KR102536927B1 - Virus-like particles comprising the merozoit surface protein-8 of malaria protozoa, and vaccine compositions using the same - Google Patents

Abstract

The present invention relates to a virus-like particle capable of inducing an immune response against malaria, a vaccine composition containing the same, an expression vector, a host cell, and a method for preparing the same.

Description

VIRUS-LIKE PARTICLES COMPRISING THE MEROZOIT SURFACE PROTEIN-8 OF MALARIA PROTOZOA, AND VACCINE COMPOSITIONS USING THE SAME}

The present invention relates to a virus-like particle capable of inducing an immune response against malaria, a vaccine composition containing the same, an expression vector, a host cell, and a method for preparing the same.

Malaria is parasitic in human blood and causes a disease, and it is a disease that causes the most deaths (2.4 million people per year) as a single disease worldwide.

About 300 to 500 million people are infected with malaria each year with relatively high morbidity and mortality. In particular, the morbidity and mortality due to malaria infection are serious not only in children but also in adults who have never been exposed to malaria.

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

In addition, the widespread incidence and increasing prevalence of malaria caused by drug-resistant parasites ( Plasmodium falciparum, Plasmodium vivax ) and insecticide-resistant vectors (Anopheles mosquitoes) have created a need to develop new methods of controlling malaria in many countries. are emphasizing

In Korea, malaria has reappeared mainly in the northern part of Gyeonggi Province and the northwestern part of Gangwon near the DMZ, and the risk of infection is gradually expanding. Although there are more than 1,000 patients in Korea every year, currently antimalarial drugs such as chloroquine and artemether cause resistance, so vaccination is evaluated as the most effective method for preventing malaria parasites.

On the other hand, virus-like particle refers to a type of antigen that is morphologically similar to the actual structure of a virus, and has been proposed as a vaccine antigen for several viruses (Roldao A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM: Expert review of vaccines 2010, 9:1149-1176; 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 are assembled in a form similar to a real virus through the combination of viral structural proteins, but are characterized by being very safe because there is no risk of infection because virus genes are not included in the assembly process.

Conventionally, studies have focused on two antigens (Pfs25 and Pvs25) provided as recombinant proteins or attenuated vaccinia viruses in relation to malaria vaccines. development is required.

Korean Patent Publication No. 10-2019-0009691

An object of the present invention is influenza virus matrix protein 1 (Influenza virus matrix protein, M1); and to provide virus-like particles capable of inducing an immune response to malaria, including merozoit surface protein-8 (MSP-8) of the malaria parasite.

Another object of the present invention is to provide a vaccine composition comprising a virus-like particle capable of inducing an immune response against malaria.

Another object of the present invention is to provide an expression vector for producing virus-like particles capable of inducing an immune response against malaria.

Another object of the present invention is to provide a host cell transformed with the expression vector for preparing the virus-like particle capable of inducing an immune response against malaria.

Another object of the present invention is influenza virus matrix protein 1; and a method for preparing virus-like particles capable of inducing an immune response against malaria, including the merozoite surface protein-8 of the protozoa malaria.

One aspect of the present invention, influenza virus matrix protein 1 (Influenza virus matrix protein, M1); and virus-like particles capable of inducing an immune response to malaria, including merozoit surface protein-8 (MSP-8) of the malaria parasite.

In the present invention, "malaria" refers to an acute febrile infectious disease caused by infection with the malaria parasite, and the symptoms and characteristics are different depending on the type of the causative pathogen. Specifically, in the present invention, the malaria parasite may be rodent malaria protozoa ( Plasmodium berghei ANKA), but is not limited thereto.

In the present invention, “Virus-Like Particles (VLPs)” means non-infectious viral subunits with or without viral proteins. For example, the virus-like particle completely lacks a DNA or RNA genome, or may undergo spontaneous self-assembly in the case of a virus-like particle containing a viral capsid protein.

In the present invention, "virus-like particles capable of inducing an immune response against malaria" means a protein structure (particle) that induces a specific immune response against malaria and has a virus-like shape. Meanwhile, in the present invention, the term "virus-like particle capable of inducing an immune response against malaria" does not mean that malaria is a disease caused by a virus, but is used in the sense that it is similar to a virus in its form and utilization. As such, it can be appropriately changed and applied as needed.

The virus-like particle contains an antigen-determining site derived from the protozoa malaria at the same time as the virus-derived structural proteins are assembled to produce a virus-like particle, so when the virus-like particle is inoculated into a specific individual It is characterized in that it can induce a specific immune response against malaria. As a specific example of the present invention, the protein construct may have a form (FIG. 1A) in which an antigenic determinant site derived from Plasmodium malaria is bound to the outside of a structural protein derived from a virus.

The virus-derived structural protein (core protein) may be influenza virus matrix protein 1.

In the present invention, "influenza virus matrix protein (M1)" is a structural protein of influenza virus, and is a matrix protein that forms a coat inside the fat layer, which is the envelope of influenza virus. means The influenza virus has 8 segmented negative strand RNA, surface proteins hemagglutinin (H), neuraminidase (N), nucleoprotein (NP) matrix (matrix, M1), proton Proton ion-channel protein (M2), polymerase acidic protein (PA), polyMerase basic protein 2 (PB2), polymerase acidic protein (PA) protein) and nonstructural protein 2 (NS2). At this time, matrix protein 1 serves as a link between the core and the envelope by surrounding the outer layer in layers. Matrix protein 1 plays an important role in assembling virus-like particles into a stable form in the production of virus-like particles.

The influenza virus matrix protein 1 is A/Puerto Rico/8/34, A/Bangkok/163/2000, A/AA/Huston/1945, A/Berlin/6/2006, A/Brandenburg/1/2006, A/ Brevig Mission/1/1918, A/Chile/8885/2001, A/DaNang/DN311/2008, A/FLW/1951, A/FW/1/1950, A/Fiji/15899/83, A/Fort Monmouth/ 1-MA/1947, A/HaNoi/TX233/2008, A/Iowa/CEID23/2005, A/Malaysia/35164/2006, A/Managua/4086.04/2008, A/Texas/VR06-0502/2007, A/ WSN/1933, A/Colorado/18/2011, A/Kentucky/04/2010, A/Maryland/28/2009, A/New Mexico/05/2012, A/Philippines/TMC10-135/2010, A/Singapore /GP4307/2010, A/Singapore/GP489/2010, A/Boston/14/2007, A/Brisbane/09/2006, A/Hong Kong/CUH34175/2002, A/Kyrgyzstan/WRAIR1256P/2008, A/Malaysia/ 12550/1997, A/Nanjing/1663/2010, A/Wyoming/08/2010, A/Berkeley/1/1968, A/Korea/426/1968, etc., but are not limited thereto. Specifically, in one embodiment of the present invention, M1 protein derived from A/Puerto Rico/8/34 was used as a structural protein of virus-like particles.

The antigenic determinant site derived from the parasite may be merozoite surface protein-8 of the parasite.

In the present invention, the term "epitope" is a basic element or minimum unit of recognition by each antibody or T-cell receptor, and refers to a specific domain, region, or molecular structure to which the antibody or T-cell receptor binds. . The antigenic determining region may be derived from malaria, and is not particularly limited as long as it can induce immune activity against the malaria.

In the present invention, "merozoit surface protein-8 (MSP-8)" is a surface protein specifically present on the surface of the malaria parasite. Merozoite surface protein-8 is a representative protein most highly expressed in the malaria parasite and is synthesized in the early stages of asexual reproduction. After attaching to erythrocytes using the synthesized merozoite surface protein-8 complex, the Malalai protozoan invades erythrocytes by targeting the merozoite surface protein-8 complex to the spectrin complex on the inner surface of the erythrocyte cell membrane. do. Accordingly, in the present invention, merozoite surface protein-8 is intended to be used as a protein of the antigenic determinant site.

The merozoite surface protein-8 may be derived from Plasmodium berghei , Plasmodium falciparum , Plasmodium vivax , Plasmodium knowlesi , Plasmodium cynomolgi , Plasmodium malariae , etc., but is not limited thereto. Specifically, in one embodiment of the present invention, Plasmodium berghei ANKA-derived merozoite surface protein-8 was utilized as an antigen-binding site of virus-like particles.

The merozoite surface protein-8 may be a polypeptide, a protein, or a nucleic acid having a sequence encoding the same, and may be codon-optimized according to a target to introduce the merozoite surface protein-8. .

"Codon optimization" refers to the replacement of at least one codon of a native sequence with a more frequent or most frequently used codon in a gene of a host cell, while maintaining the native amino phase sequence of a nucleic acid to enhance expression in a host cell of interest. refers to the process of modifying a sequence. Different species have specific biases for specific codons of specific amino acids, and codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of mRNA, which is related to the nature of the codon being translated and the availability of specific tRNA molecules. It is believed to be influenced by The predominance of selected tRNAs in cells generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

The virus-like particle capable of inducing an immune response to malaria contains merozoite surface protein-8 as an antigenic determining site derived from malaria, but does not contain any other genetic material, so it is impossible to proliferate and is not toxic. It is safe and can be used as a vaccine against malaria. The antigenic determinant site introduced on the surface of the virus-like particle has higher antigenicity than that of a purely isolated recombinant protein and can form an effective neutralizing antibody.

Specifically, the influenza virus matrix protein 1 may consist of the amino acid sequence of SEQ ID NO: 1, and the merozoite surface protein-8 may consist of the amino acid sequence of SEQ ID NO: 2. In addition, the influenza virus matrix protein 1 or merozoite surface protein-8 may include a functional equivalent of a protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2.

In the present invention, the term "functional equivalent" refers to at least 70% or more, specifically 80% or more, more specifically 90% or more of the amino acid sequence of SEQ ID NO: 1 or 2 as a result of addition, substitution or deletion of amino acids, Most specifically, it means a protein having a sequence homology of 95% or more, and having substantially the same physiological activity as the amino acid sequence of SEQ ID NO: 1 or 2.

In the present invention, the term "substantially equivalent physiological activity" refers to a virus-like substance capable of inducing a specific immune response against malaria due to structural and functional homology with the influenza virus matrix protein 1 or merozoite surface protein-8. activity as a particle.

More specifically, the influenza virus matrix protein 1 may consist of the nucleic acid sequence of SEQ ID NO: 3, and the merozoite surface protein-8 may consist of the nucleic acid sequence of SEQ ID NO: 4. For example, the influenza virus matrix protein M1 is Genebank Accession No. ABO21712 or Genebank Accession No. It may be a gene represented by EF467824. The merozoite surface protein-8 is Genebank Accession No. It may be a gene expressed as XP_022714061.1.

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

In one embodiment of the present invention, it was confirmed that IgG antibodies were formed by inoculation with the virus-like particles (FIG. 3), and it was confirmed that the number of CD4 ⁺ and CD8 ⁺ T cells significantly increased (FIGS. 4 to 6). ). In particular, as a result of treating malaria virus-like particles to mice infected with a lethal dose of the malaria parasite, it was confirmed that a high level of memory B cell population was formed (FIG. 7).

In one embodiment of the present invention, as a result of confirming the inflammatory response and parasite proliferation effect by inoculation with the virus-like particle, it was confirmed that the inflammatory response and parasite growth were significantly reduced compared to the control group not inoculated with the virus-like particle (FIG. 8).

The above results suggest that the virus-like particles of the present invention can provide a high level of immunity against malaria and can be used as a vaccine against malaria infection.

Another aspect of the present invention relates to a vaccine composition comprising the virus-like particle.

The description of the 'virus-like particle' is the same as described above.

In the present invention, "vaccine composition" means a composition capable of preventing subsequent pathogen infection by administering a protein antigen having a vaccine effect. The vaccine composition may be a composition containing virus-like particles exhibiting antigenicity against Plasmodium malaria.

Specifically, the vaccine composition may include any one selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, immune stimulants, and combinations thereof.

Such pharmaceutically acceptable carriers are known in the art and include proteins, sugars, and the like. The carriers may be aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, including drinking water and buffered media, alcoholic/aqueous solutions, emulsions or suspensions. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers include electrolyte replenishers, liquids and nutritional supplements, such as those based on Ringer's dextrose. As preservatives and other additives, antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be further included. Preservatives may include, but are not limited to, formalin thimerosal, neomycin, polymyxin B and amphotericin B.

The vaccine composition may further include an adjuvant. The adjuvant refers to a compound or mixture that enhances the immune response and/or accelerates the rate of absorption after inoculation, and includes any absorption-promoting agent. Acceptable adjuvants are Freund's preservatives, Freund's incomplete preservatives, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluton polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanin, nitrophenol and the like, but are not limited thereto.

The vaccine composition may further include an immune stimulant. The immune stimulant may include artificially synthesized levamisole, isoprenosine, and cytokines. Examples of cytokines include interferon-α, interleukin-2, GM-CSF, and G-CSF.

The vaccine composition may be in the form of containing the virus-like particles or a concentrated solution thereof, or may be used in the form of a transformed host cell itself or a dry powder form of the transformed cell. In addition, the vaccine composition can be used together with other foods or food ingredients and can be appropriately used according to conventional methods.

The vaccine composition includes a stabilizer, an emulsifier, aluminum hydroxide, aluminum phosphate, a pH adjuster, a surfactant, a liposome, an iscom adjuvant, a synthetic glycopeptide, an extender, carboxypolymethylene, a bacterial cell wall, a bacterial cell wall derivative, a bacterial vaccine, Animal poxvirus protein, viral subviral particle adjuvant, cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldiamine It may further include one or more second adjuvants selected from the group consisting of octadecyl-ammonium bromide and mixtures thereof.

In one embodiment of the present invention, a vaccine composition using virus-like particles containing merozoite surface protein-8 and influenza virus matrix protein 1 derived from the rodent malaria parasite ( Plasmodium berghei ANKA strain) is prepared, thereby preventing Plasmodium berghei ANKA strain. As a result of administering to the infected mouse, it was confirmed that immunity increased due to antibody formation and T cell proliferation (FIGS. 3 to 7), and as a result of administering the vaccine composition, it was confirmed that the inflammatory response and parasite proliferation of the mouse were significantly reduced (FIG. 8 ).

The above results suggest that the vaccine composition comprising the virus-like particles of the present invention can be used to prevent malaria infection by strengthening the immune system through antibody formation and T cell proliferation.

The vaccine composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and sterile injection solutions according to conventional methods. When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used together.

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

Liquid formulations for oral administration may include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be used in addition to water and liquid paraffin, which are commonly used simple diluents. there is.

Sterile aqueous solutions, water-insoluble agents, suspensions, emulsions, and lyophilized preparations may be used for preparations for parenteral administration. Non-aqueous preparations and suspensions may be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

Another aspect of the present invention is a nucleic acid sequence encoding influenza virus matrix protein 1 consisting of SEQ ID NO: 3; and an expression vector for producing malaria virus-like particles comprising a nucleic acid sequence encoding merozoite surface protein-8 of the malaria parasite consisting of SEQ ID NO: 4.

In the present invention, the term “vector” refers to a nucleic acid molecule used to transport linked nucleic acid fragments. As the vector, bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments (ie, fragments insertable into the host cell genome by homologous recombination) may be used, but are not limited thereto. The plasmid is a type of vector and refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated. In addition, viral vectors may have additional DNA ligated into the viral genome.

In the present invention, the term "expression vector" refers to a vector capable of directing the expression of a gene encoding a target protein operably linked thereto. Generally, expression vectors in the use of recombinant DNA technology are in the form of plasmids, so the terms plasmid and vector may be used interchangeably. However, it may also include other types of expression vectors that perform the same function, such as viral vectors.

The expression vectors are 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(+), pFastBac, 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, pSGP47, 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.

Specifically, the vector may be a Baculovirus vector. The baculovirus refers to a pathogenic virus that is not pathogenic to humans or vertebrates and has pathogenicity only to insects.

In one embodiment of the present invention, an expression vector for preparing malaria virus-like particles was obtained by cloning nucleic acid sequences encoding influenza virus matrix protein 1 and malaria merozoite surface protein-8 into pFastBac vector, one of baculovirus vectors. was prepared (Figs. 1 and 2).

The expression vector is introduced into a host cell, and the host cell transformed by the introduced vector can produce a virus-like particle capable of inducing an immune response against malaria. In this case, 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 may be selected from the group consisting of promoters, but may be different in consideration of various variables such as host cells or expression conditions.

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

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

In addition, the expression vector may contain an appropriate marker gene 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 a hygromycin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene, but is 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 fluorescent protein (red fluorescent protein, RFP) may be selected from the group consisting of genes, but is not limited thereto.

Another aspect of the present invention relates to a host cell transformed with the expression vector for preparing the virus-like particle.

In the present invention, the term "host cell" means any organism that can be infected by a virus and immunized by virus-like particles. The host cell may be metabolically engineered by transformation.

The host cell 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. The suitable host cell may be a naturally occurring or wild-type host cell, or may be a modified host cell. The wild-type host cell may be a host cell that is not genetically altered by a recombinant method.

In the present invention, "transformation" refers to a method of delivering the vector into a microorganism or a specific cell, and when the cell to be transformed is a prokaryotic cell, it may be performed by the CaCl ₂ method, the Hanhan method, and the electroporation method. there is. When the cells to be transformed are eukaryotic cells, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, etc. may be used. It is not limited. In the case of transformation of fungi such as yeast, a transformation method using lithium acetate and heat shock and an electroporation method may generally be used, but are not limited thereto.

In the present invention, the term "metabolically engineered" or "metabolic engineering" refers to biosynthetic genes, genes associated with operons, and nucleic acid sequences thereof for the production of desired metabolites such as proteins or alcohols in microorganisms. Metabolic flux by controlling and optimizing transcription, translation, protein stability and protein functionality by manipulating control elements or using appropriate culture conditions ) means to induce the optimization of

For example, a metabolically engineered host cell refers to a genetically engineered host cell in which a protein of interest is produced at or greater than the level of expression or produced in a wild-type host cell growing under essentially the same growth conditions. It can be expressed at a level of expression greater than the level of expression of the target protein of.

The type of the host cell is not particularly limited as long as it can efficiently express a specific gene by being transformed by an engineering method. Specifically, the host cell may be an insect cell.

The insect cell may be any cell developed or commercially available as a host system for gene expression, for example, Spodoptera frugiperda insect cells ( Spodoptera frugiperda ) SF21, SF9, trichoplusia ni ( Trichoplusia ni ), anti Anticarsa gemmitalis , Bombyx mori , Estigmene acrea, Heliothis virescens , Leucania separata , Lymantria dispa ( Lymantria dispar ), Malacasoma distria ( Malacasoma disstria ), Mammestra brassicae ( Mammestra brassicae ), Manduca sexta ( Manduca sexta ), Plutella zylostella ( Plutella zylostella ), Stodoptera exigua ( Spodoptera exigua ) and Spodoptera littoris ( Spodoptera littorlis ) It may be one or more selected from the group consisting of, but is not limited thereto.

Specifically, the insect cells may be SF9 cells derived from fall armyworm ( Spodoptera frugiperda ).

In one embodiment of the present invention, the recombinant baculovirus for preparing virus-like particles was transformed into SF9 insect cells to obtain transformed host cells.

Another aspect of the present invention is (a) a nucleic acid sequence encoding influenza virus matrix protein 1 consisting of SEQ ID NO: 3; and constructing an expression vector comprising a nucleic acid sequence encoding the merozoite surface protein-8 of Plasmodium malaria consisting of SEQ ID NO: 4; (b) preparing a transformant using the expression vector; (c) transfecting the transformant into a host cell; (d) culturing the transfected host cells and obtaining the culture; and (e) obtaining virus-like particles from the culture.

The virus-like particles can be produced by genetic engineering, and can be produced by genetic engineering without a separate causative agent, that is, the malaria parasite in the present invention, so that high productivity and economy can be realized.

The virus-like particles can be prepared by methods well known in the art. For example, the virus-like particle can be prepared by transforming a predetermined host cell using a recombinant DNA molecule encoding the structural protein and antigen recognition site and then culturing it, and the protein expressed in the cell is assembled on the cell surface. After that, it can be discharged into the culture supernatant. At this time, since the surface protein included in the virus-like particle maintains its natural form without undergoing an immobilization process, a desired immune response against a specific pathogen can be induced in the subject.

Specifically, the nucleic acid sequence encoding merozoite surface protein-8 in step (a) includes a primer pair comprising a first primer composed of the nucleic acid sequence of SEQ ID NO: 5 and a second primer composed of the nucleic acid sequence of SEQ ID NO: 6 can be amplified by

In addition, specifically, in step (a), the nucleic acid sequence encoding influenza virus matrix protein 1 is synthesized by a primer pair including a third primer consisting of the nucleic acid sequence of SEQ ID NO: 7 and a fourth primer consisting of the nucleic acid sequence of SEQ ID NO: 8. can be amplified.

The primers can initiate DNA synthesis in the presence of reagents for polymerization and four different nucleoside triphosphates at an appropriate buffer solution and temperature, and hybridize with nucleic acids present in the target material to generate a specific gene of the target material. can be used to amplify.

The primers may specifically be single-stranded in consideration of amplification efficiency, may be deoxyribonucleotides, naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural Can contain nucleotides. In addition, the primer may also include ribonucleotides.

The primers can incorporate additional features that do not alter the basic properties. That is, nucleic acid sequences can be modified using a number of means known in the art. Examples of such modifications are methylation, capping, substitution of nucleotides with one or more homologues, and uncharged linkages such as phosphonates, phosphotriesters, phosphoroamidates or carbamates, or phosphorothioates or phosphorodithioates. Transformation of nucleotides into charged linkages such as Nucleic acids may also contain one or more nucleic acids, such as nucleases, toxins, antibodies, signal peptides, proteins such as poly-L-lysine, intercalating agents such as acridine or psoralen, chelating agents and alkylating agents such as metals, radioactive metals, and iron oxidizing metals. It may have additional covalently linked moieties.

The primers were cloned with appropriate sequences and digested with restriction enzymes, followed by the phosphotriester method of Narang et al. (1979, Meth, Enzymol. 68:90-99), and the diethylphosphoramidite method of Beaucage et al. (1981, Tetrahedron Lett. 22: 1859-1862) and any other well-known method including direct chemical synthesis, such as the solid supported method of US Pat. No. 4458066.

The transformed host cells may be cultured under batch, fed-batch or continuous fermentation conditions, and since the host cells may express the virus-like particles by transformation, the virus-like particles from the cultured host cells may be cultured. Particle protein can be obtained.

In this case, the batch fermentation method may use a closed system, wherein the culture medium is prepared before fermentation is carried out, organisms are inoculated into the medium, and fermentation may occur without adding any components to the medium. In certain cases, the pH and oxygen content, but not the carbon source content of the growth medium, may be changed during a batch process. The metabolite and cell biomass of the batch system can be constantly changed until fermentation is stopped. In a batch system, cells may progress from a stationary lag phase through a high-growth log phase and finally reach a stationary phase where the growth rate decreases or stops. In normal terms, the cells in log phase can make most proteins.

A batch system can be transformed into a "fed-batch fermentation" system. In this system, nutrients (eg, carbon source, nitrogen source, O ₂ and typically other nutrients) can be added when the concentration of their culture falls below a threshold. A fed-batch system can be useful when catabolite inhibition inhibits the metabolism of cells and it is desirable for a medium to have a limited amount of a nutrient in the medium. A measure of the actual nutrient concentration in a fed-batch system is pH, Predictions can be made based on changes in measurable factors such as dissolved oxygen and partial pressures of off-gases such as _CO2 . Batch and fed-batch fermentations are common systems and are well known in the art.

Continuous fermentation is an open system in which a defined culture medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed during the process. Continuous fermentation is generally able to maintain a culture of constant high density where the cells are 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, a limiting nutrient such as a carbon source or a nitrogen source can be kept at a fixed rate, while all other parameters are kept appropriate. In other systems, many growth-influencing factors may change continuously while the cell concentration, as measured by media turbidity, remains constant. Continuous systems try to maintain constant growth conditions. Thus, cell loss due to medium evacuation can be balanced against the rate of cell growth in fermentation.

In one embodiment of the present invention, an expression vector is prepared using a nucleic acid sequence consisting of SEQ ID NO: 3 encoding influenza virus matrix protein 1 and a nucleic acid sequence consisting of SEQ ID NO: 4 encoding merozoite surface protein-8 of Plasmodium malaria. After the expression vector was transformed into SF9 insect cells and cultured, virus-like particles capable of inducing an immune response against malaria were obtained from the culture medium.

Unlike conventional vaccines, the virus-like particle capable of inducing an immune response against malaria of the present invention can be developed without a causative infectious agent and does not induce non-selective antibody production.

In addition, since the virus-like particles can provide immunity and preventive effects against malaria by activating the body's immune system, they are safe without causing resistance and side effects can be minimized.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

1 shows a virus-like particle capable of inducing an immune response against malaria (A: model of virus-like particle including influenza virus matrix protein 1 and merozoite surface protein-8, B: virus- Result of observing pseudoparticles under an electron microscope, C: Western blot result after reacting virus-like particles with malaria-infected serum and influenza M1 antibody).
Figure 2 shows the result of cloning the merozoite surface protein-8 of Plasmodium parasite into the pFastBac vector to prepare a virus-like particle vaccine.
Figure 3 shows the results of ELISA analysis on whether IgG, a malaria-specific antibody, is formed upon inoculation with virus-like particles.
Figure 4 shows the results of the immune response of CD4 ⁺ T cells and CD8 ⁺ T cells by inoculation with virus-like particles (AC: comparison of CD4 ⁺ T cell levels, DF: comparison of CD8 ⁺ T cell levels).
Figure 5 shows the results of observation of clusters of memory immune T cells (CD4 ⁺ T _EM ) by inoculation with virus-like particles.
Figure 6 shows the results of observation of clusters of memory immune T cells (CD8 ⁺ T _EM ) by inoculation with virus-like particles.
7 shows the results of observation of memory B cell populations by inoculation with virus-like particles.
8 shows the results of confirming the inflammatory response and parasite proliferation by inoculation with virus-like particles (A: cytokine IFN-gamma level, B: parasite proliferation).

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the present invention is not limited by the following examples.

Example 1. Preparation of vectors for preparing Virus-Like Particles (VLPs) capable of inducing an immune response against malaria

To prepare a virus-like particle comprising influenza virus matrix protein (M1) and merozoit surface protein-8 (MSP-8) of the parasite, SEQ ID NO: 4 Part of MSP-8 of rodent malaria ( Plasmodium berghei ANKA) consisting of the nucleotide sequence of was selected as an antigen gene. The gene was amplified using a pair of MSP-8 specific primers. The primers introduced restriction enzyme sites ( EcoR I and Hind III) at the underlined portions.

Meanwhile, the M1 gene was derived from the influenza A/PR/8/34 virus, and was amplified by RT-PCR using an M1-specific primer pair. The primers introduced restriction enzyme sites ( Sma I and Xba I) at the underlined portions, respectively. The M1 gene used the same sequence as the M1 gene described in Korean Patent Publication No. 10-2019-0009691 (GenBank accession number: EF467824, 1027bp).

The sequences of the primers are shown in Table 1 below.

sequence number Primer type order 5 MSP-8_Forward 5′- GAATTC ATGAAGAGGTCCAGCCAGAT-3′ 6 MSP-8_Reverse 5′- AAGCTT TTACATGATGTAGATGCAGA-3′ 7 M1_Forward 5′-TCC CCCGGG CCACCATGAGCCTTCTGACCGAGGTC-3′ 8 M1_Reverse 5'-TTACT TCTAGA TTACTTGAACCGTTGCATCTG-3'

Thereafter, the amplified sequence encoding the merozoite surface protein-8 (MSP-8) antigen using the primers as a cDNA template was introduced into a pFastBac vector (Invitrogen). In addition, a recombinant plasmid was prepared by introducing the influenza M1 protein gene amplified using primers into the pFastBac vector into which the MSP-8 coding sequence had previously been introduced. At this time, whether the M1 and MSP-8 genes introduced into the pFastBac vector were properly introduced was confirmed by DNA sequencing.

To prepare recombinant BaculoViruses (rBVs) expressing MSP-8 and M1, DNA transfection was performed using cellfectin II (Invitrogen) and SF9 cells. Transformation with the pFastBac vector containing MSP-8 and M1 was performed by white/blue screening. Recombinant baculovirus was prepared using the Bac-to-Bac expression system (Invitrogen) according to the manufacturer's manual.

Example 2. Preparation of virus-like particles capable of inducing an immune response against malaria

2-1. Preparation of virus-like particles

Virus-like particles capable of inducing an immune response against malaria were produced in SF9 insect cells co-infected with recombinant baculoviruses (rBVs) expressing MSP-8 and M1. Virus-like particles in the supernatant were pelleted using high-speed centrifugation (30 min, 45,000 xg).

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

2-2. Virus-like particle identification

The virus-like particles prepared in 2-1 above were confirmed using electron microscopy and Western blotting.

First, serum was collected from BALB/c mice 4 weeks after infection with the malaria ANKA strain. Virus-like particles were stained with medium for electron microscopy and size measurement using the same, and observed through transmission electron microscopy (TEM) (CANADA, ALBERTA).

Also, in Western blot analysis, MSP-8 protein was probed using mouse serum. Specifically, 40 μg, 8 μg, and 1.6 μg of virus-like particles were loaded for SDS-PAGE, respectively, and protozoa-infected serum and anti-M1 monoclonal antibody were used as probes.

As a result, SF9 cells producing virus-like particles were confirmed to be larger than normal cells as a control by electron microscopy, and the shape of virus-like particles was observed as an irregular sphere with spikes on the surface (Fig. 1B) . In addition, as a result of Western blot analysis, it was confirmed that the virus-like particles of MSP-8 and M1 were properly introduced (FIG. 1C).

Through the above results, it was confirmed that SF9 cells infected with recombinant baculoviruses (rBVs) expressing MSP-8 and M1 produced 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 of the present invention into mice was confirmed.

First, virus-like particles were inoculated intramuscularly using 7-week-old female BALB/c mice (Intramuscular route), and then blood was collected from the mice at a predetermined time and the degree of formation of malaria-specific IgG antibodies in serum was measured by ELISA. was measured.

Specifically, in order to measure malaria-specific antibodies, the antigen of the inactivated malaria parasite Plasmodium berghei ANKA was added to a 96-well immune plate (SPL Life Science, Korea) in 0.05 M, pH 9.6 with a final concentration of 4 μg/mL per well. Coated overnight at 4°C with carbonate bicarbonate buffer. For MSP-8 specific IgG antibody reaction, 100 mL of MSP-8 virus-like particles immunized mouse serum sample (diluted 1:100 in PBST) was added to each well and the plate was incubated at 37°C for 1 hour. cultured for a while. HRP-conjugated goat anti-mouse IgG antibody diluted in PBS (100 μL/well) was added as secondary antibody at 37°C.

As a result, it was confirmed that the total IgG response of the mice immunized by the inoculation of the virus-like particles significantly increased after the first inoculation, and in particular, the increase was excellent after the second inoculation (FIG. 3). From these results, it was confirmed that the antimalarial MSP-8-specific antibodies gradually matured by inoculation with the virus-like particles of the present invention.

The above experimental results suggest that the virus-like particles of the present invention can provide high immunity against malaria and can effectively induce an antibody response in tissue system in response to malaria infection.

Experimental Example 2. Antibody-secreting cell (B cell) response, T cell response test

Antibody-secreting cell responses following inoculation of the virus-like particles of the present invention into mice were confirmed by flow cytometry.

First, for immunization, 6-week-old female BALB/c mice were inoculated twice with the virus-like particle vaccine. Four weeks after the second immunization (boost immunization), a lethal dose of Plasmodium berghi ANKA was infected. Mice infected with the malaria parasite were sacrificed 6 days later, and blood was collected to confirm CD4 ⁺ T cell and CD8 ⁺ T cell populations.

In addition, splenocytes infected with malaria were incubated with staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1M PBS) and Fc block (BD Biosciences, CA, USA) at 4°C for 15 minutes. For surface antigen staining, cells were incubated with antibodies conjugated to fluorophores (BD Bioscience) for 30 minutes at 4°C. Antibodies used were as follows: PE-Cy7-conjugated anti-CD3e (145-2C11), PE-conjugated anti-CD4 (RM4-5), FITC or PE-conjugated anti-CD8a (53-6.7), PE- Conjugated anti-CD44 (IM7), APC-conjugated anti-CD62L (MEL-14), FITC-conjugated anti-B220 (RA3-6B2), PE-conjugated anti-IgG1 (A85-1), PE-Cy7-conjugated anti -CD27 (LG.3A10). The splenocytes were then washed from the staining buffer and the data analyzed using a BD Accuri C6 flow cytometer (BD Biosciences).

ve) 대비 현저히 증가한 CD4⁺ T세포 수준(도 4A 내지 4C) 및 CD8⁺ T세포 수준(도 4D 내지 4E)을 보이는 것을 확인하였다. As a result, in the case of mice with immunity formed by inoculation of virus-like particles, mice not treated with virus-like particles (Na

ve), it was confirmed that the CD4 ⁺ T cell level (Figs. 4A to 4C) and the CD8 ⁺ T cell level (Fig. 4D to 4E) were significantly increased compared to each other.

In addition, it was confirmed that the formation of clusters of memory immune T cells (CD4 ⁺ T _EM , CD8 ⁺ T _EM ) also increased with inoculation with virus-like particles (Figs. 5 and 6), and memory B cell cluster formation also significantly increased. It was confirmed (FIG. 7).

The above results show that mice immunized by virus-like particles can acquire a very high level of immunity against malaria infection and have excellent ability to form protective immunity against re-infection, so that they can be used as a vaccine against malaria infection. suggests that there is

Experimental Example 3. Confirmation of vaccine defense efficacy by virus-like particle inoculation

For immunization, 6-week-old female BALB/c mice were inoculated twice with the virus-like particle vaccine. 4 weeks after the second inoculation, a lethal dose of Plasmodium berghi ANKA was infected.

First, spleens were collected from mice on day 6 of infection, and splenocytes were isolated in RPMI 1640 medium (containing 10% FBS). A 96-well plate was coated with 400 ng/well of anti-mouse gamma interferon (IFN-γ capture antibody (BD Bioscience)) in 100 μL of PBS overnight at 4° C. Assay diluent (BD bioscience, CA, USA) 100 μL of 1 After blocking at 25 ° C. for a period of time, the plate was washed three times with PBS 0.01% Tween 20. Then, 100 μL of the medium solution from which the splenocytes were isolated was added per well (1:1, 1:4, 1:49 in assay diluent). ratio) and incubated for 2 hours at 25 ° C. After washing the plate 3 times with PBS 0.01% Tween 20, 100 μL of detection antibody and SAv-HRP conjugated antibody were added per well and incubated at 25 ° C for 1 hour. After washing 5 times with PBS 0.05% Tween 20, 100 μl of TMB substrate reagent (BD Biosciences, CA, USA) was added to the plate and allowed to react for 10 minutes. The reaction was stopped. The plate was measured at 490 nm on an ELISA reader. In addition, blood was collected from infected mice to check the parasite growth rate. On the 6th day of Plasmodium berghi ANKA infection, blood was collected from all mice. , 2 μL of blood was added to 100 μL of PBS, and 1 μL of staining reagent (SYBR green, ThermoFisher) was added and incubated for 30 minutes at 37 ° C. After incubation, 400 μL of PBS was added and BD Accuri C6 flow cytometry was performed. Data were analyzed using an analyzer (BD Biosciences).

As a result, as a result of treatment with virus-like particles, it was confirmed that the release of interferon gamma (IFN-γ), a type of inflammatory cytokine, was reduced compared to the control group untreated with virus-like particles, and parasitemia was also significantly reduced. was confirmed (FIG. 8).

The above results suggest that the virus-like particles of the present invention can be used in vaccines for preventing and treating malaria infection by inducing a protective immune effect against malaria infection.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. 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 present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

<110> University-Industry Cooperation Group of Kyung Hee University <120> VIRUS-LIKE PARTICLES COMPRISING THE MEROZOIT SURFACE PROTEIN-8 OF MALARIA PROTOZOA, AND VACCINE COMPOSITIONS USING THE SAME <130> 20PP30664 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 252 <212> PRT <213> artificial sequence <220> <223> M1_amino acid <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> 409 <212> PRT <213> artificial sequence <220> <223> MSP-8_amino acid <400> 2 Met Lys Arg Ser Ser Gln Ile Ile Ile Phe Leu Leu Leu Ser Leu Val 1 5 10 15 Cys Lys Phe Ser Ile Gly Ile Cys Lys Glu Asn Gly Asn Gly Asn Ile 20 25 30 Asn Lys Ser Asn Asn Asn Arg Val Ile Lys Lys Glu Arg Lys Arg Lys 35 40 45 Ala Lys Ser Asn Ala Asn Lys Asn Glu Pro Glu Asn Lys Glu His Glu 50 55 60 Ile Ile Asn Leu Tyr Asp Asp Val Gln Glu Leu Leu Gly Asn Glu Arg 65 70 75 80 Met Ser Met Leu Asp Lys Tyr Ser Ile Leu Gly Ile Asp Asp Cys Ser 85 90 95 Asn Glu Ser Glu Asn Asn Lys Ile Ile Gly Glu Tyr Asp Leu Lys Ala 100 105 110 Met Lys Ser Val Leu Leu Tyr Lys Asn Arg Ile Ser Lys Val Ser Leu 115 120 125 Glu Asn Leu Tyr Asp Val Lys Thr Val Phe Lys Arg Cys Phe Asn Lys 130 135 140 Asp Asp Pro Glu Leu Ser Lys Ser Tyr Glu His Leu Gln Asp Gln Ala 145 150 155 160 Ala Ile Glu Gly Thr Thr Ile Ile Asp Tyr Leu Ser Asn Tyr Ile Leu 165 170 175 Asn Val Tyr Val Lys Met Asn Asp Glu Phe Ile Lys Asn Asp Gly Phe 180 185 190 Lys Leu Ser Lys Tyr Ile Pro Glu Leu Glu Ile Ile Asn Tyr Ala Leu 195 200 205 Tyr Asn Gly Pro Lys Glu Leu Glu Asn Arg Ile Lys Ser Gly Leu Asn 210 215 220 Glu Ile Asn Asn Ile Ile Ile Ser Glu Ser Leu Thr Ser Ile Tyr Ser 225 230 235 240 Ser Val Val Ser Gly Leu Asn Ile Asn Cys Lys Ile Lys Asp Asp Leu 245 250 255 Ile Thr Ile Leu Asn Leu Ser Asp Gly Lys Tyr Phe Lys Val Asp Phe 260 265 270 Ser Ser Gln Ala Thr Met Ile Val Pro Gly Gln Tyr Ser His Glu His 275 280 285 Gly Asn Met Lys Lys Ile Ser Glu Tyr Phe Ile Glu Lys Asn Arg Ile 290 295 300 Cys Lys Asn Glu Lys Cys Pro Ile Asn Ser Asn Cys Tyr Val Ile Asp 305 310 315 320 Asn Val Glu Thr Cys Arg Cys Ile Pro Gly Phe Ser Lys Asn Lys Glu 325 330 335 Ser Glu Ser Leu Lys Cys Asp Ile Asp Glu Ser Thr Ser Cys Glu Asn 340 345 350 Asn Asn Gly Gly Cys Asp Val Asn Ala Thr Cys Leu Leu Leu Glu Asp 355 360 365 Lys Ile Met Cys Glu Cys Asn Asn Lys Tyr Asn Gly Asp Gly Ile Tyr 370 375 380 Cys Ser Asn Ala Ile Tyr Tyr Gly Met Asn Val Phe Val Phe Phe Leu 385 390 395 400 Ile Ser Ile Val Cys Ile Tyr Ile Met 405 <210> 3 <211> 1027 <212> DNA <213> artificial sequence <220> <223> M1_nucleic acid <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> 1230 <212> DNA <213> artificial sequence <220> <223> MSP-8_nucleic acid <400> 4 atgaagaggt ccagccagat catcatcttc ctgctgctgt ctctggtgtg caagttctca 60 atcggtatct gcaaggagaa cggaaacggt aacatcaaca agtctaacaa caacagagtc 120 atcaagaagg aacgcaagcg taaggctaag tcaaacgcca acaagaacga gcccgaaaac 180 aaggagcacg aaatcatcaa cctgtacgac gacgtgcagg agctgctggg caacgaacgc 240 atgtccatgc tggacaagta cagcatcctg ggaatcgacg actgctctaa cgagtcagaa 300 aacaacaaga tcatcggcga gtacgacctg aaggctatga agtccgtgct gctgtacaag 360 aacaggatct ccaaggtcag cctggaaaac ctgtacgacg tgaagaccgt cttcaagaga 420 tgcttcaaca aggacgaccc agagctgtct aagtcatacg aacacctgca ggaccaggct 480 gccatcgagg gaaccactat catcgactac ctgtccaact acatcctgaa cgtgtacgtc 540 aagatgaacg acgagttcat caagaacgac ggtttcaagc tgagcaagta catccctgag 600 ctggaaatca tcaactacgc tctgtacaac ggacccaagg agctggaaaa ccgcatcaag 660 tctggtctga acgagatcaa caacatcatc atctccgaaa gcctgacctc tatctactct 720 tcagtggtgt ccggtctgaa catcaactgc aagatcaagg acgacctgat caccatcctg 780 aacctgtccg acggcaagta cttcaaggtg gacttctcca gccaggccac tatgatcgtc 840 cctggacagt actcccacga gcacggcaac atgaagaaga tcagcgagta cttcatcgaa 900 aagaaccgca tctgcaagaa cgagaagtgc ccaatcaact ctaactgcta cgtgatcgac 960 aacgtcgaaa cttgccgttg catccctggc ttctcaaaga acaaggagtc tgaatcactg 1020 aagtgcgaca tcgacgagtc caccagctgc gaaaacaaca acggtggctg cgacgtgaac 1080 gctacttgcc tgctgctgga ggacaagatc atgtgcgaat gcaacaacaa gtacaacggc 1140 gacggaatct actgctccaa cgccatctac tacggaatga acgtgttcgt cttcttcctg 1200 atcagcatcg tctgcatcta catcatgtaa 1230 <210> 5 <211> 26 <212> DNA <213> artificial sequence <220> <223> MSP-8_Forward <400> 5 gaattcatga agaggtccag ccagat 26 <210> 6 <211> 26 <212> DNA <213> artificial sequence <220> <223> MSP-8_Reverse <400> 6 aagcttttac atgatgtaga tgcaga 26 <210> 7 <211> 35 <212> DNA <213> artificial sequence <220> <223> M1_Forward <400> 7 tcccccgggc caccatgagc cttctgaccg aggtc 35 <210> 8 <211> 32 <212> DNA <213> artificial sequence <220> <223> M1_Reverse <400> 8 ttacttctag attacttgaa ccgttgcatc tg 32

Claims (13)

Influenza virus matrix protein (M1); and
In the virus-like particle that induces an immune response to malaria, including merozoit surface protein-8 (MSP-8) of the malaria parasite,
The merozoite surface protein-8 consists of the amino acid sequence of SEQ ID NO: 2 or the nucleic acid sequence of SEQ ID NO: 4, virus-like particle that induces an immune response against malaria. According to claim 1,
The influenza virus matrix protein 1 consists of the amino acid sequence of SEQ ID NO: 1, a virus-like particle that induces an immune response against malaria. According to claim 1,
The influenza virus matrix protein 1 consists of the nucleic acid sequence of SEQ ID NO: 3, a virus-like particle that induces an immune response against malaria. A vaccine composition comprising the virus-like particle of any one of claims 1 to 3. According to claim 4,
The vaccine composition is a vaccine composition comprising any one selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, immune stimulants, and combinations thereof. a nucleic acid sequence encoding influenza virus matrix protein 1 consisting of SEQ ID NO: 3; and
An expression vector for preparing virus-like particles, comprising a nucleic acid sequence encoding the merozoite surface protein-8 of the parasite of SEQ ID NO: 4. According to claim 6,
The vector is a baculovirus vector, an expression vector for producing virus-like particles. A host cell transformed with the expression vector of claim 6 or 7. According to claim 8,
The host cell is 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 claim 8,
The host cell is an insect-derived SF9 cell, a transformed host cell. (a) a nucleic acid sequence encoding influenza virus matrix protein 1 consisting of SEQ ID NO: 3; and constructing an expression vector comprising a nucleic acid sequence encoding the merozoite surface protein-8 of Plasmodium malaria consisting of SEQ ID NO: 4;
(b) preparing a transformant using the expression vector;
(c) transfecting the transformant into a host cell;
(d) culturing the transfected host cells and obtaining the culture; and
(e) obtaining virus-like particles from the culture
A method for producing virus-like particles that induces an immune response against malaria. According to claim 11,
In step (a), the nucleic acid sequence encoding merozoite surface protein-8 is amplified by a primer pair including a first primer composed of the nucleic acid sequence of SEQ ID NO: 5 and a second primer composed of the nucleic acid sequence of SEQ ID NO: 6 A method for producing virus-like particles that induces an immune response against malaria. According to claim 11,
In step (a), the nucleic acid sequence encoding influenza virus matrix protein 1 is amplified by a primer pair including a third primer consisting of the nucleic acid sequence of SEQ ID NO: 7 and a fourth primer consisting of the nucleic acid sequence of SEQ ID NO: 8 A method for producing virus-like particles that induces an immune response against phosphorus and malaria.

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