JPH02502878A - Proteins and their production methods, DNA sequences, antibodies and their applications, poxviruses, transformed or infected cells and pharmaceutical formulations useful for the prevention of toxoplasmosis. - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 11. A claim in which the encoded gene consists of all or part of the DNA c in Figure 1. 9 or 10.

12, comprising a gene encoding the protein according to any one of claims 1 to 4; The poxvirus puts the poxvirus under the control of the element that expresses the gene. Virus.

13. The pox virus according to claim 12, wherein the pox virus is vaccinia virus. Swirls.

14. The element that causes the expression of the above gene is the viral protein of cotton 7.5. The poxvirus according to claim 13, having a lomotor.

15. The element for expressing the above gene is a sequence encoding a human IL2 signal sequence. The poxvirus according to claim 14.

16. A mammal infected with the poxvirus according to any one of claims 12 to 15. Mammalian cells.

17. The cells according to any one of claims 8 to 11 or claim 16 in an appropriate medium. The protein according to any one of claims 1 to 7, which is obtained by culturing the protein in How white matter is produced.

18. A DNA sequence encoding the 24Kd protein according to any one of claims 1 to 7. Column.

19, DNA sequence shown in FIG.

20, the protein according to any one of claims 1 to 7 and the protein according to claims 12 to 15 A small number of poxviruses selected from any of the listed poxviruses are used as the active drug. A pharmaceutical formulation particularly effective for the prevention of toxoplasmosis.

21. The poxvirus according to any one of claims 12 to 15 is used as an active drug. 20. The pharmaceutical preparation according to claim 19, wherein the virus is a live bacteria.

22. The poxvirus according to any one of claims 12 to 15 is used as an active drug. 21. The pharmaceutical preparation according to claim 20, wherein the virus is killed.

23. Any one of claims 2 to 22, which is in a form that can be administered to humans or animals. Pharmaceutical formulations as described.

24. Produced for the protein according to any one of claims 1 to 7, or claimed 16. An antibody induced by the poxvirus according to any one of 12 to 15.

25, the antibody according to claim 24 or the protein according to any one of claims 1 to 7 Application of toxoplasmosis diagnostic agent.

Specification Proteins and their production methods, DNA sequences, antibodies and their applications, poxviruses useful for preventing toxoplasmosis, transformed or infected cells, and toxoplasmosis. The present invention relates to the development of toxoplasmosis vaccines.

Toxoplasmosis is caused by the protozoan parasite Toxobraz 7 plasma gondii). The parasite is strictly an intracellular parasite. A eukaryotic unicellular organism.

The parasite is observed all over the world. For example, in the United States, 50% of the population have antibodies against Toxoplasma gondii.

Most infections are asymptomatic, but cause only minor damage.

However, two categories of people are at risk of developing serious disease: be exposed to Immunosuppressed patients in whom a latent infection maintained in a cyst state may be activated. person.

- A fetus whose mother is primarily infected with Toxoplasma: In fact, Toxoplasma crosses the placenta and increases in fetal tissues, particularly the central nervous system and retinal tissue. The disease grows and causes irreversible lesions.

The life cycle of the parasite consists of two phases. – Can reproduce in various mammals, including humans, and harbors “inactive” parasites. Asexual growth that ends and - sexual growth that occurs only in cats.

When a cat ingests cysts (present in the muscles of other animals), they may be called bradyzoites. An inactive parasite known as a worm is released in the intestine and enters epithelial cells, where it divides and Sexually differentiated. (Also, the above parasites are found in other mammals as well as in all mammals.) Proliferates asexually within tissues. ) The germ cells fuse and the zygote is covered with a protective wall. surrounded to form a ne sporozoite. These sporozoites have a protective integument remaining and ingesting sporozoites to any other mammals infected , sporozoites are released in the intestine, enter epithelial cells, proliferate asexually, and undergo taxa. to produce items. Taxiite disperses and causes acute disseminated infection.

Taxiite is a single cell with a size of 5μ x 2μ that can only proliferate within the cell. Proliferates in all cell types tested. Infected cells break down after 2 days. , liberating approximately 100 parasites.

In infected animals, parasite multiplication is inhibited by an immune response, and the parasites are Covered by muscles and brain.

Encapsulated forms of parasites or bradyzoites are maintained in an “inactive” state. It will be done.

Therefore, humans can be infected by two routes; - excreted by cats; ingest sporozoites.

- undercooked meat (beef and eat mutton). Branaizoites are liberated in the intestine, actively proliferate, and enter the tumor. It's going to be difficult.

In this complex life cycle of the parasite, taxiites are the first to generate an immune response. This is considered a possible target, and several studies have shown that the same membrane antigens that can elicit antibodies have been identified. It is now possible to

Proteins and polysaccharides from taxiite membrane preparations are associated with acute or chronic toxoplasmosis. It is recognized by IgM and IgG in the serum of affected patients. These membrane anti Monoclonal antibodies having specificity for the antigen are known (Kasbar). Sper et al., 1984 and Johnson et al., 1983) .

In particular, a membrane antigen with a molecular weight of 30,000 was identified by Kasper et al. (1983) and is recognized to have protective properties; was purified from the parasite by immunoadsorption using monoclonal antibodies. this Monoclonal antibodies raised against antigens are produced against the parasite in the presence of human complement. can kill.

Two other membrane antigens with molecular weights of 35,000 and 14,000, respectively, Originated by Araujo and Remington (1984); the antigen was determined by polyacrylamide gel electrophoresis. The antigen was purified from the parasite; injection of this antigen into mice Protected mice from seeds.

The immune response is repeatedly stimulated with antigens liberated by enveloped bradyzoites. The hypothesis is that these molecules must be eliminated by taxite. The excreted/secreted antigen probably has a common epitope, and therefore concomitant immunity. Based on the fact that it must be involved in the epidemic, the present invention Adopt a needle.

Therefore, from this new vaccine production strategy, the aim of the present invention is to Immediately excreted/secreted by taxiites, taxiites and bradyzoi toxoplasmosis and therefore can be recognized by serum from patients with chronic toxoplasmosis. It is a protein. Patients with chronic toxoplasmosis mentioned above have bradyzoites, i.e. It is at a stage where only the infected parasites exist.

In vitro translation of the methsenger RNA of takxite revealed that the 24Kd Along with the major antigen, another 37Kd minor antigen appears. These are reinfections human serum as a marker of the chronic disease stage when immunity is acquired. established. Furthermore, immunocompetitive experiments showed that bradyzoite antigens of the translation products of the 24Kd and 37Kd taxite metsenger RNAs, It has been found that binding by antibodies to excreted/secreted antigens can be significantly inhibited. Ta. Therefore, these antigens are probably also present in bradyzoites.

The present invention encodes an antigen of approximately 24 Kd, or the "excreted/secreted" ``Has an epitope of the 24Kd antigen that is recognized by antibodies raised against the antigen.'' The present invention relates to the determination of a cDNA sequence encoding at least a polypeptide.

Therefore, the present invention first provides a DNA encoding the 24Kd protein shown in FIG. Regarding arrays.

The invention also relates to proteins whose synthesis is controlled by this cDNA.

The above synthesis involves a vector into which the cDNA is inserted and an expression regulator governing the cDNA. Depending on the control signal, various hosts such as bacteria, yeast or higher cells Get carried out in the principal cells.

The protein of the present invention has the same primary structure as the natural protein present in Toxoplasma gondii. may be a derivative of the natural protein, or may be incompletely Proteins with epitopes that are important for recognition by antibodies and therefore important for inducing immunity It may be white matter. Furthermore, it may be fused to the above protein fragment). this Following fusion, the corresponding DNA segments are used to engineer the protein in the host cell. promote better expression or, in some cases, expel the proteins from the host. Perform genetic engineering operations designed to

Methods for expressing and cloning foreign genes in various host cells include It is obvious to those skilled in the art.

These methods, described in the Examples below, can be performed using dwarf host cells. Can be done.

The expression of the gene corresponding to the 24Kd protein in bacteria, for example E. coli, is Vector with CI[rbs as promoter PL and liposome binding site can be obtained using The obtained hybrid protein consists of a part of C■ protein and 2 4Kd protein or a part thereof.

The host cell is a yeast, such as Varnish, S. cerevisiae (S.

cerevisiae), the origin of replication for the 2μ plasmid is a functional origin of replication and a selection gene (selecta) such as URA3. A yeast plasmid vector having a ble gene) can be used. Book In this example, the bromo host of the PGK gene containing the 5' portion of the PKG gene used is mammalian. In the case of mammalian cells, a potion such as vaccinia virus is used as an expression vector. Preferred is the virus. The recombinant virus contains a gene encoding the protein of the present invention. You will have a gene. Preferably, the good gene is T of vaccinia virus. The promoter of the gene that is inserted into the K gene and encodes the protein in vaccinia 7.5 under the control of powerful promoters such as In addition, a protein encoding the protein of the present invention may also be used. A gene containing a region encoding a fragment of another protein, such as a human It may also be fused to a functional signal sequence, such as the leukin-2 signal sequence.

The present invention also provides for the expression of proteins of the present invention. A host cell and a method for producing the above-mentioned protein, which comprises culturing the host cell and recovering the protein. related.

Of course, methods for culturing, harvesting, and isolating proteins depend on the host. Although different, it is obvious to those skilled in the art.

Finally, the present invention relates to pharmaceutical formulations useful as vaccines for toxoplasmosis. . The vaccine comprises a protein or pox of the invention in a pharmaceutically acceptable excipient. Contains at least one type of virus.

In particular, the present invention comprises a live recombinant vaccinia virus expressing the protein of the present invention. Concerning live vaccines. the virus is present in a pharmaceutically acceptable excipient; Expressing the protein of the present invention.

Preferably, these vaccines are in a form that can be administered, e.g. by injection. Take. Pharmaceutically acceptable excipients are aqueous excipients for injections.

The vaccination process is tailored to the type of vaccine used (protein or live vaccine). Should.

Furthermore, according to the present invention, antibodies against the protein of the present invention can be produced. child These antibodies and proteins can be used as diagnostic agents for toxoplasmosis. can.

In the present invention, the nucleotide sequence of the gene or the amino acid of the protein shown in the drawings Although the amino acid sequence is not restated in the specification, it is a component of the invention.

The following examples are intended to demonstrate other features and advantages of the invention. It is something.

The attached FIGS. 1 to 4 will be explained.

The drawings below illustrate embodiments.

Figure 1 shows the cDNAs TG and TXl encoding the 24Kd antigen of Toxoplasma gondii. 1-3,8EQ arrangement is shown.

This applies to the inserts of clones TX8, TXIOlTXll and TX3.3. was measured.

Figure 2 shows E, col i/pTG2171 and E, eol i/pTG21. Figure 2 shows a "Western plot" analysis of 72 expression products.

Figure 3 shows recombinant vaccinia virus VV-TG.

Immunoprecipitation of proteins expressed in TX2170 is shown.

The numbers in this drawing are as follows: P - Beret S - Supernatant WT = wild type vaccinia virus + Addition of 8 tunicamycin (tunicaycin) -- No tunicamycin addition Figure 4 supplements Figure 3 and shows recombinant vaccinia viruses VV-TG, T 'Unisturn plot' analysis of serum from mice immunized with 'X2170' Show the results.

The numbers in this drawing are as follows: 1.2.3.4: VV, TG, TX2 1701:, l:, immunized mice, protein induced with pTG2171 was tested.

CI: Control: Mice tested for protein induced with an unrelated plasmid. 1.2.3.4 Serum C2:1) Tested for TG2171-induced proteins. serum from mice immunized with wild-type vaccinia virus.

Example 1: Cloning and cloning of the cDNA sequence corresponding to the 24Kd antigen of taxiite and measurement of the excreted/secreted amount of taxite as described above.

(E/S) Rabbit antibodies produced against antigen preparations are produced by in vitro translational production. Recognizes the 24Kd major band of tachyzoite messenger RNA in substances.

A cDNA corresponding to this antigen was cloned.

Using RNA extracted from taxite as a starting material, in λgt IIn A cDNA library was produced. This phage λ derivative has cDNA and large intestine After fusion with the bacterial β-galactosidase gene, under the control of the lac promoter, Expressing foreign genes (Young and Davis) , 1983). Expression of the fused protein is carried out using isopropyl β-D-thiogala. Induced at 42°C in the presence of topyranoside. Nitrocell synthesized protein adsorbed onto rolled fibers and then in the presence of rabbit antibodies raised against the E/S antigen. was cultured.

Measurement with antibodies allows identification of recombinant phages. within the phage The cDNA insert is recognized by antibodies raised against the protein or E/S antigen. Directs the synthesis of a protein fraction that has the identified epitope.

Three clones were selected and recloned from λgtll into vector pUc13. It was done.

These three clones were designated as TX8, TXlo and TXll. these places The location is shown in Figure 1. From now on, these clones will overlap considerably. I understand that.

The cDNA inserts of these three clones were assembled in the form of EcoRI fragments. was extracted, inserted into vector M137G131 in both directions, and sequenced.

The obtained phages were named as follows: M13TGTX11 and M1. 3TGTX11R-M13TGTXlO and elbow 3TGTXIOR-M13TG TX8 and M13TGTX8R sequencing )” The cyclone was made using T4 polymer The aim is to generate deletions of various sizes in single-stranded DNA using enzymes ( (using a kit commercially available from Genoflt). child Sequencing of the various sized inserts thus obtained was performed by Sanger (nucleotides are available from Pharmacia). ), 32P-dATP from Amersham, Klenow Polymerases were purchased from AppHgene (CAppHgene)-T G II 5’ AAGGCGATTAAGI IG3’ (universal block Rymer) -TG 1276 5' CAGCTTAAGGATGACA3'-TG 1299 5’ CACGAACACCGATGC3’-TG 1290 5’ TGCCTGTGGGTCTCT 3’-TG 1420 5’ GACACTCACTGTACAAC3' Analysis of the obtained sequence (IBM DN A Star compilation program ion program), the most complete loan is M13TGTX 11. However, this clone has an initiating ATG. do not have. This search for ATG was performed using other cDNA clones and the sequence 5' ACGCAAG. CTCGCTCGGCAGTTTCA 5' terminal oligonucleotide ( TG1325).

Clone 3.3 thus identified was derived from the vector M13mplO (Amarjam ) and the sequence obtained with oligonucleotide TG1420. was made into Clone 3.3 has one base added compared to clone M13TGTX11. It has two more, but it does not have a starting ATG.

The largest sequence obtained is shown in FIG. Sequence numbering follows clone TX11. Since this was a clone that was expressed in the same way, we started from the first base of this clone. Up The sequence is called TGTXII-3, SEQ and is a 785 bp oven-ready sequence. The theoretical molecular weight is about 29 Kd.

From this result and currently known research, it is clear that native genes about the N-terminal sequence of the first translation product of the protein, or about the signal that precedes the mature protein. No information is available about the possible presence of peptides and precursors.

Nevertheless, the policy adopted for the isolation and expression of this gene was A major epitope present in the 24Kd protein that is recognized by the host's immune system. The precise positioning of the first amino acid is within the scope of the present invention, as it involves the study of It is not essential.

This reasoning is used in the examples below, in which the The expression of epitopes is described.

Example 2: Expression of 24Kd antigen in E. coli clones M13TGTX11 and and M13TGTX8 are ovens in phase with the EcoR1 site of λgtll. Has a reading frame. EcoR1 fragments of these two clones is the same leading frame under the control of the heat-inducible promoter PL of λ. vector pTG1924 (French patent application 86). A protein fused with λcn (disclosed in 1986) was produced.

Fragment derived from M13TGTX11 cloned into pTG1924 generates pTG2171. The fragment derived from M13TGTX8 is , generating pTG2172.

E. coli TGE901 is transformed with pTG2171 and 2172. 42 After induction at °C, expression products are analyzed on 5DS-polyacrylamide gels.

``Western ramp'' of inducing proteins using antibodies against ``excreted/secreted'' antigens. The results of Lotpa analysis reveal the presence of specific proteins.

After running on a 15% SDS gel, 25mM) Transfer proteins to nitrocellulose membranes in 20% methanol buffer. This egg White matter migration is performed at 500mM-6V for 1 hour.

The protein bound to nitrocellulose was then dissolved in 10mM) Lys-150mN aC1-1/2000) Ridon X-100 (manufactured by Jig v (Sigma)) - Loose Rabbit anti-toxoplast diluted 100 times with buffer solution and supplemented with 3% powdered milk. React with plasmid antigen serum overnight at 4°C. Nitrocellulose (10mM) Wash three times for 10 minutes with 150 mM NaCl-buffer. 2% fetal bovine serum Biotinylation diluted 500 times with the above wash buffer supplemented with (Fe2) Add biotjnylated anti-heron Ig antibody (manufactured by Amerjam). Ru.

After standing at room temperature for 1 hour, the nitrocellulose membrane was washed three times and then treated with 2% FC5. Streptavidin-bar oil diluted 500 times with the same buffer as above was added. Oxidase (streptavidin-peroxidase) complex (flax (Made by Jam) over a period of 1 hour. After washing three times, colorant (HRP C By adding hydrogen peroxide in the presence of o1or), macroscopic observation is possible. . The results are shown in Figure 2.

A specific reaction with the 24Kd protein is observed for pTG2172. this is , considering the oven reading frame (clone TX8) shown in Figure 1. It is twice the expected size. For pTG21.71, the 33Kd protein A strong reaction with white matter was observed, and it was stronger than that with 24Kd protein (clone TX11). A weak reaction is observed.

Therefore, these results suggest that Toxoplasma gondii may Indicates the presence of a protein specifically recognized by the antibody.

Example 3: Expression of 24Kd antigen in vaccinia virus Reading frame of M13TGTX11 (Eco in phase with λgt11) R1 fragment) was excised, transfected into vaccinia virus, and the IL2 fused upstream of the GNAR and the first eight amino acids (a similar construction was (Disclosed in National Patent No. 86/11986).

For this purpose, the IL2 signal in phase with the start codon of the signal peptide is Downstream from the clone, the EcoR1 fragment is cloned into pTG188-1.

The resulting plasmid is pTG2170: Input TG 　　　　　　　　　　　　　　　　　 In EcoR1pTG2170, IL2) Xoplasma fusion The gene encoding the protein has a promoter control in vaccinia virus 7.5. It is located below the TK gene sequence of the same virus. vaccini These sequences inserted into the TK gene of A. ieny et al., 1984) into the vaccinia virus genome.

The obtained recombinant vaccinia viruses were vv, TG.

It is TX2170. BHK21 cells vv, TG.

Infect with TX2170 virus (0.1 pfu/cell) for 16 hours. in 6 hours After adding [35S] methionine, the excretion/secretion of Toxoplasma gondii The labeled protein is immunoprecipitated using a rabbit antibody directed against the labeled antigen.

Figure 3 specifically shows two proteins of 24Kd and 33Kd, respectively.

vv, TG, two forms induced by TX2170 (24Kd and 33K Since d) exists, we investigated the cause.

When labeled with [35S] methionine and [35S] cysteine, 33K It can be seen that only the d type is labeled with cysteine and methionine. All of Because cysteine is present at the N-terminal part of the sequence, p24 protein is located at the C-terminus of the cDNA. It can be seen that this corresponds to the end portion.

After the formation of a genomic library of Toxoplasma DNA, the longest cDNA ( The genomic portion containing the N-terminus of TX3.3) is sequenced. cDNA leader Upstream of the starting point of clone TX3.3, which is in phase with the I couldn't observe it. This indicates that translation initiation occurred at methionine 72. suggest Methionine 72 is consistent with being a translation initiation site in eukaryotes. Corresponds to permitted sites (Kozak's law).

It is thought to be from within the island. The true initiation appears at methionine 72, leading to p24 induces synthesis. Taxiitometsenger R in in vitro translation products The presence of the p37 type detected in NA and recognized by anti-p24 antibodies supports this hypothesis. According to the theory, this corresponds to cross-reactivity.

Furthermore, after Western blotting, we decided to culture with 45 Ca. (Maruyama et al., 1984), produced by clone TX8. It was demonstrated that the expressed recombinant protein bound calcium-45. child This is also true for the recombinant protein expressed by clone TXII: These two clones have the same C-terminal sequence, where the sequence This is the area where the lucium binding site can be confirmed.

In addition, by determining at least part of the amino acid sequence, the obtained recombinant Experiments were performed to better characterize the protein.

A specimen of 24Kd antigen obtained by purification of exoantigen released by taxiite was prepared. It was concentrated to a final volume of 50A11 and subjected to 5DS-PAGE. Then, the protein White matter is electroplotted from the gel onto a PVDF membrane. Gel and PVDF membrane Immobilized and sequenced at 24, the following sequence is read: a) Gly-Asn-Thr-Tyr-Arg-Vat-Glu-Arg -Pr.

b) Gin-Arg-Asp-Glu-Asp-11e-Phe-Leu -Arg-Ala-Leu-Asn-Lys-Gly-Gluc) Lys -Vat-11e-Asp-Asp-Vat-Glu All sequences are methionine It corresponds to the CNBr peptide obtained by cleavage at the residue.

Therefore, in particular, the object of the present invention is to obtain these amino acid sequences (a), (b) and (C) and is a protein as defined above that binds to calcium.

Example 4: In animals immunized with recombinant virus vv, TG, TX2170 Antibody induction The base of the tail of four 6-bearing Ba1b C female mice was incised, and a 5X10' p. Recombinant virus of fu vv, TG.

Inject TX2170. One row later, a second treatment is performed in the same manner. Second note Fifteen days after injection, serum was collected and analyzed using the “unisturn blotting” method described above. , for the protein induced by pTG2171 (clone TXII), Test its reactivity. Serum dilution is 100 times. Figure 4 shows all mouse 33Kd protein specifically reacts with the 33Kd protein. The immune response is particularly It was strong in mice 1 and 3, and these mice also had a slight 24Kd type. recognize. From this analysis, the vv, TG, TX2170 recombinant was expressed in E. coli. It can be seen that a response to the recombinant protein can be elicited.

Example 5: By immunizing with a recombinant vaccinia virus expressing the p24 antigen. Prevention against toxoplasmosis Recombinant vaccinia virus VVTG2170 was transformed into 5X10' plaque forming units (p 5-week-old OFI mice are immunized by oral administration of 5-week-old OFI (fu). 3 weeks later, 24 This group of mice is boosted with the same dose. toxopra Another 21 mice were immunized with recombinant vaccinia virus that does not express the Zuma antigen. This will be used as a control group. Two weeks after booster administration, mice infected with strain 76 Suspensions of brains obtained from Japanese animals (Laugier) and giraffes (Qu4) ] 1ei), 1970) (ground brain preparation was mixed into 12oo cysts/0. 3mIP B S l knee dilute) 0. Administer 3ml orally to mice . Virulence of strain 76 to OFI mice kills all mice in both groups. . Nevertheless, when compared with the control group, immunized with VVTG2170 In mice, there is a significant delay in mortality (as determined by the Mann-Whitney U test). probability p≦0.02) (the control group had a 50% mortality rate after 8 days; 50% mortality after 0.2 days).

The recombinant vaccine was administered to a group of 6-week-old BAL B/c mice (n = 105). Senior virus VVTG2170 is administered orally in a single dose of 5X108 pfu. 1.5 After a month, the plants are allowed to ingest 1200 cysts of 76 in the same manner as above. Compared to control group In comparison, mice immunized with VVTG2170 showed a significant reduction in mortality (definitely rate ≦0.05) is observed (22.8% versus 30.5% in the control group). Sara However, similar to 0F1 mice, mortality in animals immunized with VVTG2170 There is a delay. 90 days after infection, surviving mice are killed. Frontal part of the brain of these animals Take out the part with a punch (diameter 0.5 m + a) and incubate it in 80.3 ml of PB. Perform a puss test. Collect all samples from each group and homogenize them. . Two researchers then count the cysts in the Thomas cells. compared to control group , a significant increase in the number of parasite cysts was observed in the group of mice immunized with VVTG2170. A decrease is observed (by frequency comparison test, 2α=0.05: Poisson distribution).

Reference materials * Kasper Bradley and Fefe Acorn (Pf ef erkorn) - Journal of Immunology ( J. Immunol, ), 132.443-449 and Ncoh. -J, protozoo! , 30, M-356 (1983) * Kasper Crabb (Kasper Crabb) and Funitufunia Corn-J, 1mm uno1.5-2407 (1983) *Araujo and Remi Remington - Infection and Immunity nf, and 1mm, ) 45.122-128 (1984) Young and Davis-Scie nce) 222.778-782 (1983) *Kieny, Lathe, Drillien, Spe hner), Skory, Schmitt, Wiktor, Kobrowski and Luko Lecoeq-Nature 312.163-166 (1984) Kuchimigu Ro 8th day international search report international search report

Claims (25)

[Claims] 1. by antibodies produced against “excreted/secreted” antigens of Toxoplasma gondii. A protein that has a recognized epitope of the 24Kd protein. 2. It is a hybrid protein that plays a part in proteins not derived from Toxoplasma gondii. The protein according to claim 1. 3. The protein according to claim 1 or 2, having the complete sequence of a 24Kd protein. 4. A protein whose synthesis is directed by the cDNA shown in FIG. A protein corresponding to a fragment of Toxoplasma gondii that is excreted/secreted. 2. The protein according to claim 1, which is a protein recognized by an antibody produced against a ``antigen'' protein on the plate. 5. The protein according to any one of claims 1 to 4 obtained from bacteria. 6. The protein according to any one of claims 1 to 4 obtained from yeast or cell culture medium. quality. 7. Any of claims 1 to 6, which binds calcium and has the following amino acid sequence: Protein described in crab: a) [Sequence available] b) [There is an array] c) [There is an array] 8. A block for expressing the protein according to any one of claims 1 to 7 or Cells containing vectors. 9. The protein according to any one of claims 1 to 5 under the control of a bacterial promoter. Bacteria transformed with a plasmid containing genes encoding white matter The cell according to claim 8. 10. under the control of a yeast promoter, according to any one of claims 1 to 4 and 6. Claims that the yeast is transformed with a plasmid containing a gene encoding a protein The cell according to item 8. 11. Claim 9: The encoded gene consists of all or part of the cDNA shown in FIG. or the cell described in 10. 12. A protein comprising a gene encoding the protein according to any one of claims 1 to 4, The poxvirus puts the poxvirus under the control of the element that expresses the gene. Virus. 13. Pox virus according to claim 12, wherein the pox virus is vaccinia virus. Swirls. 14. The element that expresses the above gene is the virus protein of vaccinia's 7.5K gene. The poxvirus according to claim 13, having a lomotor. 15. A sequence in which the element for expressing the above gene encodes a human IL2 signal sequence The poxvirus according to claim 14. 16. A mammal infected with the poxvirus according to any one of claims 12 to 15. Mammalian cells. 17. The cells according to any one of claims 8 to 11 or claim 16 are cultured in an appropriate medium. The protein according to any one of claims 1 to 7, which is obtained by culturing the protein in How white matter is produced. 18. A DNA sequence encoding the 24Kd protein according to any one of claims 1 to 7. Column. 19. The DNA sequence shown in FIG. 20. The protein according to any one of claims 1 to 7 and the protein according to claims 12 to 15 at least one selected from the poxviruses listed in any of the above as an active drug; A pharmaceutical formulation particularly effective for the prevention of toxoplasmosis. 21. The poxvirus according to any one of claims 12 to 15 is used as an active drug. 20. The pharmaceutical preparation according to claim 19, wherein the virus is a live bacteria. 22. The poxvirus according to any one of claims 12 to 15 is used as an active drug. 21. The pharmaceutical preparation according to claim 20, wherein the virus is killed. 23. Any one of claims 20 to 22, which is in a form that can be administered to humans or animals. Pharmaceutical formulations as described. 24. produced for the protein according to any one of claims 1 to 7; 16. An antibody induced by the poxvirus according to any one of 12 to 15. 25. The antibody according to claim 24 or the protein according to any one of claims 1 to 7 Application of toxoplasmosis diagnostic agent.