KR102138938B1 - Beculovirus-based Gene Transducer Including Influenza-induced Cell-Invesive Peptide - Google Patents

Abstract

The present invention relates to a baculovirus-based gene delivery system comprising an influenza-derived cell permeable peptide and a vaccine composition using the same. When using the gene delivery system of the present invention, human, pig and chicken cells can be easily penetrated by the surface CPP protein. In addition, when using the vaccine composition of the present invention, it is possible to induce high expression of the antigen protein through high delivery efficiency, thereby effectively preventing various diseases caused by various antigens.

Description

A baculovirus-based Gene Transducer Including Influenza-induced Cell-Invesive Peptide}

The present invention relates to a baculovirus-based gene delivery system comprising an influenza-derived cell permeable peptide and a vaccine composition using the same.

Cells are divided into inside and outside based on the cell membrane, and the inside is composed of organelles that are mostly involved in the growth of cells, and the outside is a collection of foreign substances such as waste products, ions, sugars, and amino acids from the cells. As necessary, the cell transfers external substances to the inside through ion channels or carriers present in the cell membrane to receive or exchange nutrients. Intracellular delivery of foreign substances is actually closely related to intracellular delivery of drugs or other substances, and attempts to develop drugs with high delivery rates or drug delivery methods have been continuously studied.

Baculovirus is known as a biologically safe virus because it cannot replicate in animal cells and does not cause cytotoxicity (Virology 125:107-117 (1983); Hum. Gene Ther. 7:1937-1945 (1996)) ; Proc. Natl. Acad. Sci. USA 96:127-132 (1999); Trends Biotechnol. 20:173-180 (2002)). AcNPV (Autographa californica nuclear polyhedrosisvirus) belonging to the group of insect viruses is not able to replicate in various kinds of animal cells, but is known to be able to enter cells and transfer genes (Proc. Natl. Acad. Sci. USA 92:10099-10103 (1995); Proc. Natl. Acad. Sci. USA 93:2348-2352 (1996)). It has already been reported that certain genes in the autographa californica multiple nuclear polyhedrosis virus (AcMNPV) genome can be expressed at high levels in animal cells when they are regulated by animal promoters (Journal of Virology, 76(11): 5729-5736 (2002); Vaccine 26(20):2451-2456 (2008)). Unfortunately, the baculovirus itself does not have high intracellular delivery efficiency, so additional chemical/genetic improvements are needed.

The cell-penetrating peptide is a peptide composed of about 10 to 30 amino acids, and is capable of binding with various foreign substances, and is known to deliver foreign substances bound into the cell. Typically, the Tat protein of HIV (Human Immunodeficiency Virus) is known as a cell-penetrating peptide. In some cases, a small DNA to a large collagen is transferred into a cell using the Tat protein. The mechanism of action of cell-penetrating peptides having these advantages is still actively being studied, and in fact, it has been introduced by many scientists as one of the possibilities to increase the efficiency of intracellular delivery of drugs or other substances.

Influenza hemagglutinin is divided into HA1 and HA2, of which HA1 is a part involved in receptor binding, and HA2 protrudes when the virus reaches the upper and lower airways and meets a low pH and binds to the cell membranes of epidermal cells of the upper and lower airways (Fusion ) To deliver the virus into the cell. It is known that intracellular viral delivery by such binding also resembles the action of a cell-penetrating peptide.

1.Ault KA, Giuliano AR, Edwards RP, Tamms G, Kim LL, Smith JF, Jansen KU, Allende M, Taddeo FJ, Skulsky D, Barr E. (2004) A phase I study to evaluate a human papillomavirus (HPV) type 18 L1 VLP vaccine. Vaccine 22(23-24):3004-7. 2.Barsoum, J., Brown, R., McKee, M. and Boyce, F. M. (1997) Efficient transduction of mammalian cells by a recombinant baculovirus having the vesicular stomatitis virus G glycoprotein. Hum Gene Ther 8, 2011-2018. 3. Boyce, F. M. and Bucher, N. L. (1996) Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93, 2348-2352. 4. Kost, T. A. and Condreay, J. P. (2002) Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol 20, 173-180. 5.Kumar M, Bradow BP, Zimmerberg J. (2003) Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64. Hum Gene Ther. 14(1):67-77. 6. Mangor JT, Monsma SA, Johnson MC, Blissard GW. (2001) A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein. Journal of Virology 75(6):2544-2556. 7.Monsma, S. A., Oomens, A. G. and Blissard, G. W. (1996) The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol 70, 4607-4616. 8. Mulligan RC. (1993) The basic science of gene therapy. Science 260(5110):926-932. 9.Tjia, S. T., zu Altenschildesche, G. M. and Doerfler, W. (1983) Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells. Virology 125, 107-117. 10. Wilson S, Baird M, Ward VK. (2008) Delivery of vaccine peptides by rapid conjugation to baculovirus particles. Vaccine 26(20):2451-2456.

The present inventors made extensive efforts to improve the cell delivery rate of baculovirus-based gene delivery systems. As a result, a combination of a nucleotide sequence encoding a protein containing an influenza-derived cell penetrating peptide (CPP) and a combination of nucleotide sequences to prepare an expression construct and a recombinant baculovirus and processing the cells using the same In this case, the present invention has been completed by proving that the cell delivery rate is significantly improved compared to the existing baculovirus-based gene delivery system, and that it can be applied to various vaccines and gene delivery systems based on this.

Accordingly, it is an object of the present invention to provide an expression construct for producing a gene delivery system with improved cell delivery rate.

Another object of the present invention is to provide an expression vector comprising the recombinant baculovirus.

Another object of the present invention is to provide a transformant transformed with the expression vector.

Another object of the present invention is to provide a recombinant baculovirus comprising the expression construct.

Another object of the present invention is to provide a gene delivery system comprising the recombinant baculovirus.

Another object of the present invention is to provide a vaccine composition comprising the recombinant baculovirus as an active ingredient.

Another object of the present invention is to provide a method for preparing the recombinant baculovirus-based vaccine composition.

According to an aspect of the present invention, the present invention is an envelope-encoding sequence consisting of a nucleotide sequence encoding an influenza-derived HA2 protein; A first promoter operably linked to the envelope-coding sequence; Nucleotide sequence to be transported; And a second promoter operably linked to the nucleotide sequence to be transported.

The present inventors made extensive efforts to improve the cell delivery rate of baculovirus-based gene delivery systems. As a result, a combination of a nucleotide sequence encoding a protein containing an influenza-derived cell penetrating peptide (CPP) and a combination of nucleotide sequences to prepare an expression construct and a recombinant baculovirus and processing the cells using the same In the case, it was found that the cell delivery rate is significantly improved compared to the existing baculovirus-based gene delivery system, and that it can be applied to various vaccines and gene delivery systems based on this.

It is preferable that the envelope-coding sequence of the present invention and the nucleotide sequence to be transported are present in a suitable expression construct. In the expression construct, it is preferred that the nucleotide sequence encoding the influenza-derived CPP protein is operably linked to a promoter. As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoter, signal sequences, or transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence controls the transcription and/or translation of the other nucleic acid sequence.

In one embodiment of the invention, the expression construct of the invention comprises a poly adenylation sequence. For example, human elongation factor 1α (hEF1α) polyA, small growth hormone terminator (Gimmi, ER, et al., Nucleic Acids Res. 17:6983-6998 (1989)), polyadenylation sequence derived from SV40 (Schek, N , et al., Mol. Cell Biol. 12:5386-5393 (1992)), HIV-1 polyA (Klasens, BIF, et al., Nucleic Acids Res. 26:1870-1876 (1998)), β-globin polyA (Gil, A., et al, Cell 49:399-406 (1987)), HSV TK polyA (Cole, CN and TP Stacy, Mol. Cell. Biol. 5:2104-2113 (1985)) or polyoma Virus polyA (Batt, D. B and GG Carmichael, Mol. Cell. Biol. 15:4783-4790 (1995)).

In the recombinant baculovirus of the present invention, the envelope-coding sequence and the nucleotide sequence to be transported are expression constructs in the form of a first promoter-envelope-coding sequence-poly A sequence and a second promoter-transporting nucleotide sequence-poly A sequence, respectively. Trot may be included. In addition, it may be included in the form of a first promoter-CPP protein-coding sequence-second promoter-transporting nucleotide sequence-poly A sequence.

In one embodiment of the present invention, the first promoter, envelope-coding sequence, second promoter, and nucleotide sequence to be transported of the present invention are included on a single nucleotide sequence.

In one embodiment of the invention, the second promoter of the invention is located downstream of the envelope-coding sequence.

In one embodiment of the present invention, the influenza derived HA2 protein of the present invention includes a CPP (Cell Penetration Peptide) sequence in the sequence.

In the present invention, the term influenza-derived HA2 protein is interpreted to include all of various influenza-derived HA2 proteins. Specifically, for example, the HA2 protein of the present invention is interpreted to include an amino acid sequence of SEQ ID NO: 1, and also a sequence showing substantial identity with the sequence of SEQ ID NO: 1. The substantial identity above is preferably aligned when the sequences of the present invention are aligned with any other sequences as much as possible, and the analyzed sequences are analyzed using algorithms commonly used in the art. Refers to a sequence that exhibits 80% homology, more preferably at least 85% homology, even more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment can be found in Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), blastp, blasm, It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is accessible at http://wwww.ncbi.nlm.nih.gov/BLAST/. The sequence homology comparison method using this program can be found at http://wwww.ncbi.nlm.nih.gov/BLAST/blast_help.html.

As the HA2 protein used in the present invention, a biological function equivalent that is an amino acid sequence variant exhibiting equivalent biological activity to the HA2 protein containing the CPP of the present invention can also be used. These amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing mutations, the hydrophobic index of amino acids (hydropathic idex) can be considered. Each amino acid is assigned a hydrophobicity index according to hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5). The hydrophobic amino acid index is very important in conferring the protein's interactive biological function. It is well known that amino acids with similar hydrophobicity indexes must be replaced to retain similar biological activity. When introducing a variation with reference to the hydrophobic index, substitution between amino acids showing hydrophobic index difference within preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5 is performed.

On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0±1); Glutamate (+3.0±1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5±1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing a variation with reference to a hydrophilicity value, substitution is performed between amino acids showing a difference in hydrophilicity values of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in proteins that do not entirely alter the activity of the molecule is known in the art (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

In the present invention, the term "CPP" or "CPP protein" means a cell penetration peptide (Cell Penetration Peptide: CPP), and includes all of various influenza-derived CPP proteins. Specifically, for example, the CPP of the present invention may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

The nucleotide sequence encoding the CPP of the present invention can be derived from various influenza viruses. Preferably, the influenza-derived CPP protein is derived from a virus using human, porcine and avian cells as host cells, more preferably from an influenza virus capable of being delivered to human, porcine and algal cells, most preferably It can induce cell penetration into human, porcine and algal cells.

According to an embodiment of the present invention, the nucleotide sequence encoding the influenza-derived CPP protein of the present invention is derived from influenza virus type A, and more specifically, from a low pathogenic H9N2 avian influenza virus type A Can be used. The CPP protein is expressed on the surface of a recombinant virus, which energy-dependently interacts with human, porcine and avian cells to induce viral cell penetration.

Various promoters can be used as a promoter linked to the envelope-coding sequence or the nucleotide sequence to be transported of the present invention.

In one embodiment of the invention, the first promoter of the invention is a promoter that acts on insect cells.

In one embodiment of the present invention, the promoter operating in the above-described insect cells may be selected from the group consisting of baculovirus IE-1 promoter, IE-2 promoter, p35 promoter, p10 promoter, gp64 promoter and polyhedrin promoter. have.

In one embodiment of the present invention, the second promoter of the present invention is a promoter derived from a genome of a mammalian cell or a promoter derived from a mammalian virus.

In one embodiment of the present invention, the second promoter of the present invention is a U6 promoter, an H1 promoter, a cytomegalo virus (CMV) promoter, a late adenovirus promoter, a 7.5K promoter of the vaccinia virus, an SV40 promoter, a tk promoter of HSV, an RSV Promoter, human elongation factor 1α (hEF1α) promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene , Human GMCSF gene promoter, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter is selected from the group consisting of AFP promoter and albumin promoter.

In one embodiment of the invention, the influenza-derived HA2 protein of the invention consists of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence 1-23 of SEQ ID NO: 1 corresponds to the above-described CPP protein sequence.

It is understood that the envelope-coding sequence of the present invention corresponds to the nucleotide sequence encoding the HA2 protein sequence of SEQ ID NO: 1, and considering the degeneracy of the codon, it is understood that all of the various nucleotide sequences having the same encoded protein are included. For example, due to the degeneracy of codons, there are six codons for arginine or serine. It also includes all nucleic acid molecules that contain codons that encode biologically equivalent amino acids.

The "nucleotide sequence to be transported" of the present invention is not particularly limited, and the nucleotide sequence whose function has been revealed may be, for example, a predetermined target gene sequence, and may be used as a term of "target gene".

In the present invention, the target gene is, for example, a cancer treatment gene that induces the death of cancer cells and ultimately degenerates a tumor, such as a tumor suppressor gene, an immunoregulatory gene [eg, a cytokine gene, a chemokine gene, and a costimulatory factor (B7). .1 and B7.2 auxiliary molecules required for T cell activity), suicide genes, cytotoxic genes, cytostatic genes, pro-apoptotic genes, and anti-angiogenic genes. .

A suicide gene is a nucleic acid sequence that expresses a substance that induces a cell to be easily killed by an external factor or causes a toxic condition in the cell. Well known as such suicide genes are thymidine kinase (TK) genes (US Pat. Nos. 5,631,236 and 5,601,818). Cells expressing the TK gene product are susceptible to selective killing by administration of gancyclovir. A tumor suppressor gene refers to a gene encoding a polypeptide that inhibits tumor formation. Tumor suppressor genes are naturally occurring genes in mammals, and deletion or inactivation of these genes is believed to be an essential premise for tumor development. Examples of tumor suppressor genes include APC, DPC4, NF-1, NF-2, MTS1, WT1, BRCA1, BRCA2, VHL, p53, Rb, MMAC-1, MMSC-2, and retinoblastoma genes (Lee et al. Nature, 329:642 (1987)), adenomatous polyposis coli protein (US Pat. No. 5,783,666), a throat tumor suppressor gene located on chromosome 3p21.3 (Cheng et al. Proc. Nat. Acad. Sci., 95:3042-3047 (1998)), a member of the INK4 family of tumor suppressor genes including the deleted colon tumor (DCC) gene, MTS1, CDK4, VHL, p110Rb, p16 and p21, and therapeutically effective fragments thereof (Eg, p56Rb, p94Rb, etc.). Those skilled in the art will understand that all other known anti-tumor genes other than the above-exemplified genes can be used in the present invention.

The term "cytotoxic gene" as used herein refers to a nucleotide sequence expressed in a cell and exhibiting a toxic effect. Examples of such cytotoxic genes include nucleotide sequences encoding pseudomonas exotoxin, lysine toxin, diphtheria toxin, and the like.

The term "cytostatic gene" as used herein refers to a nucleotide sequence that is expressed in a cell and stops the cell cycle during the cell cycle. Examples of such cell proliferation inhibitory genes include p21, retinoblastoma gene, E2F-Rb fusion protein gene, and a gene encoding a cyclin-dependent kinase inhibitor (eg, p16, p15, p18 and p19), growth stop specificity homeo Box (growth arrest specific homeobox, GAX) genes (WO97/16459 and WO 96/30385), and the like.

In addition, many therapeutic genes that can be usefully used to treat various diseases are also carried by the system of the present invention. For example, cytokines (e.g. interferon-alpha, -beta, -delta and -gamma), interleukins (e.g. IL-1, IL-2, IL-4, IL-6, IL-7, IL-10 , IL-12, IL-19 and IL-20) and genes encoding colony stimulating factors (e.g. GM-CSF and G-CSF), chemokine groups (monocytic chemotactic protein 1 (MCP-1), monocyte chemotaxis Protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 1β (MIP-1β), Macrophage inflammatory protein 1γ (MIP-1γ), macrophage inflammatory protein 3α (MIP-3α), macrophage inflammatory protein 3β (MIP-3β), chemokine (ELC), macrophage inflammatory protein 4 (MIP-4), macrophage inflammatory protein 5( MIP-5), LD78β, RANTES, SIS-epsilon (p500), thymus activation-regulated chemokine (TARC), eotaxin, I-309, human protein HCC-1/NCC-2, human protein HCC-3, mouse Protein C10, etc.). It also includes a gene expressing tissue plasminogen activator (tPA) or urokinase and a LAL generating gene that provides a sustained thrombotic effect and prevents hypercholesterolemia. In addition, many polynucleotides for treating viral, malignant and inflammatory diseases and conditions such as cystic fibrosis, adenosine deaminase deficiency and AIDS are known.

The term "pro-apoptotic gene" as used herein refers to a nucleotide sequence that is expressed to induce programmed cell death. Examples of such pro-apoptotic genes include p53, adenovirus E3-11.6K (from Ad2 and Ad5) or adenovirus E3-10.5K (from Ad), adenovirus E4 gene, Fas ligand, TNF-, TRAIL, p53 pathway genes and genes encoding caspase.

The term "anti-angiogenic gene" in the context of the present invention refers to a nucleotide sequence that is expressed and releases an anti-angiogenic factor out of the cell. Anti-angiogenic factors include angiostatin, inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2 (PNAS, 1998, 95,8795-800), endostatin and the like.

The nucleotide sequence of the target gene described above can be obtained from a DNA sequence databank such as GenBank or EMBL.

The recombinant virus of the present invention can induce cell penetration into human, pig, and algal cells by the surface CPP protein, and induce an immune response to the antigen protein in vivo injected by the expressed antigen protein. Moreover, the recombinant baculovirus of the present invention induces not only humoral immunity but also cellular immune response. As a result, the recombinant baculovirus of the present invention can exert a good prevention effect against various diseases by this action.

In one embodiment of the present invention, the nucleotide sequence to be transported of the present invention is an antigen-coding nucleotide consisting of a nucleotide sequence encoding an antigen.

In one embodiment of the present invention, the above-mentioned antigen is selected from the group consisting of viral pathogen antigen, bacterial pathogen antigen, parasitic antigen and cancer antigen.

In one embodiment of the present invention, the viral pathogen antigen of the present invention is HPV (Human papilloma virus) antigen, HBV (hepatitis B virus) antigen, HCV (hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, MERS- CoV antigen, Zika virus antigen, HPV antigen, Norovirus antigen, Rotavirus antigen, Influenza virus antigen, Herpes simplex virus (HSV) antigen, Porcine genital and respiratory syndrome virus (PRRSV) antigen, Porcine cholera virus antigen, Porcine circovirus ( PCV) antigen, porcine diarrhea virus (PEDV) antigen, foot and mouth virus antigen and Newcastle virus antigen and viral hemorrhagic sepsis virus (VHSV) antigen.

According to another aspect of the present invention, the present invention provides an expression vector comprising the "expression construct", another aspect of the present invention described above.

Since the expression vector of the present invention includes the above-described expression construct, the content common between the two is omitted to avoid excessive complexity of the present specification.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from a genome of a mammalian cell, a promoter derived from a mammalian virus, or a promoter derived from a baculovirus (eg, a polyhedrin promoter) ) Can be used and generally have a polyadenylation sequence as the transcription termination sequence.

The vector of the present invention is a selection marker, and includes antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetra There are resistance genes for cyclins.

According to a preferred embodiment of the invention, the vector of the invention has the genetic map of Figure 8 attached. That is, in the vector of FIG. 8, gene expression of HA2 containing CPP is regulated by a polyhedrin promoter, expression of a target gene is regulated by a CMV promoter, has a BGH poly A signal as a termination signal, and is expressed on both sides of the expression cassette. Two arms of transposon 7 are located. As used herein, the term "expression construct" is defined to mean a nucleic acid molecule that contains only minimal elements for protein expression in cells.

According to another aspect of the present invention, the present invention provides a transformant transformed with an "expression vector" which is another aspect of the present invention.

In describing the'transformant' of the present invention, the content overlaps with the description of the'expression construct' or'expression vector' which is another aspect of the present invention. Repeat statements should be omitted to avoid excessive complexity.

According to another aspect of the present invention, the present invention provides a recombinant baculovirus comprising the'expression construct', another aspect of the present invention described above.

In describing the'recombinant baculovirus' of the present invention, the content overlaps with the description of the'expression construct', which is another aspect of the present invention, and the content is used to avoid excessive complexity of the present specification. For the sake of repetition, the repeated description will be omitted.

The present invention is a technology using the chimeric virus, and the chimeric virus of the present invention is based on baculovirus. Baculoviruses are rod-shaped viruses that do not express their genes from insect-specific promoters in human cells. For this reason, baculovirus is attracting attention as a basic system for gene therapy because it does not induce an immune response by viral gene expression. However, under the control of a mammalian promoter, expression of the foreign gene in the baculovirus vector occurs. Infection with baculovirus also has the advantage of not triggering the replication of the endogenous human virus. Unlike other gene therapy viruses, baculovirus can grow well in a serum-free medium, which has the advantage of being suitable for mass production.

The construction of recombinant baculovirus and the cultivation of insect cells are described in Summers and Smith. 1986. A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow. 1991. In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; U.S. Pat. No. 4,745,051; And EP0340359, which is incorporated herein by reference.

For example, a chimeric baculovirus comprising an influenza-derived CPP-containing HA2 gene and a nucleotide sequence to be transported is transformed into cells with a transfer vector carrying an influenza-derived CPP-containing HA2 gene and a nucleotide sequence to be transported. An expression construct comprising an influenza-derived CPP-containing HA2 gene and a nucleotide sequence to be transported is flanked with a transposon sequence, such as Tn7. This transfection vector is transformed into cells containing a mini-attTn7 target position (baculovirus shuttle vector) and a helper plasmid having a transposase gene, such as E. coli. When the transfer vector is transformed into the E. coli cells, transposition occurs to produce a recombinant bacmid. Subsequently, the recombinant bacmid is isolated and transformed into a suitable insect cell to generate a chimeric baculovirus. Insect cells suitable for the present invention are not particularly limited, for example, Sf9 (Spodoptera frugiperda), Spodoptera exiaua, Choristoneura fumiferana, Trichoplusia ni, and Spodoptera littoralis and Drosophila may be used as insect cells.

According to another aspect of the present invention, the present invention provides a gene delivery system comprising a recombinant baculovirus.

The gene delivery system of the present invention is characterized in that it includes another aspect of the present invention, the'expression construct' or the'recombinant baculovirus'. Will be omitted.

Recently, a method using a virus has been mainly used as a gene delivery system. As a gene delivery system using a virus, many viruses such as adenovirus, retrovirus, lentivirus, and vaccinia are used. Most of these viruses are viruses that pose new infections or dangers to humans, and are restricted to human use, whereas baculovirus is known as a biologically safe virus because it can only be replicated in limited insects. The construction of recombinant baculovirus and the cultivation of insect cells are described in Summers and Smith. 1986. A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow. 1991. In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; U.S. Pat. No. 4,745,051; And EP0340359, which is incorporated herein by reference. Most of these viruses are viruses that pose new infections or dangers to humans, and are restricted to human use, whereas baculovirus is known as a biologically safe virus because it can be reproduced only in limited insects without human infection. The virus-mediated gene delivery system is achieved through infection of the virus, which is determined through the interaction of the surface protein of the virus with the receptors of the target cells and animals, but has low efficiency.

The present invention provides an improved gene delivery system by recombining and inserting an influenza-derived CPP protein on the surface of a baculovirus, focusing on the limitations of such a baculovirus. Influenza is known to have a wide host range, including humans and all mammals including pigs, birds, cats and dogs. In the case of the gene delivery system of the present invention, since the influenza-derived CPP protein is bound to the surface, it is possible to efficiently and safely transport the desired gene into human, pig, and chicken cells. Therefore, the gene delivery system of the present invention can be usefully used for the development of gene therapeutic agents for various diseases and diseases.

According to another aspect of the present invention, the present invention provides a vaccine composition comprising the'recombinant baculovirus' which is another aspect of the present invention as an active ingredient.

The present inventors can combine the nucleotide sequence encoding a predetermined antigen with an influenza-derived CPP protein to produce an expression construct and a recombinant baculovirus and induce an improved immune response when processing the cells using the same. It was confirmed that it can be used as a vaccine.

The recombinant baculovirus of the present invention can be used to transport various antigen genes. As used herein, the term “antigen gene” or “nucleotide sequence encoding an antigen protein” means a sequence encoding an antigenic protein (eg, cell or viral surface antigenic protein) that can be recognized by the immune system.

In one embodiment of the present invention, the vaccine composition of the present invention is an anti-viral, anti-bacterial, anti-parasitic or anti-cancer vaccine composition.

The antigen of the present invention is specifically, for example, a viral pathogen antigen, a bacterial pathogen antigen, a parasitic antigen or a cancer antigen, more preferably a viral pathogen antigen or a cancer antigen, and most preferably a viral pathogen antigen.

Examples of viral pathogen antigens that can be used in the present invention include Herpesviruses such as Herpes Virus (Zarsterella-Zoster Virus), EBV (Epstein-Barr Virus) in humans; Herpesvirus antigen genes such as cytomegalovirus (CMV) or herpes simplex virus (HSV), Orthomyxoviruses containing seasonal H7N9 virus and H5 strain highly pathogenic influenza virus, viruses that cause severe respiratory diseases such as MERS or SARS, cause common cold Coronaviruses that contain coronavirus, Hepatitis A, B, C virus that causes hepatitis, Papillomavirus that causes cervical cancer and warts, Dengue, Japanese encephalitis, Zika, Yellow fever, West Nile, Flaviviruses, Norovirus and Rotavirus , Paramyxoviruses such as measles, Lentiviruses such as respiratory syncytial virus (RSV), human immunodificiency virus (HIV), and Rhabdoviruses such as rabies; Piconarviruses such as poliovirus; Contains antigens from pneumonia or tuberculosis. In particular, animal vaccines include foot and mouth disease virus, porcine genital and respiratory syndrome (PRRSV), porcine circovirus (PCV), swine pandemic virus (PEDV), and swine fever virus (CSFV). In addition, it is applicable to disease viruses that are prevalent in fish. Specifically, examples of viral pathogen antigens include, for example, HPV antigens, L1, L2, E6 or E7 proteins of HPV; Examples of antigens of HIV include T cell and B cell epitopes of nef, p24, gp120, gp41, tat, rev, pol, env and gp120 (Palker et al, J. Immunol., 142:3612-3619 (1989)). Includes. Examples of HBV surface antigens are described in Wu et al, Proc. Natl. Acad. Sci., USA, 86:4726-4730 (1989). Examples of antigens of rotavirus are VP4 (Mackow et al, Proc. Natl. Acad. Sci., USA, 87:518-522 (1990)) and VP7 (Green et al, J. Virol., 62:1819-1823 (1988); influenza virus antigens include hemagglutinin (HA), neuraminidase (NA), matrix protein (M) and nucleoprotein; HSV antigen Contains thymidine kinase (Whitley et al, In: New Generation Vaccines, pages 825-854); avian influenza virus antigens contain hemagglutinin; porcine cholera virus antigens are envelopes; foot and mouth virus antigens are envelopes And Newcastle virus antigens include HN (Hemagglutinin-neuraminidase), F (Fusion protein).

Examples of bacterial pathogen antigens that can be used in the present invention include Mycobacterium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. coli, Rickettsia spp., Listeria spp., Legionella pneumoniae, Pseudomonas spp., Vibrio spp. And antigens derived from Borellia burgdorferi. Specifically, examples of bacterial pathogen antigens that can be used in the present invention include Shigella sonnei's form-1 antigen (Formal et al, Infect. Immun., 34:746-750 (1981)); O-antigen of V. cholerae (Forrest et al, J. Infect. Dis. 159:145-146 (1989); FA/I fimbrial antigen of E.coli (Yamamoto et al, Infect. Immun., 50:925-928 (1985)) and non-toxic B-subunits of thermosensitive toxins (Klipstein et al, Infect. Immun., 40:888-893 (1983)); pertactin (Roberts of Bordetella pertussis) et al, Vacc., 10:43-48 (1992)); adenylate cyclase-hemolysine of B. pertussis (Guiso et al, Micro. Path., 11:423431 (1991)); and Clostridium Fragment C of Thetanus toxin of tetani (Fairweather et al, Infect. Immun., 58:1323-1326 (1990)).

Examples of parasite antigens that can be used in the present invention include Plasmodium spp., Trypanosome spp., Giardia spp., Boophilusspp., Babesia spp., Entamoeba spp., Eimeria spp., Laishmania spp., Schistosome spp., Brugia spp. ,Fascida spp., Dirofilaria spp., Wuchereria spp., and antigens derived from Onchocerea spp. More specifically, examples of parasitic antigens that can be used in the present invention include Plasmodium bergerii circumsporozoite antigen and P. falciparum Circumsporozoite antigens of Plasmodium spp., such as circumsporozoite antigens (Sadoff et al, Sci., 240:336-337 (1988)); Merozoite surface antigen of Plasmodium spp. (Spetzler et al, Int. J. Pept. Prot. Res., 43:351-358 (1994)); Galactose specific lectins of Entamoeba histolytica (Mann et al, Proc. Natl. Acad. Sci., USA, 88:3248-3252 (1991)); Gp63 from Leishmania spp. (Russell et al, J. Immunol., 140:1274-1278 (1988)); Paramyosin of Brugia malayi (Li et al, Mol. Biochem. Parasitol., 49:315-323 (1991)); And Schistosoma mansoni's trios-phosphate isomerase (Shoemaker et al, Proc. Natl. Acad. Sci. USA, 89:1842-1846 (1992)).

Examples of cancer antigens that can be used in the present invention include prostate specific antigens (Gattuso et al, Human Pathol., 26:123-126 (1995)), TAG-72 and carcinoembryonic antigen (CEA) (Guadagni et al, Int. J. Biol. Markers, 9:53-60 (1994)), MAGE-1 and tyrosinase (Coulie et al, J. Immunothera., 14:104-109 (1993)), p53 (WO 94/02167) ,NY-ESO1 (cancer-testis antigen), AFP (α-feto protein) and cancer antigen 125 (CA-125), and EPCA (Early Prostate Cancer Antigen).

In one embodiment of the present invention, the anti-viral vaccine composition of the present invention is anti-HPV (Human papillomavirus), anti-HBV (hepatitis B virus), anti-HCV (hepatitis C virus), anti-HIV (human immunodeficiency) virus), anti-MERS-CoV, anti-zicavirus, anti-HPV, anti-norovirus, anti-rotavirus, anti-influenza virus, anti-herpes simplex virus (HSV), anti-swine genital and respiratory syndrome virus (PRRSV), anti-swine cholera virus, anti-swine circovirus (PCV), anti-swine diarrhea virus (PEDV), anti-football virus, anti-Newcastle virus or anti-viral hemorrhagic sepsis virus (VHSV) vaccine It is a composition.

The vaccine composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carrier included in the vaccine composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. It is not. The vaccine composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

Suitable dosages of the vaccine compositions of the present invention may be variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. Can. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be applied topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. . Given that the pharmaceutical composition of the present invention is applied for obtaining immunity, it is preferable that the administration is performed in an intramuscular injection manner.

The vaccine composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating it into a multi-dose container.

According to another aspect of the present invention, the present invention provides a method for preparing a recombinant baculovirus-based vaccine composition comprising the following steps:

(a) an envelope-coding sequence consisting of a nucleotide sequence encoding an influenza derived HA2 protein; A first promoter operably linked to the envelope-coding sequence; A given antigen-coding nucleotide sequence; And producing an expression construct comprising a second promoter operatively linked to the antigen-coding nucleotide sequence;

(b) preparing a recombinant baculovirus using the expression construct prepared in step (a); And

(c) preparing a vaccine composition comprising the baculovirus prepared in step (b) as an active ingredient.

The production method of the present invention relates to a method for preparing a vaccine composition using the above-described another aspect of the present invention,'recombinant baculovirus', and this is used for overlapping contents. In order to avoid excessive complexity of the specification, the description is omitted.

In one embodiment of the invention, the influenza-derived HA2 protein of the invention consists of the amino acid sequence of SEQ ID NO: 1.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an expression construct for gene delivery system with improved cell delivery rate.

(b) The present invention provides an expression vector comprising the recombinant baculovirus.

(c) The present invention provides a transformant transformed with the expression vector.

(d) The present invention provides a recombinant baculovirus comprising the expression construct.

(e) The present invention provides a gene delivery system comprising the recombinant baculovirus.

(f) The present invention provides a vaccine composition comprising the recombinant baculovirus as an active ingredient.

(g) The recombinant baculovirus of the present invention can easily penetrate human, porcine and chicken cells by the surface CPP protein, and can induce high expression of the antigen protein through high delivery efficiency.

(h) When using the recombinant baculovirus of the present invention, various diseases caused by various antigens can be effectively prevented.

(i) When the gene delivery system or vaccine composition of the present invention is used, a safe and economic gene therapy product or vaccine can be provided.

Figure A of Figure 1a is a schematic diagram showing an Overlapping PCR made to load the influenza-derived CPP-containing HA2 gene used in the present invention into a baculovirus. In FIG. 1A, the scissors are amplified by PCR in the HA2 portion, the black square in which gp64SP is written, the signal peptide of gp64 glycoprotein, the dark gray square in which gp64TM is written, the transmembrane domain of gp64 glycoprotein, the light gray square in which gp64CT is written, gp64 glycoprotein Cytoplasmic-tail domain. Each number represents an amino acid sequence number. Figure B is a photo of the results of electrophoresis of gp64SP, HA2, and gp64TM-CT genes amplified by PCR.
Figure 1b is a diagram showing the gene sequence of the influenza-derived CPP containing HA2 is added gp64SP in the 5'direction and sp64TM-CT in the 3'direction used in the present invention.
1C is a diagram showing the gene sequence of HA2 in which influenza-derived CPP from which gp64SP was added in the 5'direction and sp64TM-CT was added in the 3'direction was used in the present invention. Figure 2 is a schematic diagram of the predicted chimeric baculovirus transfer vectors and viruses including the constructed pAc-CMV eGFP, pAc??CPP-CMV eGFP and pAcCPP-CMV eGFP. In FIG. 2, the gray square with Polh used is a polyhedrin promoter, the gray square with CMV used is a CMV promoter, and the small black square is a BGH poly A signal.
Figure 3 is a result of confirming by electrophoresis after performing PCR with HA2 specific Primer in the baculovirus DNA of the produced baculovirus Ac-CMV eGFP, Ac△CPP-CMV eGFP and Ac-CPP-CMV eGFP. "PCR Ctrl" represents the control without template, and "Ctrl" represents the baculovirus DNA of Ac-CMV eGFP.
4 is a photograph of Western blotting analysis of the expression level of GP64 and HA2 genes in Sf9 cells infected with Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP. "Ctrl" represents the control group infected with Ac-CMV eGFP. Since Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP have all GP64 envelopes based on baculovirus, it was possible to confirm the expression of GP64 protein, which was used as a measure of the criteria for expression. Expression of HA2 protein was confirmed only in Ac△CPP-CMV eGFP and AcCPP-CMV eGFP, and it can be confirmed that HA2 protein is present in baculovirus.
5 is a photograph showing the results of analyzing the expression level of the eGFP gene in HEK 293T human cells infected with Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP. "MocK" refers to the control baculovirus without eGFP. AcCPP-CMV eGFP, which contains the influenza-derived CPP protein, was confirmed to be delivered higher to HEK 293T human cells.
FIG. 6 is a photograph showing the results of analyzing the expression level of the eGFP gene in Porcine Alveolar Macrophage (PAM) pig cells infected with Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP. "MocK" refers to the control baculovirus without eGFP. AcCPP-CMV eGFP, which contains the influenza-derived CPP protein, was confirmed to be delivered higher to PAM pig cells.
7 is a photograph showing the results of analyzing the expression level of eGFP gene in Chicken Embryonic Fibroblask (CEF) chicken cells infected with Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP. "MocK" refers to the control baculovirus without eGFP. AcCPP-CMV eGFP, which contains the influenza-derived CPP protein, can be confirmed to be delivered higher to CEF chicken cells.
8 is a genetic map of a vector constructed in one embodiment of the present invention. Polh promoter, polyhedrin promoter; CPP, CPP gene derived from influenza; And, Tn7R and Tn7L are the right and left arms of transposon 7, respectively. In the case of AcCPP-CMV eGFP, the eGFP gene is located at the position of the target gene.
9 is a gene configuration for the production of recombinant baculovirus (AcHP2-HP16/18/58L1), the HA2 gene is inserted behind the polyhedrin promoter, and the target gene HPV L1 is expressed by human elongation promoter 1, respectively. It is configured to be controlled.
10 is a result of comparing the IgG antibody titer after administration of AcHP2-HP16/18/58L1 vaccine to mice at a concentration of 1X10 ⁷ or 1X10 ⁸ FFU. BALB/c mice were immunized with AcHA2-HP16/18/58L1. Antibody titers were measured by ELISA. The IgG titer for each HPV type in mice treated with AcHA2-HP16/18/58L1 vaccine with 1X10 ⁷ , 1X10 ⁸ FFU was different according to the number of times administered 1 or 2, but the IgG titer after 3 doses was similar. It was found that the IgG titer in the group intramuscularly injected with the AcHA2-HP16/18/58L1 vaccine with 1X10 ⁷ FFU was higher than the other group.
11 is a result of comparing the neutralizing antibody titer after administration of AcHP2-HP16/18/58L1 vaccine to mice at a concentration of 1X10 ⁷ or 1X10 ⁸ FFU. Neutralization analysis was performed by sera of serially diluted mice and HPV16, 18 or 58 pseudovirus. Data are expressed as the geometric mean (log) of mutual serum dilution resulting in a 50% SEAP reduction. It was confirmed that the neutralizing antibody titer for each HPV type was similar to or equal to the neutralizing antibody titer after 3 times of administration even after only 2 doses, and showed sufficient neutralizing antibody titer.
12 is a vaccination result using a baculovirus made by inserting a PRRSV GP5 gene into a pig in an embodiment of the present invention. It is possible to confirm the higher antibody value than the killed vaccine. It can be confirmed that induce specific antibodies against PRRSV GP5 compared to wild type baculovirus.
13 shows neutralizing antibody titers against PRRSV. It can be seen that neutralizing antibody titers appeared in the group that vaccination Killed vaccine and AcCPP-GP5, respectively. AcCPP-GP5 can confirm higher neutralizing antibody titers than the Killed vaccine group. This confirms that AcCCP-GP5 induces better immunity than conventional commercial vaccines.
14 is a schematic diagram of a plasmid for generating recombinant baculovirus with FMDV gene. After codon optimization of VP4 to 3D gene of foot and mouth disease virus with Bovine, it was synthesized and cloned into pcDNA vector. After cloning the gene from CMV to poly a tail through PCR, it was cloned into pFastbac-HA2 vector with KpnI and PstI, and one of the candidate groups attached SV40 Inhancer to enhance expression efficiency.
Figure 15 is a picture showing the specific IgG titer for foot and mouth virus as a result of immunizing Ac HA2-CMV FMDV in experimental mice, compared to the group in which the group immunized with Ac HA2-CMV FMDV immunized two other viruses that cloned only VP1. It showed a higher IgG antibody titer about 70%. As a result, it was found that it is more efficient to immunize the entire FMDV gene.
FIG. 16 shows the results of immunization of a recombinant baculovirus with a zicavirus prM/E gene in experimental mice. After the codon optimization of the prM gene and envelope gene of Zika virus, it was synthesized and cloned into a pcDNA vector, and designed to enhance gene expression efficiency by attaching an SV40 enhancer. The gene was cloned from SV40 enhancer to poly a tail through PCR and cloned into pFastbac-HA2 vector with KpnI. This was immunized with a laboratory mouse, and Ig G specific for Zika virus was confirmed by ELISA, and it was confirmed that it had a high antibody titer.
17 shows a CPP-containing HA2 protein and a nucleic acid sequence encoding the same.
18 shows an HA2 protein not containing CPP and a nucleic acid sequence encoding the same.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental materials and methods

Example 1: Cell preparation

Insect cell Sf9 (ATCC CRL-1711) was cultured in SF-900II medium (Gibco BRL) containing 27% at 10% FBS (fetal bovine serum, Gibco BRL) and 1% penicillin/streptomycin (Gibco BRL). . Human-derived embryonic kidney 293T cell line (ATCC), pig-derived alveolar macrophage cell line (PAM) and chicken-derived embryonic fibroblast cell line (CEF) were cultured in DMEM (Dulbecco's modified minimal essential medium) containing 10% FBS.

Example 2: HA2 gene synthesis including influenza-derived CPP protein

Viral RNA using a viral DNA-RNA extraction kit (Nucleospin RNA virus kit, Macherey-nagel) from low pathogenic H9N2 avian influenza (A/Chicken/Korea/01310/2001) secured to synthesize influenza-derived CPP gene After separation, a reverse transcription polymerization chain reaction was performed using a random hexamer. For the reverse transcription reaction, a reaction was performed at 25°C for 2 minutes, and then a reverse transcriptase (SuperScript™II Reverse Transcriptase, Invitrogen) was added, followed by a reaction at 42°C for 50 minutes and 70°C for 15 minutes to obtain a complementary base sequence (cDNA). .

PCR was performed with HA2-CPP specific primers as a template for the complementary base sequence (cDNA) generated through a reverse transcription polymerization chain reaction. The PCR result was 558 bp, and the PCR reaction was performed at 94°C for 5 minutes, followed by a total of 30 cycles of 94°C 30 seconds, 60°C 30 seconds, and 72°C 30 seconds, and then reacted at 72°C for 7 minutes. The result was obtained. After TA cloning for sequencing, it was sequenced (see FIG. 1A ). Sequence analysis result GenBank Accession No. It was confirmed that the same sequence as the JX094859.1 sequence.

Example 3: Fusion of GP64 signaling peptide, GP64 transmembrane peptide and CPP gene

The GP64 signal peptide (GP64sp) and the GP64 transmembrane domain (GP64tm) are part of the glycoprotein 64 that constitutes the baculovirus gene transporter envelope. To fuse gp64sp and GP64tm to HA2-CPP gene, specific primers were prepared and PCR was performed. Each gp64sp and GP64tm generated through PCR was fused to CPP gene fragments by overlapping PCR and sequenced (refer to FIG. 1B and Table 1).

In addition, PCR was performed by preparing specific primers to fuse gp64sp and GP64tm to the HA2-ΔCPP gene (the gene in which CPP was deleted) as a control for measuring the effect of CPP (refer to FIG. 1C, Table 2). Each gp64sp and GP64tm generated through PCR was fused to HA2-ΔCPP gene fragments by overlapping PCR and sequenced.

Example 4: Construction of baculovirus vector loaded with CPP gene and insertion of eGFP

The gp64sp_HA2-CPP_GP64tm and gp64sp_HA2-△CPP_GP64tm, which were obtained to construct the genetic vaccine, were cloned into the pFastBac1 vector, which is the basis of the vaccine, and eGFP was used as the Reporter gene to measure the cell delivery ability of the baculovirus made through this expression vector. Commonly used. Each gene was designed to be influenced by the polyhedrin promoter and the CMV promoter (see FIG. 2).

Example 5: Construction of CPP-equipped gene delivery system through baculovirus expression system

To produce a gene delivery system carrying CPP derived from influenza, a vaccine was prepared using the baculovirus expression system, Invitrogen's Bac-to-Bac system. As an experimental method, after the expression vector was prepared, transformation was performed using E. coli called DH10bac, and the gene was transferred to the bacmid by transposition from the Tn7R part to the Tn7L part in the produced vector. do. After extracting the recombinant bacmid, gene insertion is confirmed through PCR. After confirming the PCR result, Sf9 cells, which are insect cells, are transfected using Invitrogen Cellfectin. After transfection, a recombinant baculovirus is produced and recovered after 3 days. Virus was isolated from the recovered recombinant baculovirus through plaque purification. After extracting the baculovirus DNA using the viral DNA-RNA extraction kit (Nucleospin RNA virus kit, Macherey-nagel) from the separated virus, it was confirmed whether HA2 was inserted through PCR. (Reference: Figure 3)

Example 6: Confirmation of protein expression in insect cells (sf9 cells)

The produced Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP protein expression was confirmed by Western blot (see FIG. 4). As an experimental method, Ac-CMV eGFP, Ac△CPP-CMV eGFP, and AcCPP-CMV eGFP were infected with insect cells at Sf9 cells at a level of 10 MOI, and recovered after 3 days to extract proteins and electrophoresis on SDS-PAGE gel. Subsequently, anti-GP64 polyclonal antibody and anti-H9N2 polyclonal antibody were used.

Example 7: Gene delivery of CPP-loaded gene via baculovirus expression system in vitro cell Transmission ability evaluation

HEK 293T, PAM, and CEF cells were dispensed into 6 well plates and Ac-CMV eGFP, AcΔCPP-CMV eGFP and AcCPP-CMV eGFP viruses were transfected with 10 MOI, respectively. Cells 72 hours after infection were confirmed and quantified for the presence of eGFP protein using a fluorescence microscope (eclipse Ti-U, Nikon, Japan) device and a FACs device. Numericalization is obtained by calculating the Flourescent Index value by multiplying the percent (%) of eGFP positive cells with the Flourescence Geometric Mean Titer (F-GMT) produced using FACs, and then the Control (Ac-CMV eGFP) infected group is 1 After substitution, the value obtained by dividing the value of the Flourescent Index of the Ac△CPP-CMV eGFP and AcCPP-CMV eGFP virus-infected groups by the value of the Control-infected group was expressed as a Relative Flourescent Index value.

Example 8: Human papillomavirus (Human) of CPP-mounted gene delivery system through baculovirus expression system papilomavirus ; HPV ) Vaccine efficacy ( in vivo ) evaluation

To produce a vaccine for human papilomavirus (HPV) using a baculovirus-based gene delivery system, a nucleotide sequence encoding a protein containing an influenza-derived cell penetrating peptide (HA2-CPP) and HPV types 16, 58, And it was designed by combining the nucleotide sequence encoding the 18 type L1 protein. To add the protein of the baculovirus surface, the HA2 gene was inserted behind the polyhedrin promoter, and the target gene HPV L1 was configured to be regulated by human elongation promoter 1. An expression construct (pFB_HA-HP16/18/58L1) was prepared using each cloned plasmid, and a recombinant baculovirus (AcHA-HP16/18/58L1) was produced (FIG. 8).

Immuno-efficacy was compared and analyzed by immunizing the prepared AcHA2-HP16/18/58L1 vaccine with mice, and briefly explaining the method, 1X107 (Focus forming unit; FFU) of AcHA-HP16/18/58L1 produced with the vaccine was used. It was injected intramuscularly at a concentration of 1X108 FFU and administered 3 times every 2 weeks. Blood was collected 1 week after administration and serum was stored separately to measure the immune response.

Example 9: Efficacy evaluation of porcine genital respiratory syndrome (PRRS) gene vaccine using CPP-equipped gene delivery system

The eGFP gene was removed from the pAcCPP-CMV eGFP of FIG. 2 and a GPPP gene, an antigen gene of PRRS virus, was introduced to prepare a CPP-equipped gene delivery system capable of delivering the PRRS GP5 gene. Using this, the vaccine efficacy was evaluated using piglets that were 5 weeks old.

Group Vaccine substance Dose One Killed vaccine 1×10 ^4.9 TICD ₅₀ 2 AcCPP-GP5 1×10 ⁷ FFU 3 Wild type baculovirus 1×10 ⁷ FFU 4 PBS

Vaccination was performed twice at 3 week intervals, and sera was sapmling at 2 and 5 weeks to measure anti-PRRSV antibody titer through Indireted ELISA. At week 12, the sera was isolated to measure the neutralizing antibody titer.

Example 10: Evaluation of HA gene vaccine efficacy with foot and mouth virus genes

An HA2-mounted baculovirus delivery system capable of delivering the FMDV gene of FIG. 14 was prepared. This was evaluated by using the experimental rat vaccine efficacy. The experimental group was conducted in four groups, an experimental group and a negative control group, and each group was conducted using 7 BALB/c, 5-week-old female mice. Immunization was carried out with 1.0E+07 and immunization was carried out through 3 intramuscular injections every 7 days.

Example 11: Evaluation of HA gene vaccine efficacy with zicavirus gene

A CPP-mounted baculovirus delivery system capable of delivering a zicavirus prM/E gene was constructed. This was evaluated by using the experimental rat vaccine efficacy. The experimental group was conducted with the experimental group and the negative control group, and each group was conducted using 5 BALB/c, 5 week old female mice. Immunization was carried out with 1.0E+07, immunization was carried out three times through intramuscular injection at 7-day intervals, and then immunized once more with the abdominal cavity.

Experiment result

1. HA2 gene production including CPP that can be loaded into baculovirus

The HA2 gene containing CPP and the HA2 gene from which CPP has been removed were produced by PCR. The first and second sequences of the Sequence Listing are the HA2 gene sequence and amino acid sequence including 558 bp CPP prepared for use in this experiment, and the HA2 gene sequence and amino acid sequence of 489 bp CPP removed.

To fuse gp64sp and GP64tm to HA2-CPP gene, specific primers were prepared and PCR was performed. Each gp64sp and GP64tm generated through PCR was fused to CPP gene fragments by overlapping PCR and sequenced (refer to FIG. 1B and Table 1).

In addition, PCR was performed by preparing specific primers to fuse gp64sp and GP64tm to the HA2-ΔCPP gene (the gene from which CPP was deleted) as a control for measuring the effect of CPP (refer to FIG. 1C, Table 2). Each gp64sp and GP64tm generated through PCR was fused to HA2-ΔCPP gene fragments by overlapping PCR and sequenced.

2. Production of recombinant baculovirus

In order to prepare a recombinant baculovirus, three transfer vectors of pAc-CMV eGFP, pAcΔCPP-CMV eGFP and pAcCPP-CMV eGFP were planned, and the expected form of baculovirus was predicted (FIG. 2 ). To add the protein of the baculovirus surface, the polyhedrin promoter was inserted into the HA2 gene including CPP and the HA2 gene from which CPP was removed, and eGFP was constructed to be regulated by the CMV promoter.

In the case of the HA2 protein containing CPP, which is regulated by the insect virus promoter, it exhibits a very high expression level in insect cells, but does not properly express in animal cells. On the contrary, eGFP protein has a human promoter, CMV promoter, so that it is efficiently expressed in animal cells, but cannot be expressed at all in insect cells or only very fine expression is possible. Each cloned plasmid was used to create a recombinant bacmid and transfected into Sf9 cells to produce a high titer virus.

3. Measurement of transfection efficiency of eGFP gene into several cell lines

In order to determine the delivery efficiency of the eGFP gene as the baculovirus surface is modified, the Ac-CMV eGFP, Ac△CPP-CMV eGFP and AcCPP-CMV eGFP viruses were infected with HEK 293T, PAM, and CEF cells at 10 MOI, respectively. Ordered. The eGFP expression level was first confirmed with a fluorescence microscope. As shown in Fig. 5-7, when cells were infected with Ac-CMV eGFP, Ac△CPP-CMV eGFP and AcCPP-CMV eGFP through a fluorescence microscope, compared to HEK 293T cells that did not infect the virus after 72 hours, AcCPP-CMV eGFP It was confirmed that the overall fluorescence was amplified in the virus-infected cells. However, since it was not possible to accurately quantify the degree of fluorescence through a fluorescence microscope, additional experiments were conducted as follows.

In order to reliably quantify the efficiency of CPP gene delivery through infection, a Fluorescence Activated Cell Sorter (FACS) was performed. The experiment was carried out through 3 repetitions, and as shown in FIGS. 5-7, HEK infected with AcCPP-CMV eGFP virus when the Fluorescent Index (FI) of cells infected with Ac-CMV eGFP virus was viewed as 1 The number of gene copies in 293T, PAM, and CEF cells was 6.52, 2.36, and 5.88 times higher, respectively.

4. Human papillomavirus (HPV) vaccine efficacy evaluation

Immune efficacy was compared and analyzed by immunizing the produced AcHA2-HP16/18/58L1 vaccine with mice. Serum of immunized mice was collected as shown in FIG. 10, and IgG titers against HPV16, HPV18, or HPV58 were measured by ELISA, and each in a mouse that was intramuscularly administered with AcHA2-HP16/18/58L1 vaccine with 1X10 ⁷ , 1X10 ⁸ FFU The IgG titer according to the HPV type showed a difference according to the number of times of administration 1 or 2, but the IgG titer after 3 administrations was similar to 1X10 ⁴ or more. The IgG titer in the group intramuscularly injected with the AcHA2-HP16/18/58L1 vaccine with 1X10 ⁷ FFU showed the highest titer compared to the other groups.

As shown in FIG. 11, serum of immunized mice was collected and SEAP assay was performed using HPV pseudovirus, and neutralizing antibody titers against HPV16, HPV18, or HPV58 were measured to confirm the effect on humoral immune response. Neutralizing antibody titers according to each HPV type in mice treated with AcHA2-HP16/18/58L1 vaccine with 1X10 ⁷ , 1X10 ⁸ FFU are similar to or equivalent to those of neutralizing antibody titers after 3 doses with 2 doses alone. It showed the neutralizing antibody titer.

The production of specific neutralizing antibodies against vaccine antigens has been an important indicator in the evaluation of humoral immune responses and vaccine efficacy. As a result of the above animal experiment, through the produced recombinant baculovirus-based vaccine, it was confirmed that the titer of immunoglobulin and neutralizing antibody is very high, which shows the efficacy of the present invention as a vaccine that induces a strong humoral immune response. It can be said that there is. Therefore, the recombinant baculovirus loaded with the HA2 gene of the present invention was able to confirm the immune efficacy through high antibody titer as well as specific antibody formation against the antigen through in vivo delivery of the desired antigen gene. This shows that when the recombinant baculovirus loaded with the HA2 gene is applied as a vaccine, excellent immune efficacy can be expected.

5. Porcine genital respiratory syndrome (PRRS) vaccine efficacy assessment

Using Indirected ELISA, PRRSV GP5 protein group 1 and 2 showed antibody titers of 10^3, respectively, and it was confirmed that the antibody titer increased to 10^4 or higher after the second inoculation. In addition, it was confirmed that the antibody titer increased compared to the commercial Killed vaccine. (Figure 12). As a result of the measurement of the neutralizing antibody, it was confirmed that the neutralizing antibody value placed in Group 2 was higher than in Group 1 (FIG. 13). Through this, it was confirmed that AcCPP-GP5 has high gene transfer ability by loading CPP and is effective as a PRRS vaccine.

6. Foot and mouth disease (FMDV) vaccine efficacy evaluation

After inoculation in FMDV Ac HA2 CMV FMDV Group using Indirected ELISA, antibody titers of 1 X10^3 were shown, and after the second inoculation, it was confirmed that the antibody titer increased to 4X10^3 or more. The group immunized with Ac HA2-CMV FMDV showed about 70% higher antibody titer than the group immunized with the other two viruses that only cloned VP1 (FIG. 15 ). Through this, it was confirmed that Ac HA2 CMV FMDV has a high gene transfer ability by loading CPP, and it is possible to further immunize the FMDV gene as an FMDV vaccine.

7. Zika virus (ZIKV) vaccine efficacy evaluation

In the group of prM/E protein of ZIKV using Indirected ELISA, the antibody titer of about 10^3 was shown after the second inoculation, and it was confirmed that the antibody titer increased to more than 10^4 after the third inoculation (Fig. 16). Through this, it was confirmed that AcCPP-ZIKV prM/E is effective as a ZIKV vaccine.

Review

The present invention is a vaccine composition comprising a recombinant baculovirus and a recombinant baculovirus comprising a nucleotide sequence encoding an influenza-derived HA2 protein, a nucleotide sequence encoding an antigen protein, and a promoter operably linked to the antigen-coding sequence. It is about. The recombinant baculovirus of the present invention has been shown to have a very good ability in delivering genes to cells, and also has excellent antigen delivery ability into the body, and thus can be applied as a vaccine.

Since the specific parts of the present invention have been described in detail above, it is clear that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp KR Biotech Co.,Ltd. <120> Beculovirus-based Gene Transducer Including Influenza-induced Cell-Invesive Peptide <130> PN180018 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Influenza induced HA2 comprising CPP <400> 1 Gly Leu Phe Gly Ala Ile Thr Gly Phe Ile Glu Gly Gly Trp Pro Gly 1 5 10 15 Leu Val Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln Gly Val 20 25 30 Gly Ile Ala Ala Asp Lys Glu Ser Thr Gln Glu Ala Val Asp Lys Ile 35 40 45 Thr Ser Lys Val Asn Asn Ile Ile Asp Lys Met Asn Lys Gln Tyr Glu 50 55 60 Ile Ile Asp His Glu Phe Ser Glu Ile Glu Ala Arg Leu Asn Met Ile 65 70 75 80 Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Ile Trp Ala Tyr Asn Ala 85 90 95 Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu His Asp 100 105 110 Val Asn Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu Gly Ser 115 120 125 Asn Ala Ile Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys Cys 130 135 140 Asp Asp Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asp Arg Leu 145 150 155 160 Lys Tyr Lys Glu Glu Ser Lys Leu Glu Arg Gln Lys Ile Glu Gly Val 165 170 175 Lys Leu Glu Ser Glu Glu Thr Tyr Lys Ile 180 185 <210> 2 <211> 163 <212> PRT <213> Artificial Sequence <220> <223> Influenza induced HA2 without CPP <400> 2 Phe Gln His Ser Asn Asp Gln Gly Val Gly Ile Ala Ala Asp Lys Glu 1 5 10 15 Ser Thr Gln Glu Ala Val Asp Lys Ile Thr Ser Lys Val Asn Asn Ile 20 25 30 Ile Asp Lys Met Asn Lys Gln Tyr Glu Ile Ile Asp His Glu Phe Ser 35 40 45 Glu Ile Glu Ala Arg Leu Asn Met Ile Asn Asn Lys Ile Asp Asp Gln 50 55 60 Ile Gln Asp Ile Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu 65 70 75 80 Asn Gln Lys Thr Leu Asp Glu His Asp Val Asn Val Asn Asn Leu Tyr 85 90 95 Asn Lys Val Lys Arg Ala Leu Gly Ser Asn Ala Ile Glu Asp Gly Lys 100 105 110 Gly Cys Phe Glu Leu Tyr His Lys Cys Asp Asp Gln Cys Met Glu Thr 115 120 125 Ile Arg Asn Gly Thr Tyr Asp Arg Leu Lys Tyr Lys Glu Glu Ser Lys 130 135 140 Leu Glu Arg Gln Lys Ile Glu Gly Val Lys Leu Glu Ser Glu Glu Thr 145 150 155 160 Tyr Lys Ile <210> 3 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Influenza induced CPP domain <400> 3 Gly Leu Phe Gly Ala Ile Thr Gly Phe Ile Glu Gly Gly Trp Pro Gly 1 5 10 15 Leu Val Ala Gly Trp Tyr Gly 20 <210> 4 <211> 558 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding Influenza induced HA2 with CPP <400> 4 gggcttttcg gtgccataac tggattcata gaaggaggtt ggccaggact agttgcaggc 60 tggtacgggt ttcagcactc aaatgatcaa ggggttggaa tagccgcaga caaagaatca 120 actcaagaag cagttgataa aataacatcc aaagtaaata atataatcga caaaatgaac 180 aagcagtatg aaatcattga tcatgagttc agtgagattg aagccagact caatatgatc 240 aacaataaga ttgatgacca aatacaggac atctgggcgt acaatgcaga attactagta 300 ctgcttgaaa accagaaaac actcgatgag catgatgtaa atgtgaacaa tctgtataat 360 aaggtgaaga gagcattggg ttcaaatgca atagaggatg ggaagggatg cttcgagttg 420 tatcacaaat gtgatgatca atgcatggaa acaattagaa acgggactta tgacaggcta 480 aagtataaag aagaatcaaa actagaaagg cagaaaatag aaggggtaaa actggagtct 540 gaagaaactt acaagatt 558 <210> 5 <211> 489 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding Influenza induced HA2 without CPP <400> 5 tttcagcact caaatgatca aggggttgga atagccgcag acaaagaatc aactcaagaa 60 gcagttgata aaataacatc caaagtaaat aatataatcg acaaaatgaa caagcagtat 120 gaaatcattg atcatgagtt cagtgagatt gaagccagac tcaatatgat caacaataag 180 attgatgacc aaatacagga catctgggcg tacaatgcag aattactagt actgcttgaa 240 aaccagaaaa cactcgatga gcatgatgta aatgtgaaca atctgtataa taaggtgaag 300 agagcattgg gttcaaatgc aatagaggat gggaagggat gcttcgagtt gtatcacaaa 360 tgtgatgatc aatgcatgga aacaattaga aacgggactt atgacaggct aaagtataaa 420 gaagaatcaa aactagaaag gcagaaaata gaaggggtaa aactggagtc tgaagaaact 480 tacaagatt 489

Claims (20)

An envelope-coding sequence consisting of a nucleotide sequence encoding an influenza derived HA2 protein consisting of the amino acid sequence of SEQ ID NO: 1; A first promoter operably linked to the envelope-coding sequence; Nucleotide sequence to be transported; And a second promoter operably linked to the nucleotide sequence to be transported.
The expression construct according to claim 1, wherein the first promoter, envelope-coding sequence, second promoter, and nucleotide sequence to be transported are included on a single nucleotide sequence.
3. The expression construct of claim 2, wherein the second promoter is located downstream of the envelope-coding sequence.
The expression construct according to claim 1, wherein the influenza-derived HA2 protein contains a CPP (Cell Penetration Peptide) sequence in the sequence.
The expression construct of claim 1, wherein the first promoter is a promoter operating in insect cells.
The method of claim 5, wherein the promoter operating in the insect cell is characterized in that it is selected from the group consisting of baculovirus IE-1 promoter, IE-2 promoter, p35 promoter, p10 promoter, gp64 promoter and polyhedrin promoter. Expression construct.
The expression construct of claim 1, wherein the second promoter is a promoter derived from a genome of a mammalian cell or a promoter derived from a mammalian virus.
8. The method of claim 7, wherein the second promoter is U6 promoter, H1 promoter, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, human extension factor 1α(hEF1α) promoter, metallothionine promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, of human GMCSF gene Expression construct, characterized in that selected from the group consisting of a promoter, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, AFP promoter, and albumin promoter.
delete The expression construct according to claim 1, wherein the nucleotide sequence to be transported is an antigen-coding nucleotide consisting of a nucleotide sequence encoding an antigen.
11. The expression construct according to claim 10, wherein the antigen is selected from the group consisting of viral pathogen antigen, bacterial pathogen antigen, parasitic antigen and cancer antigen.
The method of claim 11, wherein the virus pathogen antigen is HPV (Human papillomavirus) antigen, HBV (hepatitis B virus) antigen, HCV (hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, MERS-CoV antigen, Zika virus antigen , HPV antigen, norovirus, rotavirus antigen, influenza virus antigen, HSV (herpes simplex virus) antigen, porcine genital and respiratory syndrome virus (PRRSV) antigen, porcine cholera virus antigen, porcine circovirus (PCV) antigen, porcine diarrhea virus (PEDV) Expression construct, characterized in that it is selected from the group consisting of antigens, foot and mouth virus antigens and Newcastle virus antigens and viral hemorrhagic sepsis virus (VHSV) antigens.
An expression vector comprising the expression construct of any one of claims 1 to 8 and 10 to 12.
A transformant transformed with the expression vector of claim 13.
A recombinant baculovirus comprising the expression construct of any one of claims 1 to 8 or 10 to 12.
A gene delivery system comprising the recombinant baculovirus of claim 15.
A vaccine composition for anti-viral, anti-bacterial, anti-parasitic or anti-cancer comprising the recombinant baculovirus of any one of claims 1 to 8 and 10 to 12 as an active ingredient.
The method of claim 17, wherein the vaccine composition is anti-HPV (Human papillomavirus), anti-HBV (hepatitis B virus), anti-HCV (hepatitis C virus), anti-HIV (human immunodeficiency virus), anti-MERS-CoV , Anti-zicavirus, anti-HPV, anti-norovirus, anti-rotavirus, anti-influenza virus, anti-herpes simplex virus (HSV), anti-swine genital and respiratory syndrome virus (PRRSV), anti-swine cholera A vaccine composition characterized by being for a virus, anti-swine circovirus (PCV), anti-swine diarrhea virus (PEDV), anti-foot-and-mouth disease virus, anti-Newcastle virus or anti-viral hemorrhagic sepsis virus (VHSV).
Method for preparing a recombinant baculovirus-based vaccine composition comprising the following steps:
(a) an envelope-coding sequence consisting of a nucleotide sequence encoding an influenza-derived HA2 protein consisting of the amino acid sequence of SEQ ID NO: 1; A first promoter operably linked to the envelope-coding sequence; A given antigen-coding nucleotide sequence; And producing an expression construct comprising a second promoter operatively linked to the antigen-coding nucleotide sequence;
(b) preparing a recombinant baculovirus using the expression construct prepared in step (a); And
(c) preparing a vaccine composition comprising the baculovirus prepared in step (b) as an active ingredient. delete

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