JP2010013361A - Antibody for norovirus, and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody against norovirus (GII-4 strain) which is very difficult to be propagated in cultured cells or in an experimental animal, and of which antigen virus is also extremely difficult to be secured, a diagnostic agent and a treating agent of the norovirus, and virus-removing materials such as a mask, an air-conditioner filter, etc., by using the antibody. <P>SOLUTION: This antibody for the VLPs (virus like particles) of the norovirus obtained by immunizing an ostrich with the VLPs of the norovirus GII-4 prepared by using baculoviruses, and collecting the antibody against the VLPs in the blood and egg york of the ostrich at or after 6 weeks from the immunization is provided. Also, the diagnostic agent and treating agent of the norovirus, and the virus-removing materials such as the mask, air-conditioner filter, etc., by utilizing the antibody are provided. The filter maintains its activity even under a severe condition such as at 120°C and 1.2 atm for 20 min. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing an antibody against Norovirus. More particularly, the present invention relates to a method of producing antibodies against norovirus from eggs using birds, preferably ostriches.

  Norovirus is a single-stranded RNA virus that does not have an envelope classified into the Caliciviridae family, and a plurality of types such as Norovirus, Sapovirus, Lagovirus, and Besivirus are recognized.

  The virus orally infects humans and develops infectious gastroenteritis. Symptoms of affected patients are mainly vomiting, diarrhea, and fever. It usually heals in 1 or 2 days, and no sequelae remain. However, it has been reported that infants and elderly people with low immunity take time to cure and die.

  Although norovirus has been confirmed to grow in the human intestine, no experimental growth method using cultured cells has been found, and the detailed research has been delayed with respect to other viruses. It is.

  There are many virus strains of norovirus, but it is GII-4 that causes many deaths in nursing homes. That is, if an antibody against this GII-4 is prepared, diagnostic agents, therapeutic agents, and virus removal materials (such as masks and air conditioner filters) that are important public health issues can be developed.

  However, although norovirus grows in the human intestine, it is difficult to grow in cultured cells and experimental animals, and it is extremely difficult to secure a virus as an antigen.

  Thus, Patent Document 1 discloses a method for obtaining a monoclonal or polyclonal antibody based on the 66th to 78th amino acid sequences common in the capsi region of genotypes 1 to 14 of Norovirus GI.

Non-Patent Document 1 discloses an enzyme using a monoclonal antibody prepared by using virus-like particles (VLPs) expressed by a baculovirus expression system using ORF2 encoding NV capsid protein as an antigen. A method for detecting norovirus GI by immunoassay (ELISA) has been introduced. The detection kit using this monoclonal antibody can detect Genogroup I (Genogroup I, GI) and Genogroup II (Genogroup II, GII), respectively.
JP 2007-145775 A “Japanese Clinical”, Vol. 60, No. 6, pp. 1188 to 1193 (2002), Japan Clinical Co., Ltd.

  It is considered effective to use an antibody as a therapeutic agent, a diagnostic agent, or a virus removal material in the case of infection with norovirus. In industrial practice, it is necessary to produce a large amount of antibodies of uniform quality.

However, due to the property that antibodies are produced using the immune activity of the living body, the same antibody cannot be obtained if the individual changes. Currently, chicken eggs are used as antibodies against influenza viruses, but only a few antibodies can be extracted from each chicken egg, and antibodies from individual chicken eggs do not always exhibit the same characteristics. Therefore, large equipment is required for mass production, and many inspections are required after purification in order to maintain uniform quality.

  The present invention has been conceived in view of such problems, and provides a method for producing antibodies against norovirus uniformly and in large quantities.

  That is, the present invention uses ostriches to produce a large amount of an antibody against a strain called GII-4 that causes many deaths among noroviruses.

  More specifically, an ostrich is immunized with VLP of norovirus GII-4 produced using baculovirus, and antibodies in the blood and yolk of ostrich are extracted.

  By immunizing ostriches, it is possible to produce large amounts of homogeneous antibodies (IgY) from egg yolk at low cost. Approximately 400g (800 rabbits) can be produced in half a year from one female ostrich. Furthermore, since the life of an ostrich is more than 60 years, it is possible to supply a uniform antibody continuously from one ostrich. By using a large number of ostriches, it is possible to use antibodies in industrial (disposable) applications.

  Since norovirus is difficult to increase in experimental animals and cultured cells, it is difficult to obtain a sufficient amount as an antigen for antibody production. However, it is possible to produce cultured cells by genetic engineering. When the structural protein region of the norovirus genome is incorporated into baculovirus and expressed in insect cells, virus particles (VLP: virus-liked particles) can be produced. The structure of VLP is Norovirus itself and has antigenicity equivalent to that of virus particles, but has no genomic RNA inside and is hollow and not infectious.

  At present, there are more than 30 types of noroviruses that are expected to have different antigenicities from each other, and VLPs covering about 60% of them have been successfully produced. By immunizing ostriches with these VLPs, a large amount of antibodies against Norovirus can be produced.

  Based on the norovirus (GII-4) gene obtained from the diarrheal stool of a patient who developed in a nursing home, a cDNA capable of efficiently producing VLP was prepared using PCR primers for the structural protein region encoded by ORF2. It is incorporated into a baculovirus vector and VLPs are produced by insect cells. GII-4 is a norovirus strain that causes infectious gastroenteritis in nursing homes.

  VLPs obtained from this baculovirus system were immunized to female ostriches, and antibodies were purified from egg yolk. The immunization program is shown below.

Immunization program Initial immunization: Freund's complete adjuvant was mixed with 30 μg of VLP and inoculated into the lumbar muscles of female ostriches.

  Booster immunization: After the first immunization, boosters were boosted 3 times every other week. Incomplete Freund's adjuvant was mixed with 30 μg VLP and inoculated into the lumbar muscles of female ostriches.

  A norovirus gene structure and primers for amplification will be described with reference to FIG. Norovirus has three open reading frames of ORF1-3. For gene amplification by RT-PCR, a highly sensitive quantitative detection (real-time PCR) primer set near the border between the ORF1 and ORF2 regions most highly conserved in the norovirus genome, and a structural protein encoded by ORF2 Use region PCR primers. In FIG. 1, the position of the base at the 5 ′ end of the primer is indicated by the base number of Norwalk / 68 (M87661) for GI and Lordsdale / 93 / UK (X86557) for GII.

Next, antibody purification methods are shown below.
Purification of antibody (IgY) from egg yolk was performed as follows.

First, 5 times the amount of TBS (20 mM Tris-HCl, 0.15 M NaCl, 0.5% NaN ₃ ) and the same amount of 10% dextran sulfate / TBS are added to the egg yolk and stirred for 20 minutes. Then, 1MCaCl ₂ / TBS is added in the same amount as egg yolk, stirred and allowed to stand for 12 hours. Thereafter, the supernatant is recovered by centrifugation at 15000 rpm for 20 minutes. Next, ammonium sulfate is added to a final concentration of 40%, and the mixture is allowed to stand at 4 ° C. for 12 hours. Then, it centrifuges at 15000 rpm for 20 minutes, and collects the precipitate. Finally, it is resuspended in the same amount of TBS as egg yolk and dialyzed with TBS. This process enabled the recovery of 90% or higher purity IgY. 2 to 4 g of IgY could be purified from one egg yolk.

Measurement by ELISA method The antigen reactivity of the obtained antibody was measured by the following ELISA.
100 μl of 2 μg / mL VLP antigen was placed in each well of a 96-well microplate for ELISA and allowed to stand at room temperature for 2 hours. Then, after washing 3 times with PBS, 100 μl of a commercially available blocking solution (Block Ace: Dainippon Sumitomo Pharma Co., Ltd.) was placed in each well and allowed to stand for 2 hours. Thereafter, after washing three times with PBS, serial dilutions of ostrich IgY antibody extracted from ostrich blood and egg yolk before and after immunization (1 to 7 weeks) (100 μm, 200 × And 2 times serial dilution) were put in each well and left at room temperature for 1 hour.

  Then, after washing 3 times with PBS, 100 μl of peroxidase-labeled anti-ostrich IgY rabbit polyclonal antibody (self-made) was placed in each well and left for 45 minutes. After washing 3 times with PBS, color was developed for 30 minutes with a commercially available color kit for peroxidase (Sumitomo Bakelite), and the absorbance (450 nm) was measured with an ELISA plate reader. The obtained result was shown by the highest dilution ratio which becomes 2 times or more of the light absorbency value of IgY before immunization.

  As a result, a significant increase in antibody titer was observed after the first immunization, and the maximum value was maintained after 6 weeks (FIG. 2). When an ostrich is used, an antibody against 400 g of Noro VLP can be produced in half a year, and a huge amount of antibody can be provided at a low cost by using a large number of ostriches. This makes it possible to develop norovirus diagnostic agents and materials for removing norovirus (spray agents, masks, air conditioner filters, rags, toilet cleaning solutions, etc.) using antibodies.

Industrial application of anti-norovirus VLP and ostrich antibody (application to filters)
The ostrich IgY obtained at the 7th week of immunization (the highest antibody titer) was supported on a non-woven fabric.
IgY100microgram was apply | coated to the nonwoven fabric of 7 cm x 15 cm, and it was made to dry at 60 to 80 degreeC. The obtained ostrich IgY-carrying filter was dipped in PBS to elute the antibody, and the antibody activity against VLP by the ELISA method was verified. Further, an ostrich IgY-carrying filter treated at 120 ° C. and 1.2 atm for 20 minutes (commonly referred to as autoclave) was eluted with PBS to verify the antibody activity.

  The antibody activity was verified by verifying the activity of the antibody eluted from the filter carrying the four antibodies (1) to (4) below against VLP.

(1) Untreated anti-norovirus VLP antibody (before loading on non-woven fabric)
(2) Antibody eluted from the antibody-carrying filter (3) Antibody eluted from the filter treated at 120 ° C. and 1.2 atm for 20 minutes (4) Ostrich antibody before immunization The results (1) and (2) are almost equivalent Further, even in (3), the activity was maintained in a high state. In (4), no activity was observed. From this, it was proved that the activity of the anti-Norovirus VLP antibody was preserved even on the filter, and that the activity remained even after heat and pressure treatment. In other words, it was expected to be industrially usable as a product for preventing norovirus infection by carrying it on a mask or air conditioner filter (FIG. 3).

  Since the antibody of the present invention has a feature that the antibody activity is not inactivated even at a temperature of 120 ° C., it can be suitably used for industrial mass production. Specifically, norovirus can protect infection from the saliva etc. of an infected person in the mask and air filter containing the antibody of the present invention. Moreover, the spray agent and toilet cleaning agent containing the antibody of the present invention can invalidate the efficacy of norovirus. Moreover, the antibody of the present invention can itself be used as a therapeutic agent. Moreover, since the food containing the antibody of the present invention prevents the growth of norovirus, it can be suitably used for foods used in summer. Further, since the antibody of the present invention is an antibody that binds to Norovirus, it can also be used as a test kit for Norovirus.

  Since the present invention is capable of mass production, it treats norovirus therapeutics (oral drugs), and even yolk itself. It can also be used as a preventive food. Furthermore, since no highly sensitive and highly specific norovirus diagnosis is currently commercialized, it is expected to be put into practical use as a norovirus detection / diagnosis kit from feces using this ostrich antibody.

It is a figure explaining the gene structure of Norovirus, and the primer for amplification. It is the graph which measured the IgY titer in the blood and yolk after the first immunization. The relationship between the antibody concentration applied to the nonwoven fabric and the emission intensity at 450 nm when the antibody eluted from the nonwoven fabric was examined by ELISA is shown.

Claims (9)

An antibody obtained by immunizing a female ostrich with Norovirus VLP produced by insect cells and purifying the blood or egg yolk of the female ostrich. A mask comprising the antibody according to claim 1. The air conditioner filter according to claim 1. A spray comprising the antibody according to claim 1. A toilet cleaning agent comprising the antibody according to claim 1. A therapeutic agent comprising the antibody according to claim 1. A food comprising the antibody according to claim 1. A test kit comprising the antibody according to claim 1. Creating a cDNA from a PCR blimmer of the structural protein region encoded by the norovirus ORF2;
Incorporating the cDNA into a baculovirus vector and causing an insect cell to produce a VLP;
Immunizing a female ostrich with the VLP;
A method for producing an antibody comprising the step of purifying an antibody from blood or egg yolk of the immunized female ostrich.

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