JPH09173053A - Yeast variant highly sensitive to immunosuppressive agents and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new yeast variant comprising Saccharomyces cerevisiae, whose multiplication is suppressed by an immunosuppressive agent FK506 of a specific concentration, and which is used for determining effects of immunosuppressive agents or screening the agents. SOLUTION: This new yeast variant highly sensitive to immunosuppressive agents comprises SU3 strain (FERM P-15362) of Saccharomyces-cerevisiae whose multiplication is suppressed by 0.1μg/ml of an immunosuppressive agent FK505, and useful as a reagent for determining effects of immunosuppressive agents or screening the agents. This yeast variant is obtained by using DV3T-A strain which is a malfunctioned variant of tonoplast, H<+> -ATPase, of Saccharomyces- cerevisiae as a parent strain, culturing the parent strain on a medium that contains ethylmethanesulfonic acid(EMS) as a mutagen to mutate the parent strain, screening the living germs on a culture medium containing the immunosuppressive agent FK506 in concentration of 0.1μg/l, and selecting the multiplication- suppressed strain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring or screening the effect of an immunosuppressant using yeast.
And a yeast used therefor.

[0002]

2. Description of the Related Art T-cells have a signal transduction system mediated by Ca ⁺⁺ / calmodulin-dependent phosphorylating enzyme, and an immunosuppressive effect is produced by specifically inhibiting this enzyme (Schreiber (1992), Cell. Vol.70, 365-368 ； Liu
(1991), Cell, Vol.66, 807-815). In addition, the yeast Saccharomyces cerevisiae
It is known that e ) also has a signal transduction system similar to that of T-cells (Cardenas et al. (1994), Drug Discov.
ery and Design, Vol.2, 102-126).

To measure the immunosuppressive action of drugs,
It is most straightforward to actually observe its growth inhibition with T-cells. However, maintaining T-cells and aseptic manipulation are difficult and costly. Therefore, it is conceivable to use yeast instead of T-cells to measure the effect of the immunosuppressant. However, no susceptibility to immunosuppressants is observed in wild strains of yeast.

[0004]

Related Art As a result of various studies, the present inventors have shown that the vacuolar membrane H ⁺ -ATPase of Saccharomyces cerevisiae yeast.
It was found that the function-deficient mutant strain has sensitivity to an immunosuppressant (Japanese Patent Application No. 6-116731).

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a novel yeast strain which is further highly sensitively suppressed by an immunosuppressant,
Another object of the present invention is to provide a method for measuring the effect of an immunosuppressive agent and a method for screening using the same, and a reagent therefor.

[0006]

In order to solve the above problems, the present invention provides 0.1 μg / ml of immunosuppressive agent FK506.
The present invention provides a Saccharomyces cerevisiae yeast, the growth of which is suppressed. As a typical yeast, Saccharomyces
Cerevisiae SU3 strain (FERM P-15362) can be mentioned.

The present invention also comprises the above yeast,
The present invention relates to a reagent for measuring the effect of an immunosuppressant or for screening. The present invention further comprises a method for measuring or screening the effect of an immunosuppressive agent, which comprises culturing the yeast in the presence of a test sample, and observing whether or not the yeast grows or the degree of growth. provide.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION To obtain yeast highly sensitive to immunosuppressants, as a starting material, vacuolar membrane H ⁺ -ATPa is used.
A se function-deficient mutant strain, for example, a yeast strain described in the specification of Japanese Patent Application No. 6-116731 can be used. In order to obtain the immunosuppressant highly sensitive strain of the present invention, the above yeast strain is used as a starting material, this is subjected to mutation treatment according to a conventional method, and then grown in the absence of the immunosuppressant, but immunosuppressed at a predetermined concentration. An agent, for example, a mutant strain that does not grow or substantially suppresses growth in a medium containing 0.1 μg / ml of FK506 may be selected.

As the mutagen, ethidium bromide, ethylmethanesulfonic acid (EMS), N-methyl-N'-
Nitro-N-nitrosoguanidine (MNNG), bromodeoxyuridine (BrdU), UV irradiation and the like can be used, for example, Sherman et al. (1986), Methods in Y.
east Genetics, Cold Spring Harbor Laboratory Pres
s, Cold Spring Harbor, New York, can be used. To test the test substance, for example, a plurality of solid medium containing the test substance at various concentrations is prepared, the surface of which is inoculated with the yeast of the present invention,
It suffices to observe at what concentration of the test substance the growth of yeast is inhibited on the solid medium. Alternatively, one kind of solid medium containing a test substance at a predetermined concentration may be prepared, and the surface of the medium may be inoculated with the yeast of the present invention to observe whether or not yeast growth occurs. Alternatively, a plurality of liquid mediums containing test substances at various concentrations are prepared, and the yeast of the present invention is inoculated and cultured in the liquid medium. Determine whether to stop growth.

Alternatively, one kind of liquid medium containing a test substance at a predetermined concentration is prepared, and the yeast of the present invention is inoculated into the medium and cultured, and the turbidity is measured to determine the extent to which the test substance inhibits the growth of yeast. You can decide. In the above measurement, a conventional medium used for culturing yeast can be used. For example, YPD medium (yeast extract 1%,
Peptone 2%, glucose 2%) can be used as basal medium, solid medium is approximately 2% above YPD medium.
It can be prepared by adding agar.

[0011]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG . Acquisition of Mutant Strains As a parent strain for the treatment of mutations, a functional defective mutant strain DV of S. cerevisiae vacuolar membrane H ⁺ -ATPase
The 3T-A strain (the same as the DV3TL-B2 strain described in Japanese Patent Application No. 6-116731) was used. The genotype of this strain is MATa, leu2, ADE2, lys2, his
3, trp1, ura3, vma3 :: TRP1.

The above yeast strain was subjected to a logarithmic growth phase (1-2 × 10 ⁶ cells / ml) in YPD medium (1% yeast extract (Difco), 2% bacto-tryptone, 2% glucose (Wako Pure Chemical Industries, Ltd.)). ), And using EMS as a mutagen, She
rman et al. (1986), Methods in Yeast Genetics, Cold Spr.
Muting treatment was carried out by the method described in ing Harbor Laboratory Press. The survival rate after mutation was 30 to 50%. After the mutagenesis treatment, 1.4 × 10 ⁷ cells were treated with 100
The cells were cultured at 30 ° C. for 5 days in YPD medium (100Ca medium) supplemented with mM CaCl ₂ .

In this way, one strain that grows in 100Ca medium is
Fifteen strains were isolated. These isolated mutants were added to YPD medium (YPD pH 5.0 medium) adjusted to pH 5.0 with 20 mM succinic acid and NaOH to obtain 0.1 μg / ml of FK50.
Culturing was carried out in a medium supplemented with 6, and a strain that could not grow on this medium was further selected to isolate one immunosuppressant hypersensitive strain. This mutant strain was named Saccharomyces cerevisiae SU3, and was founded by FERM
Deposited as P-15362.

Example 2 Characteristics of mutant strain YPD agar medium containing 0, 0.1 or 1 µg / ml of immunosuppressant FK506, and parent strain DV3T-A on YPD pH 5.0 agar medium containing 0, 0.1 or 1 µg / ml of FK506. And the mutant strain SU3 were inoculated and cultured at 30 ° C. for 72 hours, and the degree of yeast growth was observed. The results are shown in Table 1 below.

[0015]

[Table 1]

As is clear from Table 1, the DV3T-A strain suppressed growth in a YPD medium in a FK506 concentration-dependent manner and was inhibited at a FK506 concentration of 1 μg / ml, but in the YPD pH 5.0 medium, the FK506 strain was suppressed. Concentration 1 μg
/ Ml also grew. On the other hand, the growth of the mutant strain SU3 of the present invention was inhibited by 0.1 μg / ml of FK506 in any medium.

Example 3 YPD pH5.0 liquid medium containing immunosuppressant FK506 at various concentrations was prepared, and DV3T-A strain or SU3 strain was inoculated into the medium and cultured at 30 ° C. for 24 hours, and then the absorbance (turbidity at 600 nm was measured. ) Was measured, the result was as shown in FIG.
U3 is highly sensitive to immunosuppressants, and measuring the extent of its proliferation proved useful for measuring and screening the effects of immunosuppressants.

[Brief description of the drawings]

FIG. 1 shows the DV3T-A strain and the mutant strain S of the present invention.
It is a graph which shows the relationship between the proliferation of U3 and the concentration of the immunosuppressant FK506.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1: 865)

Claims (4)

[Claims] 1. An immunosuppressant FK506 at 0.1 μg / ml.
Saccharomyces cerevisiae yeast, the growth of which is suppressed by Saccharomyces cerevisiae . 2. The yeast according to claim 1, which is Saccharomyces cerevisiae SU3 strain (FERM P-15362). 3. A reagent for measuring the effect of an immunosuppressive agent or for screening, which comprises the yeast according to claim 1 or 2. 4. The effect of an immunosuppressive agent, which comprises culturing the yeast according to claim 1 or 2 in the presence of a test sample and observing whether or not the yeast has grown or the degree of growth. Measuring or screening method.