WO2020101187A1 - Vecteur recombiné pour l'expression de particules du type viral dans une plante et procédé de préparation d'une composition de vaccin contenant des particules du type circovirus l'utilisant - Google Patents

Vecteur recombiné pour l'expression de particules du type viral dans une plante et procédé de préparation d'une composition de vaccin contenant des particules du type circovirus l'utilisant Download PDF

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Publication number
WO2020101187A1
WO2020101187A1 PCT/KR2019/013581 KR2019013581W WO2020101187A1 WO 2020101187 A1 WO2020101187 A1 WO 2020101187A1 KR 2019013581 W KR2019013581 W KR 2019013581W WO 2020101187 A1 WO2020101187 A1 WO 2020101187A1
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Prior art keywords
plant
protein
pcv2
vaccine composition
recombinant vector
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PCT/KR2019/013581
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English (en)
Korean (ko)
Inventor
손은주
이용직
이상민
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주식회사 바이오앱
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Priority claimed from KR1020190127987A external-priority patent/KR102288367B1/ko
Application filed by 주식회사 바이오앱 filed Critical 주식회사 바이오앱
Priority to EP19884354.2A priority Critical patent/EP3882351A4/fr
Priority to CN201980075026.3A priority patent/CN113039275B/zh
Priority to CA3119964A priority patent/CA3119964A1/fr
Priority to JP2021526675A priority patent/JP7212968B2/ja
Publication of WO2020101187A1 publication Critical patent/WO2020101187A1/fr
Priority to US17/320,369 priority patent/US20210330780A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to a vaccine composition comprising virus-like particles (VLPs) produced using a plant, and more specifically, to a vaccine composition using a high-efficiency expression vector in a plant
  • the present invention relates to a method of manufacturing a PCV2 capsid protein produced by using virus-like particles and simultaneously increasing productivity of a usable porcine circovirus type 2 (PCV2) capsid protein.
  • the production of useful bioactive substances using transgenic plants can exclude various contaminants such as viruses, cancer genes, and enterotoxins that may occur in the method of producing proteins by synthesis from animal cells or microorganisms. If the demand for the useful material is rapidly increasing, it is absolutely advantageous compared to the existing animal cell system in terms of facility technology or cost required for mass production. It is widely used in various research fields such as vaccine development and therapeutic treatment.
  • a DNA fragment is added to the upstream of the target protein for recombination
  • the plant transformed with the recombinant vector can improve the translation of the target protein, and induce movement to a specific place in the plant, so that the target protein can be stably accumulated, thereby causing the target protein from the plant. It has been suggested that there is an effect that makes it possible to produce in large quantities.
  • Korean Patent Publication No. 10-2018-0084680 when producing the target protein in plant cells, the expression level of the target protein in the translation step is increased.
  • a small domain causing glycosylation N-glycosylation
  • the expression level of the protein is increased, and thus a method for dramatically improving the production efficiency of the target protein in a transgenic plant has been proposed.
  • a recombinant vector containing cellulose binding domain 3 and transformation using the recombinant vector It has been suggested that it is possible to separate the fusion protein containing the target protein by binding the proteins synthesized in the plant to cellulose, and to efficiently separate the target protein and the cellulosic binding domain by treating enterokinase with the fusion protein.
  • the plant system has a synthetic pathway in which a post-translational modification process, which is essential in mammals, has the advantage of being capable of producing a protein almost similar to the protein produced using animal cells. Therefore, the method for producing a protein which is a useful bioactive substance using the transformed plant as described above has the potential to replace the method for producing using animal cells or microorganisms, and has recently received great attention. have.
  • the present invention has been devised to solve the problems of the prior art as described above, in producing PCV2 (porcine circovirus type 2) to form a virus-like particle of the target protein in a transgenic plant, the protein in the translation step
  • PCV2 porosovirus type 2
  • the protein in the translation step
  • RuBisCO transit peptide Rubisco transit peptide
  • the virus-like particles are formed when the pH of the solution containing the purified PCV2 is properly adjusted as described above, and that the virus-like particles neutralize the virus.
  • the present invention was completed by demonstrating that it can be increased.
  • an object of the present invention is a polynucleotide encoding a RuBisCO transit peptide comprising the amino acid sequence represented by SEQ ID NO: 1, and PCV2 comprising an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5 (Porcine circovirus type 2) It is to provide a recombinant vector for plant expression, comprising a polynucleotide encoding a capsid protein.
  • Another object of the present invention is to provide a transformed plant transformed with the recombinant vector of the present invention.
  • Another object of the present invention is to provide a method for preparing a vaccine composition comprising virus-like particles, comprising the following steps:
  • step (S3) preparing the PCV2 capsid protein obtained in step (S2) as virus-like particles
  • step (S4) preparing a vaccine composition comprising virus-like particles obtained in step (S3).
  • an object of the present invention is to provide a vaccine composition prepared by the production method according to the present invention.
  • an object of the present invention is to provide a PCV2 virus-like particles contained in the vaccine composition prepared by the production method of the present invention.
  • an object of the present invention is to provide a method for preventing porcine circovirus infection by administering a vaccine composition prepared by the production method according to the present invention to an individual.
  • Another object of the present invention is to provide a porcine circovirus infection prevention use of the vaccine composition prepared by the production method according to the present invention.
  • Another object of the present invention is to provide a use for producing a vaccine used for the prevention of porcine circovirus infection of the composition prepared by the production method according to the present invention.
  • the present invention is a polynucleotide encoding a RuBisCO transit peptide comprising the amino acid sequence represented by SEQ ID NO: 1, and SEQ ID NO: 3 or SEQ ID NO: 5
  • a recombinant vector for plant expression comprising a polynucleotide encoding a PCV2 (Porcine circovirus type 2) capsid protein comprising the indicated amino acid sequence.
  • the polynucleotide encoding the Rubisco transit peptide may include a nucleotide sequence represented by SEQ ID NO: 2.
  • the polynucleotide encoding the PCV2 capsid protein may include a nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6.
  • the recombinant vector may further include a polynucleotide encoding a polyhistidine-tag comprising the amino acid sequence represented by SEQ ID NO: 7.
  • the recombinant vector encodes a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding a polyhistidine-tag, and a PCV2 capsid protein between a promoter and a terminator.
  • Polynucleotides may be sequentially connected, but are not limited to the order of connection.
  • the present invention also provides transformed plants transformed with the recombinant vector of the present invention.
  • the present invention provides a method for isolation and purification of a recombinant PCV2 capsid protein, comprising the following steps:
  • the protein extraction buffer is 10 to 100mM Tris (Tris), 100 to 300mM sodium chloride (NaCl), 0.01 to 0.5% Triton (Triton) X-100 (polyethylene glycol p- (1,1) , 3,3-tetramethylbutyl) -phenylether), and 5 to 300 mM imidazole.
  • Tris Tris
  • NaCl sodium chloride
  • Triton Triton
  • X-100 polyethylene glycol p- (1,1) , 3,3-tetramethylbutyl) -phenylether
  • the agarose may be Ni-NTA (nickel-nitrilotriacetic acid) agarose.
  • the step (S1) may be to transform a plant using a bacterium into which a recombinant vector has been introduced, and the bacterium is preferably Agrobacterium tumefaciens. Can be.
  • the present invention provides a method of preparing a vaccine composition comprising virus-like particles comprising the following steps:
  • step (S3) preparing the PCV2 capsid protein obtained in step (S2) as virus-like particles
  • step (S4) preparing a vaccine composition comprising virus-like particles obtained in step (S3).
  • the method for preparing the vaccine composition may further include adding an adjuvant.
  • Alum may be used for the adjuvant, but is not limited thereto.
  • the plant is dicotyledonous, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, cabbage, and dicotyledonous plants selected from the group consisting of lettuce; Or it may be a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onions.
  • the step (S1) may be to transform a plant using a bacterium into which a recombinant vector has been introduced, and the bacterium is preferably Agrobacterium tumefaciens. However, it is not limited thereto.
  • the step (S3) may be to prepare virus-like particles by changing the pH of the buffer solution containing the PCV2 capsid protein.
  • the buffer solution includes 50 to 100 mM Tris, 300 to 1000 mM sodium chloride (NaCl), and 10 to 100 mM Arginine, and the pH may be 6.9 to 7.5. .
  • the pH of the buffer solution may be preferably 7.2.
  • the present invention provides a vaccine composition prepared by the production method according to the present invention.
  • the vaccine composition may further include an adjuvant.
  • the adjuvant may be alum, but is not limited thereto.
  • the present invention provides PCV2 virus-like particles included in the vaccine composition according to the present invention.
  • the PCV2 virus-like particles have a molecular weight of about 2,000 kDa in size-exclusion chromatography and a molecular weight of 669 kDa or more in Native-PAGE (polyacrylamide gel electrophoresis), and are negative.
  • the diameter may be 10 nm or more, 40 nm or less, preferably 20 nm or more in diameter, or 30 nm or less in spherical shape.
  • the present invention provides a method for preventing porcine circovirus infection by administering a vaccine composition prepared by the production method according to the present invention to an individual.
  • the present invention provides a porcine circovirus infection prevention use of the vaccine composition prepared by the production method according to the present invention.
  • the present invention provides a use for producing a vaccine used for the prevention of porcine circovirus infection of the composition prepared by the production method according to the present invention.
  • the present invention relates to a PCV2 vaccine composition
  • a PCV2 vaccine composition comprising a virus-like particle prepared using a plant expression vector targeted to a chloroplast, it is possible to significantly reduce the production cost, a conventionally well-known method (protein in animal cells or microorganisms After production, it can block various contaminants (virus, cancer gene, enterotoxin, etc.) that may occur in the separation and purification method).
  • a synthetic pathway of eukaryotic protein that undergoes post-translational modification processes that are essentially included in animal cells, it is possible to produce proteins that maintain physiological activity. It is advantageous in that it can be managed as stock).
  • the demand for the material increases rapidly, it is more efficient and economical compared to the existing production system using animal cells or bacteria in terms of facility technology or cost required for mass production. And there is also an advantage that the supply is possible.
  • FIG. 1 is a view showing an expression cassette (expression cassette) of two recombinant PCV2 capsid proteins for expression of recombinant PCV2 capsid proteins in plants according to an embodiment of the present invention.
  • Rbc-TP Rubisco transit peptide
  • 6XHis polyhistidine-tag
  • BiP-SP chaperone binding protein signal peptide
  • FIG. 2 is a view showing the results of confirming the expression of two recombinant PCV2 capsid proteins in plants according to an embodiment of the present invention by western blotting.
  • FIG. 3 is a view showing the results of separation and purification using affinity chromatography of a recombinant PCV2 capsid protein according to an embodiment of the present invention.
  • Figure 4 is a view showing the results of the size exclusion chromatography and Native-PAGE and SDS-PAGE of the recombinant PCV2 capsid protein isolated and purified in the present invention.
  • FIG. 5 is a view showing an image taken by a transmission electron microscope of virus-like particles formed from recombinant PCV2 capsid protein isolated and purified in the present invention.
  • Figure 6 is a diagram and a schematic diagram showing the guinea pig test method for confirming the formation of an antibody of the recombinant and purified recombinant PCV2 capsid protein in the present invention (top), and a diagram showing the results confirmed by the ELISA kit (bottom) to be.
  • the present inventors in producing a PCV2 (Porcine circovirus type 2) forming a virus-like particle that is a target protein in a transgenic plant, the Rubisco transit peptide (RuBisCO transit) is targeted to the chloroplast to increase the expression level of the protein in the translation step.
  • Peptide was fused to the target protein, and when polyhistidine was attached for separation and purification, it was confirmed that the expression level of the protein and the efficiency of the separation and purification were increased, and using this, it was possible to dramatically increase the production efficiency of the target protein in transgenic plants.
  • the present invention was completed by confirming.
  • a polyhistidine-tag and a chloroplast-targeted rubisco transit peptide expressing a recombinant protein fused to a PCV2 capsid protein, a plant expression vector, and a polyhistidine-tag and biP (chaperone) targeting vesicles binding protein) A plant expression vector expressing a recombinant protein in which a signal peptide is fused to a PCV2 capsid protein was produced (see Example 1).
  • the plant expression vector was transformed into agrobacteria, and then injected into the back of Nicotiana ventamiana leaves to express the recombinant PCV2 capsid protein in plants (see Example 2).
  • the recombinant protein is separated and purified using a column filled with Ni-NTA agarose resin from Nicotiana Ventamiana leaves expressing the recombinant PCV2 capsid protein, and the virus-like particles are purified. Self-assembly of the PCV2 capsid protein was induced using a buffer solution for making (see Examples 3 to 5).
  • an antibody against PCV2 was formed by administering a composition comprising the above-obtained PCV2 virus-like particles to guinea pigs (see Example 6).
  • composition containing two genotypes of PCV2 virus-like particles was administered to guinea pigs, confirming the ability to form virus neutralizing antibodies in both PCV2a and PCV2b genotypes. 7).
  • the present invention is a polynucleotide encoding a RuBisCO transit peptide comprising an amino acid sequence represented by SEQ ID NO: 1, and PCV2 (Porcine circovirus) comprising an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5 type 2) It can provide a recombinant vector for plant expression, comprising a polynucleotide encoding a capsid protein.
  • the term “RuBisCo transit peptide” refers to an N-terminal transit peptide of a small subunit of Ribulose-1,5-Bisphosphate Carboxylase / Oxygenase (small subunit).
  • transit peptide preferably encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, most preferably by a polynucleotide represented by SEQ ID NO: 2, but the nucleotide sequence of SEQ ID NO: 2 And 80% or more, more preferably 90% or more, and even more preferably 95% or more.
  • % Of sequence homology to a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences.
  • the Rubisco transit peptide is used to transfer the recombinant protein expressed in the transformed plant into the chloroplast, and when expressed, a part of the sequence may be cut off, and only some amino acids may remain. More specifically, when the recombinant PCV2 capsid protein in which the rubisco transit peptide according to the present invention is fused is expressed in a plant, the 54 amino acids in front of the rubisco transit peptide are cut off and only the amino acid sequence represented by SEQ ID NO: 1 Can remain. In addition, in this case, an additional amino acid may be inserted between the rubisco transit peptide sequence and the polyhistidine sequence in order to align the DNA frame, and preferably glycine or isoleucine may be additionally inserted.
  • BiP chaperone binding protein
  • a gene comprising the nucleotide sequence of SEQ ID NO: 10, most preferably SEQ ID NO:
  • the gene represented by 10 but may include a nucleotide sequence having a sequence homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the base sequence of SEQ ID NO: 10.
  • a part of the sequence may be cut, and only some amino acids may remain.
  • transit peptide or “signal peptide” refers to an amino acid sequence that can induce the transport or localization of proteins to specific organelles, cell compartments, and extracellular transport sites. Refers to. The term includes both transit peptides and nucleotide sequences encoding transit peptides.
  • PCV2 refers to porcine circovirus type 2 and is a small (17-22 nm diameter) icosahedral non-enveloped DNA virus and contains a Japanese chain circular genome. do. PCV2 shares approximately 80% sequence identity with porcine circovirus type 1 (PCV1). However, in contrast to PCV1, which is generally non-virulent, pigs infected with PCV2 usually exhibit a symptom called Post-weaning Multisystemic Wasting Syndrome (PMWS). PCV2 has two main open reading frames (ORFs). ORF1 produces a viral replication protein (Rep), and ORF2 produces a "capsid protein".
  • Rep viral replication protein
  • ORF2 produces a "capsid protein”.
  • the capsid proteins produced by the transcription of ORF2 of PCV2 are grouped in three to form one surface, and an icosahedral structure in which 20 surfaces are collected is completed to form a virus-like particle (VLPs) structure.
  • VLPs virus-like particle
  • PCV2 can be divided into genotypes such as PCV2a, PCV2b, and PCV2c according to the genotype of ORF2 encoding the capsid protein.
  • the pathogenicity between these genotypes is not yet clear, and the results of artificial infection have not been able to draw conclusions about the differences in pathogenicity.
  • PCV2a and PCV2b are collectively referred to as “PCV2”, but preferably mean PCV2a.
  • PCV2a and PCV2b are collectively referred to as “PCV2”, but preferably mean PCV2a.
  • PCV2a and PCV2b are collectively referred to as “PCV2”, but preferably mean PCV2a.
  • PCV2a and PCV2b are collectively referred to as PCV2a and PCV2b, respectively.
  • polynucleotide refers to an oligomeric or polymer comprising two or more linked nucleotides or nucleotide derivatives, usually linked by phosphate ester linkages, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Shows. Polynucleotides may also be, for example, nucleotide analogues, or “skeletal” bonds other than phosphate ester linkages, such as phosphate triester linkages, phosphoramidate linkages, phosphorothioate linkages, thioester linkages or peptide linkages (peptides Nucleic acids).
  • Polynucleotides include single-stranded and / or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs of either RNA or DNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • vector refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host.
  • the vector can be a plasmid, phage particle, or simply a potential genome insert. Once transformed into a suitable host, the vector can replicate and function independently of the host's genome, or in some cases can be integrated into the genome itself.
  • plasmids are the most commonly used form of current vectors, the terms "plasmid” and “vector” can sometimes be used interchangeably.
  • the present invention includes other forms of vectors having functions equivalent to those known or known in the art.
  • the polynucleotide encoding the Rubisco transit peptide of the present invention may include the nucleotide sequence represented by SEQ ID NO: 2, and the polynucleotide encoding the PCV2 capsid protein may also include the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6. It can contain.
  • the polynucleotide encoding the rubisco transit peptide of the present invention includes variants of SEQ ID NO: 2 within the scope of the present invention.
  • the polynucleotide may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more with the base sequence of SEQ ID NO: 2.
  • polynucleotide encoding the PCV2 capsid protein of the present invention includes a variant of SEQ ID NO: 4 or SEQ ID NO: 6 within the scope of the present invention.
  • the polynucleotide may include a base sequence having a sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more with the base sequence of SEQ ID NO: 4 or SEQ ID NO: 6.
  • the recombinant vector of the present invention may further include a polynucleotide encoding a polyhistidine-tag including the amino acid sequence represented by SEQ ID NO: 7.
  • the polyhistidine-tag is included for easy separation in addition to the target protein of the present invention, PCV2, typically Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, Myc tag, S tag , SBP tag, IgG Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, and the like.
  • a polynucleotide encoding a Rubisco transit peptide between the promoter and the terminator, a polynucleotide encoding a Rubisco transit peptide, a polynucleotide encoding a polyhistidine-tag, and a polynucleotide encoding a PCV2 capsid protein sequentially It may be connected, but is not limited to the connection order.
  • promoter examples include pEMU promoter, MAS promoter, histone promoter, Clp promoter, 35S promoter derived from cauliflower mosaic virus, 19S RNA promoter derived from cauliflower mosaic virus, plant actin protein promoter, Ubiquitin protein promoter, CMV (Cytomegalovirus) promoter, SV40 (Simian virus 40) promoter, RSV (Respiratory syncytial virus) promoter, EF-1 ⁇ (Elongation factor-1 alpha) promoter, etc.
  • the terminator is, for example, nopaline Synthase (NOS), rice amylase RAmy1 A terminator, paseoline terminator, agrobacterium tumefaciens octopine gene terminator, E. coli rrnB1 / B2 terminator, etc. This is an example only and is not limited thereto.
  • NOS nopaline Synthase
  • rice amylase RAmy1 A terminator paseoline terminator
  • agrobacterium tumefaciens octopine gene terminator E. coli rrnB1 / B2 terminator, etc. This is an example only and is not limited thereto.
  • the term "transformation” refers to a change in the genetic properties of an organism by the injected DNA, and the term “transgenic plant (transgenic plant)" injects an external gene by molecular genetic method.
  • a plant produced preferably, a plant transformed with the recombinant expression vector of the present invention, the plant is not limited as long as it is intended to achieve the object of the present invention.
  • the transformed plant according to the present invention is transformed (transformation), transfection (transfection), Agrobacterium (Agrobacterium) -mediated transformation method, particle gun bombardment (particle gun bombardment), sonication (sonication), It may be prepared by a method such as an electroporation method, or a polyethylen glycol (PEG) -mediated transformation method, but there is no limitation as long as it is a method capable of injecting the vector of the present invention.
  • a method such as an electroporation method, or a polyethylen glycol (PEG) -mediated transformation method, but there is no limitation as long as it is a method capable of injecting the vector of the present invention.
  • the present invention provides a method for isolation and purification of a recombinant PCV2 capsid protein comprising the following steps:
  • Protein extraction buffer solution is 10 to 100mM Tris (Tris), 100 to 300mM sodium chloride (NaCl), 0.01 to 0.5% Triton (Triton) X-100 (polyethylene glycol p- (1,1,3,3- tetramethylbutyl) -phenylether), and 5 to 300 mM imidazole.
  • Tris Tris
  • NaCl sodium chloride
  • Triton Triton
  • X-100 polyethylene glycol p- (1,1,3,3- tetramethylbutyl) -phenylether
  • the agarose according to the present invention may be a nickel-nitrilotriacetic acid (Ni-NTA) agarose.
  • Ni-NTA nickel-nitrilotriacetic acid
  • the step (S1) may be to transform plants using bacteria into which a recombinant vector has been introduced, and the bacteria may be preferably Agrobacterium tumefaciens.
  • a method of preparing a vaccine composition comprising virus-like particles comprising the following steps:
  • step (S3) preparing the PCV2 capsid protein obtained in step (S2) as virus-like particles
  • step (S4) preparing a vaccine composition comprising virus-like particles obtained in step (S3).
  • the term "vaccine” of the present invention is a biological agent containing an antigen that causes an immune response in a living body, and refers to an immunogen that immunizes a living body by injection or oral administration to a human or animal for prevention of infection.
  • the animal is a human or non-human animal, and the non-human animal refers to pigs, cows, horses, dogs, goats, sheep, and the like, but is not limited thereto.
  • the "vaccine composition" of the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, according to a conventional method.
  • oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, according to a conventional method.
  • it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc.
  • These solid preparations include at least one excipient in the lecithin-like emulsifier, for example, starch, calcium carbonate, sucrose It can be prepared by mixing (sucrose) or lactose, gelatin, and the like. It is also possible to use lubricants, such as magnesium stearate, in addition to simple excipients.
  • a liquid preparation for oral administration suspending agents, intravenous solutions, emulsions, syrups, etc. can be used.
  • various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. Can be included.
  • Formulations for non-oral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, and lyophilized preparations.
  • Non-aqueous preparations, suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • the route of administration of the vaccine composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, This includes sublingual or rectal. Oral or parenteral administration is preferred.
  • parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the vaccine composition of the present invention can also be administered in the form of suppositories for rectal administration.
  • the dosage of the vaccine composition or pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the individual.
  • the amount required to induce an immune protective response in an individual without significant side effects may vary depending on the presence of recombinant proteins and excipients used as immunogens.
  • the method for preparing the vaccine composition may further include adding an adjuvant.
  • adjuvant refers to a substance or composition that is added to a vaccine or pharmaceutically active ingredients to increase or influence the immune response.
  • carriers or auxiliary substances for immunogens and / or other pharmaceutically active substances or compositions are generally meant.
  • the term “adjuvant” should be interpreted in a broad concept, and a broad range of substances or stratagerms that can enhance the immunogenicity of an antigen incorporated into or administered with the adjuvant.
  • the adjuvant is not limited thereto, and may be divided into an immune potentiator, an antigen delivery system, or a combination thereof.
  • the plant is Arabidopsis thaliana, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, cabbage, and dicotyledonous plants selected from the group consisting of lettuce; Or it may be a monocotyledonous plant selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onions.
  • plant as used herein may be used without limitation as long as it is a plant capable of mass-producing the recombinant protein of the present invention, and more specifically, may be one selected from the group of plants listed above, preferably tobacco.
  • tobacco in the present invention is a tobacco genus (Nicotiana genus) plant that is capable of overexpressing proteins, and is not particularly limited, and selects an appropriate variety according to the transformation method and the purpose of mass production of proteins to implement the present invention.
  • Nicotiana benthamiana L. or Nicotiana tabacum cv. Varieties such as Xanthi (Nicotiana tabacum cv. Xanthi) can be used.
  • the step (S1) may be to transform a plant using a bacterium into which a recombinant vector has been introduced, and the bacteria may be preferably Agrobacterium tumefaciens, but the tactic As one is not limited to this.
  • the step (S3) may be to prepare virus-like particles by changing the pH of the buffer solution containing the PCV2 capsid protein.
  • the replacement and concentration of the buffer solution may be performed using a filter so that the recombinant PCV2 capsid protein can form virus-like particles by self-assembly.
  • the buffer solution may include 50 to 100mM Tris, 300 to 1000mM sodium chloride (NaCl), and 10 to 100mM arginine, and the pH may be 6.9 to 7.5, preferably 7.2.
  • size exclusion chromatography can be performed to purify the self-assembled recombinant PCV2 virus-like particles.
  • the present invention provides a vaccine composition prepared by the production method according to the present invention.
  • the vaccine composition may further include an adjuvant.
  • the adjuvant may be alum, but any kind of aluminum salt suitable for use as an adjuvant can be used in the present invention.
  • Aluminum salts include aluminum hydroxide (Al (OH) 3 ), aluminum phosphate (AlPO 4 ), aluminum hydrochloride, aluminum sulfate, ammonium alum, potassium alum or aluminum silicate, and the like.
  • aluminum hydroxide or aluminum phosphate can be used as the aluminum salt adjuvant.
  • the present invention provides PCV2 virus-like particles included in the vaccine composition.
  • the PCV2 virus-like particles have a molecular weight of about 2,000 kDa in size-exclusion chromatography and a molecular weight of 669 kDa or higher in Native-PAGE (polyacrylamide gel electrophoresis), and are stained with a negative staining method to transmit electron microscope.
  • a diameter of 10 nm or more, 40 nm or less, preferably 20 nm or more, 30 nm or less may be spherical or cyclic.
  • the term “about” here means ⁇ 10%. Therefore, a molecular weight of about 2,000 kDa means 1,800 kDa to 2,200 kDa.
  • the present invention provides a method for preventing porcine circovirus infection by administering a vaccine composition prepared by the production method according to the present invention to an individual.
  • the present invention provides the use of the vaccine composition prepared by the production method according to the present invention for preventing porcine circovirus infection.
  • the present invention provides a use for producing a vaccine used for the prevention of porcine circovirus infection of a composition prepared by the production method according to the present invention.
  • PCV2 means PCV2a unless specifically indicated as PCV2b.
  • Example 1 Construction of a plant expression vector for expression of recombinant PCV2 capsid protein
  • a recombinant plant expression vector was prepared so that the plant can express the recombinant PCV2 capsid protein.
  • the genetic information for the PCV2 capsid protein was obtained, and a gene (SEQ ID NO: 4 or SEQ ID NO: 6) was synthesized with a sequence optimized for expression in Nicotiana benthamiana.
  • Recombinant PCV2 capsid protein plant expression vector targeted to chloroplast is a polynucleotide (RuBisCO transit peptide) encoding the RuBisCO transit peptide between the CaMV35S promoter gene and the NOS terminator of the pCAMBIA1300 vector, 6 consecutive sequences It was prepared by sequentially connecting a polynucleotide encoding a histidine (Histidine) (SEQ ID NO: 8) and a polynucleotide encoding a PCV2 capsid protein (SEQ ID NO: 4 or SEQ ID NO: 6).
  • Histidine histidine
  • PCV2 capsid protein SEQ ID NO: 4 or SEQ ID NO: 6
  • the recombinant PCV2 capsid protein plant expression vector targeted to chloroplast is a polynucleotide encoding a chaperone binding protein (BiP) signal peptide (SEQ ID NO: 10) between the CaMV35S promoter gene and the NOS terminator of the pCAMBIA1300 vector, Polynucleotide encoding PCV2 capsid protein (SEQ ID NO: 4 or SEQ ID NO: 6), polynucleotide encoding six consecutive histidines (SEQ ID NO: 8) and HDEL (His-Asp-Glu-Leu) peptide Polynucleotide (SEQ ID NO: 12) was prepared by connecting in sequence.
  • BiP chaperone binding protein
  • the plant expression vector prepared in Example 1 was transformed into an Agrobacterium LBA4404 strain using an electroshock method. After transforming the transformed agrobacteria in 5 mL of YEP liquid medium (10 g of yeast extract, 10 g of peptone, 5 g of NaCl, 50 mg / L of kanamycin, 25 mg / L of rifampicin) and shaking culture for 16 hours under conditions of 28 ° C., 1 ml of the primary culture solution was added. 50 ml of fresh YEP medium was inoculated and shaken for 6 hours at 28 ° C.
  • YEP liquid medium 10 g of yeast extract, 10 g of peptone, 5 g of NaCl, 50 mg / L of kanamycin, 25 mg / L of rifampicin
  • the agrobacteria cultured in this way were collected by centrifugation (7,000 rpm, 4 ° C., 5 minutes), followed by an infiltration buffer [10 mM MES (pH 5.7) so that the absorbance (OD) value was 1.0 at a wavelength of 600 nm. , 10 mM MgCl 2 , 200 ⁇ M acetosyringon].
  • Agro-infiltration was performed by injecting the Agrobacteria suspension into the back side of Nicotiana Ventamiana leaves using a syringe with a needle removed.
  • the protein in the water-soluble fraction (Supernatant; S) and the protein in the pellet (P) fraction, the fraction containing both the water-soluble fraction and the petlet ( Total; T) were separately isolated to confirm expression of the recombinant PCV2 capsid protein by Western blotting. More specifically, 30 ⁇ L of each fraction was heated after mixing with the SDS sample buffer. Then, electrophoresis was performed on a 10% SDS-PAGE gel to confirm the protein bands separated by size, and after transferring them to a PVDF membrane, after a blocking step using 5% skim milk, reaction with polyhistidine was performed. The antibody was bound and the ECL solution was treated according to the method provided by the manufacturer to confirm expression of the recombinant PCV2 capsid protein.
  • the elution solution containing the recombinant PCV2 capsid protein is a buffer solution for making virus-like particles using a filter of 30 kDa size [50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 100 mM Arginine, final pH is adjusted to 7.2] The buffer was replaced and concentrated. The isolated and purified recombinant PCV2 capsid protein was confirmed by electrophoresis (SDS-PAGE) through Coomassie staining (see FIG. 3).
  • the fraction obtained to confirm the formation of virus-like particles was subjected to Native-PAGE electrophoresis.
  • SDS-PAGE electrophoresis was performed to confirm the purification degree of the purified and purified recombinant PCV2 capsid protein.
  • the results of separation by the size exclusion chromatography and the results of Native-PAGE and SDS-PAGE electrophoresis are shown in FIG. 4.
  • the recombinant PCV2a capsid protein contained in the first and second fractions separated by size exclusion chromatography was diluted in an elution solution [50 mM Tris-Cl (pH 7.4), 300 mM NaCl, 0.5 mM EDTA] to a final concentration of 100 nM. Negative-staining was performed.
  • the 5 ⁇ L protein solution was reacted for 1 minute on a carbon-coated copper grid, and then the protein solution was removed using filter paper and washed 3 times with tertiary distilled water. At this time, the copper grid coated with carbon reacts with plasma for 1 minute to convert the hydrophobic property to hydrophilic property, so that the protein adheres well to the carbon.
  • the mixture was reacted with 1% uranyl acetate for 1 minute to perform negative staining, and then the staining solution was removed with filter paper and dried at room temperature for 12 hours.
  • the prepared grid was imaged at a magnification of 20,000 times using a transmission electron microscope (Tecnai T10, Philips, Japan).
  • the antibody is formed in the body of the guinea pig by administering a composition containing PCV2 virus-like particles.
  • the amount of the antibody formed also tended to increase as the dosage of the composition increased.
  • the presence or absence of forming a PCV2 neutralizing antibody was confirmed by using a vaccine composition in which aluminum hydroxide (Al (OH) 3 ) adjuvant was added to the composition containing the PCV2 virus-like particles identified in Example 5.
  • Al (OH) 3 aluminum hydroxide
  • the experiment was conducted according to the national drug approval standard for animal drugs (1-2-04-03 porcine circovirus type 2 genetic recombination inactivation vaccine).
  • Six guinea peaks having a weight of 300-350 g were prepared, two of them were used as a control group, and four were used as an experimental group.
  • Two weeks after the second inoculation blood was collected as in the two control groups and confirmed by an indirect fluorescence assay.
  • the results of the virus neutralization experiment are shown in Table 1 below.
  • the plant expression vector according to the present invention When the plant expression vector according to the present invention is used, production costs can be dramatically reduced, and sources of contamination such as viruses, cancer genes, enterotoxins, etc. can be blocked, and synthetic pathways of eukaryotic proteins that undergo post-translational modifications are included It is advantageous in that it is possible to produce protein that maintains physiological activity, and it can be managed as a seed stock at the commercialization stage. Furthermore, when the demand for the material increases rapidly, it is more efficient and economical compared to the existing production system using animal cells or bacteria in terms of facility technology or cost required for mass production. And it is expected that the value of industrial use will be large in that it can be supplied.

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Abstract

la présente invention porte sur une technique de préparation d'une composition de vaccin à partir du circovirus porcin de type 2 (PCV2) isolé à partir d'une plante transformée avec un vecteur recombiné ciblant la chlorophylle pour l'expression dans des plantes; et fournit un vecteur recombiné portant un polynucléotide codant pour une protéine recombinée dans laquelle une protéine ciblant la chlorophylle et une protéine capsidique du PCV2 sont fusionnées l'une à l'autre. De plus, l'invention concerne une plante transgénique transformée avec le vecteur recombiné, un procédé d'isolement et de purification d'une protéine cible à partir de la plante transgénique, un procédé de préparation d'une composition de vaccin contenant des particules du type viral l'utilisant, et une composition de vaccin préparée à l'aide dudit procédé de préparation.
PCT/KR2019/013581 2018-11-15 2019-10-16 Vecteur recombiné pour l'expression de particules du type viral dans une plante et procédé de préparation d'une composition de vaccin contenant des particules du type circovirus l'utilisant WO2020101187A1 (fr)

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EP19884354.2A EP3882351A4 (fr) 2018-11-15 2019-10-16 Vecteur recombiné pour l'expression de particules du type viral dans une plante et procédé de préparation d'une composition de vaccin contenant des particules du type circovirus l'utilisant
CN201980075026.3A CN113039275B (zh) 2018-11-15 2019-10-16 用于在植物中表达猪圆环病毒样颗粒的重组载体以及使用其的疫苗组合物
CA3119964A CA3119964A1 (fr) 2018-11-15 2019-10-16 Vecteur recombine pour l'expression de particules du type viral dans une plante et procede de preparation d'une composition de vaccin contenant des particules du type circovirus l 'utilisant
JP2021526675A JP7212968B2 (ja) 2018-11-15 2019-10-16 植物体でウイルス様粒子を発現する組み換えベクター及びこれを利用したサーコウイルス様粒子を含むワクチン組成物の製造方法
US17/320,369 US20210330780A1 (en) 2018-11-15 2021-05-14 Recombinant vector for expressing virus-like particles in plant and method for preparation of vaccine composition containing circovirus-like particles by using same

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112707953A (zh) * 2021-01-22 2021-04-27 南昌师范学院 Ev71病毒样颗粒产品、其制备方法及其应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100233191B1 (ko) * 1991-03-05 1999-12-01 제닌 페트릭 식물의 형질 전환용 키메라 유전자
KR20060011675A (ko) * 2004-07-30 2006-02-03 학교법인 포항공과대학교 셀룰로오스의 자가가수분해를 위한 셀룰라아제 발현형질전환 식물체 및 이를 이용한 수용성 당의 생산방법
WO2008151215A1 (fr) * 2007-06-04 2008-12-11 Merial Limited Production de protéines de circovirus porcin dans des plantes
KR101449155B1 (ko) 2012-12-06 2014-10-13 주식회사 바이오앱 식물체에서 목적 단백질의 번역 효율을 증진시키기 위한 염기서열
KR20150113934A (ko) 2014-03-31 2015-10-08 한미약품 주식회사 면역글로불린 Fc 단편 결합을 이용한 단백질 및 펩타이드의 용해도를 개선시키는 방법
KR20170131207A (ko) * 2016-05-20 2017-11-29 포항공과대학교 산학협력단 RbcS 융합 단백질을 이용하여 식물로부터 목적 단백질을　고발현하는 방법 및 목적 단백질 발현 식물체를 이용한 의료용 단백질의 경구투여용 조성물의 제조 방법
KR101848082B1 (ko) 2017-05-11 2018-04-12 주식회사 바이오앱 셀룰로오즈 결합 도메인을 포함하는 재조합 벡터 및 상기 벡터를 사용하여 단백질을 분리정제하는 방법
KR20180084680A (ko) 2017-01-17 2018-07-25 포항공과대학교 산학협력단 식물 세포에서의 목적 단백질 발현을 위한 재조합 벡터
KR20190127987A (ko) 2011-11-14 2019-11-13 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 고성능 frc를 형성하고 유지하는 시스템 및 방법

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100233191B1 (ko) * 1991-03-05 1999-12-01 제닌 페트릭 식물의 형질 전환용 키메라 유전자
KR20060011675A (ko) * 2004-07-30 2006-02-03 학교법인 포항공과대학교 셀룰로오스의 자가가수분해를 위한 셀룰라아제 발현형질전환 식물체 및 이를 이용한 수용성 당의 생산방법
WO2008151215A1 (fr) * 2007-06-04 2008-12-11 Merial Limited Production de protéines de circovirus porcin dans des plantes
KR20190127987A (ko) 2011-11-14 2019-11-13 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 고성능 frc를 형성하고 유지하는 시스템 및 방법
KR101449155B1 (ko) 2012-12-06 2014-10-13 주식회사 바이오앱 식물체에서 목적 단백질의 번역 효율을 증진시키기 위한 염기서열
KR20150113934A (ko) 2014-03-31 2015-10-08 한미약품 주식회사 면역글로불린 Fc 단편 결합을 이용한 단백질 및 펩타이드의 용해도를 개선시키는 방법
KR20170131207A (ko) * 2016-05-20 2017-11-29 포항공과대학교 산학협력단 RbcS 융합 단백질을 이용하여 식물로부터 목적 단백질을　고발현하는 방법 및 목적 단백질 발현 식물체를 이용한 의료용 단백질의 경구투여용 조성물의 제조 방법
KR20180084680A (ko) 2017-01-17 2018-07-25 포항공과대학교 산학협력단 식물 세포에서의 목적 단백질 발현을 위한 재조합 벡터
KR101848082B1 (ko) 2017-05-11 2018-04-12 주식회사 바이오앱 셀룰로오즈 결합 도메인을 포함하는 재조합 벡터 및 상기 벡터를 사용하여 단백질을 분리정제하는 방법

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM, S. ET AL.: "Arabidopsis thaliana Rubisco small subunit transit peptide increases the accumulation of Thermotoga maritima endoglucanase CelSA in chloroplasts of transgenic tobacco plants", TRANSGENIC RESEARCH, vol. 19, 2010, pages 489 - 497, XP019831650 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112707953A (zh) * 2021-01-22 2021-04-27 南昌师范学院 Ev71病毒样颗粒产品、其制备方法及其应用

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