KR101881013B1 - Toxoplasma gondii virus-like particle, expression vector and method for preparing the same - Google Patents

Abstract

The present invention relates to a toxoplasmosis virus-like particle, which comprises an influenza virus matrix protein (M1) and a toxoplasmosis virus-like particle comprising an antigenic determinant site derived from a toxoplasmosis.

Description

TECHNICAL FIELD [0001] The present invention relates to toxoplasmosis virus-like particles, a vector for producing the same, and a method for producing the same. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to toxoplasmosis virus-like particles, and more particularly to a toxoplasmosis virus-like particle comprising influenza virus matrix protein 1 (M1) as a structural protein.

Toxoplasma gondii is a bacterium belonging to the subclass of coccidia. Toxoplasmosis is a causative agent causing Toxoplasmosis in humans and animals distributed worldwide as obligare intercellular parasite.

Toxoplasmosis is largely classified into oocyst, tachyzoit, bradyzoit, schizont, and gametocyte, It takes five stages of development. Toxoplasma gonorrhoeae are infected by ingesting water or vegetables contaminated by oocysts of the cat's feces, or by eating toxoplasmosis which is present as a cyst in meat, such as pig, sheep, You can get infected when you do.

More than one-third of the world's population is thought to be infected with toxoplasmosis, and it is reported that infection rate is 2 to 25% depending on the group in Korea.

Toxoplasmosis can infect the fetus through the placenta when trophozoites of the toxoplasmosis are infected when the mother is infected. The initial fetus may become miscarriage or stillbirth by the infection of toxoplasmosis, and the fetus of the midgut may cause congenital malformations such as visual impairment, hydrocephalus, and mental retardation despite normal delivery.

In addition, toxoplasmosis which infects a healthy individual may cause diseases such as lymphatic inflammation, retinal choroiditis, and encephalomyelitis by destroying immune system cells and retinal endothelial cells and the like, Which can lead to meningitis or retinitis choroiditis.

On the other hand, pyrimethamine, which is also used as a therapeutic agent for malaria, is most widely used as a therapeutic agent for toxoplasmosis, but its therapeutic effect is not shown well during pregnancy. In addition, spiramycin is not effective for prevention Is used.

In addition, sulfamethoxazole, a sulfa drug that is used in combination with pyrimethamine, may reduce blood platelets by causing bone marrow suppression. In addition, the combined use of folic acid may cause allergic reactions, kidney disorders, blood disorders, Og- nosis), and vomiting.

However, currently the most widely used antitoxo-insecticides such as sulfasalazine and pyrimethamine are gradually decreasing in therapeutic effect due to the increase in resistance, and development of a vaccine capable of generating antibodies against human body and giving immunity to toxoplasmosis Has not been developed yet.

On the other hand, virus-like particles morphologically similar to the actual virus structure (hereinafter referred to as "VLP") have been proposed as vaccine antigens against several viruses (Roldao A, Mellado MC, Castilho L R, Carrondo MJ, Kushnir N, Streatfield SJ, Yusibov V: Vaccine 2012, 31: 58-83, Expert review of vaccines 2010, 9: 1149-1176; Kang SM, Kim MC, ).

Virus-like particles have received much attention in recent years as antigens for use in immunogen composition. The virus-like particle is in a form analogous to a wild-type virus, and can contain one or more surface proteins to induce an immune response in the body. Virus-like particles, unlike wild-type viruses, are inherently non-infectious and very safe despite their ability to activate the immune system.

The inventors of the present invention developed a new type of virus-like particle which induces an immune response in the body unlike a conventional therapeutic agent directly acting on toxoplasmosis and has no problem of safety and tolerance and has a preventive and therapeutic effect against toxoplasmosis .

The present invention aims to provide a novel type of virus-like particle capable of inducing an immune response against toxoplasmosis.

According to one aspect of the present invention, Toxoplasma gondii virus-like particles comprising an influenza virus matrix protein (M1) and an antigenic determinant site derived from Toxoplasma gondii are provided.

In one embodiment, the antigenic determinants derived from the toxoplasmosis may include an inner membrane complex (IMC).

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the toxoplasmosis-derived antigen recognition site is composed of the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the influenza virus matrix protein 1 is encoded by the nucleic acid sequence of SEQ ID NO: 3, and the antigen recognition site derived from the toxoplasmosis may be encoded by the nucleic acid sequence of SEQ ID NO: 4.

According to another aspect of the present invention, there is provided a vaccine composition comprising the virus-like particle as an active ingredient.

According to another aspect of the present invention, there is provided an expression vector for producing toxoplasmosis virus-like particle, which comprises a nucleic acid sequence encoding influenza virus matrix protein 1 (M1) and a nucleic acid sequence encoding an antigen recognition site derived from toxoplasmosis.

In one embodiment, the antigenic determinants derived from the toxoplasmosis may include an inner membrane complex (IMC).

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the toxoplasmosis-derived antigen recognition site is composed of the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the present invention, there is provided a host cell transformed with said expression vector.

In one embodiment, it may be a microorganism, an animal cell, a plant cell, a cultured cell derived from an animal, or a cultured cell derived from a plant.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector, comprising: transforming a host cell with the expression vector; And culturing the host cell to express the virus-like particle. The present invention also provides a method for producing toxoplasmosis virus-like particles.

According to the present invention, the toxoplasmicidal virus-like particles can be developed without a causative agent, unlike conventional vaccines, and do not induce non-selective antibody production.

In addition, the toxoplasmosis virus-like particles can provide a preventive and therapeutic effect against toxoplasmosis by activating the immune system in the body, so that it is safe without causing resistance, and side effects can be minimized.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

FIG. 1 illustrates a pFastBac vector according to an embodiment of the present invention. (A) is the amplification of the gene of Toxoplasma internode complex protein (IMC) through PCR. (B) is the amplified gene of influenza matrix protein 1 (M1). (C) and (D) are obtained by introducing each amplified gene into a plasmid using a restriction enzyme.
Figure 2 depicts a virus-like particle according to one embodiment of the present invention. (A) shows normal SF9 cells. (B) shows cells producing virus-like particles. (C) is an electron microscopic observation of the virus-like particle according to an embodiment of the present invention and measurement of the size thereof. (D) shows the results of Western blot analysis. 20 μg, 10 μg, and 5 μg of virus-like particles were individually loaded for SDS-PAGE, and anti-toxoplasma polyclonal antibodies and anti-M1 monoclonal antibodies were used as probes.
FIG. 3 shows the degree of production of toxoplasmosis-specific antibody upon induction of an immune response. (A), (B) and (C) show the results of immunoassay using virus-like particles according to one embodiment of the present invention and the anti-toxigenic IgG, IgG1 and IgG2a antibodies Respectively. (D) and (E) are the IgA and IgG antibody responses of the feces.
FIG. 4 shows the toxoplasmosis-specific antibody response to challenge infection. (A), (B) and (C) show mice infected with toxoplasmosis virus-like particles according to an embodiment of the present invention, and after 4 weeks, the mice were treated with Toxoplasma- specific IgG, IgG1 and IgG2a antibodies (* P < 0.05). (D), (E), (F), and (G) show the toxin-specific IgA and IgG antibody responses of feces and intestine (* P <0.05).
FIG. 5 is a graph showing changes in cerebral cyst and body weight due to toxoplasmosis infection. (A) and (B) are graphs showing the number and size of brain cysts after 4 weeks of infection with mice infected with virus-like particles and infected with toxoplasmosis virus according to an embodiment of the present invention. (C) is the measurement of change in body weight and survival rate after infection.
Figure 6 shows the reaction of splenocytes secreting antibody. (A), (B), (C) and (D) show that mice immunized with virus-like particles according to an embodiment of the present invention were infected with toxoplasmosis, and IgG, IgG1, IgG2a (* P < 0.05). &Lt; / RTI &gt;
Figure 7 confirms T cell response. (A), (B), and (C) show that mice infected with virus-like particles according to an embodiment of the present invention are infected with toxoplasmosis, and after 4 weeks, , IL-6, and IL-10 levels were measured. (D) and (E) are the levels of CD4 + T cells and CD8 + T cells (* P <0.05).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

When an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements unless specifically stated otherwise.

Unless otherwise defined, can be performed by molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the art of recombinant DNA within the skill of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. It is not intended that the invention be limited to the particular methodology, protocols, and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, 5 'to 3', amino acid sequences from left to right, amino to carboxyl.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention provides Toxoplasma gondii virus-like particles comprising an influenza virus matrix protein (M1) and an antigenic determinant site derived from Toxoplasma ganoderma .

The above-mentioned &quot; Virus-Like Particle (VLP) &quot; means a noninfectious viral subunit with or without a viral protein. For example, the virus-like particle may be completely deficient in the DNA or RNA genome, or the virus-like particle containing the viral capsid protein may undergo spontaneous self-assembly.

In particular, the virus-like particle according to one embodiment of the present invention can be produced by a genetic engineering method, and can include matrix protein 1 (M1) derived from influenza virus as a structural protein. The virus-like particle can be produced by a genetic engineering method without a separate causative agent, that is, toxoplasmosis, so that high productivity and economy can be realized.

The &quot; influenza virus matrix protein 1 &quot; is a structural protein of influenza virus and refers to a matrix protein that forms a coat inside the fat layer, which is an envelope of influenza virus. Influenza viruses consist of a subtype named A, B and C. The influenza virus is surrounded by a layer of matrix protein 1 (M1) acting as a link between the core and the viral envelope, and the matrix protein 1 is widely used as a structural protein in the development of influenza virus-like particles.

The virus-like particle may contain the influenza virus matrix protein 1 as a structural protein, and the surface of the influenza virus matrix protein 1 may include one or more antigenic determinants derived from toxoplasmosis.

Since the virus-like particle contains an antigenic determinant derived from toxoplasmosis on the surface, it can induce a specific immune response to toxoplasmosis when it is introduced into a specific individual. Thus, the virus-like particle may confer immunity to toxoplasmosis to the subject.

Such virus-like particles can be produced by methods well known in the art. For example, the virus-like particle may be prepared by transforming a predetermined host cell using a recombinant DNA molecule encoding the structural protein and the antigen-recognizing site, and then culturing. The protein expressed in the cell is assembled on the cell surface And can be discharged into post-culture supernatant. At this time, the surface proteins contained in the virus-like particles are maintained in a natural state without being subjected to an immobilization process, so that a desired immune response against a specific virus can be induced in the individual.

The virus-like particle acts as an antigen in the individual and can be used to label an antigen with T or B immune cells through reaction with antigen-presenting cells such as dendritic cells. The virus-like particle contains an antigenic determinant derived from Toxoplasma gonorrhea. However, since the virus-like particle does not contain any genetic material, it can not be propagated and is not toxic. Therefore, it can be used as a vaccine against toxoplasmosis. The antigenic site introduced on the surface of the virus-like particle has a higher antigenicity than that of the purely isolated recombinant protein and can form an effective neutralizing antibody.

The &quot; epitope &quot; is a basic unit or minimum unit of recognition by each antibody or T cell receptor, and means a specific domain, region or molecular structure to which the antibody or T cell receptor binds. The antigenic determinant site may be derived from toxoplasmosis, and is not particularly limited as long as it can induce immune activity against the toxoplasmosis.

In one embodiment, the antigenic determinant is selected from the group consisting of SAG1 (membrane-associated surface antigen), SAG2, secreted dense-granule protein (GRA1), GRA2, GRA4, GRA7, ROP1 And at least one selected from the group consisting of MIC1 (Micronemal protein), MIC2, MIC4, MIC6, MIC7, MIC8, MIC9, MIC10, MIC11, MIC2 associated protein, AMA1 (Plasmodium apical membrane antigen 1) But it may preferably include an inner membrane complex (IMC).

The &quot; inner membrane complex protein (IMC) &quot; is a surface membrane system constituting a flattened alveoli forming the basis of a plasma membrane, and is connected to a cytoskeletal network. The endosteal complex protein plays an important role in genome replication, cell movement, and host cell infiltration. The endosteal complex protein is involved in the formation of new cells in Toxoplasma gondii, and the replicated chromatin and cytoplasm are closely related to the assembly of the skeleton as a cell division process (Sheffield and Melton, 1968).

Particularly, the inventors of the present invention found that the endosteal complex protein effectively induces the production of anti-toxoplasia antibodies by its structural characteristics, and thus, as a form fused with influenza virus matrix protein 1, a high level of immunity against toxoplasmosis And that it is possible to provide it.

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the toxoplasmosis-derived antigen recognition site is composed of the amino acid sequence of SEQ ID NO: 2. In addition, the influenza virus matrix protein 1 is encoded by the nucleic acid sequence of SEQ ID NO: 3, and the antigen recognition site derived from the toxoplasmosis can be encoded by the nucleic acid sequence of SEQ ID NO: 4.

The influenza virus matrix protein 1 or the antigenic determinants derived from the toxoplasmosis include functional equivalents of proteins comprising the amino acid sequence of SEQ ID NO: 1 or 2. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Refers to a protein having a homology of at least 95% and having a physiological activity substantially equivalent to the amino acid sequence of SEQ ID NO: 1 or 2. The "substantially homogenous physiological activity" means that the influenza virus matrix protein 1 and / Refers to an activity as a virus-like particle capable of inducing a specific immune response to toxoplasmosis due to the structural and functional homology of the antigenic determinant site derived from toxoplasmosis.

According to another aspect of the present invention, there is provided a vaccine composition comprising the virus-like particle as an active ingredient.

The vaccine composition may be in the form of a virus-like particle or a concentrate thereof, or may be used in the form of a transformed host cell itself or a dry powder of transformed cells. In addition, the vaccine composition can be used together with other food or food ingredients and can be suitably used according to conventional methods.

The vaccine composition may further comprise one or more excipients selected from the group consisting of stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, bacterial cell walls, An animal phoxvirus protein, a subviral particle adjuvant, a cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, Octadecyl-ammonium bromide, and mixtures thereof. &Lt; Desc / Clms Page number 7 &gt;

In addition, the vaccine composition may comprise a medically acceptable carrier. The "medically acceptable carrier" may include any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, It is not.

Examples of carriers, excipients and diluents that can be included in the vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

In addition, the vaccine composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, and sterilized injection solutions according to a conventional method. In the case of formulation, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are commonly used may be used together.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate), sucrose or lactose, gelatin Etc. can be used together. In addition to these excipients, lubricants such as magnesium stearate talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are simple diluents commonly used, various excipients such as wetting agents, sweeteners, connect. For parenteral administration, sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents may be used. Examples of the water-insoluble preparation and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

According to another aspect of the present invention, there is provided an expression vector for producing toxoplasmosis virus-like particle, which comprises a nucleic acid sequence encoding influenza virus matrix protein 1 (M1) and a nucleic acid sequence encoding an antigen recognition site derived from toxoplasmosis.

The vector refers to a nucleic acid molecule used to carry a linked nucleic acid fragment. Such vectors may be, but are not limited to, bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments (i.e., fragments insertable into the host cell genome by homologous recombination). The plasmid means a circular double-stranded DNA loop in which a DNA fragment can be additionally inserted as a vector. In addition, viral vectors can link additional DNA into the viral genome.

The expression vector means a vector capable of directing the expression of a gene encoding an operably linked target protein. Generally, in the use of recombinant DNA technology, the expression vector is in the form of a plasmid, so that the term plasmid and vector may be used interchangeably. However, other types of expression vectors that perform the same function as a viral vector may also be included.

For example, the expression vector may be selected from the group consisting of pET-3a-d, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET- (+), pET-21b (+), pET-22b (+), pET- pET-30a-c (+), pET-30a-c (+), pET-26b (+), pET- (+), pET-32a-c (+), pET-32 Supplement / LIC, pET-32 Xa / LIC, pET-33b (+), pET- (+), pET-35b (+), pET-36b (+), pET-37b (+), pET- ), pET-41a-c (+), pRSETA, pRSETB, pRSETC, pESC- PPIC6A, pPIC6B, pPIC6B, pGAPZ C, pGAP? A, pGAP? B, pGAP? C, pPIC3.5K, pPIC6A, pPIC6B, pPIC6, HIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZA, CT, pYES2 / CT, pYES2 / NTA, pYES2 / NTB, pYES2 / NTC, pYES2 / CT, pYES2 / CT, pYIC2 / CT, pYC2 / CT, , pYES-DEST52, pTEF1 / Zeo , pFLD1, PichiaPink TM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP PSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 or pYJ406.

Meanwhile, the expression vector is introduced into a host cell, and a host cell transformed with the introduced vector can produce the toxoplasmosis-like particle. Wherein the vector may comprise a promoter that is recognized by the host organism.

The promoter may be selected from the group consisting of SBE4, 3TP, PAI-1, p15, p21, CAGA12, hINS, A3, NFAT, NFKB, AP1, IFNG, IL4, IL17A, IL10, GPD, TEF, ADH, CYC, INU1, PGK1, , GAL1, GAL10, GUT2, tac, T7, T5, nmt, fbp1, AOX1, AOX2, MOX1 and FMD1 promoters but may be varied in consideration of various variables such as host cells or expression conditions.

The nucleic acid sequence encoding the toxoplasmotic virus-like particle may be operably linked to the promoter sequence. The term "operably linked" means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. That is, the gene encoding the toxoplasmosis-like particle can be operatively linked to a promoter in the vector so that its expression can be regulated.

Alternatively, the expression vector may further comprise additional regulatory sequences. The regulatory sequence may be a Shine-Dalgano sequence of the replicase gene of phage MS-2 and a Shine-Dalkano sequence of cII of bacteriophage lambda, but is not limited thereto.

In addition, the expression vector may contain an appropriate marker gene necessary for screening the transformed host cells. The marker gene may be an antibiotic resistance gene or a fluorescent protein gene, and the antibiotic resistance gene may be selected from the group consisting of a hygromycin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene and a tetracycline resistance gene, It is not. The fluorescent protein gene may be selected from the group consisting of a yeast-enhanced green fluorescent protein (yEGFP) gene, a green fluorescent protein (GFP) gene, a blue fluorescent protein (BFP) And a red fluorescent protein (RFP) gene, but the present invention is not limited thereto.

According to another aspect of the present invention, there is provided a host cell transformed with said expression vector. The host cell refers to any organism that can be infected by a virus and which can be immunized by virus-like particles. The host cell may be metabolized by transformation.

In one embodiment, the host cell can be a microorganism, an animal cell, a plant cell, a cultured cell derived from an animal, or a cultured cell derived from a plant. The suitable host cell may be naturally occurring or may be a wild type host cell, or it may be a modified host cell. The wild-type host cell may be a host cell that has not been genetically altered by recombinant methods.

The type of the host cell is not particularly limited as long as it can be transformed by an engineering method and efficiently express a specific gene, and it may be preferably an insect cell. The insect cell may be any cell developed or marketed as a host system for gene expression, such as Spodoptera frugiperda SF21, SF9, Trichoplusia ni, Such as Anticarsa gemmitalis, Bombyx mori, Estigmene acrea, Heliothis virescens, Leucania separata, (Lymantria dispar), Malacasoma disstria, Mammestra brassicae, Manduca sexta, Plutella zylostella, Stodoptera exigua Spodoptera exigua, and Spodoptera littorlis. However, the present invention is not limited thereto.

The term "metabolically engineered" or "metabolic engineering" as used herein refers to biosynthetic genes, genes associated with operons, and control elements of these nucleic acid sequences for the production of desired metabolites such as alcohol or proteins in microorganisms can involve rational path design and assembly of control elements.

The term "metabolically engineered" as used herein is intended to encompass metabolic fluxes that are regulated and optimized by transcription, translation, protein stability and protein functionality using genetic engineering and appropriate culture conditions, As shown in FIG. A biosynthetic gene may be heterologous to a host (e. G., A microorganism) by being foreign to the host or transformed by mutagenesis, recombination or by association with a heterologous expression control sequence in an endogenous host cell. Suitable culture conditions include conditions such as culture medium pH, ionic strength, nutrient content, temperature, oxygen, carbon dioxide, nitrogen content, humidity, and other culture conditions that allow the production of compounds by the metabolic metabolism of the microorganism . Culture conditions suitable for microorganisms capable of functioning as host cells are well known in the art.

Thus, the "engineered " or" modified "host cells may be produced by introducing genetic material into a selected host or parent microorganism to alter or alter cellular physiology and biochemistry. Through the introduction of genetic material, the parent microorganism can acquire new properties, for example, the ability to produce new intracellular metabolites or higher amounts of intracellular metabolites.

For example, the introduction of genetic material into the parent microorganism can result in new or altered ability to produce chemicals. The genetic material introduced into the parent microorganism comprises a gene or a portion of a gene encoding one or more enzymes involved in the biosynthetic pathway for chemical production and further components for modulation of the expression or expression of these genes, Promoter sequence.

The term "altered host cell" refers to a genetically engineered host cell in which the target protein is produced at a level of expression or is produced at a level greater than the level of expression, or grown under essentially the same growth conditions Can be expressed at a level of expression that is greater than the level of expression of the desired protein in a modified or wild-type host cell. The "modified host cell" means a wild-type or modified host cell that is genetically engineered to overexpress a gene encoding the protein of interest. The modified host cell can express the target protein at a higher level than the wild type or modified host cell.

The term &quot; transformation &quot; refers to a method of transferring the vector into a microorganism or a specific cell. When the cell to be transformed is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Acac. Sci. USA, 9: 2110-2114 (1973), and Hanahan, D (1973)), one method (Cohen, SN et al., Proc. (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)), and the like. have.

(Graham, FL et al., Virology, 52: 456 (1973)) when the cell to be transformed is a eukaryotic cell, the microinjection method (Capecchi, MR, Cell, 22: 479 (Wong, TK et al., Gene, 10: 87 (1980)), DEAE - dextran treatment (Gopal, Mol. Cell. Biol., 5: 1188-1190 (1985)), and gene bendardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 1990)), but the present invention is not limited thereto.

In the case of transformation of yeast-like fungi, generally, lithium acetate (RDGietz, Yeast 11, 355360 (1995)) and heat shock (Keisuke Matsuura, Journal of Bioscience and Bioengineering, Vol. (Nina SkoluckaAsian, Pacific Journal of Tropical Biomedicine, 94-98 (2011)), but the present invention is not limited thereto.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector, comprising: transforming a host cell with the expression vector; And culturing the host cell to express the virus-like particle. The present invention also provides a method for producing toxoplasmosis virus-like particles.

The transformed host cells can be cultured under batch, feed-batch, or continuous fermentation conditions, and the host cells can express the toxoplasmosis-like particles by transformation. Therefore, -Like particle protein.

At this time, the classical batch fermentation method can use a closed system, the culture medium is prepared before fermentation is carried out, the organism is inoculated into the medium, and fermentation can take place without adding any ingredient to the medium have. In certain cases, the pH and oxygen content, not the carbon source content of the growth medium, can be varied during the batch process. The metabolites and cellular biomass of the batch system may be constantly changing until fermentation is stopped. In a batch system, the cells can progress over the highly grown log phase on stationary retard, and the growth rate may be reduced or stopped to finally reach a stationary phase. In the normal period, the cells on the log can make most of the protein.

A variation of the standard batch system is the "feed-batch fermentation" system. In such a system, nutrition (e.g., carbon source, nitrogen source, O ₂ , and typically, other nutrients) may be added when the concentration of these cultures falls below a threshold. A feed-and-batch system may be useful when inhibition of catabolism is desired to inhibit cell metabolism and it is desirable for the medium to have a limited amount of nutrients in the medium. Measurement of the actual nutrient concentration in the feed- Can be predicted based on changes in measurable factors such as dissolved oxygen and partial pressure of the waste gas such as CO ₂ . Batch and feed-batch fermentation is well known in the art as a general system.

Continuous fermentation is an open system in which the defined culture medium is continuously added to the bioreactor and the same amount of conditioned media is removed simultaneously during the process. Continuous fermentation generally allows the cells to maintain a constant high density of cultures that are initially in log phase growth. Continuous fermentation may be able to manipulate one or any number of factors that affect cell growth or final product concentration.

For example, a nutrient such as a carbon source or a nitrogen source can be maintained at a fixed rate, and all other parameters can be appropriately maintained. In other systems, many growth-affecting factors can continue to change while the cell concentration measured by the medium turbidity remains constant. Continuous systems try to maintain constant growth conditions. Thus, cell loss due to media excretion can be balanced against cell growth rate in fermentation. Methods for maintaining nutrients and growth factors during the continuous fermentation process as well as techniques for maximizing the rate of product formation are known in the art.

It will be apparent to those skilled in the art that various changes and modifications may be made thereto without departing from the spirit and scope of the present invention as set forth in the appended claims. It is encompassed in the technical idea of.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Preparation Example 1: Preparation of toxoplasmosis virus-like particles

The gene of the toxoplasmosis complex protein (IMC) was amplified by PCR using a forward primer (5'-AAAGAATTCACCATGGGGAACACGGCGTGCTG-3 ') and a reverse primer (5'-TTACTCGAGTTAGTTTCTGTCGTTGCTTGC-3').

The influenza matrix protein 1 (M1) gene was amplified by RT-PCR using a forward primer (5'-AAAGAATTCACCATGAGTCTTCTAACCGAGGT-3 ') and a reverse primer (5'-TTACTCGAGTTACTCTAGCTCTATGTTGAC-3'). The primers were introduced with restriction enzyme sites (EcoR I and Xho I, respectively) (FIGS. 1A and 1C).

Each of the above genes was introduced into the pFastBac vector and cut with EcoR I and Xho I restriction enzymes to confirm the introduction of IMC and M1 genes (FIG. 1B, 1D). The nucleic acid sequence of the introduced gene was confirmed to be identical to the previously published sequence (IMC: HQ012579, M1: EF467824) by DNA sequencing (Eurofins MWG Operon).

In order to produce recombinant baculoviruses (rBVs) expressing IMC and M1, DNA transfection was carried out by cellfectin II (Invitrogen) and SF9 cells, and transformation using pFastBac vector containing IMC and M1 was carried out in white / blue screening. Baculovirus was produced according to the manufacturer's instructions via a Bac-to-Bac expression system (Invitrogen).

Preparation Example 2: Preparation of toxoplasmosis virus-like particles

Toxoplasma virus-like particles were produced in SF9 insect cells co-infected with recombinant rBVs expressing IMC and M1. The virus-like particles released into the cell culture supernatant were harvested and purified through a discontinuous sucrose gradient (15% -30% -60%) at 4 ° C, 28000 rpm for 1 hour.

Virus-like particle bands between 30% and 60% were collected, diluted with phosphate buffered saline (PBS) and centrifuged at 4 ° C, 28000 rpm for 1 hour. Virus-like particles were resuspended in PBS overnight at 4 ° C.

Virus-like particles were confirmed by Western blotting and electron microscopy. In Western blot analysis, antibodies were collected from mice infected with Toxoplasma gondii ME49 strain. The content of M1 protein was determined by anti-M1 antibody. For electron microscopy and size measurements, virus-like particles were stained medium and observed by transmission electron microscopy (TEM).

SF9 cells producing virus-like particles were found to be larger than control cells (Fig. 2A, 2B). The size and shape of the virus-like particles were observed by electron microscopy in the form of spheres with spikes on the surface (FIG. 2C). Western blot was carried out using anti-toxoplasic polyclonal antibody and anti-M1 monoclonal antibody to confirm that it was introduced into virus-like particles of IMC and M1 (Fig. 2D).

As a result, SF9 cells infected with rBVs expressing IMC and M1 were found to produce particles with similar morphology and size to virions.

Experimental Example 1: Immunoinduction reaction test in an animal model

The immune response induced by inoculation of the virus-like particles of Preparation Example 2 in mice was confirmed.

After the virus-like particle vaccination (Intranasal route) of Preparation Example 2, blood was collected at predetermined time from the mice. ( Toxoplasma gondii , ME49) challenge infection (Oral, Intraperitoneal route), and the degree of Toxoplasma specific IgG, IgG1, IgG2a and fecal IgA and IgG antibody formation of serum was measured by ELISA method (FIG. 3) .

The total IgG, IgG1, and IgG2a responses of the mice immunized with the virus-like particles were significantly increased at 1 and 4 weeks after inoculation suggesting that the Toxoplasma IMC-specific antibody was progressively matured (Figures 3B, 3C, 3D). In addition, the IgA and IgG antibody responses in the feces were significantly increased compared to the negative control, indicating that mucosal immunity was induced (Fig. 3E, 3F).

In order to confirm the antibody response profile of serum, feces, and intestine due to challenge infection, four weeks after the virus-like particle inoculation, toxoplasmosis seed (ME49) was orally infected. Very high levels of IgG, IgG1, IgG2a antibody responses were observed in the sera by toxoplasmosis infection (P <0.05, Figs. 4A, 4B, 4C).

In addition, since intranasal immunization was predicted to be induced by the mucosal route, toxoplasmosis-specific IgA and IgG antibody responses were measured in the feces and intestinal mucosa. As a result, very high levels of toxoplasmosis-specific IgA and IgG antibodies were detected in feces (Fig. 4D, 4E, * P < 0.05) and intestine (Fig. 4F, 4G, * P < The results indicate that the IgA and IgG antibodies were formed very rapidly by infection of toxoplasmosis.

These results suggest that the virus-like particles of Preparation Example 2 provide high immunity against toxoplasmosis and can effectively induce antibody responses in tissue system and mucosa in response to infection with toxoplasmosis.

Experimental Example 2: Protective Immunity Test

The protective immunity effect induced by inoculation of the virus-like particles of Preparation Example 2 in mice was confirmed.

Mice challenged with Toxoplasma gondii (ME49) after the virus-like particle vaccination of Preparation Example 2 were sacrificed after 4 weeks and the amount and size of brain cysts were analyzed.

The mice immunized with the virus-like particles exhibited higher protective immunity after oral or laparoscopic (IP) infection compared to the negative control. In particular, the number of brain cysts was significantly reduced (Fig. 4A, * P < 0.05) and the cyst size was also significantly decreased (Fig. 5A, 5B, reduction rate: 60%, * P <

Further, as a result of measuring the change in body weight by Toxoplasma infection, the immunized mice showed an increase in body weight despite the infection, whereas the negative control group lost weight or died (FIG. 5C). The survival rate of mice not immunized with the negative control, i.e., the virus-like particles, was only 60% (Fig. 5D).

These results suggest that virus-like particles can induce a protective immune effect against toxoplasmosis infection.

Experimental Example 3: Antibody-secreting cell reaction, cytokine production and T cell response test

After the virus-like particles of Preparation Example 2 were inoculated into the mice, the antibody-secretory cell response was confirmed.

Splenocytes infected with toxoplasmosis were collected and cultured in vitro . Spleen cells of mice immunized by infection with Toxoplasma gondii secreted significantly higher levels of toxoplasmosis-specific IgG, IgG1, IgG2a into the supernatant as compared to the negative control (Figs. 6A, 6B, 6C, * P < 0.05). Furthermore, analysis of the IgA antibody contained in the supernatant of the same culture medium revealed that the level of toxoplasmosis-specific IgA antibody was very high after 4 days.

In addition, the virus-like particles were inoculated into mice, and cytokine production and T cell response following toxoplasmosis infection were confirmed.

One month after infection, splenocytes were obtained to measure cytokine production. The levels of IFN-y, IL-6, and IL-10 in cytokine-secreting cells were measured. The spleen cells of mice immunized by toxoplasmosis-antigen stimulation produced significantly higher levels of IFN-y, IL-6, IL-10 compared to negative control (Fig. 7A, 7B, 0.05).

Immunized mice induced higher levels of Th1 / Th2 cytokine response by infection and significantly increased levels of CD4 + T cells and CD8 + T cells (Fig. 7D, 7E, * P < 0.05).

That is, mice immunized by the virus-like particles can acquire a very high level of immunity against infection with toxoplasmosis.

Experimental Example 4: Immunological induction effect test by antigenic site replacement

The IMC proteins used in Preparation Examples 1 and 2 were mixed with SAG1 (secretion dense-granule protein 1), GRA1 (secreted dense-granule protein 4), ROP1 (Rhoprotein protein 1), ROP2 Rhoptic protein 2), MIC1 (Micronemal protein 1), and MIC8 (Micronemal protein 8), and Experimental Examples 1 to 3 were repeated.

As a result, there was some difference in the effect of the immune response induced when the antigen-specific region of the virus-like particle was different, but the immunological reaction and the antibody-secretory cell response were effectively induced in the animal model, and the cytokine Generation and T cell responses were confirmed.

In particular, mice immunized with the virus-like particles produced high levels of IFN-y, IL-6 and IL-10, and the levels of CD4 + and CD8 + T cells were markedly increased.

That is, since the virus-like particle includes the matrix protein 1 (M1) derived from influenza virus as a structural protein and contains an antigenic determinant site derived from toxoplasmosis on the surface, the virus-like particle is specific to toxoplasmosis An immune response can be induced.

In addition, the virus-like particle effectively induces an immune response even when the antigen-determining region is different, so that the virus-like particle can be used as a vaccine against toxoplasmosis.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

<110> Kyung-Hee University <120> TOXOPLASMA GONDII VIRUS-LIKE PARTICLE, EXPRESSION VECTOR AND          METHOD FOR PREPARING THE SAME <130> 2016-PP-10047 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 252 <212> PRT <213> Influenza virus <400> 1 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro   1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe              20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr          35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe      50 55 60 Thr Leu Thr Val Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val  65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala                  85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala             100 105 110 Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met         115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe     130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu                 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met             180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln         195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Ser Ser     210 215 220 Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys                 245 250 <210> 2 <211> 164 <212> PRT <213> Toxoplasma gondii <400> 2 Met Gly Asn Thr Ala Cys Cys Gly Phe Asp Ser Asp Ser Thr Ala Asp   1 5 10 15 Leu Glu Ile Gly Arg Glu Gly Glu Val Arg Ser Ser Lys Pro Ile Gln              20 25 30 Val Ser Lys Glu Ala Phe Asp Asn Trp Met Asn Arg Tyr Glu Ala Gly          35 40 45 Asp Thr Met Glu Val Leu Phe Pro Asp Gly His Arg Ile Glu Cys Asn      50 55 60 Leu Lys Ile Asp Arg Pro Lys Asn Phe Met Asn Leu Thr Phe Asn Gln  65 70 75 80 Lys Val Arg Pro Ile Gln Leu Asp Asp Ile Ala Ala Val Leu Tyr Gly                  85 90 95 Ser Asp Pro Arg Ser Ser Glu Cys Ala Asp Ser Lys Met Leu Arg Asn             100 105 110 Pro Cys Val Val Gly Phe Arg Leu Ala Ser Ser Gly Arg Ala Ile Ala         115 120 125 Phe Ser Phe Lys Asp Ile Thr Asp Ala Gln Cys Phe Val Ser Phe Leu     130 135 140 Asp Asp Glu Ile Lys Lys Asn Gln Glu Ser Asn Lys Ser Ser Ala Ser 145 150 155 160 Asn Asp Arg Asn                 <210> 3 <211> 1027 <212> DNA <213> Influenza virus <400> 3 agcgaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact 60 ctctatcatc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg aagatgtctt 120 tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct 180 gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg 240 aggactgcag cgtagacgct ttgtccaaaa tgcccttaat gggaacgggg atccaaataa 300 catggacaaa gcagttaaac tgtataggaa gctcaagagg gagataacat tccatggggc 360 caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg gcctcatata 420 caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga 480 acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact 540 aatcagacat gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat 600 ggctggatcg agtgagcaag cagcagaggc catggaggtt gctagtcagg ctagacaaat 660 ggtgcaagcg atgagaacca ttggaactca tcctagctcc agtgctggtc tgaaaaatga 720 tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc aacggttcaa 780 gtgatcctct cactattgcc gcaaatatca ttgggatctt gcacttgaca ttgtggattc 840 ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc 900 cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg 960 ctgtggatgc tgacgatggt cattttgtca gcatagagct ggagtaaaaa actaccttgt 1020 ttctact 1027 <210> 4 <211> 495 <212> DNA <213> Toxoplasma gondii <400> 4 atggggaaca cggcgtgctg cggtttcgac agtgactcta ctgctgacct cgagatcggt 60 cgagaggggg aagtgcggag tcgcaaacca attcaggtat ccaaagaggc gtttgacaac 120 tggatgaatc gttatgaggc cggagacacg atggaagtgc tttttcctga tggtcaccga 180 attgagtgta acttgaaaat cgaccgaccg aaaaacttca tgaatctcac cttcaatcag 240 aaagtaagac ccatccagct ggatgacatt gcagctgtcc tatatggctc ggatcctcgc 300 agttccgaat gcgcagatag caaaatgctg cgaaacccct gtgtcgtggg cttccgcctc 360 gcgagctctg gacgagccat cgcgttttct tttaaagaca tcacggacgc gcagtgtttt 420 gtgtctttcc tggacgacga aatcaagaag aatcaggagt caaacaagtc ttcagcaagc 480 aacgacagaa actaa 495

Claims (11)

Influenza virus matrix protein &lt; RTI ID = 0.0 &gt; (M1) &lt;
And an inner membrane complex (IMC) derived from a toxoplasmosis formed on the surface of the influenza virus matrix protein 1,
Toxoplasma gondii virus-like particles that increase toxoplasmosis-specific IgG, IgG1, IgG2a levels. delete The method according to claim 1,
The influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the antigenic determinant derived from the toxoplasmosis is the amino acid sequence of SEQ ID NO: 2. The method according to claim 1,
The influenza virus matrix protein 1 is encoded by the nucleic acid sequence of SEQ ID NO: 3, and the antigenic determinant site derived from the toxoplasmosis is encoded by the nucleic acid sequence of SEQ ID NO: 4. A toxoplasmicidal vaccine composition comprising the virus-like particle of any one of claims 1, 3, and 4 as an active ingredient. A nucleic acid sequence encoding influenza virus matrix protein 1 (M1) and
A nucleic acid sequence encoding an internal membrane complex (IMC) derived from toxoplasmosis,
Expression vectors for the production of toxoplasmosis virus-like particles which increase toxoplasma specific IgG, IgG1, IgG2a levels. delete The method according to claim 6,
Wherein said influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and said toxoplasmosis-derived antigenic determinant comprises the amino acid sequence of SEQ ID NO: 2. 8. A host cell transformed by the expression vector of claim 6 or 8. 10. The method of claim 9,
A host cell that is a microorganism, an animal cell, a plant cell, a cultured cell derived from an animal, or a cultured cell derived from a plant. Transforming the host cell with the expression vector of claim 6 or 8; And
And culturing the host cell to express the virus-like particle.

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