JPH03236747A - Artificial feed for silkworm to be infected with virus, viral infection promoting agent for silkworm and production of useful substance using the same - Google Patents

Abstract

PURPOSE:To obtain an artificial feed for efficiently infecting a host silkworm with a virus acting as a vector for the production of a valuable substance by adding chloramphenicol to an artificial feed for silkworm. CONSTITUTION:The objective artificial feed for silkworm contains chloramphenicol. The amount of the chloramphenicol is 30-200mg titer or thereabout based on 100g of dried feed. The amount is unbelievably large, that is, as large as 3 to 20 times the amount in conventional feed for preventing the infection of silkworm with common pathogenic microorganisms. Since chloramphenicol is an antibiotic substance having an action to inhibit the synthesis of cell wall, it is principally uninfluential to the proliferation of virus caused by the replication of DNA in the cell nucleus. It is expected that the abnormal metabolism of cell is induced in the silkworm used as the host of the virus and an intracellular environment suitable for the proliferation of virus is formed by the action of an extremely large amount of chloramphenicol compared with the ordinary amount.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to artificial feed and virus infection for efficiently infecting silkworms, which are hosts, with viruses, which are vectors for producing useful substances, in genetic engineering. This invention relates to accelerators and methods for producing useful substances using the same.

[Prior Art] In recent years, techniques for producing useful substances in silkworms using gene recombination techniques have been actively researched. For example, a method using silkworm nuclear polyhedrosis virus as a vector has been reported (Japanese Patent Laid-open Nos. 61-9288 and 62-20).
No. 8276, etc.).

In these methods, a recombinant virus is prepared by replacing the polyhedral gene of silkworm nuclear polyhedrovirus with a gene encoding another useful substance, and this is inoculated into 5th instar silkworms to infect them. After a few days, the recombinant virus is produced in silkworm cells during the multiplication process, and the useful substances secreted into body fluids are separated and purified.

Compared to E. coli and yeast, which have traditionally been used as hosts, this gene recombination technology using silkworms allows the production of useful substances to be secreted outside the cells, making it easier to extract useful substances. Good production efficiency (E. coli and yeast are not released outside the cells), ■ Since there is no protease in Kai3 body fluids, the useful substances produced are not degraded (
Escherichia coli and yeast are very good at producing useful substances because their cell structure is closer to that of humans than microorganisms, and the processing of the useful substances they produce is similar to that of natural products. It is seen as a promising and innovative method.

[Problems to be Solved by the Invention] However, conventional genetic recombination methods using silkworms have had major problems.

In other words, methods for inoculating silkworms with recombinant viruses include transdermal inoculation, in which the virus solution is injected into each silkworm one by one to infect them, and oral inoculation, in which the virus solution is fed along with a tolerant and the silkworms are infected through the gastrointestinal tract. Two methods are being considered.

Among these methods, the oral inoculation method is extremely efficient in the mass production of useful substances because, for example, the inoculation is completed by applying the virus solution to the artificial feed and warming it to -10°C. However, when using this oral inoculation method, the inoculated virus taken into the digestive tract of the silkworm is inactivated by the silkworm digestive fluid, and the silkworm is often not infected with the virus.

For these reasons, the current practice is to inoculate recombinant viruses transdermally, but this transdermal inoculation requires that each silkworm be injected immediately after they have molted their skin. In addition, it required skill and effort to carry out the process without damaging the silkworms, so it could not be used in the production of useful substances in the sole industry.

Therefore, in order to carry out genetic recombination technology using silkworms in industrial production, it has been desired to develop an efficient virus inoculation method.

[Means for Solving the Problems] The present inventor screened a wide range of compounds for controlling agents for silkworm virus diseases, and unexpectedly discovered that chloramphenicol, an antibiotic, promotes viral infection. I found out.

In other words, by administering chloramphenicol, ■ the infectivity of the virus does not decrease, but on the contrary increases (completely different from the effect on bacteria), and ■ the weight of silkworms increases rapidly, even abnormally, even after being infected with the virus. , ■ We found that virus proliferation occurs rapidly in a short period of time.

It has also been found that silicic acid also causes a similar effect.

The present inventors believe that if this knowledge is applied to genetic recombination methods,
We thought that we could develop an oral inoculation method that efficiently inoculates recombinant viruses, and conducted further research.

As a result, they discovered that if the recombinant virus was orally administered together with a certain amount of the antibiotic chloramphenicol, silkworms were easily infected with the recombinant virus, and the silkworms also grew rapidly. completed.

That is, an object of the present invention is to provide an artificial feed for silkworms for promoting virus infection, which is characterized by containing chloramphenicol.

Another object of the present invention is to provide a virus infection promoter characterized by carrying a recombinant virus in an artificial feed for silkworms containing chloramphenicol.

Furthermore, another object of the present invention is to raise silkworms after oral administration of a viral infection promoter carrying a recombinant virus,
The present invention provides a method for producing a useful substance, which is characterized by collecting a desired useful substance from the body fluid of a silkworm.

Chloramphenicol, which is added to the artificial feed of the present invention, is a conventionally known antibiotic.
(10 mg titer per 100 g of dry matter) is used.

However, the purpose of using chloramphenicol according to the present invention is to actively infect the virus and increase the weight of silkworms, which is completely different from its conventional use. Therefore, the amount of chloramphenicol added in the artificial feed of the present invention is also 30 g per 100 g of dry matter.
The titer is about 200 mg, which is about 3 to 20 mg of the usual amount added.
This is twice the amount, which is completely unthinkable in the past.

The chloramphenicol used in the present invention may be purified or triturated, or used for pharmaceuticals, livestock, fisheries, etc., regardless of its type, as long as it is not repellent to silkworms.

The amount of chloramphenicol added may be as described above, but it is preferable to change the amount depending on the growth status of the silkworm.
g titer, 70 to 150 per 100 g of dry matter in the 5th instar.
It is desirable to add mg titer.

In addition to the above-mentioned chloramphenicol, the silkworm artificial feed for promoting virus infection of the present invention contains various ingredients that are mixed in ordinary silkworm artificial feed, such as mulberry leaf powder, cellulose powder, defatted soybean powder, corn powder ( cornmeal)
, potato starch, defatted rice bran, various sugars, various sterins, various organic acids, various inorganic salt mixtures, various vitamins, various contact attractants, various amino acids, etc. can be added.

Among these ingredients, mulberry leaf powder promotes the feeding of artificial feed, but on the other hand, it tends to suppress virus infection and proliferation, so if the silkworm is a wide-feeding variety, the amount added should be reduced. This is desirable.

Furthermore, in addition to the above-mentioned components, the artificial feed for virus infection of the present invention may contain silicic acid for the purpose of further promoting virus infection.

This silicic acid may be in its anhydride or various crystal forms, and is blended in artificial feed (in dry matter) at about 0.1 to 5%, preferably at about 1 to 2% for 4th instar silkworms. It is preferable to add about 1 to 3% to 5th instar silkworms.

Although silicic acid may be added alone to an artificial feed that does not contain chloramphenicol, it is particularly effective when used in combination with the artificial feed of the present invention that contains chloramphenicol.

The virus infection promoter of the present invention is prepared by allowing the artificial feed obtained as described above to carry a recombinant virus.

A recombinant virus is prepared by a known method, for example, by replacing the polyhedral gene of the vector NPV with a gene encoding a target substance, and
The propagation is also carried out by known methods or in accordance therewith.

Carrying the recombinant virus is carried out by coating or impregnating the artificial feed with a solution containing the recombinant virus.

In order to infect silkworms with the recombinant virus, it is necessary to remove about 103 recombinant viruses per silkworm, preferably about 10 or more recombinant viruses per silkworm. It is necessary to determine the amount of recombinant virus to be carried on the recombinant virus.

Although this virus infection promoter can be used during the fourth instar of the silkworm, it is preferably administered after the silkworm enters the fifth instar, when the body of the silkworm increases in size and body fluid volume increases.

In order to produce a desired useful substance using the above-mentioned virus infection promoter, for example, the virus infection promoter is orally administered to 5-year-old silkworms, and after continued rearing for 6 to 8 days, body fluids are collected from the silkworms. Then, the useful substances contained in this body fluid can be separated and made into N. As a method for separating and purifying useful substances, a combination of various purification means can be employed depending on the physical properties of the intended useful substance.

[Effect] Although the effect of the present invention has not yet been elucidated, it is presumed to be based on the following mechanism.

That is, since chloramphenicol is an antibiotic that has the effect of inhibiting cell wall synthesis, it basically does not affect the proliferation of viruses due to DNA replication within the cell nucleus.

On the other hand, in the silkworm, which is the host of the virus, the action of extremely large amounts of chloramphenicol compared to the amount normally used induces abnormal cellular metabolism, resulting in an intracellular environment favorable for the proliferation of the virus. is estimated to have been formed.

Furthermore, since infection with pathogenic microorganisms is completely inhibited by administering a large amount of chloramphenicol, only the target virus can propagate advantageously within the silkworm.

Furthermore, although the details of the action of silicic acid are unknown, it is thought that it works to increase the sensitivity of silkworms to viruses more than it exerts a nutritional effect.

[Effect of the invention] The present invention facilitates virus infection by feeding silkworms with artificial feed containing chloramphenicol,
Moreover, it promotes the growth of the silkworms themselves.

Therefore, oral inoculation of recombinant viruses to silkworms, which has been considered extremely difficult in the past, can be easily carried out, and genetic recombination technology using silkworms as hosts can be carried out on an industrial scale.

Based on the present invention, the virus-inoculated silkworms grow significantly and develop disease in a short period of time by comprehensively examining and considering (1) the progress of the test silkworms, (2) virus concentration, (2) the timing of inoculation, (3) feed composition, etc. It will be more effective if you set the conditions like this.

[Examples] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 Using the artificial feed for silkworms shown in Table 1 below, a second experiment was conducted based on the results of preliminary experiments in order to investigate which ingredients of the artificial silkworm feed contribute to virus infection.
The factors shown in the table were set, and a diluted solution of Nucleopolyhedrosis NPV51-5 strain was applied to the surface of each feed, and it was orally inoculated to 2-day-old silkworms of the 4th instar for 24 hours. M
Orthogonal experiments of viral infection were performed using exchange feeding with tR food.

The silkworm variety used was N9XC9, and 2
0 animals were tested.

As a result, it was found that the administration of the antibiotic chloramphenicol, the addition of silicic acid, etc. were factors that greatly contributed to the infection of the virus.

(Feed) Table 1 [Amount per 100g of dry matter] (Setting factors) Table 2 Symbol factor 1st level 2nd level A Administration of antibiotics (per 100g of dry matter) TP'20 Synthesis from 4th to 5th instar 1st day from 5 antibiotics Umbrella: K, HPO,, Mg5O, 7HeO%Fe
PO,・4H20 and CaCO5 mixture umbrella*: Vitamin B1, Vitamin B2, Vitamin B・,
Mixture of nicotinic acid, calcium pantothenate, folic acid, choline chloride and inositol 5th age 2 to 4 days Synthetic juvenile hormone administration No E 4th age Biotin supplementation None Yes (0,0
003%> F Warm quality of silicic anhydride None With (1,5
%>TP": Antibiotic thiopeptin cp": Antibiotics chloramphenicol A to F were added to the basic feed and fed to the silkworms. In addition, reduce the amount of cornmeal equivalent to this addition amount!
jl! I did it.

Example 2 Using the polyhedral virus NPV51-6 strain as a virus strain, the ease of virus infection and body weight of silkworms with and without the addition of chloramphenicol were compared. The number of silkworms (N9XC9) used in the test was 20 per section, and the virus was 1 '3X10' per silkworm as a nuclear polyhedron.
A range of ˜1,3×10′ cells was inoculated. Two types of basic artificial feed for silkworms shown in Table 3 were used. Also,
For artificial feeds without added chloramphenicol, add 20 m of the antibiotic thiobentin per 100 g of dry feed.
g titer was added.

In addition, when producing useful substances using viruses as vectors, the absolute amount of virus multiplication becomes a problem, and furthermore, it takes 5 to 8 days from virus infection to onset of disease, so in this experiment, we used silkworms of 5th instar and 1st day. Oral inoculation was performed. The results are shown in Table 4.

(Feed) Table 3 [Containing amount per 100g of dry matter] (Leave below) (Results) From the results in Table 4, the median infectious dose log ID5o (median 1nfec-
-tive dosage) When calculated, it was 3.23 for chloramphenicol-free feed A, and the same for additive feed A.
2.05 for feed B, and 3.7 for feed B.
5.2.42. Based on this log ID5s, when comparing the difficulty of virus infection in feeds with added chloramphenicol and feeds without chloramphenicol, feed A without mulberry leaf powder is 15.1 times (101·1) more likely to have mulberry leaf powder added. It was found that Feed B was 21.4 times more infectious for the <101.33> virus. It was also revealed that feed supplemented with mulberry leaf powder has the effect of inactivating viruses.

Furthermore, the weight of silkworms was heavier in the chloramphenicol-treated group than in the non-treated group, and administration of chloramphenicol has the effect of increasing the number of silkworm cells and speeding up the growth of silkworms. The matter became clear. This results in an increase in the absolute amount of virus within each silkworm, indicating that administration of chloramphenicol is more advantageous in producing useful substances within the silkworm body. .

Example 3 Using a virus that does not have polyhedra, the improvement in virus infectivity due to the addition of chloramphenicol was investigated in the same manner as in Example 2.

As the virus, a polyhedron of the virus strain NPV51-5 was dissolved in a 0.5% NaCO solution to obtain a free state.

The results are shown in Table 5.

(Results) (Margins below) From these results, it is clear that even with the free virus obtained by dissolving the viral polyhedra, the group treated with chloramphenicol had the following differences compared to the group not treated with chloramphenicol. became.

■The onset of the disease tends to get earlier over time.

■The incidence rate is relatively high, with 10gIDao in the administration group.
The value was 4.83, whereas the logIDso of the non-administered group
The value is 6.33 (31.6 times more infectious (IQl, 5)).

■The weight of silkworms increased significantly in the treated group.

From this, it is clear that administration of chloramphenicol is effective against viral infection of silkworms and subsequent virus proliferation, and it should be used effectively for the production of useful substances using viruses as vectors and silkworms as hosts. Rukoto has been shown.

that's all

Claims (9)

[Claims] (1) Artificial feed for silkworms for promoting virus infection, characterized by containing chloramphenicol. (2) The amount of chloramphenicol in the dry matter feed is 10
The artificial feed for silkworm infection for virus infection according to claim 1, which has a titer of 30 to 200 mg per 0 g. (3) The artificial feed for silkworms for promoting viral infection according to claim 1 or 2, which does not contain mulberry leaf powder. (4) Claim 1 further comprising silicic acid
Artificial feed for silkworms for promoting viral infection as described in any one of Items 1 to 4. (5) The artificial feed for silkworms for promoting virus infection according to claim 4, wherein the content of silicic acid in the dry feed is 0.1 to 5%. (6) A virus infection promoter, characterized in that the silkworm artificial feed for promoting virus infection according to claim 1 carries a recombinant virus. (7) The virus infection promoter according to claim 6, wherein the recombinant virus is derived from silkworm nuclear polyhedrosis virus. (8) After orally administering the virus infection promoter according to claim 6 to silkworms and raising them, body fluids of the virus-infected silkworms are collected, and useful substances are isolated and purified from the body fluid. Production method. (9) Claim 8, wherein the silkworms to be administered are of 5th age.
2. Methods for producing useful substances described in Section 1.