JPS6047686A - Anti-influenza-virus substance agalactin, and its preparation - Google Patents

Abstract

PURPOSE:To produce novel substance, agalactin, which is a novel anti-influenza- virus substance, by culturing a Streptococcus B-group II-type strain, and extracting from the cultured cells. CONSTITUTION:Known Streptococcus B-group II-type strain is cultured in known Tod-Hewitt medium, etc., the bacterial cell is treated with N-acetylmuramidase, and the water-soluble fraction is collected to obtain the agalactin. Agalactin has the following physical properties. Appearance, white powder; molecular weight, 200,000-500,000; UV-absorption spectrum, shoulder at 205nm; color reactions, positive to anthrone, Bayer, diphenylamine-acetic acid, and Molish reactions, and negative to thiobarbituric acid reaction; solubility, easily soluble in water, slightly soluble in ethanol and methanol, and hardly soluble in chloroform, acetone and benzene.

Description

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a novel anti-influenza virus substance agalactin and a method for producing the same.

Many anti-influenza virus substances have been reported so far, but very few have been put into practical use.

The inventors have been involved in research on anti-influenza virus agents for a long time, and have now succeeded in isolating a new anti-influenza virus substance from the bacteria of Stregutokotka group 18 type U, and are calling it " It was named ``Agalactin''.

The uninvented agalactin is produced from Streptococcus group 8 type 0 bacteria as follows.

The Streptococcus group B type I bacterium used in the invention is already a known bacterium, Streptococcus B#...
It is available to many people as type/18R821 (Wl(050-59)).

According to the uninvented method, Streptococcus group B type I bacteria are first grown in a medium commonly used for culturing the bacteria, such as Todd-Hewitt.
Culture medium: Plain Heart Infusion (BE Engineering) medium. Cultivation is preferably carried out at a temperature around 37°C for 15 to 20 hours.

This culture solution is sterilized by adding formalin or the like, and the bacterial cells are collected by centrifugation or the like. The image is crushed by ultrasonication, butenase treatment, etc., and then enzymatically treated with N-acetylmuramidase. In doing so, agalactin is extracted into the aqueous solvent as a water-soluble component.

The agalactin thus obtained can be treated with enzymes such as deoxyribonuclease and ribonuclease, gel filtration, dialysis, ion exchange chromatography, electrophoresis, etc.
Further purification can be achieved using ultrafiltration or the like, alone or in combination.

The thus obtained agalactin of the present invention has the following physical properties.

a) Physicochemical properties ■Properties Two-white powder ■Specific rotation: [α] 2 knees 6 degrees (0 = 0.5.H, o
) ■Molecular weight: 200,000 to 300,000 ■Ultraviolet absorption spectrum: Has a shoulder at 205 nm (
Figure 1) Color reaction: positive for Anthrone, vial, diphenylamine-acetic acid and Molisch reactions, negative for thiobarbic acid reaction ■Solubility: easily soluble in water, slightly soluble in ethanol and methanol, chloroform, acetone and benzene dissolution.

■Thermal stability: No deactivation after heat treatment at 260°C for 24 hours and No□C for 110 minutes.

■Enzymatic degradation resistance: various glycosidases, neuraminases, pegtidase, trypsin.

Not inactivated by pronase enzymatic treatment.

■Chemical resistance: Periodic acid, 2 M+! ! , [+1DT
A.

Not deactivated by 8DS.

(2) Biological properties ■ Antiviral activity is not antagonized by concanavain a (con-A).

■It does not show toxicity to hatched chicken eggs at lQI+1g/N and to MDOK cells at 1■/ml.

■Minimum effective amount for suppressing virus proliferation: <1μf/rrtl■
Minimum effective amount for inhibiting red blood cell aggregation: <2 onq/me Minimum effective amount for inhibiting red blood cell hemolysis: <10 nf7ml Next, feeding will be explained.

Example 1 (1) Streptococcus group B type II/18R821 strain was inoculated into Todo-Hewitt medium and cultured at 37° C. for 16 hours to increase the number of bacteria. 8. Add formalin to the culture solution to a final concentration of 0.01% to sterilize it. OOOrp
Bacteria were collected into pellets by centrifugation at m for 30 minutes. Add 1 portion of Hank's liquid to this beret! The medium was then centrifuged under the same pressure to remove the medium. This operation was repeated until the culture medium lost its color. Finally, 30,
After centrifugation at 000 rpm for 2 hours, dried bacterial cells i, st' were obtained from the culture solution of 51. The cells were suspended in 100ml of Hank's solution, subjected to ultrasonication for 10 minutes, added with 1 t of pronase, and cultured with shaking at 37°C for 16 hours. At this time, the pH of the bacterial solution rapidly decreases to 0. The pH was adjusted to 7.5 with IN caustic soda, all of pronase 12 was newly added, and culture was performed in the same manner as above. This pronase treatment was carried out again, and the treatment solution k was
The cells were collected by ultracentrifugation for an hour. This bacterial cell 't'0.0
After suspending in 1M phosphate buffer (PBS) and sonicating, 1 g of bacterial cells was suspended in 1M phosphate buffer (PBS). against N-acetylmuramidase 111
9 was added, and enzyme treatment was performed at 37°C for 16 hours. This bacterial solution was ultracentrifuged for 2 hours at 30,00 Orpm to obtain a slightly viscous and transparent liquid. 100ml of this supernatant
Ethanol 4QQmA was added gently under stirring at 12-4℃, left at -20℃ for 4 hours, and 10.00Orp
The mixture was cooled and centrifuged for 30 minutes at m, and the precipitate was collected. This sediment'
f. Dissolved in 50rrLe of distilled water to obtain a slightly brown transparent agalactin solution.

(11) Deoxyribonuclease and ribonuclease were added to the agalactin solution obtained above to carry out sufficient enzymatic decomposition (no decrease in the antiviral activity of agalactin was observed due to this decomposition). This treatment solution (185% salt added 0.01M) was added to Liss buffer (pH 7,
5) Cephacryl C equilibrated with! L-6B column (2
, 5X9Q6n) and fractionation was performed at a rate of 1 ml per minute. Agalactin is eluted between the first peak (vO) and the second peak exhibiting UV (λ=254) absorption. The active fractions were collected and 61% reduced by freeze-drying. This concentrate was dialyzed overnight against oIM Tris buffer (pH 7,5) and then equilibrated with the same buffer for 4°C.
i! l Trophine (AM) column (2, OX 10
cm).

Most of the agalactin was recovered in the flowthrough, and some (nonspecifically adsorbed to impurity proteins) was adsorbed to the column. Agalactin adsorbed on the column was eluted with a salinity of around 0.1M by elution using a 1.0M NaOJ linear salt concentration gradient. After dialyzing this eluate against 0.01M Tris buffer, DI! It was layered on an iA[Ii cell p fine column, and all of the flow-through was collected. This permeate and the previously collected permeate were combined. This product was subjected to the column chromatography described above repeatedly to obtain pure agalactin.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of uninvented agalactin. The applicant Sendai Microbiological Research Institute is 200 250 300 wavelengths (nm)

Claims (1)

[Claims] 1. An anti-influenza virus substance aganctin having the following physical properties. ■Properties: White powder @ Specific rotation: [α] α knee 6° (Q = 0.5, H,
o) ■Molecular weight: 200,000 to 300,000 ■Ultraviolet absorption spectrum: Has debris at 205 nm ■Color reaction: Positive for Anthrone, Vial, diphenylamine-acetic acid and Molisch reactions, negative for thiobarbic acid reaction ■Solubility: In water Easily soluble, slightly soluble in ethanol and methanol, slightly soluble in chloroform, acetone and benzene. 2. Cultivate Stregutokotka group 18 type bacteria, treat the cultured bacteria with N-acetyl muramidase, and incubate the water with M
A method for producing agalactin, an anti-influenza virus substance, characterized by collecting its sexual components;