KR101835705B1 - Trichinella spiralis virus-like particle, expression vector and method for preparing the same - Google Patents

Abstract

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

Description

FIELD OF THE INVENTION [0001] The present invention relates to a fungus virus-like particle, a vector for producing the fungus virus-like particle, and a method for producing the same. BACKGROUND ART [0002]

The present invention relates to a fowl virus-like particle comprising an influenza virus matrix protein 1 (M1) as a structural protein.

Trichinella spiralis is a parasitic common parasite that is parasitic to the muscles of mammals such as humans, pigs, rats, and causes vomiting, hari, fever, stiffness and pain of infected muscles, dyspnea, facial edema and conjunctivitis.

The tiger bug is parasitic in the male and female adult moths in the small intestine mucus of an animal, and the parasite is parasitized in muscle fibers distributed in the rhabdomyosus of the whole body through the blood vessels and lymph nodes of the barrier.

When the infected meat is consumed, the endocervical plug (first repellant) is liberated from the stomach by the digestive process, and after being removed from the small intestine of the host, it is grown in the mucosa of the lower intestine under the colon in 5 to 7 days do.

Males die after copulation and females give birth to electron-pack after 5 days of infection. The electrons enter the rhabdomyoside fibers to form a coating of muscle fibers, and the cysts in the cysts can no longer grow, so they can multiply by ingestion by other hosts.

Mebendazole or albendazole is mainly used for the treatment of the tiger bug, but it is effective only when the tiger bug is in the intestine and does not function when the tiger bug is reached in the cucumber larva stage in the muscle .

Symptoms caused by infestation of the tiger bug can lead to death of the infected person in severe cases, but currently available therapeutic agents can be used in a limited manner and the therapeutic effect is gradually diminished by tolerance. In addition, a vaccine capable of generating a specific antibody against a typhoon and imparting immunity thereto has not been developed.

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

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

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

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

According to one aspect of the present invention, there is provided an influenza virus matrix protein (M1) and a Trichinella spiralis virus-like particle comprising an antigenic determinant site derived from a tiger mosquito .

In one embodiment, the triticale-derived antigenic determinant may comprise a 53 KDa excretory secretory protein (T653K).

In one embodiment, the influenza virus matrix protein 1 comprises the amino acid sequence of SEQ ID NO: 1, and the antigens derived from the tiger bug may comprise 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 antigenic determinant derived from the tiger bug can be encoded by the nucleic acid sequence of SEQ ID NO: 4.

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

According to another aspect of the present invention, there is provided an expression vector for producing a fowl virus-like particle comprising a nucleic acid sequence encoding influenza virus matrix protein 1 (M1) and a nucleic acid sequence encoding an antigenic determinant site derived from a tiger mollusk.

In one embodiment, the triticum-derived antigenic determinant may comprise a 53 KDa excretory-secretory protein.

In one embodiment, the influenza virus matrix protein 1 comprises the amino acid sequence of SEQ ID NO: 1, and the antigens derived from the tiger bug may comprise the amino acid sequence of SEQ ID NO: 2.

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

In one embodiment, the host cell can be a microorganism, an animal cell, a plant cell, a cultured cell derived from an animal, or a cultured cell derived from a plant.

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

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

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

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

1 is a PCR analysis of T653K and M1 genes of virus-like particles according to an embodiment of the present invention. (A) and (B) were obtained by amplifying T653K of the brassica by PCR and introducing it into the pFastBac vector. (C) and (D) are amplifications of the influenza M1 gene and introduced into the pFastBac vector.
Figure 2 depicts a virus-like particle according to one embodiment of the present invention. (A) is an electron microscopic observation and size measurement of the virus-like particles according to an embodiment of the present invention. (B) shows the results of Western blot analysis. Twenty micrograms, 10 micrograms, and 5 micrograms of virus-like particles were respectively loaded for SDS-PAGE, and anti-tympanic polyclonal mouse antibodies and anti-M1 monoclonal antibodies were used as probes.
FIG. 3 shows the tyrosine-specific IgG response upon induction of an immune response. (A) is a diagram of the experiment schedule. (B) is the measurement of IgG antibody response in the serum through ELISA analysis (after prime and after boost).
FIG. 4 shows the results of confirming the tyrosine-specific IgG1 and IgG2a responses upon induction of the immune response. (A) is a diagram of the experiment schedule. (A) is a measurement of the IgG1 antibody reaction in the serum. (B) is a measurement of the IgG2a antibody reaction in the serum.
FIG. 5 shows the typhoon specific antibody response to challenge infection. (A), (B), and (C) show the results obtained by infecting a mouse having immunity with the fowl virus-like particle according to an embodiment of the present invention with a tiller, and after 1 week, - specific IgG, IgG1, and IgG2a antibody responses (* P <0.05, ** P <0.01). (D) is a measurement of the ratio of IgG1 and IgG2a.
FIG. 6 shows the protective effect of virus-like particle inoculation according to an embodiment of the present invention. The degree of parasite reduction between the mice immunized with virus-like particles and the control group was measured.

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

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

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

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

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

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

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

One aspect of the present invention provides an influenza virus matrix protein (M1) and a Trichinella spiralis virus-like particle comprising an antigenic determinant site derived from the tiger mosquito .

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

In particular, the virus-like particle according to an embodiment of the present invention may be produced by a genetic engineering method and may include matrix protein 1 (M1) derived from influenza virus as a core protein. The virus-like particles can be produced by a genetic engineering method without a separate causative agent, i.e., a typhoon, so that high productivity and economical efficiency can be realized.

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

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

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

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

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

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

In one embodiment, the antigenic determinant is selected from the group consisting of excretory secretory proteins, TspSP-1, Ts23-2, serine protease, enolase, 5'-nucleotidase, putative trypsin, heat shock protein 70 (Hsp 70), paramyosin, and glutamic acid-rich protein (NBL) , But it may preferably contain an excretory secretory protein (T653K) of 53 KDa.

The "excretory secretory protein" is known to play an important role in the immune regulation of parasites, and has been studied as a candidate for the immunostimulatory antigen of the tiger bug. Especially, the 53KDa excretion-secretion protein is contained in a large amount in the ES product, and since the homologous protein of the excretory-secretory protein of 53KDa exists in the ES product of another species of the Trichinella genus, it can be used as an antigen for the detection of the anti-follicle antibody.

In particular, the present inventors have found that the 53KDa excretory-secretion protein effectively induces the production of anti-femoral antibody by its structural characteristics, and therefore, as a form fused with influenza virus matrix protein 1, a high level of immune response And that it is possible to provide it.

In one embodiment, the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the 53 kDa excretory-secretion protein derived from the tiger bug can be composed of the amino acid sequence of SEQ ID NO: 2. In addition, the influenza virus matrix protein 1 is encoded by the nucleic acid sequence of SEQ ID NO: 3, and the 53 kDa excretory-secretory protein derived from the tiger bug can be encoded by the nucleic acid sequence of SEQ ID NO: 4.

The influenza virus matrix protein 1 or the 53 kDa excretory-secretory protein derived from the tiger bug includes a functional equivalent of a protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2.

Is at least 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, more preferably 90% or more, Means a protein having a homology of at least 95% with a physiological activity substantially equivalent to the amino acid sequence of SEQ ID NO: 1 or 2.

The "substantially homogenous physiological activity" refers to a virus-like particle capable of inducing a specific immune response to the typhoon due to the structural and functional homology with the influenza virus matrix protein 1 or the 53KDa excretory- &Lt; / RTI &gt;

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

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

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

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

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

In addition, the vaccine composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, or sterilized injection solution according to a conventional method.

In the case of formulation, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are commonly used may be used together.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate), sucrose or lactose, gelatin Etc. can be used together. In addition to these excipients, lubricants such as magnesium stearate talc may also be used.

As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are simple diluents commonly used, various excipients such as wetting agents, sweeteners, have. For parenteral administration, sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents may be used. Examples of the water-insoluble preparation and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

According to another aspect of the present invention, there is provided an expression vector for producing a fowl virus-like particle comprising a nucleic acid sequence encoding influenza virus matrix protein 1 (M1) and a nucleic acid sequence encoding a 53 kDa excretory-secretion protein derived from a tiger mosquito.

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

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

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

On the other hand, the expression vector is introduced into a host cell, and a host cell transformed with the introduced vector can produce the fowl virus-like particle. Wherein the vector may comprise a promoter that is recognized by the host organism.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The transformed host cells can be cultured under batch, feed-batch, or continuous fermentation conditions, and the host cells are capable of expressing the fowl virus-like particles by transformation, A similar particle protein can be obtained.

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

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

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

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

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

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

Preparation Example 1: Preparation of fowl virus-like particles

The 53 kDa excretory-secretory protein (T653K) gene was amplified by PCR using a forward primer (5'-AAA GAATTC ACCATGTTCAGCATCACATTAAA-3 ') and a reverse primer (5'-TTA CTCGAG TTAGAACAACAACTGTAGT-3'). The primers were introduced with underlined restriction enzyme sites (EcoR I and Xho I, respectively) (FIG. 1A, 1B). The sequence encoding T653K amplified as a template for cDNA was introduced into the pFastBac vector (Invitrogen).

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

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

The accession numbers (EF467824, 1027bp) and T653K (accession number: DQ399914.1) genes introduced into the pFastBac vector were confirmed by DNA sequencing.

In order to prepare recombinant baculoviruses (rBVs) expressing T653K and M1, DNA transfection was performed using cellfectin II (Invitrogen) and SF9 cells. Transformation with pFastBac vector containing T653K and M1 was performed by white / blue screening. Baculovirus was produced according to the manufacturer's instructions via a Bac-to-Bac expression system (Invitrogen).

Preparation Example 2: Preparation of fowl virus-like particles

Brassica virus-like particles were produced in SF9 insect cells co-infected with recombinant rBVs expressing T653K and M1.

The cell culture supernatant was separated on the third day of infection and the cells were removed by centrifugation (6000 rpm, 4 ° C) for 30 minutes. Virus-like particles in the supernatant were pelleted by high speed centrifugation (30 min, 45,000 x g).

The pelleted precipitate was resuspended in phosphate buffered saline (PBS) overnight at 4 ° C and harvested and purified at 4 ° C, 45,000 xg for 1 hour through 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, T653K protein was probed with mouse serum. Serum was collected from BALB / c mice infected by the rhizoma ME49 strain 4 weeks later. The content of M1 protein was determined by anti-M1 monoclonal antibody. For electron microscopy and size measurement, virus-like particles were stained with media and observed by transmission electron microscopy (TEM) (KAIST, Tecnai G2 spirit, USA).

Virus-like particles were observed by electron microscopy in the form of spheres with spikes formed on the surface. The size of the virus-like particle was confirmed to be about 40 to 120 nm (Figure 2A).

Western blots were performed using anti-typhoid polyclonal antibodies and anti-M1 monoclonal antibodies to confirm that they were introduced as virus-like particles of T653K and M1 (Fig. 2B).

In other words, SF9 cells infected with rBVs expressing T653K and M1 produced particles with similar morphology and size to virions.

Experimental Example 1: Humoral Immune Induction Reaction Test of Animal Model

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

40 female BALB / c mice (6-8 weeks old) were divided into 4 groups (naive control, TS control group infected with T. spiralis , T653k VLP group, T653k VLP / CT group).

Mice were immunized by intramuscular injection of the T653k virus-like particles alone or with cholera toxin (CT) (25 μg VLPs / mouse, 2 μg CT / mouse).

All experimental groups were inoculated 2 times (0th and 4th weeks) and after 4 weeks of the last inoculation, T. spiralis larvae were orally administered.

After the virus-like particle vaccination of Preparation Example 2, sera were collected from mice at different time points, and the levels of specific IgG, IgG1, and IgG2a were measured by ELISA (FIG. 3A).

IgG, IgG1, and IgG2a levels of the mice immunized with the virus-like particles were significantly higher than those of the naive control (0, 1, 5, and 8 weeks) The immune response was further increased (Fig. 3B).

At this time, the level of IgG2a was higher than that of IgG1, and the above results suggest that the IgG2a response is relatively dominant. In addition, IgG2a levels were relatively higher than IgG1 levels, but IgG1 antibody responses were also confirmed, suggesting that the Th1 / Th2 mixed immune response can be induced by the virus-like particles.

That is, the virus-like particle of Preparation Example 2 provides a high immunity to the tiger bug, and can effectively induce the antibody response in response to infection of the tiger bug.

Experimental Example 2: Tincture-specific antibody reaction test

The typhoon specific antibody response induced by the virus-like particle inoculation of Production Example 2 was tested.

Four weeks after the inoculation of the virus-like particles, the tiger larva was infected orally through the oral cavity in order to confirm the antibody response profile according to the challenge infection of the mice.

IgG, IgG1, and IgG2a levels were measured at 1 week, 4 weeks, and 6 weeks after the infection with the tiller bats to determine the level of the tritonium-specific antibody induced by the virus-like particle vaccine (FIGS. 5A, 5B, 5C ).

Mice immunized with virus-like particle infections produced significantly higher levels of triton-specific IgG, IgG1, IgG2a (* P <0.05, ** P <0.01 compared to negative control (TS control) ), The antibody response of mice administered with cholera toxin was further increased.

In other words, the serum-level of IgG, IgG1, and IgG2a antibodies can be significantly increased by typhimurium infection because the virus-like particle induces a tiller-specific immune response.

In addition, the immune response was further increased in the mice to which virus-like particles and cholera toxin were administered together, and the IgG2a response was relatively dominant (Fig. 5D).

These results suggest that the Th1 / Th2 mixed immune response can be effectively induced by the virus-like particles. That is, a mouse that has been immunized with the virus-like particle can acquire a very high level of immunity against infestation of the tinebrush.

Experimental Example 3: Protective Immunity Test

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

After the virus-like particle vaccination of Production Example 2, mice infected with the tiger bug were sacrificed after 6 weeks, and the tiger larva larvae were isolated from the diaphragm.

The mice immunized with cholera toxin and virus-like particles showed a 53.2% reduction in parasites and a 34% reduction in parasites in mice vaccinated with virus-like particles alone (* P <0.05, ** P &Lt; 0.01).

These results suggest that the virus-like particle can induce a protective immune effect against the tritonbrush infection and that the cholera toxin can enhance the protective immune effect as an adjuvant.

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

The T653K protein used in Preparation Examples 1 and 2 was treated with TspSP-1, Ts23-2, serine protease, enolase, 5'-nucleotidase, Putative trypsin, and heat shock protein 70 (Hsp 70), respectively, and Experimental Examples 1 to 3 were repeated.

As a result of the experiment, the virus-like particles had some differences in the immunoreactivity induced by different antigenic sites, but they induced immune responses and antibody-secretory cell responses in animal models.

The IgG, IgG1, and IgG2a levels of the mice immunized with the virus-like particles were markedly increased as compared with the naive control, and it was confirmed that the Th1 / Th2 mixed immune response could be induced.

Particularly, mice immunized with the virus-like particles effectively produced trichogang-specific IgG, IgG1, and IgG2a by typhimurium infection, and the immune response was observed when the cholera toxin was administered together with the virus-like particles More.

That is, since the virus-like particle includes the matrix protein 1 (M1) derived from influenza virus as a structural protein and contains an antigenic determinant derived from a tiger bug on its surface, the virus-like particle has a specific immune response Can be effectively induced.

In addition, since the virus-like particle effectively induces an immune response even if the antigen-crystal region is different, the virus-like particle can be used as a vaccine to produce an anti-fowl antibody by fusing the matrix protein 1 derived from the influenza virus and the tuna .

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

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

<110> kyung hee university <120> TRICHINELLA SPIRALIS VIRUS-LIKE PARTICLE, EXPRESSION VECTOR AND          METHOD FOR PREPARING THE SAME <130> 16PP10334 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 252 <212> PRT <213> Influenza virus <400> 1 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro   1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe              20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr          35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe      50 55 60 Thr Leu Thr Val Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val  65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala                  85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala             100 105 110 Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met         115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe     130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu                 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met             180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln         195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Ser Ser     210 215 220 Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys                 245 250 <210> 2 <211> 412 <212> PRT <213> Trichinella spiralis <400> 2 Met Phe Ser Ile Thr Leu Asn Leu Phe Ile Ile Ala Phe Val Asn Phe   1 5 10 15 Gln Leu Cys Thr Cys Ser Thr Asp Asn Glu Asn Val Ala Ile Lys Glu              20 25 30 Met Thr Phe Ser Leu Pro Ile Ser Val Leu Gln Asp Glu Arg Gln Phe          35 40 45 Asp Gly Lys Lys Leu Lys Lys Leu Leu Lys Pro Leu Gly Lys Leu Tyr      50 55 60 Asn Thr Ser Ser Asp Glu Gly Ile Pro Ile Asn Thr Asn Glu Ala Thr  65 70 75 80 Leu Ser Val Glu Lys Met Met Val Glu Leu Asn Arg Leu Ile Gln Lys                  85 90 95 Glu Tyr Ser Phe Leu Tyr Lys Gln Tyr Gln Lys Leu Lys Thr Val Leu             100 105 110 Gln Ala Glu Lys Cys Asp Ser Thr Thr Asn Val Tyr Thr Val Thr Leu         115 120 125 Gln Asn Thr Asp Cys Glu Ser Lys Ser Ile Glu Gly Ser Pro Ala     130 135 140 Thr Asn Cys Ser Asp Val Glu His His His Pro Leu Ser Cys Ser Ile 145 150 155 160 Leu Ser Lys Val Ala Ser Ala Asp Glu Lys Ile Ile Gly Ala Tyr Cys                 165 170 175 Ser Val Gly Leu Glu Glu Ser Phe Pro Lys Lys Lys Ser Ile Cys Lys             180 185 190 Leu Ser Arg Tyr Pro Gly Glu Glu Lys Phe Lys Thr Phe Val Pro Glu         195 200 205 Glu Val Ser Ser Trp Phe His Asp Ala Ile Val Tyr Val Pro Thr Gly     210 215 220 Asn Arg Pro Leu Ser Asn Ser Met His Ser Asn Asn Tyr Arg Gly Arg 225 230 235 240 Gln Gly Ile Val Gly Leu Gly Met Leu Pro His Leu Gly Ala Val Gln                 245 250 255 Met Asn Val Val Thr Ile Phe Arg Lys Asn Gly Lys Thr Thr Glu Val             260 265 270 Leu Ser Leu Ile Asn Ala Asn Gly Ser Ile Glu Ile Pro Lys Val Phe         275 280 285 Val Thr Asn Pro Ile Gln Lys Pro Phe Gly Asp Glu Ile Asp His Ile     290 295 300 Leu Arg Glu Ala Phe Gly Thr Met Lys Leu Ser Ser Ser Asp Ile Glu 305 310 315 320 Asp Lys Leu Gln Lys Leu Tyr Asn Ala Thr Ile Ser Thr Lys Val Lys                 325 330 335 His Arg Ala Thr Pro Tyr Asp Thr Asp Asp Ala Tyr Val Ile Thr Glu             340 345 350 Val Ala Gly Val Ile Asp Glu Ser Lys Glu His Ile Gly Ser Ile Asn         355 360 365 Thr Phe Pro Ser Tyr Gly Lys Phe Gln Ile Gly Trp Lys Glu Ala Asp     370 375 380 Lys Ser Ala Leu Arg Leu Lys Arg Phe Ala Lys Pro Pro Lys Gly Val 385 390 395 400 Phe Gln Asp Ile Phe Ser Glu Leu Gln Leu Leu Phe                 405 410 <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> 1239 <212> DNA <213> Trichinella spiralis <400> 4 atgttcagca tcacattaaa cttattcatt attgcttttg taaattttca attgtgcaca 60 tgttctacag acaatgagaa tgttgcaata aaggaaatga cattttcact tcctatttcc 120 gtgctacaag atgagcgtca attcgatgga aaaaaactga aaaaattatt gaagcccctt 180 ggaaaattat acaatacatc aagtgatgaa ggtataccga taaatacaaa tgaagctact 240 ctgtcggttg agaaaatgat ggttgaattg aatcgcttaa tacaaaaaga atacagtttt 300 ctttacaagc agtatcagaa attaaagacc gtcctacaag cagaaaaatg tgacagcaca 360 acaaacgtgt acaccgtaac actgcaaaat acagactgtg aatccaaatc gattatcgaa 420 ggaagccctg ctacgaattg cagtgatgtc gaacatcacc atcctctaag ctgcagcatt 480 ttaagtaaag tcgcttctgc agacgaaaaa attattggag catactgtag cgttggtttg 540 gaggaatcat ttccaaagaa aaaatctata tgcaaactgt ccagatatcc aggagaagag 600 aaatttaaaa cattcgttcc tgaagaagtt tcatcttggt ttcatgatgc aattgtatac 660 gtaccaactg gaaatcgtcc gctgtcgaac tcaatgcata gtaacaatta ccgtgggcga 720 caaggtattg ttggtcttgg aatgctacct catttgggcg cagttcaaat gaatgttgta 780 actattttca ggaaaaatgg taaaactact gaagttctat cacttataaa tgcaaatggt 840 tcaatagaaa tcccgaaggt ttttgttaca aatccaattc agaagccatt tggtgatgaa 900 atagatcata ttttaagaga agcttttggt accatgaaat taagcagttc tgacatagaa 960 gataaacttc aaaagttgta caatgcaaca attagcacta aggttaaaca cagggcaaca 1020 ccgtacgata cggacgatgc ttacgtaata actgaagtag ccggagtgat agatgaaagc 1080 aaagaacaca ttggcagcat taatacattt cccagttatg gaaaatttca gattggttgg 1140 aaagaggctg ataaatcggc actacgttta aagcgctttg caaagccccc aaaaggggtt 1200 ttccaagata ttttttcaga actacagttg ttgttctaa 1239

Claims (11)

Influenza virus matrix protein &lt; RTI ID = 0.0 &gt; (M1) &lt;
And a 53KDa excretory secretory protein (T653K) derived from the tiger bug bound to the surface of the influenza virus matrix protein 1,
Trichinella spiralis virus-like particles that increase the level of the tiger-specific IgG, IgG1, IgG2a. delete The method according to claim 1,
The influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the antigenic determinant site derived from the tiger bug is composed of the amino acid sequence of SEQ ID NO: 2. The method according to claim 1,
The influenza virus matrix protein 1 is encoded by the nucleic acid sequence of SEQ ID NO: 3, and the antigenic determinant site derived from the tiger bug is encoded by the nucleic acid sequence of SEQ ID NO: 4. 6. A vaccine composition for vomiting of the herpes virus, comprising the virus-like particle of any one of claims 1, 3, and 4 as an active ingredient. A nucleic acid sequence encoding influenza virus matrix protein 1 (M1) and
A nucleic acid sequence encoding a 53 kDa excretory secretory protein (T653K) derived from the tiger moth,
Expression vectors for the production of fowl virus-like particles which increase the level of the tiger-specific IgG, IgG1, IgG2a. delete The method according to claim 6,
Wherein the influenza virus matrix protein 1 is composed of the amino acid sequence of SEQ ID NO: 1, and the antigenic determinant site derived from the tiger bug is composed of the amino acid sequence of SEQ ID NO: 2. 8. A host cell transformed by the expression vector of claim 6 or 8. 10. The method of claim 9,
A host cell that is a microorganism, an animal cell, a plant cell, a cultured cell derived from an animal, or a cultured cell derived from a plant. Transforming the host cell with the expression vector of claim 6 or 8; And
And culturing the host cell to express the virus-like particle.

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