JPH11225783A - Toxoplasma gondii antigen, antibody to the antigen, and immunoassay using the antigen or antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antigen having a specific amino acid sequence and also having an amino acid sequence exhibiting antigenicity specific to Toxoplasma gondii, and useful as e.g. a marker for diagnosing Toxoplasma gondii infections and judging the therapeutic effect of such infections. SOLUTION: This antigen is a new Toxoplasma gondii antigen having an amino acid sequence of the formula, or an amino acid sequence wherein one or more amino acid residues are substituted, deleted, inserted and/or added, in the above amino acid sequence, and also having an amino acid sequence exhibiting antigenicity specific to Toxoplasma gondii, being useful as e.g. a marker for diagnosing Toxoplasma gondii infections such as toxoplasmosis, and judging the therapeutic effect of such infections. The antigen is obtained by the following procedure: a cDNA library derived from Toxoplasma gondii RH strain is screened using human hsp70 DNA as probe, and the resulting hsp-like proteinic gene is integrated into an expression vector followed by expression in host cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel Toxoplasma gondii antigen, a nucleic acid encoding the antigen, a recombinant vector containing the nucleic acid, an antibody against the antigen, and the use of the antigen or the antibody. The present invention relates to a method for immunoassay of an anti-Toxoplasma gondii antibody or a Toxoplasma gondii antigen. INDUSTRIAL APPLICABILITY The present invention can be used for diagnosis of an infection caused by Toxoplasma gondii.

[0002]

2. Description of the Related Art At present, several antigen regions have been defined for antigens specific to each type of Toxoplasma gondii.
8, 64, 61, 54, 52, 42, 30, 24, 6K
Da proteins are known. As cyst-type insect-specific antigens, 36, 34, 21, and 18 KDa proteins are known. Further, sporozoite-specific antigens include 25 and 67 KDa. Among these various antigens, the lesion in toxoplasmosis is caused by the proliferation of proliferative parasites, so from the clinical aspect, various anti-toxoplasma specific antibody detection reagents use proliferative parasite antigens There are many examples. Also recently,
A reagent using a synthetic peptide containing an antigenic epitope of P30, which is a toxoplasma membrane antigen, which is one of the proliferative insect body antigens, using a molecular biological technique, and an antigen epitope of those regions also exists.

[0003] Toxoplasma is the blood of mammals and birds,
It is an intracellular protozoan parasitizing the cells of tissues and is widely distributed throughout the world. In its life history, oocysts are produced by sexual reproduction in the mucosal epithelial cells of the cat, the ultimate host, and are excreted in the stool. The oocyst who came to the outside world eventually produces eight sporozoites and becomes a mature oocyst and becomes infectious. Sporozoites are released in the intestinal tract by oral infection of mature oocysts, invade from the intestinal wall, become proliferative parasites, and propagate throughout the body while proliferating.

[0004] In the acute phase of toxoplasmosis, the proliferative body produces fever and lymphadenopathy, causing chorioretinitis, pneumonia, myocarditis, and the like. A cyst containing cyst-type worms is formed in the stomach, and the infection is continued quietly with almost no growth.

[0005] Toxoplasma gondii vary depending on the region and age, but also in Japan, from several tens percent to 70 to 70%.
It is an important protozoan infectious disease with an infection rate of 80%. The most disastrous toxoplasmosis is congenital toxoplasmosis.

[0006] Toxoplasmosis, which is attracting attention as an opportunistic infection of AIDS, has recently been said to be important as an iatrogenic disease. That is, when an untransfected organ transplant patient receives a transplant of an organ infected with toxoplasma, acute toxoplasmosis is caused. Immunosuppressants used to reduce organ transplant rejection also reduce the immune response of the transplant patient to infection, resulting in severe toxoplasmosis when infected by transplantation of infected organs, which is fatal. Often.

[0007] Toxoplasma gondii can proliferate and survive by intracellular parasitism. Macrophage infection is important from the aspect of protective immunity in the early stage of infection. When macrophages phagocytose foreign substances, they form phagosomes, which eventually fuse with lysosomes, become phagolysosomes (secondary lysosomes), and undergo enzymatic degradation by proteases. Intracellular parasites escape this foreign body digestion mechanism.

In the case of Toxoplasma gondii, upon invasion and infection of macrophages, the phagosome membrane is modified by the Toxoplasma gondii that has entered the cells, and
-Creates specific intracellular structures called phorous vacuoles. This parasitophorous vacuole membrane lacks host cytoplasmic membrane molecules. Eventually, a cord-like structure called a parasito phorous network is formed by substances secreted from the leptory. It is thought that it functions to maintain the nutrition and environment necessary for the survival and growth of Toxoplasma gondii. In addition, parasitophorous vacuoles infected with living Toxoplasma gondii are not attacked by proteases that fuse with lysosomes. On the other hand, in the case of dead Toxoplasma, vacuoles (phagosomes) taken up by macrophages fuse with lysosomes and undergo enzymatic digestion.

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel Toxoplasma gondii antigen which has not been known hitherto. Further, an object of the present invention is a nucleic acid encoding the novel Toxoplasma gondii antigen of the present invention,
An object of the present invention is to provide a recombinant vector containing the nucleic acid, an antibody against the antigen, and a method for immunoassaying an anti-Toxoplasma gondii antibody or a Toxoplasma gondii antigen using the antigen or the antibody.

[0010]

Means for Solving the Problems As a result of earnest study, the present inventors have found that Toxoplasma gondii contains human hsp7
0 protein (Hunt, C., and RI Morimoto. 1985. Conse
rved features of eukaryotic hsp70 genes revealed b
y comparison with the nucleotide sequenceof human
hsp70. Proc. Natl. Acad. Sci. USA. 82; 6455-6459.
The present inventors have found that there is a novel antigen somewhat similar to the above), succeeded in the cloning and expression of DNA encoding the antigen, and purified the antigen, thus completing the present invention.

[0011] That is, the present invention relates to SEQ ID NO: 1 in the sequence listing.
Or the amino acid sequence represented by
Or an amino acid sequence in which a plurality of amino acid residues have been substituted, deleted, inserted and / or added, and comprising an amino acid sequence exhibiting antigenicity specific to Toxoplasma gondii. Provide an antigen. The present invention also provides a nucleic acid encoding the above-mentioned Toxoplasma gondii antigen of the present invention. Furthermore, the present invention provides a recombinant vector containing the nucleic acid of the present invention and capable of expressing the Toxoplasma gondii antigen according to any one of claims 1 to 3 in a host cell. Furthermore, the present invention provides an antibody against the above-mentioned Toxoplasma gondii antigen of the present invention. Further, the present invention provides an immunoassay method for measuring an anti-Toxoplasma gondii antibody in a sample using an antigen-antibody reaction between the above-mentioned Toxoplasma gondii antigen of the present invention and an anti-Toxoplasma gondii antibody in the sample. . Furthermore, the present invention provides an immunoassay method for measuring Toxoplasma gondii antigen in a sample by utilizing an antigen-antibody reaction between the antibody of the present invention and a Toxoplasma gondii antigen in the sample.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION
One preferred example of the novel Toxoplasma gondii antigen of the present invention has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

[0013] Generally, in a protein antigen,
Even if a small number of amino acid residues are substituted or deleted, or a small number of amino acid residues are inserted or added in the amino acid sequence of the protein, it has almost the same antigenicity as the original protein. It is well known to those skilled in the art. Accordingly, an antigen having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and which is specific for Toxoplasma gondii An antigen consisting of an amino acid sequence that exerts a sex is also included in the scope of the present invention. Here, “antigenicity specific to Toxoplasma gondii” means that an antigen-antibody reaction is specifically performed with an antibody against a protein having the amino acid sequence represented by SEQ ID NO: 1. Especially,
Since a polypeptide having about 5 amino acid residues or more is known to exhibit antigenicity, a part of the amino acid sequence represented by SEQ ID NO: 1 is naturally included in the scope of the present invention. In this case, if the number of amino acid residues is too small, the possibility of cross-reaction with other proteins increases, so the number of amino acid residues is preferably 30 or more, more preferably 5 or more.
It is 0 or more, more preferably 100 or more. The amino acid sequence of the antigen obtained by modifying the amino acid sequence of SEQ ID NO: 1 by substitution, deletion, insertion, addition, or the like of amino acid residues is 80% or more of the amino acid sequence of SEQ ID NO: 1 or a corresponding portion thereof, More preferably 90% or more,
More preferably, it has a homology of 95% or more.

[0014] The present invention also provides a nucleic acid encoding the above-mentioned Toxoplasma gondii antigen of the present invention. The nucleotide sequence of the DNA encoding the Toxoplasma gondii antigen having the amino acid sequence represented by SEQ ID NO: 1 cloned in the following Examples is shown in SEQ ID NO: 2 of the Sequence Listing together with the deduced amino acid sequence encoded by the DNA. The amino acid sequence shown in SEQ ID NO: 2 is the same as the amino acid sequence shown in SEQ ID NO: 1. Any nucleic acid encoding the above-described Toxoplasma gondii antigen of the present invention (including one in which one or more amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1) Are also included in the scope of the present invention.

Further, the present invention provides a recombinant vector containing the above-described nucleic acid of the present invention and capable of expressing the above-mentioned T. gondii antigen of the present invention in a host cell. This recombinant vector inserts and ligates the nucleic acid of the present invention to a cloning site of an expression vector having at least a replication origin enabling replication in a host, a promoter, and a cloning site downstream of the promoter. It can be manufactured by the following. Such expression vectors are well known in the art, and various types are commercially available. The recombinant vector of the present invention can be easily prepared by inserting and ligating the nucleic acid of the present invention into the multicloning site of such a commercially available expression vector by a conventional method. An example of the procedure for producing a recombinant vector is specifically described in the following Examples.

The Toxoplasma gondii antigen of the present invention can be obtained by transforming a host cell with the above-mentioned recombinant vector by a conventional method, and purifying the target protein from the resulting transformant. The nucleic acid to be inserted into the recombinant vector can also be prepared by the method specifically described in the following examples, and since the nucleotide sequence of the nucleic acid has been clarified by the present invention, Gondi cells from RT
-It can be easily prepared by the PCR method.

The present invention further provides an antibody against the Toxoplasma gondii antigen of the present invention. This antibody may be a polyclonal antibody or a monoclonal antibody, but is more preferably a monoclonal antibody from the viewpoint of the reliability of the measurement results when used for immunoassay. Both polyclonal and monoclonal antibodies can be obtained by conventional methods. That is, a polyclonal antibody can be obtained by immunizing an animal with the above antigen by a conventional method, and further purifying it as an antiserum or from the antiserum. In addition, a monoclonal antibody is obtained by immunizing an animal with the above antigen by a conventional method, collecting spleen cells or lymphocytes, fusing the myeloma cell with a conventional method, selecting a fused cell, and performing an antigen-antibody reaction with the antigen It can be obtained by selecting a clone producing an antibody, culturing the clone and collecting a monoclonal antibody. An example of a method for producing a monoclonal antibody is specifically described in the following Examples.

The present invention further provides a method for immunoassay of an anti-Toxoplasma gondii antibody or a Toxoplasma gondii antigen in a sample using the above-mentioned Toxoplasma gondii antigen or the above-mentioned antibody of the present invention. The immunoassay method itself is well known in this field, and various methods are known, and any of these methods can be adopted. That is, for example, when classified according to the reaction format, there are a sandwich method, a competitive method, an agglutination method, etc., and any of these methods may be used. Method, fluorescent immunoassay, biotin immunoassay and the like, and any of these may be used. In short, the Toxoplasma gondii antigen of the present invention, and an immunoassay method for measuring an anti-Toxoplasma gondii antibody in a sample using an antigen-antibody reaction with an anti-Toxoplasma gondii antibody in the sample, and the antibody of the present invention, Toxoplasma in the sample
All immunoassay methods for measuring Toxoplasma gondii antigen in a sample utilizing an antigen-antibody reaction with Gondi antigen are included in the scope of the present invention.

Samples to be subjected to the immunoassay method of the present invention include, but are not limited to, blood and serum of a person suspected of being infected by Toxoplasma gondii.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

Example 1 Coding of hsp70-like protein of Toxoplasma gondii
Cloning of the gene to be cloned MOSEI cDNA library (Amersham LIFE SCIE
Commercially available from NCE) of Toxoplasma gondii RH strain.
It is composed of poly (A) ⁺ RNA isolated from tayzoite. A probe (ATCC accession number 57494 human gD) obtained by labeling human hsp70 DNA with a radionuclide ( ³² P) against these cDNA libraries.
(Derived from NA) was screened for a Toxoplasma gondii gene that specifically reacts with the human hsp70 gene region by a cross-hybridization method. The respective compositions and reaction temperatures of the hybridization buffer and the washing buffer used at the time of hybridization were as follows. That is, 3 × SSC, 5
× Denhardt's solution, 50 mM Tris base (pH 7.
5) 1 mM EDTA, 0.5% SDS, and 20 μg / μl of salmon sperm denatured at 60 ° C. were added to each and used for hybridization. Also, 0.1 × SSC,
0.1% SDS was added, and the reaction and washing were performed at 60 ° C. By this method, 1 × 10 ⁵ cDNAs
From the clones, 15 clones specifically reacting with the human hsp70 DNA probe were obtained.

In addition, among these 15 clones, the most cD
A pTH14 clone with a large NA size was used to ligate the restriction enzyme site BamHI in the multiple cloning site of the cloning vector pBluescript SKII (+) (Stratagene).
And cloned.

Example 2 Determination of Nucleotide Sequence of Cloned DNA: pTH14 The nucleotide sequence was determined using Genetic Analyzer 310.
(Perkin Elmer). On this occasion,
The labeling of the sequence primer was performed by a dye terminator method using a cycle sequence kit FS (manufactured by PerkinElmer). For sequencing, a total of 12 primers were synthesized (ABI DNA Synthesizer 391, PerkinElmer).
(Type) It tested for sequencing by primer walking. The determined nucleotide sequence is shown in SEQ ID NO: 2, together with the deduced amino acid sequence encoded by the nucleotide sequence.

Example 3 Data Analysis of Nucleotide Sequence Analysis of gene nucleotide sequence and amino acid sequence was performed using Genetyx-Ma
c Performed using software (Software development).

Example 4 Recombinant Toxoplasma gondii hsp70-like protein
Expression and Purification (1) Construction of Expression Vector When constructing an expression vector, T. cerevisiae was cloned by PCR using the pTH14 clone as a template . gondii hsp7
Amplifying the 0 region, the expression vector pET15b (Nova, USA)
gen)) XhoI-BamH
I was recloned. At this time, the 5 'end transcription initiation codon was deleted by excluding it from the set primers. The primers used for amplifying the target region at this time are shown below.

TH: 5′-CGGGATCCTTAACCGAGAGAGAGAGGCGC-3 ′ T7: 5′-CATCTCGAGCTGGAGACAGCTGGTGGT-3 ′

(2) Expression of Target Gene and Purification of Expressed Protein The host cell Escherichia coli BL21 transformed with an expression vector pET15b containing a gene encoding a Toxoplasma gondii hsp70-like protein was cultured to express the target gene. . At that time, an LB medium (manufactured by BIO101) was used as a culture solution, and an antibiotic ampicillin was added to 50 μg / μl. The culture was performed at a temperature of 37 ° C. Furthermore, the expression of the protein was 1 m final concentration.
M IPTG (manufactured by Nakarai Task Co.) was added and induced for 3 hours.

(3) Recovery and purification of expressed protein 5 mM imidazole, 0.5 M NaCl, 20 mM Tr
The expressed antigen is suspended in 10 ml of is (pH 8.0), and 200 W, 5 W is applied by an ultrasonic crusher (Olympus).
The cells were crushed at 0 ° C. for minutes. Next, in order to exclude these cell debris, 12000 rpm, 10 minutes, 4 ° C.
Under the conditions described above.

Next, the cell lysate was placed in a binding buffer (60
mM imidazole, 0.5 M NaCl, 20 mM Tri
(s-HCl pH 8.0). The supernatant after centrifugation is used for Probond for purification of nickel chelate column.
10 ml was applied to a column (Invitrogen, San Diego, CA). Next, the Probond column is washed with a washing buffer (60 m
M imidazole, 0.5 M NaCl, 20 mM Tris
-HCl pH 8.0).

The purified expression protein finally bound to the column was dissolved in an elution buffer (1M imidazole, 0.5
Elution was carried out using 5 ml of (NaCl, 20 mM Tris-HCl). These proteins are centriprep-
The mixture was centrifuged using a 30 (Amicon) concentrator. As a result, the yield of these recombinant proteins was about 1 mg in 1 L of culture. Furthermore, the degree of purification of these purified expression proteins was confirmed by Coomassie Brilliant Blue protein staining as three major bands of 76 KDa, 46 KDa, and 31 KDa.

Example 5 Toxoplasma gondii hsp70-like recombinant purified protein
Recombinant T. gondii h purified confirmation Example 4 antigenic quality
Antigen analysis by Western blotting was performed on the immunological specificity of the sp70-like protein using clinical materials. The specific procedure of this operation was as follows. That is, recombinant Toxoplasma gondii hs
1 μg of the p70-like protein was electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred to a nitrocellulose membrane again by electrophoresis. After coating the active groups of the nitrocellulose membrane with skim milk powder (Meiji Dairy), the serum sample 1 was diluted 100-fold with a phosphate buffer (0.01 M, pH 7.2).
After reacting for 2 hours at room temperature, phosphate buffer (0.01
M, pH 7.2) four times. Thereafter, a peroxidase-conjugated anti-human immunoglobulin antibody (manufactured by Amersham) was reacted and exposed to a film (Fujifilm) using an ECL kit (manufactured by Amersham).

As a result, the results of western blotting were completely consistent with the detection status of various specific antibodies. The results are shown in Table 1 below.

[0033]

[Table 1] *: Using immunoglobulin class IgG and IgM antibodies

Example 6 Toxoplasma gondii hsp70-like purified expressed protein
Production of Antibodies Using Toxoplasma gondii HR Tachyzoites was generated in the human B cell line ARH. In addition, Toxoplasma
Gondi FUKYA strain B10. Generated in A (4R) mice.

(1) Anti-Toxoplasma gondii hsp
Preparation of 70-like Protein Monoclonal Antibody For BALB / c mice, 200 μg / ml of purified recombinant Toxoplasma gondii hsp70-like protein was mixed 1: 1 with Freund's complete adjuvant, and applied to three subcutaneous parts of the trunk. Each immunization was performed by inoculating 0.25 ml of each. Next, two weeks later, induced immunization was performed with Freund's incomplete adjuvant using the same antigen as used for the first immunization. At this time, the mixing ratio of the adjuvant and the antigen, the inoculation volume and the inoculation site are the same as in the first immunization.

Further, three days after the induced immunization, mouse spleen cells were taken out to prepare hybridomas, and BAL was obtained using a cell fusion device type SSH-1 (manufactured by Shimadzu Corporation, Kyoto).
It was fused with SP2 myeloma cells derived from B / c mice. The removed spleen cells were obtained by the method of Kohler et al.
al., Nature, vol. 256, p495-497 (1975)).

Further, the fused cells were selected twice every two days in a HAT selection medium (Boehringer Mannheim). From the culture supernatant at this time, ELISA
As a result, the presence of an anti-Toxoplasma gondii hsp70-like protein monoclonal antibody was confirmed. Positive hybridoma cells were cloned by the limiting dilution method. In addition, a monoclonal antibody showing a non-specific reaction to human hsp70 was selected by the same method except that a cell fusion method using polyethylene glycol was used. Further, the ascites fluid obtained was purified IgG by hydroxyapatite chromatography to obtain a monoclonal antibody.

(2) Construction of ELISA The ELISA performed in the above steps was performed as follows. ELISA is for Current Protocols in immunology
1997, vol. 1, 2.1.2-2.1.20. That is, a flat bottom microtiter plate (manufactured by Dynatech, Z
ug, Switzerland) to a concentration of 2 μg / ml in PBS (-)
Recombinant Toxoplasma gondii hsp prepared by
The 70-like expressed protein was immobilized. The solid phase conditions are 12 at 4 ° C.
Let stand for more than an hour. The solid phase plate described above contains a blocking solution (borate buffer containing 0.05% Tween 20).
1 mM EDTA, 0.25% BSA, 0.05% NaN
Blocking was performed using ₃ ). The blocking condition was left at 4 ° C. overnight.

The process of the anti-Toxoplasma gondii IgG antibody ELISA prepared under these conditions is shown below.

1. After appropriately diluting the non-test serum and the culture supernatant, 100 μl is dropped into each hole, and the mixture is shaken so that an immune reaction occurs evenly, and then allowed to stand at room temperature (15 to 25 ° C.).
For 1 hour.

2.1. After the operation, the washing solution (0.05%
Tween 20, 1.4 mM potassium dihydrogen phosphate 3.
6 mM disodium hydrogen phosphate dodecahydrate, 146 m
After washing three times with M sodium chloride), 100 μl of a 2000-fold diluted alkaline phosphatase-labeled anti-mouse IgG conjugate (Tago, Camarillo, CA) was dispensed into each hole, shaken, and incubated at room temperature for 1 hour.

3.2. After the above operation, the plate was washed three times with the same washing solution described above, and then 100 μl of 1 mM p-nitrophenylphosphoric acid was dispensed into each hole.
Allow to develop color for minutes. Thereafter, 100 μl of 1.5N sulfuric acid was added to stop the reaction.

4. The developed plate was measured for absorbance using a wavelength of 492 nm.

Example 7 Construction of Enzyme Immunoassay for Detection of IgG Antibody (Human) Against Toxoplasma gondii hsp70-like Protein A flat-bottomed microtiter plate (Zug, manufactured by Dynatech)
Switzerland) 0.01MP to a concentration of 10μg protein / ml
50 μl of the purified recombinant Toxoplasma gondii hsp70-like protein adjusted with BS (pH 7.0) was immobilized. The solid phase condition was left at 4 ° C. overnight. The above plate was prepared using a blocking solution (0.2% BSA, 0.1% Tween).
en20, 0.01M PBS (pH 7.0) 200
Blocking was performed using μl. The blocking condition was left at room temperature (15 to 25 ° C.) for 2 hours. 200 μl of washing solution after blocking (same as blocking solution)
And washed three times.

The process of the anti-Toxoplasma gondii hsp70-like protein IgG antibody (human) enzyme immunoassay prepared under these conditions is shown below.

1. 50 μl of the test serum and cerebrospinal fluid diluted 100-fold with 0.01 M PBS (pH 7.0) are dropped into each hole, shaken so that an immune reaction occurs evenly, and allowed to stand still and incubated at room temperature for 2 hours. did.

2. 1. After the above operation, 200 μl of washing solution
After washing twice with 200 μl of distilled water twice using
Alkaline phosphatase-labeled anti-human IgG (manufactured by Tago) diluted 1000-fold with 1M PBS (pH 7.0)
50 μl was dispensed into each hole, shaken, and then incubated at room temperature for 2 hours.

3. 2. After the above operation, 200 μl of washing solution
After washing twice with 200 μl of distilled water twice using
100 μl of p-nitrophenyl phosphate diluted to 3 mM with a solution containing 5 M sodium carbonate and 0.5 mM magnesium chloride was dispensed into each hole, shaken, and then incubated at room temperature for 30 to 60 minutes.

4. After the completion of the reaction, the absorbance was measured using a wavelength of 405 nm. At this time, an OD of 0.110 or less was regarded as negative, an OD of 0.200 or more as positive, and 0.110 to 0.200 was suspended.

5. Table 2 shows the results.

[0051]

[Table 2]

6. These are hydrocephalus, including detection of Toxoplasma gondii-specific IgM antibody in serum and Toxoplasma gondii-specific DN by PCR in placenta.
A case in which congenital toxoplasmosis was diagnosed by the detection of A, the neonatal congenital toxoplasmosis newborn and its mother had a higher OD than two healthy subjects, and it is useful as a marker for determining the acute phase of Toxoplasma gondii infection and the therapeutic effect. .

Example 8 Toxoplasma gondii hs
Construction of enzyme-linked immunosorbent assay for detection of p70-like protein Flat bottom microtiter plate (Zug, manufactured by Dynatech)
Switzerland) 0.01MP to a concentration of 10μg protein / ml
One monoclonal antibody against Toxoplasma gondii hsp70-like protein prepared with BS (pH 7.0)
00 μl was solid-phased. The solid phase condition was left at 4 ° C. overnight.
The plate described above contains blocking solution (0.2% BSA,
0.1% Tween 20 added, 0.01M PBS (pH
7.0)) Blocking was performed using 200 μl.
The blocking condition was left at room temperature (15 to 25 ° C.) for 2 hours. After blocking, the plate was washed three times with 200 μl of a washing solution (similar to the blocking solution).

The process of the enzyme immunoassay for detection of Toxoplasma gondii hsp70-like protein prepared under these conditions is described below.

1. 100 μl of the test serum and cerebrospinal fluid diluted 10-fold with 0.01 M PBS (pH 7.0) are dropped into each hole, shaken so that an immune reaction occurs evenly, then allowed to stand, and incubated at room temperature for 1 hour. did.

2. 1. After the above operation, 200 μl of washing solution
After washing twice with 200 μl of distilled water twice using
50 μl of biotin-labeled Toxoplasma gondii hsp70-like protein diluted with 1 MPBS (pH 7.0)
(0.1 μg protein) was dispensed into each hole, shaken, and then incubated at room temperature for 2 hours.

3.2. After the above operation, the plate was washed twice with 200 μl of a washing solution and once with 200 μl of distilled water,
100 μl of alkaline phosphatase-labeled avidin (1 μg / ml, manufactured by Sigma, Mo, USA) diluted with MPBS (pH 7.0) was dispensed into each hole, shaken, and then incubated at room temperature for 30 minutes.

4.3. After the operation described above, after washing twice with 200 μl of the washing solution and once with 200 μl of distilled water, 0.05
100 μl of p-nitrophenyl phosphate diluted to 3 mM with a solution containing M sodium carbonate and 0.5 mM magnesium chloride was dispensed into each hole, shaken, and then incubated at room temperature for 30 to 60 minutes.

5. After the completion of the reaction, the absorbance was measured using a wavelength of 405 nm. At this time, the OD was 1.000 or more negative, 0.800 or less positive, and 0.800 to 1.000 judgment pending.

6. Table 3 shows the results.

[0061]

[Table 3]

7. These are hydrocephalus, including detection of Toxoplasma gondii-specific IgM antibody in serum and Toxoplasma gondii-specific DNA by PCR in placenta.
In a case where congenital toxoplasmosis was diagnosed by the detection of, the OD of a newborn with congenital toxoplasmosis and its mother was lower than that of two healthy subjects, and it is useful as a marker for determining the acute phase of Toxoplasma gondii infection and the therapeutic effect.

Example 9 Toxoplasma gondii hs
Construction of enzyme-linked immunosorbent assay for detection of p70-like protein Flat bottom microtiter plate (Zug, manufactured by Dynatech)
Switzerland) 0.01MP to a concentration of 10μg protein / ml
Monoclonal antibody against Toxoplasma gondii hsp70-like protein prepared with BS (pH 7.0) (Y
A-1) was immobilized on 100 μl of solid phase. The solid phase condition was left at 4 ° C. overnight. The plate described above contains blocking solution (0.
2% BSA, 0.1% Tween 20, 0.01MP
Blocking was performed using 200 μl of BS (pH 7.0). Blocking conditions are room temperature (15 to 25).
C) for 2 hours. After blocking, the plate was washed three times with 200 μl of a washing solution (similar to the blocking solution).

The process of the enzyme immunoassay for detecting Toxoplasma gondii hsp70-like protein prepared under these conditions is described below.

1. One time with 0.01M PBS (pH 7.0)
100 μl of the test serum and cerebrospinal fluid diluted 0-fold were dropped into each hole, shaken so that an immune reaction occurred uniformly, and allowed to stand still and incubated at room temperature for 1 hour.

After the operation of 2.1, after washing twice with 200 μl of the washing solution and once with 200 μl of distilled water, 0.01MP was applied.
50 μl (0.1 μg protein / ml) of a monoclonal antibody (YA-2) against biotin-labeled Toxoplasma gondii hsp70-like protein diluted with BS (pH 7.0)
Was shaken after dispensing into each hole, and incubated at room temperature for 2 hours.

3.2. After the above operation, the plate was washed twice with 200 μl of a washing solution and once with 200 μl of distilled water,
100 μl of alkaline phosphatase-labeled avidin (1 μg / ml, manufactured by Sigma, Mo, USA) diluted with MPBS (pH 7.0) was dispensed into each hole, shaken, and then incubated at room temperature for 30 minutes.

4.3. After the operation described above, after washing twice with 200 μl of the washing solution and once with 200 μl of distilled water, 0.05
100 μl of p-nitrophenyl phosphate diluted to 3 mM with a solution containing M sodium carbonate and 0.5 mM magnesium chloride was dispensed into each hole, shaken, and then incubated at room temperature for 30 to 60 minutes.

5. After the completion of the reaction, the absorbance was measured using a wavelength of 405 nm. At this time, the OD was determined to be negative when the OD was 0.150 or less, positive when the OD was 0.500 or more, and suspended from 0.150 to 0.500.

6. Table 4 shows the results.

[0071]

[Table 4]

7. These are hydrocephalus, including detection of Toxoplasma gondii-specific IgM antibody in serum and Toxoplasma gondii-specific DNA by PCR in placenta.
In a case where congenital toxoplasmosis was diagnosed by the detection of T. gondii, a newborn with congenital toxoplasmosis and its mother had a higher OD than two healthy subjects, and it is useful as a marker for judging the acute phase of Toxoplasma gondii infection and the therapeutic effect.

[0073]

According to the present invention, a novel Toxoplasma
Gondi antigen was provided. According to the method of the present invention, infection of Toxoplasma gondii can be diagnosed by immunoassaying this antigen or an antibody against this antigen. In particular, according to the immunoassay using the antigen of the present invention, antibodies generated in the early stage of infection can be measured with high sensitivity.

[0074]

[Sequence List] <110> YANO Akihiko <120> Toxoplasma gondii antigen, antibody to the antigen and method for immunoassay using the antigen or antibody <130> 98579 <160> 4

<210> 1 <211> 647 <212> PRT <213> Toxoplasma gondii RH <400> 1 Met Ala Asp Ser Pro Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser 1 5 10 15 Cys Val Gly Val Trp Lys Asn Asp Ala Val Glu Ile Ile Ala Asn Asp 20 25 30 Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu 35 40 45 Arg Leu Val Gly Asp Ala Ala Lys Asn Gln Val Ala Arg Asn Pro Glu 50 55 60 Asn Thr Ile Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Asp Asp 65 70 75 80 Pro Ser Val Gln Ser Asp Met Lys His Trp Pro Phe Lys Val Ile Ala 85 90 95 Gly Pro Gly Asp Lys Pro Leu Ile Glu Val Thr Tyr Gln Gly Glu Lys 100 105 110 Lys Thr Phe His Pro Glu Glu Val Ser Ala Met Val Leu Gly Lys Met 115 120 125 Lys Glu Ile Ala Glu Ala Tyr Leu Gly Lys Glu Val Lys Glu Ala Val 130 135 140 Ile Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys 145 150 155 160 Asp Ala Gly Thr Ile Ala Gly Leu Ser Val Leu Arg Ile Ile Asn Glu 165 170 175 Pro Thr Ala Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Gly Cys Gly 180 185 190 Glu Met Asn Val Leu Ile Phe Asp Met Gly Gly Gly Thr Phe Asp Val 195 200 205 Ser Leu Leu Thr Ile Glu Asp Gly Ile Phe Glu Val Lys Ala Thr Ala 210 215 220 Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Val Asp 225 230 235 240 Phe Cys Val Gln Asp Phe Lys Arg Lys Asn Arg Gly Lys Asp Ile Ser 245 250 255 Thr Asn Ser Arg Ala Leu Arg Arg Leu Arg Thr Gln Cys Glu Arg Thr 260 265 270 Lys Arg Thr Leu Ser Ser Ser Ile Gln Ala Thr Ile Glu Ile Asp Ser 275 280 285 Leu Phe Glu Gly Ile Asp Tyr Ser Val Ser Ile Ser Arg Ala Arg Phe 290 295 300 Glu Glu Leu Cys Met Asp Tyr Phe Arg Asn Ser Leu Leu Pro Val Glu 305 310 315 320 Lys Val Leu Lys Asp Ser Gly Ile Asp Lys Arg Ser Val Ser Glu Val 325 330 335 Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Gln Leu Ile 340 345 350 Thr Asp Phe Phe Asn Gly Lys Glu Pro Cys Arg Ser Ile Asn Pro Asp 355 360 365 Glu Ala Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Lys Gly 370 375 380 Val Thr Ser Ser Gln Val Gln Asp Leu Leu Leu Leu Asp Val Ala Pro 385 390 395 400 Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr Lys Leu Ile 405 410 415 Glu Arg Asn Thr Thr Ile Pro Thr Lys Lys Ser Gln Thr Phe Thr Thr 420 425 430 Tyr Ala Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr Glu Gly Glu 435 440 445 Arg Ala Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe His Leu Asp 450 455 460 Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe 465 470 475 480 480 Asp Ile Asp Ala Asn Gly Ile Met Asn Val Thr Ala Gln Asp Lys Ser 485 490 495 Thr Gly Lys Ser Asn Gln Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu 500 505 510 Ser Ala Ser Glu Ile Asp Arg Met Val Gln Glu Ala Glu Lys Tyr Lys 515 520 525 Ala Glu Asp Glu Gln Asn Lys His Arg Val Glu Ala Lys Asn Gly Leu 530 535 540 Glu Asn Tyr Cys Tyr His Met Arg Gln Thr Leu Asp Asp Glu Lys Leu 545 550 555 560 Lys Asp Lys Ile Ser Ser Glu Asp Arg Asp Thr Ala Asn Lys Ala Ile 565 570 575 Gln Glu Ala Leu Asp Trp Leu Asp Lys Asn Gln Leu Ala Glu Lys Glu 580 585 590 Glu Phe Glu Ala Lys Gln Lys Glu Val Glu Ser Val Cys Thr Pro Ile 595 600 600 605 Ile Thr Lys Leu Ty r Gln Ala Gly Ala Ala Ala Gly Gly Met Pro Gly 610 615 620 620 Gly Met Gly Gly Met Pro Gly Gly Met Gly Gly Met Pro Gly Gly Met 625 630 635 640 640 Gly Gly Met Pro Gly Gly Met 645

<210> 2 <211> 1941 <212> DNA <213> Toxoplasma gondii RH <400> 2 atg gcg gac tct cct gct gtg ggt att gac ctt ggc acc acc tat tct 48 Met Ala Asp Ser Pro Ala Val Gly Ile Asp Leu Gly Thr Thr Tyr Ser 1 5 10 15 tgc gta ggt gtg tgg aag aac gat gct gtg gaa atc atc gcg aac gac 96 Cys Val Gly Val Trp Lys Asn Asp Ala Val Glu Ile Ile Ala Asn Asp 20 25 30 cag gga aac agg acg acc ccg tcc tac gtc gcg ttc acc gac acg gag 144 Gln Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu 35 40 45 aga ctt gtc ggt gat gct gcg aag aac caa gtc gca cgc aac ccg gaa 192 Arg Leu Val Gly Asp Ala Ala Lys Asn Gln Val Ala Arg Asn Pro Glu 50 55 60 aac acc att ttc gat gcc aag cgc cta atc ggt cgc aag ttt gat gat 240 Asn Thr Ile Phe Asp Ala Lys Arg Leu Ile Gly Arg Lys Phe Asp Asp 65 70 75 80 ccc tcg gtc cag tcg gac atg aag cat tgg cca ttc aag gtc att gct 288 Pro Ser Val Gln Ser Asp Met Lys His Trp Pro Phe Lys Val Ile Ala 85 90 95 ggt ccg gga gac aag ccc ctc att gaa gtc acg tac cag gga gag aag 336 G ly Pro Gly Asp Lys Pro Leu Ile Glu Val Thr Tyr Gln Gly Glu Lys 100 105 110 aag acg ttc cac cct gaa gag gtt tcc gcc atg gtt ttg ggc aaa atg 384 Lys Thr Phe His Pro Glu Glu Val Ser Ala Met Val Leu Gly Lys Met 115 120 125 aag gaa atc gcg gag gct tac ctc ggc aag gaa gtg aag gag gcc gtc 432 Lys Glu Ile Ala Glu Ala Tyr Leu Gly Lys Glu Val Lys Glu Ala Val 130 135 140 att acc gtt cct gcg tac ttc ac tcg cag cgt cag gct acc aag 480 Ile Thr Val Pro Ala Tyr Phe Asn Asp Ser Gln Arg Gln Ala Thr Lys 145 150 155 160 gat gct ggt acc att gcc ggc ctc agc gtc ctc cgc att atc aac gag 528 Asp Ala Gly Thre Ala Gly Leu Ser Val Leu Arg Ile Ile Asn Glu 165 170 175 ccc aca gcg gct gcc att gct tat ggt ctg gac aag aag ggc tgc ggt 576 Pro Thr Ala Ala Ala Ala Ile Ala Tyr Gly Leu Asp Lys Lys Gly Cys Gly 180 185 190 gag atg aac gtc ctc atc ttc gac atg ggt ggc ggt acg ttc gat gtg 624 Glu Met Asn Val Leu Ile Phe Asp Met Gly Gly Gly Gly Thr Phe Asp Val 195 200 205 tcg ctg ctt aca atc gaa gac ggt atc ttt gcacc gct 672 Ser Leu Leu Thr Ile Glu Asp Gly Ile Phe Glu Val Lys Ala Thr Ala 210 215 220 ggt gac acc cat ctt ggt ggt gaa gat ttc gac aac cgt ttg gtg gac 720 Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Leu Val Asp 225 230 235 240 ttc tgc gtc cag gac ttc aag cgc aag aac cgc gga aag gac atc agc 768 Phe Cys Val Gln Asp Phe Lys Arg Lys Asn Arg Gly Lys Asp Ile Ser 245 250 255 acc aac agcc ctt cgt cgc ctg cgt acc cag tgc gag cgc acc 816 Thr Asn Ser Arg Ala Leu Arg Arg Leu Arg Thr Gln Cys Glu Arg Thr 260 265 270 aag aga act ctc tct agc agc atc cag gca acc atc gaa att gac tct 864 Lys Thr Leu Ser Ser Ser Ile Gln Ala Thr Ile Glu Ile Asp Ser 275 280 285 ctt ttt gag ggc att gac tac tct gtg tct atc tct cgt gcg cgc ttt 912 Leu Phe Glu Gly Ile Asp Tyr Ser Val Ser Ile Ser Arg Ala Arg Phe 290 295 300 gag gag ctt tgc atg gac tac ttc cgc aac tcc ctg ttg ccc gtc gag 960 Glu Glu Leu Cys Met Asp Tyr Phe Arg Asn Ser Leu Leu Pro Val Glu 305 310 315 320 aag gtc ctc aag gac tct ggt atgcgc tcg gtc agc gaa gtt 1008 Lys Val Leu Lys Asp Ser Gly Ile Asp Lys Arg Ser Val Ser Glu Val 325 330 335 gtg ttg gtt ggt gga tct acc cgt atc ccc aag att cag cag ctc atc 1056 Val Leu Val Gly Gly Ser Thr Arg Ile Pro Lys Ile Gln Gln Leu Ile 340 345 350 act gac ttc ttc aac gga aag gag ccg tgc agg tcg atc aac ccc gat 1104 Thr Asp Phe Phe Asn Gly Lys Glu Pro Cys Arg Ser Ile Asn Pro Asp 355 360 365 gag gcc gtt gcg tac ggt gct gct gtc cag gca gcg atc ttg aag gga 1152 Glu Ala Val Ala Tyr Gly Ala Ala Val Gln Ala Ala Ile Leu Lys Gly 370 375 380 gtt acc agc tct cag gtg cag gat ttg ctt ctt gtt 1200 Val Thr Ser Ser Gln Val Gln Asp Leu Leu Leu Leu Asp Val Ala Pro 385 390 395 400 400 ctc tct ctc ggt ctg gag aca gct ggt ggt gtc atg acc aag ctg att 1248 Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr Lys Leu Ile 405 410 415 gaa aga aac aca acg atc ccg acc aag aag tct cag acc ttc acc acg 1296 Glu Arg Asn Thr Thr Ile Pro Thr Lys Lys Ser Gln Thr Phe Thr Thr 420 425 430 tac gcg gac aac cag c ca gga gtg ctg att cag gtg tac gaa ggt gag 1344 Tyr Ala Asp Asn Gln Pro Gly Val Leu Ile Gln Val Tyr Glu Gly Glu 435 440 445 cgt gcg atg acc aaa gac aac aac ctc ctg ggc aaa ttc cac ctg ata c g Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe His Leu Asp 450 455 460 ggt atc ccc ccc gcc ccc cgt ggt gtc ccc caa atc gaa gtc act ttc 1440 Gly Ile Pro Pro Ala Pro Arg Gly Val Pro Gln Ile Glu Val Thr Phe 465 470 475 480 gat atc gac gct aac ggt atc atg aac gtc aca gcg caa gac aag tcc 1488 Asp Ile Asp Ala Asn Gly Ile Met Asn Val Thr Ala Gln Asp Lys Ser 485 490 495 acc gga aag agc aac caa atc acc atc ac aac gac aag ggc cgc ctc 1536 Thr Gly Lys Ser Asn Gln Ile Thr Ile Thr Asn Asp Lys Gly Arg Leu 500 505 510 agt gcg tcc gaa atc gac cgc atg gtg caa gag gca gag aag tac aaa 1584 Ser Ala Ser Glu Ile As Met Val Gln Glu Ala Glu Lys Tyr Lys 515 520 525 gcc gaa gac gaa cag aac aag cac cgt gtg gag gcg aag aat ggc ctg 1632 Ala Glu Asp Glu Gln Asn Lys His Arg Val Glu Ala Lys Asn Gly Leu 530 535 ag aac tac tgc tac cac atg aga cag acc ttg gat gac gag aag ctt 1680 Glu Asn Tyr Cys Tyr His Met Arg Gln Thr Leu Asp Asp Glu Lys Leu 545 550 555 560 aag gac aag atc tcc tct gag gac aga gac act gcc ag aag gcc atc 1728 Lys Asp Lys Ile Ser Ser Glu Asp Arg Asp Thr Ala Asn Lys Ala Ile 565 570 575 cag gag gcc ctt gac tgg ctg gac aag aac caa cta gca gag aag gag 1776 Gln Glu Ala Leu Asp Trp Leu Asp Lysn Gln Leu Ala Glu Lys Glu 580 585 590 gaa ttc gag gcg aag cag aag gaa gtt gag tcc gtc tgc aca cca atc 1824 Glu Phe Glu Ala Lys Gln Lys Glu Val Glu Ser Val Cys Thr Pro Ile 595 600 600 605 atc acc aag ct cag gca ggt gcg gct gca ggt ggc atg cct ggt 1872 Ile Thr Lys Leu Tyr Gln Ala Gly Ala Ala Ala Gly Gly Met Pro Gly 610 615 620 ggt atg ggc ggt atg cct ggt ggt atg ggc ggt atg cct ggt ggt atg 1920 G Gly Gly Met Pro Gly Gly Met Gly Gly Met Pro Gly Gly Met 625 630 635 640 ggc ggt atg ccc ggc ggg atg 1941 Gly Gly Met Pro Gly Gly Met 645

<210> 3 <211> 29 <212> DNA <213> Artificial Sequence <400> 3 cgggatcctt aaccgagaga gagaggcgc 29

<210> 4 <211> 27 <212> DNA <213> Artificial Sequence <400> 4 catctcgagc tggagacagc tggtggt 27

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/569 G01N 33/569 A 33/577 33/577 B // (C12N 15/09 ZNA C12R 1:90) (C12P 21 / 02 C12R 1:19) (C12P 21/08 C12R 1:91)

Claims (10)

[Claims] An amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence, And a Toxoplasma gondii antigen comprising an amino acid sequence exhibiting antigenicity specific to Toxoplasma gondii. 2. The Toxoplasma gondii antigen according to claim 1, comprising an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing or a part thereof and exhibiting an antigenicity specific to Toxoplasma gondii. 3. The Toxoplasma gondii antigen according to claim 2, which comprises the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 4. A nucleic acid encoding the Toxoplasma gondii antigen according to any one of claims 1 to 3. 5. The nucleic acid according to claim 4, which has the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 6. A recombinant vector comprising the nucleic acid according to claim 4 or 5 and capable of expressing the Toxoplasma gondii antigen according to claim 1 in a host cell. 7. An antibody against the Toxoplasma gondii antigen according to any one of claims 1 to 3. 8. The antibody according to claim 7, which is a monoclonal antibody. 9. An anti-Toxoplasma gondii antibody in a sample is obtained by utilizing an antigen-antibody reaction between the Toxoplasma gondii antigen according to any one of claims 1 to 3 and an anti-Toxoplasma gondii antibody in the sample. The immunoassay method to be measured. 10. An immunoassay method for measuring Toxoplasma gondii antigen in a sample by utilizing an antigen-antibody reaction between the antibody according to claim 7 and a Toxoplasma gondii antigen in the sample.