JPH05306238A - Specific antibody and composition for controlling infectious disease of silkworm - Google Patents

Abstract

PURPOSE:To obtain a specific antibody effective in controlling silkworm infectious disease and having binding activity to silkworm virus used in passive immunity and a silkworm infectious disease control composition containing this antibody as an active ingredient. CONSTITUTION:Objective specific antibody against pathogen (one or more kinds of viral pathogens selected from nuclear polyhedrosis virus, cytoplasmic polyhedrosis virus and flacherie) of silkworm infectious disease, preferably a hen egg antibody obtained from an egg of a hen hyper-immunized with the pathogen or a milk antibody obtained from milk of mammal such as cow, sheep and goat hyper-immunized with the pathogen, especially hen egg antibody. The objective silkworm infectious disease control composition contains its antibody as an active ingredient. The silkworm infectious disease is effectively controlled using a feed in which this antibody is blended.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibody having a specific binding activity to an infectious disease pathogen of Bombyx mori and a feed containing the antibody.

[0002]

2. Description of the Related Art Silkworms have been a source of silk since ancient times.
It is bred in large quantities all over the world. In addition, as a technique for supporting large-scale sericulture, the development of an artificial feed containing no mulberry leaves or powdered mulberry leaves added at a fixed ratio has been actively promoted. With the remarkable development of artificial feed for silkworms in recent years,
It can be said that a mass-rearing method that is comparable to the growth rate of silkworms, the uniformity of growth, the quality of the resulting cocoons, etc. has been established, compared with the case of using natural mulberry leaves.
However, on the other hand, it is said that silkworms bred with artificial feed tend to be susceptible to various infectious diseases. There are various diseases in silkworms, but viral diseases are most contagious and the damage is great. Currently, silkworm pathogenic viruses include nuclear polyhedrosis virus, cytoplasmic polyhedrosis virus,
Three types of softening virus have been reported.

Antibiotics and the like have been studied as a control measure against these viral infectious diseases, but a drug that does not affect the growth of silkworms and effectively prevents or treats the disease has not been developed yet. The only method for suppressing nuclear polyhedrosis of the silkworm by adding transfer-ribonucleic acid extracted from yeast cells to the feed (Japanese Patent Publication No. 62-3
6488) has only been reported.

As a means of preventing viral infections,
Vaccination is generally performed in humans or livestock. That is, by inoculating a human or livestock with an inactivated virus or virus constituents, the immune capacity of the individual is activated and the resistance to pathogens is increased. Vaccinated individuals prepare their own specific antibodies against the virus in preparation for subsequent infection. This specific antibody-producing ability occupies an important position in the self-defense function of higher animals such as vertebrates.

On the other hand, it is said that insects including silkworms have no ability to prepare such a specific antibody in the body.
Therefore, there is no point in vaccination for the purpose of preventing silkworm virus disease.

[0006] In addition to vaccination, passive immunization is another means for controlling viral infections. Passive immunization is a method in which a specific antibody against a virus is orally administered in advance and the subsequent infection is suppressed.

As a known example of passive immunization, prevention of dental caries using a specific antibody against dental caries (Streptococcus mutans) (J. Dent. Res., 70, 162-166, 1991)
, Rotavirus diarrhea prevention using specific antibodies against rotavirus (Microbiol. Immunol., 34, 617-
629, 1990) etc. have been reported.

[0008] In addition, in a known example of passive immunity in the field of aquaculture, the effectiveness of specific antibodies against fish infectious diseases such as prevention of yellowtail nodule disease and prevention of flounder fry intestinal tract opacification has been reported (Japanese Patent Laid-Open No. Hei 10 (1999) -242945). 1-168246).

However, there is no report on whether passive immunity is effective against viral infections of insects such as silkworms. It is difficult to analogize the effectiveness of the passive immunization method in which specific antibodies are administered to silkworms, which are completely different from vertebrates both in terms of biological function and life cycle.

[0010]

[Problems to be Solved by the Invention] The dramatic development of artificial feed that does not use natural mulberry leaves has enabled large-scale sericulture. However, there is a problem that viral infections occur more frequently than when mulberry leaves are used, which causes great damage to the sericulture industry. Antibiotics are being developed for viral infections, but
It isn't enough to see a noticeable protective effect. Also,
It has also been pointed out that the use of drugs affects the growth of silkworms.

[0011] An object of the present invention is to prevent viral infection of silkworms by the passive immunization method, that is, by administering specific antibodies to silkworms. Another object is to provide a specific antibody having a binding activity to the silkworm virus used in the passive immunization. Further, it is to provide a composition containing the specific antibody, which is effective for controlling silkworm-borne infectious diseases.

[0012]

[Means for Solving the Problems] In order to solve the above problems, the present inventors focused their attention on passive immunity as a method for controlling silkworm infectious diseases, and as a result of conducting research, The preparation was successful. In addition, the silkworm feed was prepared as a composition containing the specific antibody, and the oral immunization of the specific antibody demonstrated the passive immunity effect on the viral infection of the silkworm for the first time. That is, the present invention relates to a specific antibody used for controlling silkworm infectious disease and a composition for controlling silkworm infectious disease containing the antibody.

The silkworm infectious disease of the present invention means an infectious disease caused by a viral pathogen such as a nuclear polyhedrosis virus, a cytoplasmic polyhedrosis virus, an emollient virus and the like.

The specific antibody of the present invention means an antibody that specifically binds to a pathogen of a silkworm infectious disease, and the type of antibody is obtained from eggs of laying hens hyperimmunized with a pathogen of a silkworm infectious disease. It is a chicken egg antibody or a milk antibody obtained from milk of a mammal hyperimmunized with a silkworm infectious disease pathogen, and the antibody purity is not limited. That is, it may be a pure antibody, or in the case of a chicken egg antibody, whole egg powder, egg yolk powder, or water-soluble protein fraction powder of egg yolk containing it. In the case of a milk antibody, it may be whole milk powder, skim milk powder containing it, or whey protein powder.

The animal hyperimmunized with the pathogen of the silkworm infectious disease may be any animal capable of producing a specific antibody against the pathogen, but the antibody and its antibody used for controlling the silkworm infectious disease which is the object of the present invention. From the practical point of view of providing the composition, it is limited to laying hens or mammals such as cows, goats, and sheep that can produce large amounts of specific antibodies. Among these, hyperimmunity workability, antibody production ability,
From the viewpoint of animal breeding costs and the like, the most preferable method is to immunize a laying hen with a pathogen and obtain an antibody from the hen's egg.

As a method of hyperimmunizing laying hens, it is advisable to repeatedly administer the infectious disease pathogen of silkworm to the laying hens as an antigen to increase the specific antibody in the eggs.
As a method for hyperimmunizing mammals, it is advisable to repeatedly administer the antigen to animals such as cows, goats, and sheep to increase the specific antibody in milk.

The antigen used in this case may be prepared by a known method. For example, a large amount of antigen can be prepared by culturing a large amount of a pathogenic virus for silkworm infectious disease and then treating it with a known inactivating agent such as formalin. Alternatively, it is also possible to collect silkworms killed by an infectious disease, homogenize the affected part by a method such as homogenization, and use it as an antigen. Alternatively, for the purpose of increasing the purity of the pathogenic virus contained in the homogenate of the affected part,
It is also possible to purify the pathogenic virus and use it as an antigen by combining known methods such as centrifugation, column chromatography and filtration.

As a method for administering the antigen to the laying hen or mammal, a general administration method such as intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, or oral administration by drinking water is used, but operability and specificity are used. Intramuscular injection is preferable from the viewpoint of production efficiency of specific antibody. The antigen used for administration is generally a solution or suspension of silkworm infectious disease pathogen, or a mixture of the solution and an aluminum hydroxide gel or Freund's adjuvant.

The administration of the antigen is repeated until the amount of the antibody reaches the maximum value while examining the amount of the specific antibody appearing in the egg or the milk by a method such as enzyme immunoassay. The amount of the antibody can be maintained at a certain level or more throughout the spawning period or the lactation period by repeatedly administering the antigen to the hens or mammals at appropriate intervals.

Since the amount of the antigen to be administered depends on the type of animal to be administered, the type of antigen, etc., it is necessary to select it in a timely and preliminary test, but in the case of administration to laying hens,
Generally, an antigen amount of 10 μg to 1 mg / bird / dose is selected as the viral amount.

After the silkworm infectious disease pathogen is administered as an antigen to laying hens or mammals, hen's eggs or milk containing a specific antibody against the antigen is collected and used to control the silkworm infectious disease of the present invention. Specific antibodies to the pathogen can be prepared. When using chicken eggs, whole egg liquid or yolk liquid is separated after egg splitting, homogenized with a homogenizer, etc., and sterilized and dried by hot air or freeze drying. Can be obtained. Further, a known method for purifying an egg egg antibody from the egg yolk liquid or egg yolk powder (Japanese Patent Laid-Open No. 6-58242)
4-38098, Agric. Biol. Chem. 54 (10), 2531-253.
5, 1990, etc.), an egg yolk water-soluble protein powder or a pure antibody powder having a high specific antibody purity against the used antigen can be prepared. When milk is used, the whole milk powder containing a specific antibody against the antigen used after sterilizing the skim milk obtained by separating the milk or the lipid component in the milk with a cream separator, etc., and then using hot air drying or freeze drying Alternatively, skim milk powder is obtained. Further, from the milk or the skim milk, a whey protein powder having a high specific antibody purity against an antigen used by a known method or a pure specific antibody can be prepared.

The silkworm infection control composition of the present invention means whole egg powder, egg yolk powder, egg yolk water-soluble protein powder, chicken egg antibody pure product obtained as a specific antibody against the pathogen of silkworm infection, from eggs of hyperimmune chickens. It refers to a composition in which powder, or powdered whole milk powder, skim milk powder, whey protein powder, milk antibody pure product powder, etc., obtained from milk of a hyperimmune mammal is mixed as an active ingredient for controlling silkworm infections. As other auxiliary ingredients of the composition, vitamins, minerals, silkworm feeding attractants, antibiotics and the like can be simultaneously added.

The composition of the present invention is preferably fed in the form of slices or ribbons during the juvenile period, or ribbons or blocks during the period of larval silkworms. Whatever form the silkworm can eat. But it's okay. Alternatively, a powder containing a specific antibody against a pathogen of a silkworm infectious disease may be suspended in water together with other auxiliary components to prepare a liquid agent, which may be applied to a silkworm feed for use.

[0024]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples. Example 1. Preparation of chicken egg antibody against Bombyx mori polyhedrosis virus (antigen) Bombyx mori polyhedron (virus inclusion body) 500 mg
Was suspended in 50 ml of 0.5% aqueous sodium carbonate solution,
The mixture was stirred at 0 ° C for 30 minutes. After adjusting the pH to 8.5, 4000 rpm,
The cells were centrifuged for 15 minutes, and the supernatant was used as a crude virus purification solution to further purify the virus by the ultracentrifugation method.
That is, after ultracentrifugation at 48,000 g for 90 minutes, the obtained precipitate (virus) was suspended in 10 mM Tris-HCl (pH 8.5). The solution was centrifuged again at 4,000 rpm for 15 minutes, and the supernatant was ultracentrifuged at 48,000 g for 90 minutes. A suspension of the obtained precipitate in 10 mM Tris-HCl (pH 8.5) was finally used as a purified virus antigen solution (1 mg / ml). (Immunity) Purified virus antigen solution (1m
(g / ml) was mixed and emulsified with Freund's complete adjuvant in the same amount, and 2 ml was intramuscularly injected per animal. Immune injection was performed once a week for a total of 4 times. (Preparation of Chicken Egg Antibody) Eggs (for 5 months from 1 month after immunization) having an increased titer of specific antibody against the antigen were cleaved to obtain 4.4 kg of yolk liquid. The egg yolk liquid was pulverized by a spray-dry method (air temperature of 150 ° C., air temperature of 80 ° C.) to obtain 2.1 kg of chicken egg antibody (yolk powder) against silkworm nuclear polyhedrosis virus.

Example 2. Preparation of feed for controlling silkworm virus infection <Basic artificial feed for silkworm> Mulberry leaf powder 31% Skim soybean powder 20% Cellulose powder 20% Agar 10% Potato starch 10% Vitamin C 2% Citric acid 2% Mineral mixture 2% Propionic acid 1% Vitamin B group 1% Soybean oil 1% To 100 g of a basic artificial diet for silkworms, 240 g of water was added and mixed well, followed by autoclaving with an autoclave. 17 ml of a suspension (20 mg / ml) of chicken egg antibody (yolk powder) against silkworm nuclear polyhedrosis virus was added to 170 g of the artificial feed.
The mixture was added dropwise and thoroughly soaked to obtain 187 g of a feed for controlling silkworm virus infection.

Test Example 1. Antibody titer measurement of chicken egg antibody The antibody titer of chicken egg antibody was measured by enzyme-linked immunosorbent assay (ELISA). The basic procedure is shown below. (1) ELISA plate: 96-well polystyrene plate
(Manufactured by Nunc) was used. (2) Antigen coating: The purified virus antigen solution used for immunization was 0.05 μM carbonate buffer (pH 9.6) at 10 μg / m 2.
It was diluted to 1 and 100 μl was added to each well of the ELISA plate and left overnight at 5 ° C. (3) Washing: 200 μl / well of 20 mM phosphate buffer containing 0.5% Tween 20 (pH 7.4, abbreviated as PBS-Tween hereinafter) was added and washed. Washing was performed 4 times. (4) Blocking: 100 μl of PBS-Tween containing 10 mg / ml ovalbumin was added per well, and 37
It was left at ℃ for 1 hour. (5) Washing: The same as (3). (6) Addition of sample: A sample solution diluted with PBS-Tween was added at 100 μl / well and left at 37 ° C. for 1 hour. (7) Washing: The same as (3). (8) Addition of secondary antibody: anti-chicken IgG rabbit IgG-
Alkaline phosphatase conjugate (manufactured by Zymet) was diluted 2,000 times with PBS-Tween to give 100 μl /
The wells were added and left at 37 ° C for 1 hour. (9) Washing: The same as 3). (10) Enzymatic reaction: P-nitrophenyl phosphate.210 manufactured by Sigma was used as a substrate. Substrate 1 mg / m
0.1 M ethanolamine buffer solution (pH 9.8)
) Was added to each well and the enzyme reaction was carried out at 37 ° C. for 20 minutes, and then 50 μl / well of 2M NaOH solution was added to stop the reaction. (11) Absorbance measurement: The absorbance at 405 nm of each well was measured, and a blank (instead of the sample, PBS-Tw was used).
The value obtained by subtracting the absorbance of the well to which een was added is E
It was set as the LISA value.

The egg yolk obtained as a result of immunization in Example 1 was assayed for the specific antibody titer against silkworm nuclear polyhedrosis virus by ELISA. That is, after immunization, 1
Egg yolk is collected every week and 500 with PBS-Tween
The double-diluted product was used as a sample for ELISA. Figure 1 shows the changes in the specific antibody titer against the silkworm nuclear polyhedrosis virus. It was found that an increase in antibody titer was observed after a total of four immunizations each week.

Test Example 2. Effect of hen egg antibody on silkworm nuclear polyhedrosis Disease feed for controlling silkworm virus infection prepared in Example 2 187
Twenty silkworms on the third day of the third day were placed on the g and fed for 2 days. At this time, as a control, a group was also provided in which a chicken egg yolk powder not immunized with the antigen was added to the basic artificial diet for the silkworm at the same concentration. In addition, a group to which only the artificial artificial diet for silkworm was given was also prepared. On the other hand, disinfect the purified polyhedra (virus inclusion bodies) with penicillin and streptomycin,
The suspension in sterile water was further diluted with sterile water in 10 steps. 1 ml of this virus diluted solution 2 g of basic artificial diet for silkworm
The silkworms, which had been soaked in and were sufficiently soaked, were given to the silkworms when they turned into silkworms at the 4th instar, and they were fed for 24 hours, after which the onset of nuclear polyhedrosis was observed. Table 1 shows the incidence of nuclear polyhedrosis in the group administered with the specific egg yolk antibody (yolk powder) -added feed, the group administered with the control egg yolk powder-added feed and the group administered with no additive egg yolk powder.

[0029]

[Table 1]

When the virus inoculum was 1 × 10 ⁸ cells / ml, the control egg yolk powder-added group and the non-added group showed 100% incidence, whereas the specific egg yolk antibody-added group had an incidence of 40%. .. At the same inoculation dose of 1 × 10 ⁷ cells / ml, the incidence was 40% in the control egg yolk powder addition group and the non-addition group, while it was suppressed to 0% in the specific egg egg antibody addition group.

[0031]

EFFECTS OF THE INVENTION As the breeding form of silkworms has become larger and larger, once an infectious disease has occurred, it has been seriously damaged. Among silkworm infectious diseases, viruses are particularly damaging, and there is currently no means to completely prevent them. Antibiotics are being developed, but they have not reached a remarkable protective effect. In addition, there are still problems such as affecting the growth of the silkworm itself.
Under such circumstances, the present invention has demonstrated for the first time the effect of controlling silkworm infectious disease by passive immunization using a specific antibody against the pathogen of silkworm infectious disease.

[Brief description of drawings]

FIG. 1 shows the change in specific antibody titer against silkworm nuclear polyhedrosis virus.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takehisa Kin, 9-5 Akahori Shinmachi, Yokkaichi-shi, Mie, Taiyo Kagaku Co., Ltd. (72) Hajime Mori 27- 1 (72) Inventor Michio Himeno 23, Onoshiba, Onoshiba-cho, Sakai-shi, Osaka 3-58 (72) Inventor Keizo Hayashiya 31-1 Kita-Shirakawa-Higashiiori-cho, Sakyo-ku, Kyoto-shi, Kyoto

Claims (5)

[Claims] 1. A silkworm infection control composition and / or silkworm feed comprising a specific antibody against a silkworm infection pathogen used for controlling silkworm infection and the antibody. 2. The specific antibody according to claim 1, wherein the specific antibody is a chicken egg antibody obtained from eggs of laying hens hyperimmunized with a silkworm infectious disease pathogen. 3. The specific antibody according to claim 1, wherein the specific antibody is a milk antibody obtained from milk of a mammal hyperimmunized with a pathogen of a silkworm infectious disease. 4. The specific antibody according to claim 1, 2 or 3, wherein the pathogen of Bombyx mori infection is a viral pathogen. 5. The specific antibody according to claim 4, wherein the viral pathogen is one kind or two or more kinds selected from nuclear polyhedrosis virus, cytoplasmic polyhedrosis virus, and softening disease virus.