KR102037451B1 - A plasmodium berghei virus-like particle, vaccine composition, expression vector and method for preparing the same - Google Patents

Abstract

The present invention relates to a malaria virus-like particle, which comprises an influenza virus matrix protein (M1) and a malaria virus comprising a malaria-derived Okonote surface antigen-like protein Pfs28. Provide analogous particles

Description

Malaria virus-like particle, vaccine composition comprising the same as an active ingredient, a vector for producing the same, and a method for preparing the same.A PLASMODIUM BERGHEI VIRUS-LIKE PARTICLE, VACCINE COMPOSITION, EXPRESSION VECTOR AND METHOD FOR PREPARING THE SAME

The present invention relates to malaria virus-like particles, and more particularly to malaria virus-like particles comprising influenza virus matrix protein 1 as structural proteins.

Malaria is a parasitic disease in human blood that causes malaria and is the single most common cause of death worldwide (2.4 million annually).

About 300 to 500 million people are infected with malaria each year with relatively high morbidity and mortality. The prevalence and mortality rates of malaria infection are particularly high in children, as well as adults who have moved to areas where malaria has been previously exposed to malaria.

The World Health Organization (WHO) estimates that 2-3 million children die of malaria each year in Africa alone.

In many countries, the widespread incidence and increased prevalence of malaria caused by drug-resistant parasites ( Plasmodium falciparum , Plasmodium vivax ) and pesticide-resistant media (Anopheles mosquitoes) underscores the need to develop new control methods for the disease. (Nussenzweig and Long 1994).

In Korea, malaria has reemerged around the armistice line and the northern part of Gyeonggi Province and northwestern Gangwon Province, and the area of infection risk is gradually expanding. More than 1,000 patients occur every year in Korea, but the anti-malarial drugs chloroquine and artemether cause resistance, which is why vaccination is the most effective way to prevent malaria.

On the other hand, virus-like particles that are morphologically similar to the actual virus structure have been proposed as vaccine antigens for several viruses (Roldao A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM: Expert review of vaccines 2010, 9: 1149). Kang SM, Kim MC, Compans RW: Expert review of vaccines 2012, 11: 995-1007; Kushnir N, Streatfield SJ, Yusibov V: Vaccine 2012, 31: 58-83).

Virus-like particles have received a lot of attention recently as antigens for use in immunogen compositions. Virus-like particles are similar in form to wild type viruses and may contain one or more surface proteins to induce an immune response in the body. Unlike wild-type viruses, virus-like particles are very safe because they lack genetic material and are non-infectious, although they can activate the immune system.

The present inventors have tried to develop a novel type of virus-like particle that induces an immune response in the body unlike a conventional therapeutic agent that directly acts on malaria, is safe and has no problems of resistance, and is excellent in preventing the infection of malaria.

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

According to one aspect of the invention, Influenza virus matrix protein 1 (Influenza virus matrix protein; M1); And malaria virus-like particles (Ookinete surface antigen-like protein Pfs28) derived from malaria.

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, the motor conjugate surface antigen-like protein derived from malaria may be 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 motion conjugate surface antigen-like protein derived from malaria 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 malaria virus-like particle as an active ingredient.

According to another aspect of the invention, the nucleic acid sequence of SEQ ID NO: 3 that encodes influenza virus matrix protein 1 (M1) and the sequence number that encodes a malaria-derived Ookinete surface antigen-like protein Pfs28 An expression vector for preparing malaria virus-like particles comprising the nucleic acid sequence of 4 is provided.

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, the motor conjugate surface antigen-like protein derived from malaria may be composed of the amino acid sequence of SEQ ID NO: 2.

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

In one embodiment, the host cell may be derived from microorganisms, animal cells, plant cells, culture cells derived from animals, or plants.

According to another aspect of the invention, transforming the host cell with the expression vector; And culturing the host cell to express a virus-like particle. There is provided a method for producing a malaria virus-like particle comprising a.

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

In addition, the malaria virus-like particles may activate the body's immune system and provide immunity and preventive effects against malaria, thus causing no resistance and minimizing side effects.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 depicts a malaria virus-like particle according to one embodiment of the present invention. (B) is an electron microscope to observe the malaria virus-like particles according to an embodiment of the present invention. (C) is the result of Western blot analysis of malaria virus-like particles reacted with influenza M1 antibody. (D) is the result of Western blot analysis in which malaria virus-like particles reacted with malaria protozoa. 50 μg, 10 μg, 2 μg of malaria virus-like particles were loaded for SDS-PAGE, respectively, and anti-malaria polyclonal antibodies and anti-M1 monoclonal antibodies were used as probes.
Figure 2 confirms the formation of malaria specific IgG, IgG2a, IgG1 and IgG2b through ELISA analysis after nasal inoculation (Fig. 2A) and muscle inoculation (Fig. 2B) of the virus-like particles according to an embodiment of the present invention.
Figure 3 shows the infection of malaria protozoan Plasmodium berghei (ANKA) by the intraperitoneal route after induction of an immune response by a virus-like particle according to an embodiment of the present invention and malaria specific IgG, IgG2a, IgG1 and IgG2b antibodies The degree of formation is measured.
Figure 4 shows the effect of protective immunity (protective immunity) by virus-like particle inoculation according to an embodiment of the present invention. Weight change and survival rate were compared between the mouse and the control group immunized with the virus-like particles according to an embodiment of the present invention.
5 and 6 confirm the immune response by virus-like particle inoculation according to an embodiment of the present invention.
Figure 7 confirms the protective immunity (protective immunity) effect of the virus-like particle inoculation according to an embodiment of the present invention. Malaria infection rates were compared between mice and controls in which immunity was formed by virus-like particles according to an embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

When a part is said to "include" a certain component, this means that it may further include other components, without excluding other components, unless specifically stated otherwise.

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

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

Various scientific dictionaries that include the terms included herein are well known and available in the art. While any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, some methods and materials have been described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents, because it can be used in various ways.

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

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

One aspect of the invention provides an influenza virus matrix protein (M1) and a malaria virus-like particle comprising an antigen determination site from malaria.

In one embodiment, the virus-like particles include Influenza virus matrix protein 1 (M1); And a kinetic surface antigen-like protein (Pfs28) derived from parasitic protozoan mouse malaria ( Plasmodium berghei , ANKA strain).

The term "virus-like particle (VLP)" refers to a non-infectious viral subunit with or without viral protein. For example, the virus like particles may be completely devoid of DNA or RNA genomes, or virus like particles comprising viral capsid proteins may undergo spontaneous self assembly.

In particular, the virus like particles may be prepared by genetic engineering methods, and may include matrix protein 1 (M1) derived from influenza virus as a core protein. The virus analogous particles can be produced by genetic engineering methods without a separate causative agent, that is, malaria, so that high productivity and economy can be realized.

The “influenza virus matrix protein 1” is a structural protein of influenza virus, and means a matrix protein that forms a coat inside a fat layer, which is an envelope of influenza virus. The influenza virus consists of subtypes named A, B and C. The influenza virus is surrounded by a layer of matrix protein 1 (M1) that serves as a linkage between the core and the viral envelope, and the matrix protein 1 may be used as a structural protein in the development of influenza virus-like particles.

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

Since the virus-like particle includes an antigen determination site derived from malaria on its surface, it can induce a specific immune response against malaria when introduced into a specific individual. Thus, the virus-like particle can impart immunity to malaria to the subject.

The virus-like particles can be prepared 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 an antigen recognition site, followed by culturing, wherein the protein expressed in the cell is assembled at the cell surface. It can then be drained into the culture supernatant. At this time, since the surface protein contained in the virus-like particle maintains its natural form without undergoing immobilization, it can induce a desired immune response against a specific pathogen in the individual.

The virus-like particles act as antigens in an individual and can label antigens on T or B immune cells through reaction with antigen-labeled cells such as dendritic cells.

The virus-like particle contains an antigen determination site derived from malaria, but since it does not contain any genetic material, it can be used as a vaccine against malaria because it is impossible to proliferate and is safe because of no toxicity. The antigen-determining site introduced into the surface of the virus-like particle has higher antigenicity than the purely isolated recombinant protein and can form an effective neutralizing antibody.

The “antigen determination site (epitope)” is the basic element or minimum unit of recognition by each antibody or T cell receptor and refers to the specific domain, region or molecular structure to which the antibody or T cell receptor binds. The antigenic determining site may be derived from malaria, and is not particularly limited as long as it can induce immune activity against the malaria.

In one embodiment, the antigenic determining site is EBA, GLURP, RAP1, RAP2, Sequestrin, PO32, STARP, SALSA, PfEXPl, Pfs25, Pfs28, PFS27 / 25, Pfs48 / 45, Pfs230, or their derived from malaria It may include an analogue, but may preferably include an Okotete surface antigen-like protein Pfs28 derived from the parasitic protozoan mouse malaria ( Plasmodium berghei , ANKA strain).

The "Ookinete surface antigen-like protein Pfs28" is a protein essential for host cell parasites of malaria, and its viability based on cell recognition, invasion, and genetic destruction. Closely related to

In particular, the inventors have shown that a virus-like particle vaccine comprising a motor conjugate surface antigen-like protein (Pfs28) derived from the parasitic protozoan mouse malaria ( Plasmodium berghei , ANKA strain) is fused with virus matrix protein 1 in a subject. It has been shown that it can provide a high level of immunity to malaria when introduced.

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, the motor conjugate surface antigen-like protein derived from malaria may be composed of the amino acid sequence of SEQ ID NO: 2. In addition, the influenza virus matrix protein 1 may be encoded by the nucleic acid sequence of SEQ ID NO: 3, and the motion conjugate surface antigen-like protein derived from malaria may be encoded by the nucleic acid sequence of SEQ ID NO: 4.

The influenza virus matrix protein 1 or the motion conjugate surface antigen-like protein derived from malaria comprises a functional equivalent of a protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2.

The “functional equivalent” is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 70% of the amino acid sequence of SEQ ID NO: 1 or 2 as a result of the addition, substitution or deletion of amino acids Is 95% or more of sequence homology, and means a protein having substantially the same biological activity as the amino acid sequence of SEQ ID NO: 1 or 2.

The "substantially equivalent physiological activity" is a virus-like that can induce a specific immune response against malaria due to its structural and functional homology with the influenza virus matrix protein 1 or a motor conjugate surface antigen-like protein derived from malaria. Activity as a particle.

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

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

The vaccine composition includes stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylenes, bacterial cell walls, derivatives of bacterial cell walls, bacterial vaccines, Animal poxvirus protein, viral subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldi It may further comprise one or more second adjuvants selected from the group consisting of octadecyl-ammonium bromide and mixtures thereof.

In addition, the vaccine composition may comprise a medically acceptable carrier. The “medically acceptable carrier” may include any and all solvents, dispersion media, coatings, antigen adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. It is not.

Carriers, excipients, and diluents that may 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, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto.

In addition, the vaccine composition can be used in the form of oral formulations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and sterile injectable solutions according to conventional methods.

When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like may be used together.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin in the lecithin-like emulsifier. And the like can be used together. In addition to the above excipients, lubricants such as magnesium styrate talc may also be used.

As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, and the like may be used, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used in addition to water and liquid paraffin, which are commonly used simple diluents. connect. For parenteral administration, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations can be used. Water-insoluble preparations, suspending agents may be used propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like.

According to another aspect of the invention, the nucleic acid sequence of SEQ ID NO: 3 that encodes influenza virus matrix protein 1 (M1) and the sequence number that encodes a malaria-derived Ookinete surface antigen-like protein Pfs28 An expression vector for preparing malaria virus-like particles comprising the nucleic acid sequence of 4 is provided.

The vector refers to a nucleic acid molecule used to carry a linked nucleic acid fragment. The vector may include, but is not limited to, bacteria, plasmids, phages, cosmids, episomes, viruses and insertable DNA fragments (ie, fragments insertable into the host cell genome by homologous recombination). The plasmid is a type of vector, which means an annular double stranded DNA loop capable of additionally connecting DNA fragments therein. Viral vectors can also link additional DNA into the viral genome.

The expression vector refers to a vector capable of directing the expression of a gene encoding a target protein operably linked. In general, since the expression vector is in the form of a plasmid in the use of recombinant DNA technology, the terms plasmid and vector can be used interchangeably. However, it may also include other forms of expression vectors that perform the same function, such as viral vectors.

For example, the expression vector is pET-3a-d, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET-16b, pET-17b, pET-17xb, pET-19b, pET-20b (+), pET-21a-d (+), pET-22b (+), pET-23a-d (+), pET-24a-d (+), pET-25b (+ ), pET-26b (+), pET-27b (+), pET-28a-c (+), pET-29a-c (+), pET-30a-c (+), pET-30 Ek / LIC, pET-30 Xa / LIC, pET-31b (+), pET-32a-c (+), pET-32 Ek / LIC, pET-32 Xa / LIC, pET-33b (+), pET-34b (+) , pET-35b (+), pET-36b (+), pET-37b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a-c (+ ), pET-41 Ek / LIC, pET-42a-c (+), pET-43.1ac (+), pET-43.1 Ek / LIC, pET-44a-c (+), pRSETA, pRSETB, pRSETC, pESC- HIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2 / CT, pYD1 Yeast Display Vector, pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, pYES2 / NT C, pYES2 / CT, pYES2.1 , pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPink ^TM , p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP 47, 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, but is not limited thereto.

Meanwhile, the expression vector is introduced into the host cell, and the host cell transformed by the introduced vector can produce the malaria virus-like particle. At this time, the vector may include a promoter recognized by the host organism.

The promoter is SBE4, 3TP, PAI-1, p15, p21, CAGA12, hINS, A3, NFAT, NFKB, AP1, IFNG, IL4, IL17A, IL10, GPD, TEF, ADH, CYC, INU1, PGK1, PHO5, TRP1 , GAL1, GAL10, GUT2, tac, T7, T5, nmt, fbp1, AOX1, AOX2, MOX1 and FMD1 promoter may be selected from the group consisting of, but may be varied in consideration of various variables such as host cell or expression conditions.

The nucleic acid sequence encoding the malaria virus-like particle can be operably linked with the promoter sequence. By “operably linked” is meant that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by another nucleic acid fragment. That is, the gene encoding the malaria virus-like particle can be operably linked to a promoter in a vector to regulate expression.

On the other hand, the expression vector may further comprise an additional regulatory sequence. The regulatory sequence may be, but is not limited to, a shine-dalgano sequence of a replicase gene of phage MS-2 and a shine-dalgano sequence of cII of bacteriophage lambda.

In addition, the expression vector may comprise appropriate marker genes necessary for selecting 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 hygromycin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, and tetracycline resistance gene, but is not limited thereto. It is not. The fluorescent protein gene is a yeast-enhanced green fluorescent protein (yEGFP) gene, a green fluorescent protein (GFP) gene, a blue fluorescent protein (BFP) gene, and a red fluorescence The protein may be selected from the group consisting of red fluorescent protein (RFP) genes, but is not limited thereto.

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

In one embodiment, the host cell may be a microorganism, an animal cell, a plant cell, a culture cell derived from an animal, or a culture cell derived from a plant. Such suitable host cells may be naturally occurring or wild type host cells, or may be altered host cells. The wild-type host cell may be a host cell that has not been genetically changed by a recombinant method.

The host cell is not particularly limited as long as it can be transformed by an engineering method to efficiently express a specific gene, and preferably may be 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 , anti Anticarsa gemmitalis ), Bombyx mori mori ), Estigmene acrea ), Heliotis bireth ( Heliothis virescens ), Leucania separata ), Lymantria dispar ), Malacasoma Distria the disstria), mamme Strasbourg Braga's car (Mammestra brassicae), Manduca other sex (Manduca sexta ), Plutella zylostella , Spodoptera exigua ) and Spodoptera littorlis may be selected from one or more groups, but is not limited thereto.

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

The "metabolized" is metabolic flux by regulation and optimization of transcription, translation, protein stability and protein functionality using genetic engineering and appropriate culture conditions. Optimization may further include. Biosynthetic genes may be foreign to the host or may be heterologous to a host (eg, a microorganism) by being modified by mutagenesis, recombination or association with heterologous expression control sequences in an endogenous host cell. Suitable culture conditions may 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 metabolism of the microorganisms. Can be. Suitable culture conditions for microorganisms that can function as host cells are well known in the art.

Thus, such "engineered" or "modified" host cells can be produced by introducing genetic material into a selected host or parental microorganism to modify or alter cell physiology and biochemistry. Through the introduction of genetic material, parental microorganisms can acquire new properties such as new intracellular metabolites or the ability to produce higher amounts of intracellular metabolites.

For example, the introduction of genetic material into parental microorganisms can result in new or modified ability to produce chemicals. Genetic materials introduced into the parental microorganism include genes or portions of genes encoding one or more enzymes involved in biosynthetic pathways for chemical production, and additional components for the expression or expression control of these genes, such as Promoter sequences may also be included.

By “altered host cell” is meant a genetically engineered host cell, wherein the target protein is produced at a level of expression, or at a level greater than the level of expression, or grown under essentially the same growth conditions. It may be expressed at a level of expression that is greater than the level of expression of the protein of interest in the altered or wild-type host cell. The term "modified host cell" refers to a wild type or altered host cell that is genetically designed to overexpress a gene encoding a protein of interest. The modified host cell can express the protein of interest at higher levels than the wild type or changed parent host cell.

On the other hand, the "transformation" refers to a method of transporting the vector into a microorganism or a specific cell, when the cell to be transformed is a prokaryotic cell, CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acad. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D , J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. have.

If the cells to be transformed are eukaryotic cells, micro-injection (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (1973)) , Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE Dextran treatment (Gopal, Mol. Cell. Biol., 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 ( 1990), etc., but is not limited thereto.

In the case of transformation of fungi such as yeast, lithium acetate (Rithium acetate, RD Gietz, Yeast 11, 355360 (1995)) and heat shock (Keisuke Matsuura, Journal of Bioscience and Bioengineering, Vol. 100, 5; 538) -544 (2005)), but may be performed by transformation and electroporation (Nina SkoluckaAsian, Pacific Journal of Tropical Biomedicine, 94-98 (2011)), but is not limited thereto.

According to another aspect of the invention, transforming the host cell with the expression vector; And culturing the host cell to express a virus-like particle. There is provided a method for producing a malaria virus-like particle comprising a.

The transformed host cell may be cultured under batch, feed-batch or continuous fermentation conditions, and the host cell may express the malaria virus-like particle by transformation so that the virus- from the cultured host cell Similar particle proteins can be obtained.

At this time, the classical batch fermentation method may use a closed system, wherein the culture medium is prepared before the fermentation is carried out, and the fermentation may take place without inoculating the organism with the medium and without adding any component to the medium. have. In certain cases, the pH and oxygen content, but not the carbon source content of the growth medium, may vary during the batch process. The metabolites and cell biomass of the batch system can constantly change until fermentation is stopped. In a batch system, cells may progress over a high growth log on stationary retardation and finally reach a stationary phase where growth rate is reduced or stopped. In the general period, the cells on the log can make most of the protein.

A variant of the standard batch system is the "feed-batch fermentation" system. In such systems, nutrients (eg, carbon sources, nitrogen sources, O ₂ , and typically other nutrients) may be added when the concentration of their culture drops below the limit. Feed-batch systems may be useful when catabolism inhibits cell metabolism and it is desirable for the medium to have a limited amount of nutrients in the medium. It can be predicted based on changes in measurable factors such as dissolved oxygen and partial pressure of waste gas such as CO ₂ . Batch and feed-batch fermentations are well known in the art as a general system.

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

For example, limiting nutrients, such as carbon or nitrogen sources, at a fixed rate, all other parameters can be maintained as appropriate. In other systems, factors that affect a lot of growth may continue to change while the cell concentration, as measured by medium turbidity, remains constant. Continuous systems seek to maintain steady state growth conditions. Thus, cell loss by the withdrawal of the medium can be balanced against the rate of cell growth in fermentation. Techniques to maximize the rate of product formation, as well as methods of maintaining nutrients and growth factors during the continuous fermentation process are known in the art.

Those skilled in the art to which the present invention pertains will be able to apply by changing the type, introduction ratio, etc. of each configuration based on the description of the present invention, if the equivalent technical effect is implemented in spite of the above modification, the present invention It will be included in the technical idea of the.

Through the following examples, the present invention will be described in more detail, but the present invention is not limited by the following examples.

Production Example  1: Preparation of a vector for preparing malaria virus-like particles

The malaria zygote surface antigen-like protein (Pfs28) gene was amplified by PCR using forward primer (5'-AAA GAATT CACCATGAATTTTAAATACAGTTT-3 ') and reverse primer (5'-TTA CTCGAG TTACATTACTATCACGTAAA-3'). The primers were introduced with underlined restriction enzyme sites (EcoR I and Xho I), respectively (FIGS. 1A, 1B). The sequence encoding Pfs28 amplified cDNA as a template was introduced into the pFastBac vector (Invitrogen).

Influenza matrix protein 1 (M1) genes were amplified via RT-PCR using forward primers (5'-TCC CCCGGG CCACCATGAGCCTTCTGACCGAGGTC-3 ') and reverse primers (5'-TTACT TCTAGA TTACTTGAACCGTTGCATCTG-3'). The primers were introduced with underlined restriction enzyme sites (SmaI and XbaI), respectively. A / PR / 8/34 virus was inoculated into MDCK cells, and viral RNA was extracted through the RNeasy Mini kit.

Amplified influenza M1 protein was introduced into the pFastBac vector. Recombinant plasmids were introduced into E. coli DH5-alpha, and DH10-Bac.

The M1 (accession number: EF467824, 1027bp) and MIC8 (accession number; AF353165, 2.055bp) genes introduced into the pFastBac vector were identified by DNA sequencing determination.

In order to prepare recombinant baculoviruses (rBVs) expressing Pfs28 and M1, DNA transfection was performed using cellfectin II (Invitrogen) and SF9 cells. Transformation with pFastBac vector containing MIC8 and M1 was performed by white / blue screening. Baculoviruses were prepared according to the manufacturer's manual via the Bac-to-Bac expression system (Invitrogen).

Production Example  2: Preparation of Malaria Virus-Similar Particles

Malaria virus-like particles were produced in SF9 insect cells co-infected by recombinant rBVs expressing Pfs28 and M1. Virus-like particles in the supernatants were pelleted by high-speed centrifugation (30 min, 45,000 μg).

Virus-like particles were resuspended in phosphate buffered saline (PBS) overnight at 4 ° C. and harvested and purified for 1 hour at 45,000 μg 4 ° C. via a discontinuous sucrose gradient (20-30-60%). Protein concentration was determined by QuantiPro BCA Assay Kit (Sigma-Aldrich).

Virus like particles were identified by Western blot and electron microscopy. In Western blot analysis, mouse serum was used to probe the Pfs28 protein. Serum was collected after 4 weeks in BALB / c mice infected with malaria ME49 strain. The content of M1 protein was determined by anti-M1 monoclonal antibody. Virus-like particles were media stained for electron microscopy and size measurements and observed via transmission electron microscopy (TEM) (CANADA, ALBERTA).

SF9 cells producing virus-like particles were found to be larger than normal cells as controls.

The morphology of virus-like particles was observed by electron microscopy in the form of amorphous spheres with spikes on the surface (FIG. 1B).

Western blots were performed using anti-malaria polyclonal antibodies and anti-M1 monoclonal antibodies to confirm that they were introduced into the viral analogs of Pfs28 and M1 (FIG. 1C, 1D).

As a result, SF9 cells infected with rBVs expressing Pfs28 and M1 were found to produce particles similar in shape and size to virions.

Experimental Example  1: Immune Induction Activity Test in Animal Model

The immune response induced after inoculation of the virus-like particles into the mice was confirmed.

The virus-like particles of the above examples were inoculated intranasally (Intranasal route, FIG. 2A) and intramuscular (Intramuscular route, FIG. 2B), respectively, and blood was collected at predetermined times from mice.

After inoculating virus-like particles in each route, serum malaria-specific IgG, IgG2a, IgG1 and IgG2b antibody formation was measured by ELISA (FIG. 2).

Total virus and IgG2a antibodies in mice were significantly increased after 1 to 2 weeks of inoculation by the virus-like particles (FIGS. 2A and 2B).

In addition, malaria-specific IgG, IgG2a were challenged after infection with the malaria protozoan Plasmodium berghei (ANKA) to mice inoculated with virus-like particles to confirm the antibody response profile according to challenge infection. , IgG1 and IgG2b antibody formation was measured (Fig. 3A, 3B).

Malaria infection resulted in high levels of IgG, IgG2a antibody response in serum, and the results showed that mice immunized by virus-like particle inoculation could effectively produce malaria prototypic IgG and IgG2a antibodies by malaria infection. That means you can.

Experimental Example  2: protective immune effect test (survival rate)

The protective immune effects induced after inoculation of the virus-like particles into mice were tested.

The weight and survival rate of mice vaccinated with the malaria progeny (Plasmodium berghei, ANKA) and then vaccinated by intranasal and intramuscular routes, respectively, for 2 weeks Measured.

Mice immunized (IN + Cha) by intranasal inoculation of virus-like particles lost 55% less weight than mice infected with malaria (Naive + Cha) and survived for 6 more days (Figure 4A, 4B).

Mice immunized with intramuscular route (IM + Cha) had a 35% less weight and survived four more days compared to infected mice (Naive + Cha) (Figures 4C, 4D).

The results suggest that the virus-like particles can induce a protective immune effect against malaria infection, and can more effectively induce an immune response when administered intranasally.

Experimental Example  3: immune response test

The immune response induced after inoculation of the virus-like particles into the mice was confirmed.

After infecting mice with malaria protozoan Plasmodium berghei (ANKA) via the peritoneal route, the immune response to malaria was determined by flow cytometry using CD4 + T-cell, CD8 + T-cell and Total B-cell, Germinal. The center was measured.

Mice immunized by intranasal route of virus-like particles (IN + Cha) increased about 16% and 10% CD4 + T-cell and CD8 + T-cell compared to normal mice (Naive). It was increased about 9% and 4% compared to (Naive + Cha) (Figs. 5A and 5B), and about 4% and 3% compared to mice (Naive + Cha) infected at Total B-cell and Germinal center (Fig. 5C). And 5D).

Mice immunized by intramuscular route (IM + Cha) increased CD4 + and CD8 + T-cells by 8% and 6%, respectively, compared to normal mice (Naive), and compared with infected mice (Naive + Cha). 5% and 3% increase (FIGS. 6A and 6B). In addition, the total B-cell and Germinal centers increased by 4% and 2%, respectively, compared to mice infected (Navie + Cha) (Figs. 6C and 6D).

Experimental Example  4: protective immune activity test according to infection ( Infection rate )

Mice infected with malaria protozoan Plasmodium berghei (ANKA) intraperitoneally after the virus-like particle vaccination were sacrificed after 2 weeks and parasitemia of malaria protozoa was compared.

Immunized mice (IN + Cha) immunized by the intranasal route of virus-like particles (IN + Cha) reduced malaria infection rate to about 1 minute, and immunized mice (IM + immunized by intramuscular route) Cha) reduced the rate of malaria infection by about 50% (FIGS. 7A and 7B).

The results suggest that the virus-like particles can induce a protective immune effect against malaria infection.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

<110> Kyung-Hee University <120> A PLASMODIUM BERGHEI VIRUS-LIKE PARTICLE, VACCINE COMPOSITION,          EXPRESSION VECTOR AND METHOD FOR PREPARING THE SAME <130> 17PP10784 <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 Pro 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 Pro 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> 213 <212> PRT <213> Plasmodium berghei <400> 2 Met Asn Phe Lys Tyr Ser Phe Ile Phe Leu Phe Phe Ile Gln Leu Ala   1 5 10 15 Ile Arg Tyr Asn Asn Ala Lys Ile Thr Val Asp Thr Ile Cys Lys Gly              20 25 30 Gly Lys Leu Ile Gln Met Ser Asn His Tyr Glu Cys Lys Cys Pro Ser          35 40 45 Gly Tyr Ala Leu Lys Thr Glu Asn Thr Cys Glu Pro Ile Val Lys Cys      50 55 60 Asp Lys Leu Glu Asn Ile Asn Lys Val Cys Gly Glu Tyr Ser Ile Cys  65 70 75 80 Ile Asn Gln Gly Asn Phe Gly Leu Glu Lys Ala Phe Val Cys Met Cys                  85 90 95 Thr Asn Gly Tyr Met Leu Ser Gln Asn Ile Cys Lys Pro Thr Arg Cys             100 105 110 Tyr Asn Tyr Glu Cys Asn Ala Gly Lys Cys Ile Leu Asp Ser Ile Asn         115 120 125 Pro Asn Asn Pro Val Cys Ser Cys Asp Ile Gly Lys Ile Leu Gln Asn     130 135 140 Gly Lys Cys Thr Gly Thr Gly Glu Thr Lys Cys Leu Leu Lys Cys Lys 145 150 155 160 Ala Ala Glu Glu Cys Lys Leu Thr Gly Lys His Tyr Glu Cys Val Ser                 165 170 175 Lys Pro Gln Ala Pro Gly Thr Gly Ser Glu Thr Pro Ser Asn Ser Ser             180 185 190 Phe Met Asn Gly Met Ser Ile Ile Ser Ile Ila Ala Leu Leu Val Ile         195 200 205 Tyr Val Ile Val Met     210 <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> 642 <212> DNA <213> Plasmodium berghei <400> 4 atgaatttta aatacagttt tattttttta ttttttatcc aacttgcaat acgatataat 60 aatgcaaaga tcactgtaga cacgatatgt aaaggtggaa aactaattca aatgagtaat 120 cattacgaat gtaaatgtcc ttctggatat gcattaaaaa ctgaaaacac ttgcgagcca 180 attgttaagt gtgataaact cgaaaatata aataaagtat gtggtgaata ttctatatgt 240 ataaaccagg gaaattttgg cttagaaaaa gcttttgtat gtatgtgtac aaatggatat 300 atgttatcac aaaatatatg taagccaaca agatgttata actatgaatg taacgctgga 360 aaatgtatac ttgattctat taaccctaac aatccagtat gctcatgtga tataggaaaa 420 attttacaga atggtaaatg cacaggtaca ggagaaacta aatgtttatt gaaatgtaaa 480 gctgcagaag aatgcaaatt gactggaaaa cattatgagt gtgtttccaa accccaagca 540 ccaggtactg gtagcgaaac gccatcaaat agcagtttta tgaacggaat gtcaataatc 600 agtattattg cattacttgt tatttacgtg atagtaatgt aa 642

Claims (9)

Influenza virus matrix protein 1 (M1) consisting of the amino acid sequence of SEQ ID NO: 1; And
Motorok surface antigen-like protein derived from malaria protozoa consisting of the amino acid sequence of SEQ ID NO: 2 (0okinete surface antigen-like protein Pfs28); Virus-like particles that can induce an immune response to malaria, including. delete The method of claim 1,
The influenza virus matrix protein 1 is encoded by the nucleic acid sequence of SEQ ID NO: 3, the motor conjugate surface antigen-like protein derived from the malaria protozoa can induce an immune response against malaria encoded by the nucleic acid sequence of SEQ ID NO: 4 Virus-like particles. A vaccine composition comprising a virus-like particle capable of inducing an immune response against malaria of claim 1 as an active ingredient. The nucleic acid sequence of SEQ ID NO: 3 encoding influenza virus matrix protein 1 (M1); And
An expression vector for preparing a virus-like particle capable of inducing an immune response against malaria comprising a nucleic acid sequence of SEQ ID NO: 4 encoding a malaria protoplast derived ookinete surface antigen-like protein Pfs28. The method of claim 5,
The influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, the motor conjugate surface antigen-like protein derived from the malaria protozoa virus capable of inducing an immune response against malaria consisting of the amino acid sequence of SEQ ID NO: Expression Vectors for Preparation of Similar Particles. A host cell transformed with the expression vector of claim 5. The method of claim 7, wherein
A host cell which is a microorganism, an animal cell, a plant cell, a culture cell derived from an animal, or a culture cell derived from a plant. Transforming the host cell with the expression vector of claim 5; And
Culturing the host cell to express a virus-like particle; a method of producing a virus-like particle capable of inducing an immune response to malaria comprising a.

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