JP2002165589A - Protozoa-producing cultured cell line, method for preparing the same and antigenic substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antigenic substance for antemortem diagnosis of an animal suffering from encephalitozoonosis. SOLUTION: This cultured cell line of RK-13 is parasitized and proliferated with Encephalitozoon cuniculi derived from a rabbit. The method for preparing the cultured cell line is provided. The antigen is obtained by treating the produced protozoa spore. Furthermore, the immunoassay reagent is prepared by adsorbing the resultant spore antigen on an insoluble carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a cultured cell line that produces the protozoan encephalitozone zone permanently and productively, a method for producing this parasitic cell line, and a method for treating a protozoan spore obtained from the parasitic cell line. The present invention relates to an immunoassay reagent comprising the antigen thus obtained and the protozoan spore antigen.

[0002] The encephalitozoon is a protozoan that widely infects various animals across species. In humans, it was discovered in 1993 as a new pathogen [World Health Report 19
96 (The World Health Repo
rt 1996), World Health Organization (1996)]. The parasites are of particular concern in mice, rats, guinea pigs, rabbits, and dogs, and when laboratory animals are subclinically infected, they have a significant effect on the results of animal studies conducted for various purposes. Therefore, there is a demand for the supply of completely free animals in the Ensephalit Zone.

[0003] In the case of pets, the supply of subclinically infected pets is likely to become a source of infection to humans. In particular, since rabbits are extremely sensitive and easily cause neurological symptoms and renal damage, a test for encephalitozoonosis is required in the veterinary medical field.

[0004]

2. Description of the Related Art Conventionally, in the case of infected animals, the most reliable method is to examine histopathology of a specimen after evil or euthanasia, but it is useful only for identifying the cause when a disease occurs. is there. For this reason, an immunoserum test is conceivable for prenatal diagnosis, but the test requires encephalitozoon parasite, which is a raw material of a diagnostic antigen. The easiest way to achieve this is through production of protozoa using laboratory animals.However, since this protozoa is released in the form of spores, which are the final growth body in the urine, there is always concern about contamination during infection experiments, There is a problem with the indoor washing and fumigation after the experiment because isolation from breeding animals is a problem, and spores are highly resistant to heat, drugs or chemical treatment and are not inactivated by ordinary disinfection. Become.

[0005] In addition, a special safety animal experiment facility is required, and a skilled technician who can handle both pathogens and animals at the same time even if the protozoa can be produced by animals is required. There is also a problem that it is difficult to remove a large amount of contaminants derived from animals having a strong influence.

[0006] In order to solve the above problems, it is necessary to develop a protozoan production system that does not depend on animal infection experiments.
Since the Encephalitozoon is a protozoan that grows in cells, the use of cultured cells enables the production of protozoa in vitro. If such an in vitro protozoan production system is established, not only can biohazard be prevented, but also the production of biologically uniform protozoa, the adjustment of production, and the separation and purification of antigens with extremely few contaminants can be achieved. it can.

[0007] In the past, there have been numerous reports of successful in vitro cell culture of the encephalitozon zone [Canning & Lom,
Vertebrate microsporia (The Microsp
oridia of Vertebrates, Academic Press, 1985], useful culture cells that can maintain parasite cells at a high rate and cultivate them permanently without killing host cells are Waller, Laboratory Animals (Laboratory). Animals),
9, p. 61 (1975)], only examples of MDCK cells (canine kidney cells). Rabbit-derived cells that showed a high parasite infestation rate were RCP (choroid plexus) cells, but were very sensitive and had the problem of being easily killed by passage (Canning and Rom, ibid. ).

[0008]

However, since MDCK cells are an established cell line of canine kidney cells, they are a source of antigens in immunoassays for rabbits, which are considered to be a major source of human infection. Has a problem in antigenicity, and it is preferable to use a rabbit-derived protozoan antigen produced in a rabbit-derived cell line as much as possible in terms of specificity, sensitivity, and reliability. However, as described above, no report has been found on rabbit-derived cultured cells that stably produce rabbit-derived protozoa in a prolific manner.

[0009] Therefore, the present invention provides a rabbit-derived cultured cell line that stably produces spores of rabbit-derived Encephalitozoon protozoa in a prolific and permanent manner, a method for producing the same, and a method of treating the produced protozoan spores. And an immunoassay reagent using the antigen.

[0010]

In order to achieve the above object, the present inventors first focused on a typical kidney of a rabbit with encephalitozoonosis, and obtained a cell line in which a parasite was parasitized from this organ by a primary culture method. Since the host cells eventually die in this system, the protozoan spores produced next are infected to an established cell line of the same species (from rabbits). We succeeded in producing a cultured cell line that can be produced in a continuous manner. Next, an antigen was prepared from the produced protozoan spores, an immunoassay method using the antigen was established, and it was found to be useful for serodiagnosis of rabbits with encephalitozoonosis, and the present invention was completed.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION (1) Method for Producing Protozoan-Producing Cultured Cell Lines First, kidneys are aseptically removed from rabbits with encephalitozoonosis and cut into small pieces with scissors according to the primary culture method for animal tissues. Trypsin (0.25%, Gibco, USA)
, And the enzyme digest is inoculated into a 60-mm plastic petri dish, and cultured statically in a 5% carbon dioxide incubator at 37 ° C.

Since the growing kidney cells become full of the petri dish in 3 to 4 weeks, the cells are subcultured once a week by trypsin dispersion treatment. After the third passage, the cells are cultured in a 25 cm ² plastic flask, and the presence or absence of parasitic cells is observed with an inverted microscope. Usually, parasitic cells are visually observed in the fifth to eighth generations, and a large number of protozoan spores are observed after the eighth generation. 4 in 11-14 generations
0% or more of the cells become parasitic cells and cell destruction proceeds, and it becomes difficult to maintain the cells because many cells are hyperparasitized.

[0013] In the eighth generation, the protozoa that have developed are Encephalitozonic
PCR that is known to be
(Polymerase chain reaction) and genetic diagnosis by direct sequencing. If it is Encephalitozoon kunicli, for example, purified spore DNA
Small ribosomal RNA (small ribosomal)
lRNA) gene region has a gene sequence of GenBank (G
enBank) and the gene sequence (accession No.
X98467 / L39107 / L17072 / AJ00
5581 / X98470 / L07255 / X98469
/ AF067144) should be 100% complete. When it is confirmed that the protozoa that have developed are Encephalitozoon kunikuri, proceed to the following steps.

The established cell line RK-13 cells (ATC
C number CCL-37, which can be purchased from Dainippon Pharmaceutical Co., Ltd.)
² Prepare a plastic flask. Next, the flask is inoculated with a sediment of a culture supernatant containing an appropriate number (for example, about 1 × 10 ⁵ ) of protozoan spores (the above-mentioned culture supernatant of the 11th to 14th generations is good) and cultured at a temperature of 37 ° C. The first passage (by trypsin dispersion) is performed 4 days after inoculation and the second passage is performed 7 days after the first passage.

The third passage is performed 7 days after the second passage, and the culture temperature is changed as follows. That is, the first three
Culture for 37 days is performed at 37 ° C, and culture for the following 4 days is performed at 27 ° C. The fourth culture is performed at 37 ° C. for the first three days after the passage, at 27 ° C. for the next two days, and at 30 ° C. for the remaining two days. Fifth
In the third round, the culture is performed at 37 ° C. for the first three days, and at 30 ° C. for the subsequent four days.

Usually, the parasitism rate is 20 to 4 at passages 5 to 6
In order to prevent cell destruction due to hyperparasitism, the culture temperature is kept constant at 37 ° C. for the sixth and subsequent times, and subculture is continued once a week. In this way,
A cell line that stably produces protozoan spores can be obtained at a parasitic rate of 30%. The method can also be applied for the purpose of producing a rat culture cell line that permanently produces Encephalitozoon parasites from other species, such as rats.

The growth medium that can be used for the RK-13 cell line is penicillin (500 iu / ml), streptomycin (500 μg / ml), 2 mM L-glutamine, 1 mM Na-pyruvate and 10% fetal bovine serum. Eagle's minimum essential medium (Eagl
e's Minimum Essential Med
ium) (manufactured by Gibco Corporation, USA).

(2) Preparation of antigen Protozoan spores produced from the RK-13 cell line prepared as described above have a size of 1.1 to 1.5 × 2.0.
Since it is about 2.6 μm, it can be easily recovered from the culture solution by low-speed centrifugation (for example, 3,000 rpm, 10-minute centrifugation). Furthermore, it can be easily separated and purified by a Percoll density gradient centrifugation method or the like, and is used as a raw material for an encephalitozoon antigen.

However, Encephalitozoon spores can be obtained by a conventional method for destroying animal cells such as Triton X-100, NP-
It is difficult to solubilize by treatment with a surfactant such as 40, ultrasonic waves, freeze-thaw, etc., and the antigenicity is easily lost by treatment under more severe conditions. However, it is difficult to obtain a solution such as 2-mercaptoethanol or dithiothreitol. Heat treatment for several minutes in the presence of a suitable reducing agent and sodium dodecyl sulfate (SDS) can destroy spores while maintaining antigenicity and obtain a soluble antigen.

(3) Immunoassay Reagent The antigen prepared as described above is immobilized on a suitable insoluble carrier (plastic plate, nitrocellulose membrane, latex particles, etc.) and subjected to an enzyme-linked immunosorbent assay (ELISA: Enz).
yme-linked immunosorbent
assay), Western blot (Western)
Blot) method, latex agglutination test method, and the like. In addition, for immunochromatography method, labeled substances such as various colloid particles are prepared by physical adsorption method or chemical bonding method. It can also be introduced into the components.

(4) Immunoassay methods Various immunoserum tests for detecting encephalitozoon antibodies have already been devised. For example, indirect fluorescent antibody method, carbon immunoassay (carbon immunoassay)
assay), a complement fixation test, an immunoperoxidase test, an enzyme-linked immunosorbent assay, an indirect particle agglutination test, etc. (Canning and Rom, ibid.). Because protozoa are expected to spread widely and inapparently to the animal kingdom, a measurement that eliminates as much as possible cross-reactive antibodies derived from other parasite infections as well as nonspecific reactions The method is desirable.

For example, the competitive enzyme antibody method (competi
five enzyme-linked immuno
In a sorbent assay, an appropriate amount of a serum sample is mixed with a purified specific antibody that has been labeled in advance with biotin, a fluorescent dye (fluorescein, rhodamine dye) or an isotope, and the mixture is placed in a small container in which the antigen is immobilized. After reacting for a certain period of time, the sample is positively or negatively determined by quantitatively measuring the labeled antibody that has competitively bound to the antigen. That is, since competitive inhibition is a phenomenon caused by the presence of a reactant having substantially the same binding constant thermodynamically, it can be said that a substance capable of inhibiting a labeled antibody is an encephalitozone-specific antibody contained in a serum sample.

In the Western blot method,
First, various components constituting the antigen are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to the molecular size. Next, it is immobilized electrically on an insoluble carrier such as a nitrocellulose (NC) membrane. Furthermore, the antigen-antibody complex formed by reacting the immobilized antigen with the serum sample is converted into a visible colored band by utilizing an enzyme reagent or an isotope, and finally, the presence or absence of a band and the pattern of the sample are positive or negative. This is a negative judgment, and is very reliable as a confirmation test method.

[0024]

Embodiment 1 The present invention will be described in more detail with reference to an embodiment. (B) Preparation of FFP-RK13 cultured cell line As described in “Method for preparing protozoan-producing cultured cell line”, RK-13 cells are infected with rabbit-derived encephalitozoon protozoa, and the protozoa are continuously produced. A viable cultured cell line was obtained. We named this cell line FFP-RK13. The FFP-RK13 cultured cell line has the following characteristics. (A) Culturing at 37 ° C. suppresses hyperparasitization of Encephalitozoon protozoa leading to destruction of host cells and maintains a stable infestation rate, but at a lower temperature (eg, 30 ° C. or 27 ° C.).
C) increase parasitization and increase spore production. (B) The produced protozoa are genus (genu) by genetic diagnosis.
s) is the Encephalit Zone
zoo), species are kunicuri (cun)
iculi). (C) The number of protozoan spores released into the culture solution is 25 cm ²
When cultured in a flask culture for one week, 1
0 ^6, which is a 10 ^7. (D) This cell line can be cryopreserved under ultra-low temperature of liquid nitrogen.

(B) Preparation of FFP-RK13 antigen From the culture supernatant of FFP-RK13 cultured cells, 3,000 r
Protozoan spores were collected by centrifugation at pm for 10 minutes. further,
Kilic [Kilick, Analytical Biochemistr
y), Vol. 114, p. 46 (1981)] according to the method of Percoll density gradient centrifugation.
07 g / ml, 1.12 g / ml and> 1.14 g / ml
The spores were concentrated in three ml layers, collected and washed with sterile water.

Next, the purified spores (mixed with the spores collected from the above three layers) are added to a Laemmli sample buffer (manufactured by Bio-Rad, USA) containing an appropriate amount of a reducing agent (2-mercaptoethanol). Suspended 95
The mixture was centrifuged at 10,000 rpm for 2 minutes, the supernatant was collected, the same amount of the above buffer was added to the sediment, the mixture was re-processed under the same conditions, and the supernatant was combined after centrifugation. After gel desalting, it was used as a diagnostic antigen.

(C) Preparation of antiserum Since the competitive enzyme antibody method requires a labeled-specific antibody reagent as described above, purified healthy encephalitozoon spores are frequently injected into experimental healthy rabbits without adjuvant by intravenous injection. Hyperimmunization was performed to obtain a high titer antiserum (the experiment was performed at a special safety animal experimental facility).

(D) Preparation of Biotin-Labeled Antibody An IgG fraction was obtained from the above high titer antiserum by protein A-column chromatography, and then the antibody was subjected to Gretsch et al. [Gretch et al. Chemistry (Analytical Biochemis)
try), Vol. 163, p. 270 (1987)], and biotinyl N-hydroxysuccinimide ester (biotinyl N-hydroxysuccinimide ester).
Cinimide ester, manufactured by EY Laboratories, USA) was reacted and subjected to gel filtration with a NAP-5 column.

(E) Method of antagonistic enzyme antibody method Measurement of the amount of labeled antibody bound to the antigen-sensitized well (immobilized antigen): 200 μl of the prepared antigen (1 μg / ml) dissolved in 50 mM Tris buffer was untreated. PBS was added to each well of a microtiter plate (96-well Nunc plate, manufactured by Intermed, Denmark) and left at 37 ° C. for 3 hours, followed by PBS containing Tween 20 (0.5 ml / l).
(Phosphate buffered saline) three times, and 250 μl of Super Block (blocking buffer, manufactured by Pierce USA)
and blocked at 37 ° C. for 30 minutes. Next, the biotin-labeled antibody-test serum mixture [Tween 20-PBS]
The solution contains a biotin-labeled antibody and a test serum at a final concentration of 12.5 μg / ml, each containing a 1: 100 dilution.]
0 μl was added, and reacted at 37 ° C. for 30 minutes. Tween 20
-Washed three times with PBS. In addition, streptavidin-bound peroxidase (streptavidin-p
eroxidase conjugate (manufactured by Molecular Probes, The Netherlands)) solution (1 μg / ml)
After adding 0 μl and reacting at 37 ° C. for 30 minutes, Tween 20-
Washed 4 times with PBS. 200 μl of a substrate ABTS solution [2,2-azinodi- (3-ethylbenzthiazolinesulfonic acid) -diammonium salt, manufactured by Zymed, USA] was added thereto, and the absorbance was measured at a wavelength of 415 nm using a microplate reader (Bio-Rad, Model 550, USA). (Product name).

Measurement of the amount of labeled antibody bound to the antigen-unsensitized well: Antigen-free buffer (50 mM Tris buffer) 20
The reaction was carried out under the same conditions using the same samples described above, except that wells treated with only 0 μl were used.

FIG. 1 shows a model diagram of the results obtained by the competitive enzyme antibody method.

[0032]

## EQU1 ## The competitive inhibition rate was calculated according to the following equation. Inhibition rate (%) = (TM) / T × 100 T: [measured value of (labeled antibody + negative control serum) in antigen-sensitized well] − [(labeled antibody +
Measuring value of negative control serum) M: [(labeled antibody + test serum) in antigen-sensitized wells
-[Measured value of (labeled antibody + test serum) in unsensitized wells]

(F) Results of field cases of rabbits with encephalitozoonosis by the enzyme-linked immunosorbent assay Field examples of rabbits with spontaneous onset of encephalitozoonosis 1
Table 1 shows the results of a serum sample from 0 animals and a serum sample from 20 healthy experimental rabbits tested by the enzyme-linked immunosorbent assay. The inhibition rate of 20 samples of normal rabbit serum was 0%, while the inhibition rate of 10 samples of rabbit serum of encephalitozoonosis was 3%.
2.0 to 75.3%. In other words, a clear antagonistic effect indicating the presence of the specific antibody only in the serum of the infected rabbit was observed.

[0034]

[Table 1]

(G) Method of Western Blot Approximately 100 μg of the antigen used in the above-mentioned competitive enzyme antibody method was subjected to reducing SDS-PAGE (10% mini gel, 2D well-
After developing with a size of 72 mm (W) × 1 mm, manufactured by Japan Bio-Rad Company, it was electrically transferred to an NC (0.2 μm, manufactured by Schleierschell, Germany) film. In the inspection, the antigen-adsorbed NC film was used as a strip having an appropriate width. After treatment with Super Block (trade name, manufactured by Pierce USA), immunostaining was performed. The conditions for immunostaining were the primary antibody (serum sample 1:
The secondary antibody (alkaline phosphatase-labeled anti-rabbit IgG (1,000-fold dilution from Binding Site, UK)) was for 2 hours at room temperature for 2 hours at room temperature, and washed 4 times with Tween 20-PBS. The substrate was 5-bromo-4-chloro-3-indolyl-phosphate / nitrobluetetrazolium (BCIP / NB
T) (KPI, USA) solution was used.

(H) Results of field samples of rabbits with encephalitozoonosis by Western blotting In the same manner as above, serum samples from 10 rabbits with spontaneously developing encephalitozoonosis and serum samples from 20 healthy experimental rabbits were used. Serum samples were tested by Western blot. Only antigen-adsorbed strips reacted with 10 specimens of infected rabbit serum that showed an inhibitory effect by the competitive enzyme antibody method were approximately 47
A positive pattern consisting of multiple bands centered on the band at kDa was shown. No band formation was observed in antigen-adsorbed strips reacted with normal rabbit serum samples. The results are summarized in Table 1, and a typical stained band pattern is shown in FIG.

As described above, the result of confirming the positive diagnosis by the competitive enzyme antibody method by Western blotting was obtained. That is, antibodies against various antigenic substances of Encephalitozoon protozoa were produced in the serum samples of Encephalitozoonosis rabbits, and the Western blot indicates that the specific antibody was detected as a colored band.

Further, Western blotting showed that the effective antigen component of the purified spores was a multicomponent having a major band of about 47 kDa. That is, the cultured cell line of the protozoan parasite of the present invention produces spores containing an antigen component useful for serodiagnosis.

[0039]

Industrial Applicability The cultured cell line of the present invention has a temperature-dependent protozoan-producing ability and can efficiently produce protozoan spores derived from rabbits. It has the property of being able to specifically capture antibodies. Thus, by using protozoan spore antigens as immunoassay reagents, it is possible to provide high-accuracy immunoassay reagents, and to conduct prenatal diagnosis and health surveys of pets or experimental animals, mainly rabbits, for encephalitozoonosis. Application is expected.

[Brief description of the drawings]

FIG. 1 shows a model diagram of the results obtained by the competitive enzyme antibody method using a microtiter plate as a solid phase. The degree of color tone of each well (small circle) indicates a change in color tone after the addition of the substrate, and the absorbance at 415 nm is assumed to be A1 measured value =
1.1; A2 measurement = 0.1; B1 measurement = 0.6; B
2 measured values = 0.1; C1 measured value = 0.3; C2 measured value =
0.1; D1 measured value = 0.2; D2 measured value = 0.1.

FIG. 2 shows a typical positive staining band pattern when Western blotting is applied to a field case of rabbit with encephalitozoonosis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 5/06 (C12N 1/10 C12R 1:91) C12R 1:91) (C12N 1/10 (C12N 5/02 C12R 1:91) C12R 1:91) (C12N 5/02 C12N 5/00 E C12R 1:91) C12R 1:91)

Claims (6)

[Claims] 1. A cultured cell line that produces Encephalitozoon parasites permanently and productively. However, this cultured cell line must be a cell line that has the property of suppressing hyperparasitosis at 37 ° C. after parasitism has been established, and of adjusting parasitism promotion at lower temperatures. 2. The cultured cell line according to claim 1, which is an FFP-RK13 cultured cell line. 3. A method for producing the parasitic cell line according to claim 1 or 2. 4. An antigen obtained by treating a protozoan spore produced from the cultured cell according to claim 1 or 2. 5. An immunoassay reagent comprising the antigen according to claim 4 bound to a solid phase or a chemical substance. 6. The immunoassay reagent according to claim 5, wherein the immunoassay reagent is an immunoassay reagent for a competitive enzyme antibody method and a Western blot method.