaatgc cctgagagct gaggacactg ccatttatta ctgtgcaaga 300 gatggtcctt attactacgg tactagcggt gctatggact actggggtca aggaacctca 360 gtcaccgtct cctca 375 <210> 7 <211> 112 <212> PRT <213> Calisivirus <400> 7 Lys Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Ser Ser Thr 20 25 30 Tyr Leu His Trp Tyr Arg Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Tyr Met Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala 100 105 110 <210> 8 <211> 336 <212> DNA <213> Calisivirus <400> 8 aaaatagttc tcacccagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60 atgacctgct ctgccacctc aagtgtaagt tccacttact tgcactggta ccgacagaag 120 ccaggatcct cccccaaact ctggatttat ggcacatcca acctggcttc tggagtccca 180 gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240 gctgaagatg ctgccactta ttactgccac cagtatcatc gttccccgta catgttcgga 300 ggggggacca agctggaaat aaaacgggct gatgct 336 < 210> 9 <211> 118 <212> PRT <213> Calisivirus <400> 9 Glu Val Lys Leu Val Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Gly Asp Lys Arg Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 10 <211> 354 <212> DNA <213> Calisivirus <400> 10 gaagtgaagc tggtggagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60 tcctgcaagg cttctggata cacattcact gactacaaca tggactgggt gaagcagagc 120 catggaaaga gccttgagtg gattggatat atttatccta acaatggtgg tactggctac 180 aaccagaagt tcaagagcaa ggccacattg actgtagaca aatcctccag cacagcctac 240 atggagctcc acagcctgac atctgaggac tctgcagtct attactgtgc aacaggggat 300 aaacggtggt acttcgatgt ctggggcgca gggaccacgg tcaccgtctc ctcg 354 <210> 11 <211A <l> A <P> A <sup> 11 <211A> Thr Leu Gly Thr 1 5 10 15 Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn 20 25 30 Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu 85 90 95 Glu Leu P ro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala 115 <210> 12 <211> 345 <212> DNA <213> Calisivirus <400> 12 tctcctacat agtaatggca tcacttattt gtattggtat 120 ctgcagaagc caggccagtc tcctcagctc ctgatttatc agatgtccaa ccttgcctca 180 ggagtcccag acaggttcag tagcagtggg tcaggaactg atttcacact gagaatcagc 240 agagtggagg ctgaggatgt gggtgtttat tactgtgctc aaaatctaga acttcctcgg 300 acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgct 345 <210> 13 <211> 119 <212> PRT <213> Calisivirus <400 > 13 Glu Val Lys Leu Met Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Asp Tyr 20 25 30 Phe Met Asn Trp Met Gln Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asn Gly Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Asn Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asp Thr Ala His 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Lys Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Asp Asp Tyr Asp Asp Gly Thr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 14 <211> 357 <212> DNA <213> Calisivirus <400> 14 gaggtgaagc tgatggaatc tggacctgaa ctggtgaagc ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattaacg gactatttta tgaattggat gcagcagagc 120 catggaaaga gccttgagtg gattggacgt attaatggtt ataatggtga tactttctac 180 aaccagaact tcaagggcaa ggccacatta actgtagaca aatcttctga cacagcccac 240 atggagctcc ggagcctgac atctaaggac tctgcagtct attattgtgc aagattggat 300 gattacgacg acgggacgga ctactggggt caaggaacct cagtcaccgt ctcctca 357 <210> 15 <211> 111 <212> PRT <213> Calisivirus <400> 15 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe T hr Leu Ser Ile Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala 100 105 110 <210> 16 <211> 333 <212> DNA <213> Calisivirus <400> 16 gacattgtga tgacacagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60 ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca 120 catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc 180 aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtggaacct 240 gaagatgttg gagtgtatta ctgtcaaaat ggtcacagct ttccgctcac gttcggtgct 300 gggaccaagc tggagctgaa acgggctgat gct 333 <210> 17 <211> 16 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: signal sequencel <400> 17 Met Gly Trp Ser Trp Phe Leu Phe Leu Leu Ser Gly Gly Thr Ser 1 5 10 15 <210> 18 <211> 48 <212> DNA <213> Unknown Organism <220> <223> Description of Unknown Organism: signal sequence <400> 18 atgggttggt cttggatttt tctttttctt ctttctggtg gta cttct 48 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: spacer <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: spacer <400> 20 ggcggtggcg gttctggtgg cggtggctct ggcggtggcg gttct 45 <210> 21 <211> 10 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: antibody recognition site <400> 21 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <210> 22 <211> 30 < 212> DNA <213> Unknown Organism <220> <223> Description of Unknown Organism: antibody recognision site <400> 22 gaacagaagt tgatttctga agaagatttg 30 <210> 23 <211> 4 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: KDEL signal <400> 23 Lys Asp Glu Leu 1 <210> 24 <211> 12 <212> DNA <213> Unknown Organism <220> <223> Description of Unknown Organism: KDEL signal < 400> 24 aaggatgaac tt 12 <210> 25 <211> 279 <212> PRT <213> Calisivirus <400> 25 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Gly Thr Ser 1 5 10 15 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 20 25 30 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 35 40 45 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 50 55 60 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 65 70 75 80 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 85 90 95 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 100 105 110 Arg Gly Tyr Tyr Glu Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Asp Ile Val Thr Gln Ala Ala Phe Ser Asn 145 150 155 160 Pro Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys 165 170 175 Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln 180 185 190 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu 19 5 200 205 Ala Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 225 230 235 240 Tyr Cys Ala Gln Asn Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu Glu Ile Lys Arg Ala Asp Ala Glu Gln Lys Leu Ile Ser Glu 260 265 270 Glu Asp Leu Lys Asp Glu Leu 275 <210> 26 <211> 840 <212> DNA <213> Calisivirus <400> 26 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga ggtgcagctt 60 caggagtcgg gacctggcct ggtggcgccc tcacagagcc tgtccattac ctgcactgtc 120 tctgggttct cattaaccag ctatgatata agctggattc gccagccacc aggaaagggt 180 ctggagtggc ttggagtaat atggactggt ggaggcacaa attataattc agctttcatg 240 tccagactga gcatcagcaa ggacaactcc aagagccaag ttttcttaaa aatgaacagt 300 ctgcaaactg atgacacagc catatattac tgtgtaagag gttactacga gggttactat 360 gctatggact actggggtca aggaacctca gtcaccgtct cctcaggcgg tggcggttct 420 ggtggcggtg gctctggcgg tggcggttct gatattgtga cgcaggctgc attctccaat 480 ccagtcactc ttggaacatc agcttccatc t cctgcaggt ctagtaagag tctcctgcat 540 agtaatggca tcacttattt gtattggtat ctgcagaagc caggccagtc tcctcagctc 600 ctgatttatc agatgtccaa ccttgcctca ggagtcccag acaggttcag tagcagtggg 660 tcaggaactg atttcacact gagaatcagc agagtggagg ctgaggatgt gggtgtttat 720 tactgtgctc aaaatctaga acttcctcgg acgttcggtg gaggcaccaa gctggaaata 780 aaacgggctg atgctgaaca gaagttgatt tctgaagaag atttgaagga tgaactttaa 840 <210> 27 <211> 282 <212> PRT <213> Calisivirus <400> 27 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Gly Thr Ser 1 5 10 15 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Gly Leu Val Gln Ser Gly Gly 20 25 30 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Leu Ser Asp Phe 35 40 45 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 50 55 60 Ala Ala Ser Ser Sern Lys Val Asn Asp Tyr Thr Thr Glu Tyr Ser Val 65 70 75 80 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 85 90 95 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 100 105 110 Tyr Cys Ala Arg Asp Gly Pro Tyr Tyr Tyr Gly Thr Ser Gly Ala Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Ile Val Leu 145 150 155 160 Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Arg Val Thr 165 170 175 Met Thr Cys Ser Ala Thr Ser Ser Val Ser Ser Thr Tyr Leu His Trp 180 185 190 Tyr Arg Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr Gly Thr 195 200 205 Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 210 215 220 Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala 225 230 235 240 Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro Tyr Met Phe Gly 245 250 255 Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Glu Gln Lys Leu 260 265 270 270 Ile Ser Glu Glu Asp Leu Lys Asp Glu Leu 275 280 <210> 28 <211 > 849 <212> DNA <213> Calisivirus <400> 28 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga ggtgcagctt 60 caggagtcgg gaggaggctt ggtacagtct gggggttttcc tgagactctc ctgtcgttca t ctgtcagtca t120 catg gagtgggtcc gccagcctcc agggaaggga 180 ctggagtgga ttgctgcaag cagtaataaa gttaatgatt atacaacaga gtacagcgtc 240 tctgtgaagg gtcggttcat cgtctccaga gacacttccc aaagcatcct ctacctgcag 300 atgaatgccc tgagagctga ggacactgcc atttattact gtgcaagaga tggtccttat 360 tactacggta ctagcggtgc tatggactac tggggtcaag gaacctcagt caccgtctcc 420 tcaggcggtg gcggttctgg tggcggtggc tctggcggtg gcggttctaa aatagttctc 480 acccagtctc cagcaatcat gtctgcatct ctaggggaac gggtcaccat gacctgctct 540 gccacctcaa gtgtaagttc cacttacttg cactggtacc gacagaagcc aggatcctcc 600 cccaaactct ggatttatgg cacatccaac ctggcttctg gagtcccagc tcgcttcagt 660 ggcagtgggt ctgggacctc ttactctctc acaatcagca gcatggaggc tgaagatgct 720 gccacttatt actgccacca gtatcatcgt tccccgtaca tgttcggagg ggggaccaag 780 ctggaaataa aacgggctga tgctgaacag aagttgattt ctgaagaaga tttgaaggat 840 gaactttaa 849 <210> 29 <211> 278 <212> PRT <213> Calisivirus <400> 29 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Gly Thr Ser 1 5 10 15 Glu Val Lys Leu Val Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 20 25 30 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 35 40 45 Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 50 55 60 Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe 65 70 75 80 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 85 90 95 Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 100 105 110 Ala Thr Gly Asp Lys Arg Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 115 120 125 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Asp Ile Val Thr Gln Ala Ala Phe Ser Asn Pro 145 150 155 160 Val Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser 165 170 175 Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys 180 185 190 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala 195 200 205 Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe 210 215 220 Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 225 230 235 240 Cys Ala Gln Asn Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys 245 250 255 Leu Glu Ile Lys Arg Ala Asp Ala Glu Gln Lys Leu Ile Ser Glu Glu 260 265 270 270 Asp Leu Lys Asp Glu Leu 275 < 210> 30 <211> 837 <212> DNA <213> Calisivirus <400> 30 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga agtgaagctg 60 gtggagtctg gacctgagct ggtgaagcct ggggcttcag tgaagatatc ctgcaaggct 120 tctggataca cattcactga ctacaacatg gactgggtga agcagagcca tggaaagagc 180 cttgagtgga ttggatatat ttatcctaac aatggtggta ctggctacaa ccagaagttc 240 aagagcaagg ccacattgac tgtagacaaa tcctccagca cagcctacat ggagctccac 300 agcctgacat ctgaggactc tgcagtctat tactgtgcaa caggggataa acggtggtac 360 ttcgatgtct ggggcgcagg gaccacggtc accgtctcct cgggcggtgg cggttctggt 420 ggcggtggct ctggcggtgg cggttctgat attgtgacgc aggctgcatt ctccaatcca 480 gtcactcttg gaacatcagc ttccatctcc tgcaggtcta gtaagagtct cctacatagt 540 aatggcatca cttatttgta ttggtatctg cagaagccag gccagtctcc tcagctcctg 600 atttatcaga tgtccaacct tgcctcagga gtcccagaca ggt tcagtag cagtgggtca 660 ggaactgatt tcacactgag aatcagcaga gtggaggctg aggatgtggg tgtttattac 720 tgtgctcaaa atctagaact tcctcggacg ttcggtggag gcaccaagct ggaaatcaaa 780 cgggctgatg ctgaacagaa gttgatttct gaagaagatt tgaaggatga actttaa 837 <210> 31 <211> 275 <212> PRT <213> Calisivirus <400> 31 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Gly Thr Ser 1 5 10 15 Glu Val Lys Leu Met Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 20 25 30 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Asp Tyr 35 40 45 Phe Met Asn Trp Met Gln Gln Ser His Gly Lys Ser Leu Glu Trp Ile 50 55 60 Gly Arg Ile Asn Gly Tyr Asn Gly Asp Thr Phe Tyr Asn Gln Asn Phe 65 70 75 80 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asp Thr Ala His 85 90 95 Met Glu Leu Arg Ser Leu Thr Ser Lys Asp Ser Ala Val Tyr Tyr Cys 100 105 110 Ala Arg Leu Asp Asp Tyr Asp Asp Gly Thr Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr 145 150 155 160 Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser 165 170 175 Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu 180 185 190 Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile 195 200 205 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser 210 215 220 Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn 225 230 235 240 Gly His Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 245 250 255 Lys Arg Ala Asp Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys 260 265 270 270 Asp Glu Leu 275 <210> 32 <211> 828 <212> DNA <213> Calisivirus <400> 32 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga ggtgaagctg 60 atggaatctg gacctgaact ggtgaagcct ggggcttcag tgaagatatc ctgcaaggct 120 tctggttact cattaacgga ctattttatg aattggatgc agcagagcca tggaaagagc 180 cttgagtgga ttggacgtat taatggttat aatggtgata ctttctacaa ccagaacttc 240 aagggcaagg ccacattaac tgtagacaaa tcttctgaca cagcccacat ggagctccgg 300 agcctg acat ctaaggactc tgcagtctat tattgtgcaa gattggatga ttacgacgac 360 gggacggact actggggtca aggaacctca gtcaccgtct cctcaggcgg tggcggttct 420 ggtggcggtg gctctggcgg tggcggttct gacattgtga tgacacagtc tccagccacc 480 ctgtctgtga ctccaggaga tagagtctct ctttcctgca gggccagcca gagtattagc 540 gactacttac actggtatca acaaaaatca catgagtctc caaggcttct catcaaatat 600 gcttcccaat ccatctctgg gatcccctcc aggttcagtg gcagtggatc agggtcagat 660 ttcactctca gtatcaacag tgtggaacct gaagatgttg gagtgtatta ctgtcaaaat 720 ggtcacagct ttccgctcac gttcggtgct gggaccaagc tggagctgaa acgggctgat 780 gctgaacaga agttgatttc tgaagaagat ttgaaggatg aactttaa 828 <210> 33 <211> 44 <212> DNA <213> mouse immunoglobulin gene <400> 33 aaggcccaccagg 44ag 33c > DNA <213> mouse immunoglobulin gene <400> 34 aaggcccaac cggccatggc ccaggtrcag ctgaagsagt cagg 44 <210> 35 <211> 44 <212> DNA <213> mouse immunoglobulin gene <400> 35 aaggcccaac cggccatggc cgaggtycag ctgcarcagt ctgg 44 <210> <211> 44 <212> DN A <213> mouse immunoglobulin gene <400> 36 aaggcccaac cggccatggc ccaggtccar ctgcagcagy ctgg 44 <210> 37 <211> 44 <212> DNA <213> mouse immunoglobulin gene <400> 37 aaggcccaac cggccatggc cgargtgaag ctgrtggart <ctgg 44 <210> 211> 44 <212> DNA <213> mouse immunoglobulin gene <400> 38 aaggcccaac cggccatggc cgaggtgaag cttctcsagt ctgg 44 <210> 39 <211> 44 <212> DNA <213> mouse immunoglobulin gene <400> 39 aaggcccaac cggccatggc cgaggttcag cttcagcagt 44 <210> 40 <211> 39 <212> DNA <213> mouse immunoglobulin gene <400> 40 gggcggccgc ccagatccag gggccagtgg atagacaga 39 <210> 41 <211> 39 <212> DNA <213> mouse immunoglobulin gene <400> 41 gggcggccgc ccacacacag gggccagtgg atagaccga 39 <210> 42 <211> 39 <212> DNA <213> mouse immunoglobulin gene <400> 42 gggcggccgc ccacacccag gggccagtgg atagactga 39 <210> 43 <211> 39 <212> DNA <213> mouse immunoglobulin gene <400> 43 gggcggccgc ccgcagccag ggaccaaggg atagacaga 39 <210> 44 <211> 34 <212> DNA <213> mouse immunoglobulin gene <400> 44 ccgctagcgg g gacattgtg atgwcwcagt ctcc 34 <210> 45 <211> 29 <212> DNA <213> mouse immunoglobulin gene <400> 45 ccgctagcga tgttktgrtg acccaract 29 <210> 46 <211> 29 <212> DNA <213> mouse immunoglobulin gene <400 > 46 ccgctagcga tattgtgatg ackcaggct 29 <210> 47 <211> 29 <212> DNA <213> mouse immunoglobulin gene <400> 47 ccgctagcga trttgtgatr acccaggat 29 <210> 48 <211> 34 <212> DNA <213> mouse immunoglobulin gene <400> 48 ccgctagcgg tracattgtg ctgacmcart ctcc 34 <210> 49 <211> 35 <212> DNA <213> mouse immunoglobulin gene <400> 49 ccgctagcgg asaaawtgtk ctcacccagt ctcca 35 <210> 50 <211> 32 <212> DNA <213 > mouse immunoglobulin gene <400> 50 ccgctagctg tgacatymag atgachcagt ct 32 <210> 51 <211> 32 <212> DNA <213> mouse immunogloblin gene <400> 51 ccgctagctg tgatatmcag atgacacaga ct 32 <210> 52 <211> 32 <212 > DNA <213> mouse immunoglobulin gene <400> 52 ccgctagctg tgatattgtg ctaactcagt ct 32 <210> 53 <211> 35 <212> DNA <213> mouse immunoglobulin gene <400> 53 ccgctagctg tsaaattgtk ctcwcccagt ctcca 35 <21 0> 54 <211> 37 <212> DNA <213> mouse immunoglobulin gene <400> 54 ggggcgcgcc taactgctca ctggatggtg ggaagat 37 <210> 55 <211> 68 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 55 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga ggtgcagctt 60 caggagtc 68 <210> 56 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 56 ttaaagttca tccttcaaat cttcttcaga aatcaacttc tgttcagcat cagcccgttt 60 tatttc 66 <210> 57 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 57 ccgccagagc caccgccgac cc gacct cc gcc cct cc 210> 58 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 58 tctggtggcg gtggctctgg cggtggcggt tctgatattg tgacgcag 48 <210> 59 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 59 tctggtggcg gtggctctgg cggtggcg gt tctaaaatag ttctcacc 48 <210> 60 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 60 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga agtgaagctg 60 gtggag 66 <210> <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 61 atgggttggt cttggatttt tctttttctt ctttctggtg gtacttctga ggtgaagctg 60 atgga 65 <210> 62 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 62 tctggtggcg gtggctctgg cggtggcggt tctgacattg tgatgac 47 <210> 63 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 63 ttaaagttca tccttcaaat cttcttcaga aatcaacttc tgttcagcat cagcccgttt 60 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 64 cccgggatgg gttggtcttg gatt 24 <210> 65 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 65 gagctcttaa agttcatcct tcaaatct 28 <210> 66 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 66 gagctcttac aaatcttctt caga 24 <210> 67 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 67 gaattcaatg ggttggtctt ggat 24 <210> 68 <211> 24 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 68 aagcttaagt tcatccttca aatc 24 <210> 69 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 69 cccgggttaa agttcatcct tcaaatcttc ttcagaaatc 40

[Brief description of the drawings]

FIG. 1 is a drawing confirming the reactivity of antibodies produced by baculovirus to calicivirus.

FIG. 2 is a drawing showing the results of ELISA for a tobacco-derived product into which an anti-caliche antibody gene has been introduced.

FIG. 3 is a drawing showing an electrophoresis image of a Western assay for a tobacco-derived product into which an anti-caliche antibody gene has been introduced.

FIG. 4 shows the results of purification of plant-expressed antibodies by SDS-PAGE.
It is the drawing shown by silver staining later.

FIG. 5 is a drawing showing a calibration curve obtained by ELISA constructed using the present anti-caliche antibody.

FIG. 6 is a drawing showing a color image showing a detection result by immunochromatography using a gold colloid-labeled anti-caliche antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/569 C12P 21/08 4H045 33/577 C12N 15/00 ZNAA // C12P 21/08 5/00 C (71) Applicant 599089664 Ichiro Ueda 9-chome, Kita 9-Jo Nishi, Kita-ku, Sapporo City, Hokkaido (72) Inventor Takeshi Ken Matsumura Hokkaido Green Bio Research Institute, Inc. (72) Inventor Noriko Ichimachida No.15, Higashi 5 Line, Naganuma-cho, Yubari-gun, Hokkaido Inside the Hokkaido Green Bio Research Institute Co., Ltd. Inventor Mihoko Kato 1777-13 Shinko Nishi Nishi, Ishikari-shi, Hokkaido Inside Science Stanaka Co., Ltd. (72) Akiha Chihiro Sugimoto 13 Odahiro, Odahiro-shi, Inada-machi Nishi 2 Line 13 Utsuda Ojukusha 5 (72) Inventor Ichiro Ueda 9-9 Kita-ku, Kita-ku, Hokkaido Kita-ku, Kita 18-Jo 9-chome, Hokkaido University (72) Inventor Li Seiichi Kita-ku, Sapporo, Kita-ku, 9-chome 9-chome Hokkaido University F-term (reference) 2B030 AA02 AB03 AD08 CA06 CA17 CA19 CB02 CD03 CD06 CD10 CD13 CD17 CD21 4B024 AA01 AA08 AA11 BA51 CA01 DA01 EA04 GA30 HA15 4B063 QA01 QA18 QA19 QQ03 QQ10 QQ96 QR48 QR55 QS33 QX01 4B064 AG27 CA11 CA19 CC24 DA01 DA15 4B065 AA89X AA90Y AB01 CA14 ACA4 CA45 ACA4

Claims (8)

[Claims] 1. A plant somatic cell which has at least a gene encoding a variable region thereof among genes encoding an anti-human calicivirus monoclonal antibody in its own cell. 2. A gene encoding an anti-human calicivirus monoclonal antibody, wherein at least a gene encoding a variable region thereof comprises a gene encoding an H chain and an L chain of the anti-human calicivirus monoclonal antibody and a spacer sequence. The plant somatic cell according to claim 1, wherein the plant somatic cell is a construction gene linked through a chain. 3. An expression vector for producing an anti-human calicivirus monoclonal antibody, comprising a gene encoding the H chain and L chain of the anti-human calicivirus monoclonal antibody. 4. A plant comprising the whole or a part of the plant somatic cell according to claim 1 or 2. 5. A method for growing a plant, comprising producing an F1 plant by crossing the plant according to claim 4. 6. A gene encoding an anti-human calicivirus monoclonal antibody, which comprises at least a gene encoding a variable region thereof from a plant somatic cell, or from a plant somatic cell, An anti-human calicivirus monoclonal antibody, which is isolated and produced from a plant that is wholly or partially composed. 7. A gene encoding an anti-human calicivirus monoclonal antibody, wherein at least a gene encoding a variable region thereof is replaced with a gene encoding an H chain and an L chain of the anti-human calicivirus monoclonal antibody and a spacer sequence. 7. The anti-human calicivirus monoclonal antibody according to claim 6, which is a construction gene linked through the intermediary. 8. A virus for detecting a human calicivirus in a test object by associating an antigen-antibody reaction with the anti-human calicivirus monoclonal antibody according to claim 6 with the presence of the human calicivirus in the test object. Detection method.