KR101256181B1 - Fusion protein comprising antigen of Toxoplasma gondii and diagnosing kit comprising the same - Google Patents

Abstract

The present invention Toxoplasma worms ( Toxoplasma) The present invention relates to a fusion protein in which SAG1 (surface antigen 1) and GST (glutathione-S-transferase) of gondii are fused through a linker, a composition for diagnosis, and a diagnostic kit for toxoplasmosis infection comprising the fusion protein. The present invention also relates to a gene encoding the fusion protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and a method for producing a fusion protein comprising culturing the host cell. .

Description

Fusion protein comprising antigen of Toxoplasma gondii and diagnosing kit comprising the same

The present invention Toxoplasma worms ( Toxoplasma) It relates to a fusion protein in which antigen antigen SAG1 (GAGDI) and glutathione-S-transferase (GST) of a gondii are fused through a linker, a composition for diagnosis of toxoplasmosis infection comprising the fusion protein, and a diagnostic kit. The present invention also relates to a gene encoding the fusion protein, a recombinant vector comprising the gene, a host cell transformed with the recombinant vector, and a method for producing a fusion protein comprising culturing the host cell. .

Toxoplasma gondii ) is an opportunistic protozoan parasite that invades macrophages and infects the central nervous system. Toxoplasma worms are infected by various warm-blooded animals, including humans, through oocysts in cat feces, which are cats as host strains, and are present as tissues in infected animal tissues when humans feed on them. In addition, when a mother is infected with Toxoplasma worm, its trophozoite is passed through the placenta to the fetus. In the case of congenital infections, early fetuses develop pseudoacids, and in late fetuses, even if normal delivery occurs, symptoms such as visual impairment, hydrocephalus, and mental retardation occur. Acquired infections are asymptomatic in most individuals, but serious symptoms, such as retinal choroidal meningitis and meningitis, occur in immunocompromised patients such as AIDS patients following HIV infection or immunodeficiency caused by medical treatment. Currently, about one third of the world's population has toxoplasmosis antibodies.

A method for diagnosing a toxoplasma worm infection, wherein the parasite is isolated in blood or body fluid, the parasite is identified in tissue, a specific nucleotide sequence is detected by PCR, or a specific anti-product produced by the host in response to the infection. Methods for detecting toxoplasmosis immunoglobulins (Beaman et al., 1995 Principles and Practice of Infectious Diseases 4th Ed., Churchill Livingstone Inc., New York, 2455-75; Remington JS et al., 1995, Infectious Diseases) of the Fetus and Newborn Infant, WB Saunders, Philadelphia, PA, 140-267). In addition, as a method of diagnosing a congenital infection of a fetus during pregnancy, a method for detecting the presence of various classes of anti-toxoplasma worm immunoglobulins (binding capacity of IgG, IgM, IgA, IgG) and comparing the immunological profile of mother to child Etc.

Furthermore, in connection with the diagnosis of toxoplasma worm infection, International Publication No. 2003/080839 discloses a method based on phage-display technology for identifying antigen fragments of toxoplasma worm protein and its use as a diagnostic and immunological agent. , International Publication No. 2006/09665, provides chimeric recombinant antigens fused with three or more different antigenic sites of toxoplasma spp. As diagnostic and immunologic agents for toxoplasma spp. Infection.

However, the diagnosis of such toxoplasma worm infection does not provide sufficient sensitivity and specificity to diagnose accurately and quickly in all patients. Therefore, there is a need for the provision of a diagnostic agent that is more specific, sensitive and quick to identify.

In the previous study, the present inventors verified the antigenicity by fusion-expressing SAG1 (or P30), one of the major surface antigens of Toxoplasma spp., With the GST protein, and the N-terminal region among the SAG1 proteins of Toxoplasma spp. (SAG1A) has been confirmed to aggregate antigenicity (The Korean Journal of Parasitology, 34 (2): 135-141, 1996). However, when prokaryotes such as Escherichia coli are used to recombinantly express the antigen or its fusion protein, there has been a problem in that proteins are accumulated in the cytoplasm in the form of an inclusion body and are difficult to separate and purify. In addition, it has been found that when the antigen is directly linked with GST and expressed, GST has a problem that the tertiary structure of the antigen protein is modified or its activity is weakened.

Accordingly, the present inventors can easily supply and mass-provide when the recombinant expression is easy to isolate and purify, the structure and activity of the antigen is well maintained, to provide an antigen for diagnosing toxoplasma pellucida that can specifically and to diagnose toxoplasma worm infection. As a result of the study, the introduction of a specific linker led to the design of a fusion protein with a significant improvement in the water solubility and antigenicity of the toxoplasma spp. Antigen.

Specifically, the present inventors confirmed that when a specific linker is inserted between SAG1A and GST when the GST is fused with the SAG1 antigen of toxoplasma spp. By means of separation and purification, the water solubility is greatly increased and the antigenicity is improved. In addition, the insertion of such linkers greatly increases the water solubility of the fusion protein, thereby making it easier to obtain recombinant antigens, as well as providing flexibility to the fusion protein attached to the substrate when instilled in ELISA or rapid diagnostic kits. The present invention was completed by confirming that the tertiary structure of the antigen was well maintained to increase specificity and sensitivity in the antigen-antibody reaction.

One object of the present invention is Toxoplasma surface antigen 1 (SAG1) and glutathione-S-transferase (GST) of gondii ) provide a fusion protein fused via a linker. Preferably, the linker is a linker having an amino acid sequence of SEQ ID NO: 3.

It is another object of the present invention to provide a gene encoding the fusion protein.

It is another object of the present invention to provide a recombinant vector comprising the gene.

Still another object of the present invention is to provide a transformant transformed from a host cell with the recombinant vector.

Another object of the present invention to provide a method for producing a fusion protein comprising the step of culturing the transformant.

Another object of the present invention to provide a composition and diagnostic kit for diagnosing toxoplasma worm infection comprising the fusion protein.

As one embodiment for achieving the above object, the present invention is Toxoplasma gondii ) SAG1 (surface antigen 1) and GST (glutathione-S-transferase) relates to a fusion protein fused through a linker. Preferably, the linker is a linker having an amino acid sequence of SEQ ID NO: 3.

As used herein, the term "fusion protein" refers to a protein in a form in which two or more proteins are artificially linked, and in the present invention, a protein in a form in which SAG1 and GST of the toxoplasma worm are linked through a linker. Such fusion proteins can be obtained by chemical synthesis after each partner has been determined or by expression and purification by genetic recombination methods. Preferably, a nucleic acid encoding SAG1, a nucleic acid encoding a linker, and a fusion gene joining the nucleic acid encoding GST can be obtained by expression in a cell expression system. In a preferred embodiment, the amino acids constituting the fusion protein of the present invention are shown by the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "SAG1 (surface antigen 1)" refers to a major immunogenic protein present on the membrane surface of toxoplasma worms and is known to function in the invasion of host cells. In the present invention, SAG1 is used to include not only the entire protein but also a functional fragment thereof that retains antigenicity. Preferably, in the present specification, SAG1 may refer to 171 amino acids of the N-terminal except for the signal sequence, which may be expressed by replacing SAG1 or SAG1A. As a specific example, the amino acid sequence of SAG1A is shown in SEQ ID NO: 2.

As used herein, the term "GST (glutathione-S-transferase)" refers to an enzyme that is involved in the detoxification and metabolism of various harmful toxic substances in vivo, and in the present invention, GST expresses and isolates the target protein as water-soluble. It was used as a means to facilitate purification. GST increases the expression and water solubility of the fusion protein and has the advantage of being easy to purify and detect using immobilized glutathione (eg, glutathione affinity chromatography) or commercially available GST antibodies. In a specific embodiment of the present invention, GST was expressed using a commercialized GST expression vector, pGEX-4T-1, and the amino acid sequence of GST is shown in SEQ ID NO: 4.

As used herein, the term "linker" refers to a peptide inserted between the protein and the protein so that the activity can be enhanced by increasing structural flexibility when the fusion protein is prepared by linking SAG1 and GST. Preferably, the present invention is characterized by using a protein consisting of 40 amino acids at the N-terminus of GRA2, which is a dense granule protein of toxoplasma spp. As a linker protein. In the present invention, GRA2, a protein derived from toxoplasma spp., Was selected in order to prevent damage to specificity of a target for diagnosis, and a linker was selected between the GST and SAG1A by selecting the amino acid sequence having the highest atypical probability among GRA2 proteins as a linker. Inserted in Preferably, the linker has the amino acid sequence of SEQ ID NO: 3.

Due to the use of such a linker, it was possible to physically separate between GST and SAG1A to minimize the SAG1A intrinsic properties and the effect on the protein tertiary structure, as a result of the fusion protein of the present invention compared to the case without using the linker It was confirmed that the water solubility of (see FIG. 2) was greatly improved, and when reacted with the serum of the toxoplasma worm infection patient, it showed a strong antigen-antibody response (see FIG. 3). At this time, it was also confirmed that the fused GST did not affect the antigen-antibody response of SAG1A (see FIG. 3). In addition, the use of the linker allows SAG1A to maintain spatially three-dimensional structure and ensure fluidity from the bottom of the container or the nitrocellulose membrane when the fusion protein is attached to the bottom of the diagnostic kit or to the nitrocellulose membrane. This can increase specificity and sensitivity in antigen-antibody reactions.

Fusion proteins of the invention include variants of fusion proteins as well as proteins having their wild type amino acid sequences. A variant of a fusion protein means a protein in which the normal amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Such variants have a protein which preferably has at least 75%, more preferably 85%, even more preferably 90% and most preferably at least 95% homology with the amino acid sequence of SEQ ID NO. It may include.

In addition, the fusion protein may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like. The variant or modifier is a functional equivalent that exhibits the same biological activity as the native protein, but may be a variant or modifier that modifies the properties of the protein as needed. Preferably, the structural stability against heat, pH, etc. of the protein is increased or protein activity is increased by the variation and modification on the amino acid sequence.

The fusion protein of the present invention may be chemically synthesized or produced through genetic recombination methods. Preferably, the recombinant protein may be transformed into a host cell to isolate and purify the protein expressed therefrom.

Thus, as another aspect, the present invention relates to a gene encoding the fusion protein and a recombinant vector comprising the gene.

As another aspect, the present invention relates to a transformed host cell transformed with the recombinant vector and a method for producing a fusion protein comprising culturing the host cell.

The process of producing the fusion protein of the present invention by the genetic recombination method includes the following steps.

First, a recombinant vector containing a gene encoding a fusion protein is prepared.

DNA sequences encoding SAG1A and linker can be prepared by various methods known in the art, such as chemical synthesis, RT-PCR, respectively, and preferably a primer capable of amplifying the target gene using the DNA of the target protein as a template. It can be prepared via PCR using. The obtained SAG1A and linker nucleic acid can be operably linked to a vector comprising a nucleic acid encoding GST to produce a recombinant expression vector. In the present invention, any expression vector containing a GST gene generally used in the art can be used without limitation, and for example, when E. coli is used as a host cell, a pGEX vector (eg, pGEX-2T, pGEX-4T, pGEX-KG, etc.) or pET vectors can be used. For the purposes of the present invention, expression vectors may further comprise signal sequences for membrane targeting or secretion in addition to expression control elements such as promoters, initiation codons, termination codons, introns, polyadenylation signals and enhancers.

Next, the host cell is transformed using the recombinant vector.

A host cell that can be transformed with a recombinant vector according to the present invention includes both prokaryotic and eukaryotic cells, and a host having high DNA introduction efficiency and a high expression efficiency of introduced DNA is commonly used. Bacteria, for example, known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera fruitgifer (SF9), CHO, COS 1, COS 7 Animal cells such as, BSC 1, BSC40, BMT 10 and the like can be used, preferably Escherichia coli can be used.

Methods for preparing transformants by introducing recombinant vectors into host cells include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, and agrobacterial mediated transformation. , PEG, dextran sulfate, lipofectamine mediated transformation methods, and the like.

Next, the transformant is cultivated in a suitable medium and conditions to obtain a fusion protein.

The medium and the culture conditions can be used by appropriately selecting the tolerable according to the host cell. Conditions such as temperature, pH of the medium and incubation time can be appropriately adjusted to be suitable for growth of cells and mass production of proteins during the culture. The expressed fusion protein can be isolated from the cell's lysate. Specifically, cell pellets may be obtained by centrifugation or filtration, and an eluate including cell lysate may be obtained by removing media components including a ruptured cell wall or cell membrane. Specifically, cell pellets are well known in the art, such as alkalis, surfactants (CHAPS and SDS, etc.), the use of organic solvents and enzymes (lysozyme, etc.), sonication and the use of French Presses. And lysis by periodic high temperature, pressure, application of freezing and thawing cycles, and the like. The cell filtrate obtained above can obtain a clean eluate by removing the minimum area of biomass in the cell filtrate through cell fractionation, e.g., the unbroken part of the cell or the supernatant free of insoluble cell debris by centrifugation. Clear eluents can be obtained by taking these and, if necessary, can be subjected to additional purification procedures known in the art.

In addition, since the fusion protein expressed according to the present invention contains GST, it can be easily isolated and purified. Separation and purification methods that can be used at this time include affinity chromatography using a substance capable of specifically binding to the GST, such as an antibody to GST or glutathione (GSH), and most preferably using glutathione affinity chromatography. Do.

As another aspect, the present invention relates to a composition and diagnostic kit for diagnosing Toxoplasma worm infection comprising the GST-Linker-SAG1A fusion protein described above.

The fusion protein of the present invention can be used for diagnosing toxoplasmosis infection by forming and detecting an immune complex through an antigen-antibody complex reaction against an antibody present in a specimen of a suspicious toxoplasmosis infection. The sample may be a biological sample obtained from a human or animal, such as a body fluid (acupuncture, blood, serum, plasma, urine, semen, amniotic fluid, etc.), tissue or cells.

In the diagnostic kit of the present invention, the fusion protein may be used in a form bound to a solid substrate or support. Examples of solid supports that can be used include nitrocellulose (eg in the form of membranes or microtiter wells), polyvinylchloride (eg in the form of sheets or microtiter wells), polystyrene latex (eg beads or microtiter plates), Polyvinylidene fluoride, diazolated paper, nylon membrane, activated beads, and Protein A beads, and the like.

Such diagnostic kits may further include tools, reagents, and the like, commonly used in immunological assays in the art. Such tools or reagents include, but are not limited to, suitable carriers, labeling materials capable of producing detectable signals, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, polyesters, Polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharides, polymers such as magnetic fine particles plated with latex metal, other papers, glass, metals, agarose and combinations thereof.

Antigen-antibody complex formation includes tissue immunostaining, radioimmunoassay (RIA), enzyme immunoassay (ELISA), western blotting, immunoprecipitation assay, immunodiffusion assay, complement fixation assay (Complement Fixation Assay), FACS and protein chip (protein chip), etc., but is not limited thereto.

Labels that enable qualitative or quantitative measurement of antigen-antibody complex formation include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes. . Enzymes available as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, and glucose oxy Multidase, Hexokinase and GDPase, RNase, Glucose Oxidase and Luciferase, Phosphofructokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphopyruvate Decarboxylase, β- Latamases and the like, but are not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include acridinium esters, luciferin, luciferase, and the like, but are not limited thereto. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4- , and the like, but are not limited thereto. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. .

Preferably, the diagnostic kit of the present invention may be used as a rapid diagnostic kit that is downsized to enable on-site inspection using body fluids and the like and is economically improved. Such rapid diagnostic kits may include a strip-like structure, in which an analyte in a sample causes an antigen-antibody reaction in a signal band located in a migration pathway and visually confirms the result of the reaction by chemical color development, etc. The diagnosis can be made quickly and easily.

The GST-linker-SAG1A fusion protein of the present invention has a large increase in water solubility, thereby securing a large amount of recombinant antigens through expression and separation purification, and greatly improved antigenicity can be usefully used for diagnosing toxoplasmosis. In addition, when the fusion protein is attached to the solid support of the diagnostic kit, it has flexibility through atypical sites (linkers) and preserves the tertiary structure of the antigen well, thereby increasing specificity and sensitivity in the antigen-antibody reaction. have.

1 illustrates the structure of the fusion protein of the present invention.
2 shows the results of confirming the expression of GST-SAG1A fusion protein and GST-linker-SAG1A fusion protein through Coomassie blue staining (a) and Western blot (b). T represents the total cell eluate, S represents the upper fraction centrifuged at 13,000 rpm for 10 minutes, and P represents the precipitate fraction from centrifugal precipitation.
Figure 3 shows the results confirmed by Western blot after reacting the serum (serum number 7-81 and 4-10) of toxoplasmosis patients with GST protein, GST-SAG1A fusion protein and GST-linker-SAG1A fusion protein, respectively.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Preparation of Fusion Proteins

1-1. Toxoplasma worms SAG1A  Preparation of Nucleic Acids

Toxoplasma worms (RH strain) stored in the abdominal passage in mice were isolated by centrifugation (1800 g, 30 minutes) in 40% Percol (Pharmacia Biotech, Sweden) in phosphate buffered saline. 5 x 10 ⁸ tachyzoite was washed three times with PBS and washed with 1 ml K buffer (0.1 M Tris-Cl, pH7.5, 12.5 mM EDTA, 0.15 M NaCl) at 58 ° C. for 1 hour and at 37 ° C. 16 hours pretreatment. Next, the DNA was extracted with phenol / chloroform / isoamyl alcohol and washed with 70% alcohol.

To amplify the nucleic acid encoding the N-terminal 171 amino acid except for the signal sequence of SAG1, a sense primer (5'-GTTGAATTCGATCCCCCTCCTTGTTGCC-3 ', SEQ ID NO: 5) and an antisense primer (5'-GTGGAATTCGACTCCATCTTTCCCGCA-3' , SEQ ID NO: 6), 10 mM Tris-Cl, pH8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM dNTPs, 0.2 mM primers, and 0.5 U Taq DNA polymerase (Boehringer) as a reaction mixture. Mannheim Biochemica, Germany) was constructed and PCR was performed with a thermal cycler (M / J, USA). PCR was performed for 1 minute at 94 ° C, 1-2 minutes at 58 ° C, and 1-2 minutes at 72 ° C, and finally 5 minutes at 72 ° C for total elongation. The amplified DNA was electrophoresed at 1.2% agarose.

1-2. Preparation of Linker Nucleic Acids

In order to amplify the nucleic acid encoding the N-terminal 40 amino acids excluding the signal sequence of GRA2 from the DNA of the toxoplasma worm extracted in Example 1-1, a sense primer (5'-CGG GAT CC C AGG GAC CAG TCG) AC-3 ', SEQ ID NO: 7) and antisense primer (5'-CGG GAT CCA ACC GGT TCT TCT GGC T-3, SEQ ID NO: 8) were used, and the reaction mixture and reaction conditions described in Example 1-1 above. PCR was performed to amplify the linker nucleic acid.

1-3. Preparation of Fusion Proteins

A nucleic acid encoding the N-terminal 40 amino acid of GRA2 prepared in Example 1-2 was inserted into the BamHI restriction enzyme site of the pGEX-4T-1 vector expressing GST (glutathione-S-transferase) (Pharmacia Biotech, Sweden) After inserting the nucleic acid encoding SAG1A prepared in Example 1-1 into the EcoRI site, E. coli DH5a was transformed to clone the GST-linker-SAG1A vector. Next, the isolated vector was transformed into E. coli BL21 (DE3), and then 0.5 mM IPTG was added at 30 ° C. for 3 hours to induce the expression of the GST-linker-SAG1A fusion protein.

On the other hand, the same method as above by inserting the SAG1A encoding nucleic acid encoding in Example 1-1 into the EcoRI site of the pGEX-4T-1 vector (Pharmacia Biotech, Sweden) expressing GST (glutathione-S-transferase) as a control Expression of the GST-SAG1A fusion protein.

Example  2. Confirmation of Water Soluble Expression of Fusion Proteins

In Example 1-3, the cell eluate obtained by pulverizing BL21 cells induced by IPTG expression in physiological saline by ultrasound (5 times for 10 seconds at 10% intensity) was analyzed by 11% SDS-PAGE, and the protein component was analyzed. To confirm, Coomassie blue was stained and the results are shown in FIG. 2 (a). In addition, the cell eluate was western blotted with an anti-GST antibody and the results are shown in FIG. 2 (b).

In FIG. 2, T is the total cell eluate, S is the upper fraction centrifuged at 13,000 rpm for 10 minutes, and P is the result of fusion protein expression in the precipitate fraction at centrifugation. As shown in FIG. 2 (a), both GST-SAG1A fusion protein and GST-linker-SAG1A fusion protein expressed well, but in the case of GST-SAG1A fusion protein without linker, bands appeared in the precipitation fraction (P). It was found that some of the (arrow) fusion proteins formed an inclusion body. On the other hand, in the case of the GST-linker-SAG1A fusion protein of the present invention, almost no band was found in the precipitation fraction (arrow), and the band appeared mainly in the upper fraction (S). In addition, in the Western blot result of FIG. 2 (b), the GST-Linker-SAG1A fusion protein, unlike the GST-SAG1A fusion protein, was reconfirmed that most of them were soluble in water.

Example  3. Antigen Identification to Patient Serum

GST protein and serum GST-SAG1A fusion protein and GST-linker-SAG1A prepared in the serum of toxoplasmosis patients (serum numbers 7-81 and 4-10) possessed by the Department of Parasitology, Catholic University College of Medicine. After reacting the fusion protein, the anti-GST antibody (1: 100) and the anti-human IgG antibody (1: 1000) (Cappel Co., USA) bound to the peroxidase with the secondary antibody were treated with a primary antibody and a chromogenic substrate. After performing a Western blot using H ₂ O ₂ and 4-chloro-1-naphthol (4C1N, Sigma Chem. Co., USA), the results are shown in FIG. 3.

As shown in FIG. 3, the serum of the toxoplasmosis patient did not react with GST, but reacted with both the GST-SAG1A fusion protein and the GST-linker-SAG1A fusion protein (arrow), but not with the GST-SAG1A fusion protein. Compared with the GST-linker-SAG1A fusion protein including a linker it was confirmed that the stronger reaction.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Fusion protein comprising antigen of Toxoplasma gondii and          diagnosing kit comprising the same <130> DPP20114271KR <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> GST-linker-SAG1A fusion protein <400> 1 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro   1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu              20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu          35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys      50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn  65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                  85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Ser Ile Ala Tyr Ser             100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu         115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn     130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr             180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala         195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg     210 215 220 Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp Gly 225 230 235 240 Lys Pro Leu Asp Glu Arg Ala Val Gly Gly Lys Gly Glu His Thr Pro                 245 250 255 Pro Leu Pro Asp Glu Arg Gln Gln Glu Pro Glu Glu Pro Tyr Ser Gln             260 265 270 Arg Ala Ser Arg Val Ala Glu Pro Pro Leu Val Ala Asn Gln Val Val         275 280 285 Thr Cys Pro Asp Lys Lys Ser Thr Ala Ala Val Ile Leu Thr Pro Thr     290 295 300 Glu Asn His Phe Thr Leu Lys Cys Pro Lys Thr Ala Leu Thr Glu Pro 305 310 315 320 Pro Thr Leu Ala Tyr Ser Pro Asn Arg Gln Ile Cys Pro Ala Gly Thr                 325 330 335 Thr Ser Ser Cys Thr Ser Lys Ala Val Thr Leu Ser Ser Leu Ile Pro             340 345 350 Glu Ala Glu Asp Ser Trp Trp Thr Gly Asp Ser Ala Ser Leu Asp Thr         355 360 365 Ala Gly Ile Lys Leu Thr Val Pro Ile Glu Lys Phe Pro Val Thr Thr     370 375 380 Gln Thr Phe Val Val Gly Cys Ile Lys Gly Asp Asp Ala Gln Ser Cys 385 390 395 400 Met Val Thr Val Thr Val Gln Ala Arg Ala Ser Ser Val Val Asn Asn                 405 410 415 Val Ala Arg Cys Ser Tyr Gly Ala Asn Ser Thr Leu Gly Pro Val Lys             420 425 430 Leu Ser Ala Glu Gly Pro Thr Thr Met Met Leu Val Cys Gly Lys Asp         435 440 445 Gly Val     450 <210> 2 <211> 171 <212> PRT <213> Toxoplasma gondii <220> <221> PEPTIDE <222> (1) .. (171) <223> SAG1A <400> 2 Pro Pro Leu Val Ala Asn Gln Val Val Thr Cys Pro Asp Lys Lys Ser   1 5 10 15 Thr Ala Ala Val Ile Leu Thr Pro Thr Glu Asn His Phe Thr Leu Lys              20 25 30 Cys Pro Lys Thr Ala Leu Thr Glu Pro Pro Thr Leu Ala Tyr Ser Pro          35 40 45 Asn Arg Gln Ile Cys Pro Ala Gly Thr Thr Ser Ser Cys Thr Ser Lys      50 55 60 Ala Val Thr Leu Ser Ser Leu Ile Pro Glu Ala Glu Asp Ser Trp Trp  65 70 75 80 Thr Gly Asp Ser Ala Ser Leu Asp Thr Ala Gly Ile Lys Leu Thr Val                  85 90 95 Pro Ile Glu Lys Phe Pro Val Thr Thr Gln Thr Phe Val Val Gly Cys             100 105 110 Ile Lys Gly Asp Asp Ala Gln Ser Cys Met Val Thr Val Thr Val Gln         115 120 125 Ala Arg Ala Ser Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr Gly     130 135 140 Ala Asn Ser Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro Thr 145 150 155 160 Thr Met Thr Leu Val Cys Gly Lys Asp Gly Val                 165 170 <210> 3 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> linker peptide <400> 3 Gly Lys Pro Leu Asp Glu Arg Ala Val Gly Gly Lys Gly Glu His Thr   1 5 10 15 Pro Pro Leu Pro Asp Glu Arg Gln Gln Glu Pro Glu Glu Pro Tyr Ser              20 25 30 Gln Arg Ala Ser Arg Val Ala Glu          35 40 <210> 4 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> glutathione S-transferase from pGEX-4T-1 vector <400> 4 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro   1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu              20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu          35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys      50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn  65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu                  85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Ser Ile Ala Tyr Ser             100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu         115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn     130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu                 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr             180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala         195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg     210 215 220 Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg Asp 225 230 235 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> sense primer for SAG1A <400> 5 gttgaattcg atccccctcc ttgttgcc 28 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for SAG1A <400> 6 gtggaattcg actccatctt tcccgca 27 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sense primer for linker <400> 7 cgggatccca gggaccagtc gac 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for linker <400> 8 cgggatccaa ccggttcttc tggct 25

Claims (10)

A fusion protein wherein SAG1 (surface antigen 1) and GST (glutathione-S-transferase) of Toxoplasma gondii are fused through a linker consisting of the amino acid sequence of SEQ ID NO.
The fusion protein of claim 1, wherein said fusion protein is expressed in water solubility.
According to claim 1, wherein the surface antigen 1 (SAG1) is a fusion protein consisting of the amino acid sequence of SEQ ID NO: 2.
The fusion protein of claim 1, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 1.
Gene encoding the fusion protein of any one of claims 1 to 4.
Recombinant vector comprising the gene of claim 5.
The transformant transformed the host cell with the recombinant vector of claim 6.
Comprising the step of culturing the transformant of claim 7,
A method for preparing a fusion protein in which surface antigen 1 (SAG1) and glutathione-S-transferase (GST) of Toxoplasma gondii are fused through a linker consisting of the amino acid sequence of SEQ ID NO.
A composition for diagnosing toxoplasma worm infection comprising the fusion protein of claim 1.
Toxoplasma worm infection diagnostic kit comprising the fusion protein of claim 1.

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