JPH0816119B2 - Protein polysaccharide KV-071 and human rotavirus infectious disease preventive agent containing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a protein polysaccharide KV-071 having a human rotavirus infection inhibitory activity, and more specifically, for example, a culture obtained by culturing a filamentous fungus belonging to the genus Aspergillus or a human rotavirus infection obtained therefrom. The present invention relates to a protein polysaccharide KV-071 which is a component showing inhibitory activity, and a human rotavirus infection preventive agent containing the same as an active component.

BACKGROUND ART It has been confirmed that the important pathogenic virus for diarrhea of infants is rotavirus, and vaccines are being developed to control it. However, it was reported that it was completely ineffective in the trial administration of the live vaccine in the high diarrhea zone in Africa (DeMol, P. Lancet 2; 10.
8,1986). Thus, the present situation is that no effective means for preventing human rotavirus infection has been found.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention has been made in view of the above-mentioned current situation, and the present inventors have cultivated filamentous fungi, and as a result of studying the anti-human rotavirus activity of the culture solution and the microbial cell extract. Found that filamentous fungi belonging to the genus Aspergillus produce a protein polysaccharide having anti-human rotavirus activity in a culture solution. An object of the present invention is to provide such a protein polysaccharide KV-071 and a human rotavirus infection preventive agent containing the same as an active ingredient.

Means for Solving the Problems The structural feature of the present invention is, for example, a protein polysaccharide KV-0 obtained from a culture obtained by culturing a filamentous fungus that produces a substance showing a human rotavirus infection inhibitory activity belonging to the genus Alpergillus.
71 and a human rotavirus infectious disease preventive agent containing the same as the active ingredient, and a human rotavirus obtained by the active ingredient of such preventive agent as it is in the culture of such microorganism as it is or by removing cells, concentrating, fractionating, etc. It exists in a culture of a filamentous fungus containing a protein polysaccharide showing an infection-inhibiting activity.

 Hereinafter, the present invention will be described in detail.

The characteristics of an example of a filamentous fungus producing an anti-human rotavirus component belonging to the genus Alpergillus used in the present invention are described in
Although described in the specification of No. 58-35162, this strain has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 3100 (June 6, 1975).

For example, defatted soybean flour (including soybeans and soybean meal), carbon sources such as sugars, nitrogen sources, inorganic salts such as phosphates, sulfates and metal salts, and growth substances such as vitamins and amino acids are added to this strain. After inoculating various kinds of appropriately added medium, under aerobic conditions such as shaking culture, aeration culture, and static culture, for example, pH 3 to 1
The desired culture can be obtained by culturing at 0 to 30 to 40 ° C. for 5 to 7 days.

This culture has a human rotavirus infection inhibitory effect as it is, but in order to separate the active ingredient in a concentrated state, for example, a culture solution obtained by removing cells from the culture is used.
It can be obtained by sterilizing at 120 ° C. for 5 minutes in an autoclave, dialyzing the culture broth against cold water at 4 ° C. for 1 day to desalt, centrifuging, and freeze-drying the supernatant. Further, the active ingredient substance can be obtained not only by such separation but also by gel filtration, ultrafiltration and various chromatographies.

The substance of the present invention obtained as described above is the protein polysaccharide KV-071 which is a brownish-colored powder.

From the above culture, the AP described in Japanese Patent Publication No. 58-33240
The sugar composition of the protein polysaccharide separated by the same method as the method for separating S from the culture was galactose 8.02 to 8.12, mannose 2.01 to 2.35, glucose 1.98 to 2.28, arabinose 1, fucose 0.02 to 0.06, xylose 0.08. APS is different from KV-071 in that it is ~ 0.39, contains a lot of galactose, and contains fucose and xylose.

The characteristic items of the protein polysaccharide KV-071 will be described step by step below. The protein polysaccharide was obtained by the above-mentioned method such as excess of culture, dialysis of liquid, centrifugation, lyophilization of supernatant.

(A) Physical and chemical properties (1) Elemental analysis The results of elemental analysis are shown in Table 1.

(2) Color reaction KV-071 was dissolved in water and the color reaction was performed. The results are shown in Table 2.

It is clear from the color reaction in Table 2 that KV-071 contains a saccharide and a protein, the saccharide having aldohexose as the main component and no 2-deoxy sugar, amino sugar, and uron sugar.

(3) Solubility KV-071 is methanol, ethanol, acetone, chloroform, ethyl acetate, ethyl ether, benzene, n
-Insoluble in organic solvents such as hexane, but easily soluble in water and up to about 10%. Not hygroscopic.

(4) pH value KV-071 was dissolved in 100 ml of water and the pH was measured to be 6.7.
~ 7.0, almost neutral.

(B) Structural characteristics KV-071 is approximately 43% detected by the phenol-sulfuric acid method.
Saccharides detected by the lowry foreign method
% Protein part.

(1) Constitution of carbohydrate part Concerning the constituent sugar of the carbohydrate part of KV-071, after adding hydrochloric acid and methanol to the sample and performing methanolysis at 100 ° C for 16 hours, trimethylsilylation was carried out by a conventional method. It contained fucose, arabinose, xylose, mannose, galactose, and glucose. The ratios are shown in Table 3.

(2) Constitution of protein part The protein part of KV-071 was hydrolyzed according to a conventional method, and the amino acid composition was analyzed using an amino acid analyzer. The results are shown in Table 4.

(3) Molecular weight The molecular weight of KV-071 is 10,000-
It was 100,000.

As described above, the substance protein polysaccharide KV-071 according to the present invention,
Not only human Tavirus serotype 1 Wa strain, but also human Rotavirus serotype 2 KUN strain, human Rotavirus serotype 3 M
o The strain also has an infection-preventing effect and can prevent human rotavirus infection.

The following shows the test results of human rotavirus infection inhibitory effect according to the present invention, the preparation of the sample used in the test, using the defatted soybean flour medium described above strain Microindustrial Research Institute No. 3100
The cells were cultivated by aeration at 38 ° C for about 7 days, and sterilized by autoclaving at 120 ° C for 5 minutes after the culture. Further, after removing the bacterial cells with a centrifuge, the culture solution was dialyzed against cold water at 4 ° C. for 1 day for desalting. The desalted culture broth was further centrifuged to remove insoluble matter, and the supernatant was lyophilized. This freeze-dried preparation was dissolved in distilled water to give a 5% aqueous solution,
A sterile test sample was passed through a 22 μm sterilizing filter to test the effect of inhibiting humanrovirus infection.

Defatted soybean flour medium Defatted soybean flour 25 g Glucose 50 g Magnesium sulfate 0.2 g Dipotassium carbonate 0.2 g Water 1000 ml, pH 7.0 The cultured cells used for the growth of humanrovirus are monkey embryonic kidney cell-derived cell lines (hereinafter referred to as MA104). The toxicity of KV-071 aqueous solution to this cell is 0.01-1.0%.
The cell degeneration (hereinafter referred to as CPE) was observed at the concentration of 4 days. As a result, 1% showed about 30% cytopathicity from the third day, and 0.5% or less did not show CPE at all. Therefore, the test concentration of KV-071 was 0.5% or less.

Test Example 1 1 × 10 ⁵ MA104 at 100 / ml was put on a 96-well microplate.
After being dispensed in an amount of μl, the cells were cultured in a carbon dioxide incubator at 37 ° C. for 3 days, and then replaced with a culture medium in a microplate with a MEM culture medium containing 10% FCS (fetal calf serum). Next, human rotavirus serotype 1 Wa strain and various concentrations of KV-071 aqueous solution were added at 37 ° C.
Mix 100 μl of MA104 for 30 minutes, 60 minutes, or 120 minutes with MA104
The mixture was dispensed into a microplate containing the mixture and cultured at 37 ° C. for 1 hour in a carbon dioxide incubator. After 20 minutes, add 50 μl of trypsin solution at a concentration of 50 μg / ml, incubate for another 20 minutes, and then
Was replaced with the culture medium in the microphone plate. After culturing for 15-20 hours in a carbon dioxide incubator, human rotavirus-infected MA104 was measured.

Human rotavirus infection of MA104 is human rotavirus W
The number of human rotavirus-infected cells was measured with FITC fluorescent antibody using anti-guinea pig serum of strain a.

Table 5 shows the inhibition rate of human rotavirus (Wa strain) infection of KV-071 solution by such a test.

Test Example 2 The same procedure as in Test Example 1 was carried out, except that the human rotavirus serotype 1 Wa strain and KV-071 were mixed at temperatures of 37 ° C and 4 ° C.

 The results are shown in Table 6.

Test Example 3 The same procedure as in Test Example 1 was carried out, except that KUN strain of human rotavirus serotype 2 was used and the mixing conditions with KV-071 were 6 ° C and 6 ° C.
0 minutes. The number of MA104 infected with human rotavirus is KU
The measurement was performed by using FITC fluorescent antibody with N strain of anti-guinea pig serum.

The results, that is, the inhibition rate of human rotavirus (KUN strain) infection by the KV-071 solution is shown in Table 7.

Test Example 4 The same procedure as in Test Example 1 was carried out, except that the human rotavirus serotype 3 Mo strain was used and the mixing conditions with KV-071 were 37 ° C. for 60 minutes. The number of MA104 infected with human rotavirus was measured by FITC fluorescent antibody using anti-guinea pig serum of Mo strain.

The results, that is, the inhibition rate of human rotavirus (Mo strain) infection by the KV-071 solution is shown in Table 8.

Human rotavirus is one of the causes of digestive system infections and is transmitted by fecal mouth infection, so KV as a preventive agent
It is desirable to administer -071 orally as an aqueous solution, powder, tablet or capsule, and an effective amount is 3 g or less for humans, preferably 1 to 2 g. The preventive agent of the present invention is
Thus, it is used as an external disinfectant, an internal therapeutic agent, and the like.

Tables 9 and 10 show the types and results of toxicity test and the results of mutagenicity test of the dried product of the culture in which the above filamentous fungus was cultured, respectively.

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Claims (4)

[Claims] 1. A filamentous fungus of the genus Aspergillus, which has an activity of inhibiting human rotavirus infection, exhibits a molecular weight of 10,000 to 100,000 as measured by a gel filtration method, and has a sugar moiety of galactose, mannose, glucose, arabinose, fucose, It is composed of xylose, and the protein portion is composed of at least aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and arginine. The protein polysaccharide KV-071. 2. A filamentous fungus of the genus Aspergillus is a microorganism
The protein polysaccharide KV according to claim 1, which is No. 00.
-071. 3. A preventive agent for human rotavirus infection comprising the protein polysaccharide KV-071 as an active ingredient. 4. A human obtained by culturing a filamentous fungus that produces an anti-human rotavirus infectious disease component belonging to the genus Aspergillus, in which the protein polysaccharide KV-071 is cultured as it is, or obtained by removing, concentrating or fractionating the cells. The preventive agent for human rotavirus infection according to claim 3, which is a culture containing a protein polysaccharide showing rotavirus infection inhibitory activity.