WO2004029259A1 - バキュロウイルスベクター、バキュロウイルスベクター製造方法及び遺伝子導入方法 - Google Patents
バキュロウイルスベクター、バキュロウイルスベクター製造方法及び遺伝子導入方法 Download PDFInfo
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- 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
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- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
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- C12N2760/20011—Rhabdoviridae
- C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
- C12N2810/6072—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses
- C12N2810/6081—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses rhabdoviridae, e.g. VSV
Definitions
- the present invention relates to a baculovirus vector that can be suitably used for gene therapy and the like, a method for producing the baculovirus vector, and a method for introducing a gene using the baculovirus vector.
- retroviruses adenoviruses, and adeno-associated viruses have been mainly used as gene therapy vectors.
- safety issues such as the emergence of self-sustaining culturing viruses and the activation of cancer genes by random integration of genes, and the improvement of gene transfer efficiency and specific gene transfer methods was not sufficient.
- problems such as induction of cytotoxicity and immune response, and inactivation by neutralizing antibodies.
- baculovirus an insect virus, is considered to have no virulent infection in insects until recently, and has been used as a system that can express large amounts of transgenes exclusively using insect cells.
- Baculovirus is an insect pathogenic virus that infects insects such as Lepidoptera, Hymenoptera, and Diptera, and has a circular double-stranded DNA as a gene.
- a group of viruses called Nuclear Polyhedorosis Virus (NPV)
- NDV Nuclear Polyhedorosis Virus
- the polyhedron is essential for the virus to survive in nature, but is not necessary for the virus to grow itself, so even if a foreign gene to be expressed is substituted for the polyhedron gene, the virus will not Infects, multiplies, and produces large amounts of foreign gene products without any hindrance.
- AcNPV Autographs caUfornica NPV
- BmNPV Bombyx mori NPV
- the present inventors have succeeded in developing the world's highest expression efficiency vector, pAcYMl, for paculovirus vectors (for example, see Non-Patent Document 1). He expressed hepatitis C virus structural protein in a system using this vector, and succeeded in developing an early antibody diagnostic system using that vector as an antigen. The introduction of this diagnostic system led to the development of post-transfusion hepatitis C in Japan. Was almost suppressed.
- Non-Patent Documents 2 and 3 gene transfer into mammalian cells using paculovirus has been reported (for example, see Non-Patent Documents 2 and 3). Initially, gene transfer using baculovirus was considered to be specific for hepatocytes, but the present inventors have shown that gene transfer is possible in a wide range of animal cells (e.g., Patent Document 4).
- baculovirus vectors are able to efficiently express foreign genes without infecting and replicating a wide range of mammalian cells and to replicate them has been highlighted as a potential gene therapy vector. It became so.
- Baculovirus vectors 1 have a viral gene of 130kbp and can insert a large (less than 15kbp) foreign gene. 2) no viral gene is expressed in mammalian cells, so there is little cytotoxicity and adverse immune response. 3) A recombinant virus can be produced in a short time, and 4) Humans do not have neutralizing antibodies against baculovirus. Having. In order to improve the infection efficiency, the envelope proteins of other viruses such as the vesicular stomatitis virus G protein have been used in the baculovirus itself to improve its efficiency. With gP 64 protein is Putanpaku protein have been reported attempts to present on the particle surface (e.g., see Non-patent document 5) (Fig. 3 left). This drastically improved the efficiency of gene transfer into mammalian cells.
- Non-Patent Document 6 Fig. 3, right.
- Non-Patent Document 3 (Non-Patent Document 3)
- Non-Patent Document 5 (Non-Patent Document 5)
- Non-Patent Document 6 (Non-Patent Document 6)
- An object of the present invention is to solve the conventional problems and achieve the following objects. That is, it is an object of the present invention to provide a novel baculovirus vector capable of presenting a desired protein on the surface of a virus particle without having to show infectivity to insect cells, a method for producing the same, and a method for gene transfer using the baculovirus vector. With the goal.
- the present inventors have achieved the present invention based on the development of a method for deleting the gp64 gene of the paculovirus and, instead, efficiently incorporating the target protein into the surface of the virus particle.
- a cotransfectant that cotransfects at least a plasmid containing a gene encoding a protein that can be expressed on the cell surface and any of wild-type, mutant and recombinant paculovirus DNA into insect cells Including a Yong process, and A method for producing a baculovirus vector comprising producing at least a part of a culovirus DNA and producing a pseudotype paculovirus covered with a protein that can be expressed on the cell surface.
- Baculovirus DNA is a recombinant baculovirus DNA, in which homologous recombination between the polyhedron gene and the first foreign gene and homologous recombination between the gp64 protein and the second foreign gene have been performed.
- the method for producing a baculovirus vector according to ⁇ 1>, which is a baculovirus DNA is a baculovirus DNA.
- paculovirus DNA according to ⁇ 1>, wherein the baculovirus DNA is a recombinant baculovirus DNA, and the baculovirus DNA is a paculovirus DNA obtained by homologous recombination between a polyhedrin gene and a first foreign gene. This is a method for producing a paculovirus vector.
- the vector containing the second foreign gene is simultaneously cotransfected, homologous recombination of the gp64 protein and the second foreign gene occurs, and the baculovirus DNA containing the foreign gene is removed.
- the method for producing a baculovirus vector according to ⁇ 3>, wherein the pseudotype baculovirus comprising the protein capable of being expressed on the cell surface is produced.
- ⁇ 5> The method for producing a paculovirus vector according to any one of ⁇ 2> to ⁇ 4>, wherein the first foreign gene is at least one of a transgene and a marker gene.
- ⁇ 6> The method for producing a baculovirus vector according to any one of ⁇ 2> and ⁇ 4>, wherein the second foreign gene is at least one of a transgene and a marker gene.
- ⁇ 7> The method for producing a paculovirus vector according to any one of ⁇ 1> to ⁇ 6>, wherein the protein that can be expressed on the cell surface is a protein that is incapable of infecting insect cells.
- ⁇ 8> The method for producing a paculovirus vector according to any one of ⁇ 1> to ⁇ 6>, wherein the protein that can be expressed on a cell surface is a protein that can infect insect cells.
- ⁇ 9> Proteins that can be expressed on the cell surface after the co-transfection step A cell expressing a plasmid containing a gene encoding baculovirus is infected with the first pseudotyped baculovirus generated by the cotransfection step, the virus is amplified, and the second pseudotyped baculovirus is amplified.
- the paculovirus DNA is a recombinant baculovirus DNA deficient in the gp64 gene, and the cotransfection step further comprises the step of cotransfecting a vector having an introduced gene. This is a method for producing a culovirus vector.
- Insect cells that transiently express a protein that can be expressed on the cell surface on the cell surface are infected with a pac64 virus deficient in the gp64 gene, which has the gp64 protein covered on the particle surface, and is infected with the cell surface.
- a method for producing a Bacchus virus vector comprising at least a step of causing a virus particle covered with a highly expressible protein to germinate from the cell surface.
- ⁇ 12> A paculovirus vector produced by the method for producing a paculovirus vector according to any one of ⁇ 1> to ⁇ 11>.
- Baculovirus is a pseudotyped virus in which the DNA of the paculovirus lacks the gp64 gene and is covered with a protein that cannot infect insect cells.
- Baculovirus virus is a pseudotyped virus that lacks the gp64 gene and has a protein that can infect insect cells, and that the protein is not a protein expressed from the baculovirus DNA. It is a characteristic paculovirus vector.
- ⁇ 15> The baculovirus vector according to any one of ⁇ 12> to ⁇ 14>, wherein the gene can be specifically introduced by selecting cells.
- ⁇ 16> Using the baculovirus vector according to any one of ⁇ 12> and ⁇ 15> to introduce a gene into any cell in a living body (excluding human) and outside the living body A gene transfer method characterized by the following. BRIEF DESCRIPTION OF THE FIGURES
- FIG. 1 is a diagram illustrating the infection of baculovirus polyhedra into insect cells.
- Figure 2 shows the life cycle of baculovirus.
- FIG. 3 is a diagram showing a conventional recombinant vector.
- FIG. 4 is a diagram showing a conventional exchange vector.
- FIG. 5 is a diagram showing the production of a new pseudotyped baculovirus.
- FIG. 6 is a diagram showing the production of a novel pseudotyped paculovirus.
- FIG. 7 is a diagram showing the production of a novel pseudotyped PacuMouth virus.
- FIG. 8 is a diagram showing production of a gp64 gene knockout vector.
- FIG. 9 is a diagram showing the production of PacuMouth virus AcA64 / GFP / LacZ in which the gp64 gene has been deleted.
- FIG. 10 shows the construction of a plasmid capable of transiently expressing gp64 protein in insect cells.
- FIG. 11 is a diagram showing the production of a gp64 gene-deficient virus Ac ⁇ 64 / GFP / LacZ * 64 transiently covered with a gp64 protein.
- FIG. 12 is a diagram showing amplification of AcA64 / GFP / LacZ * 64.
- FIG. 13 is a diagram showing the production of Ac ⁇ 64 / GFP / LacZ * 64 having a luciferase gene transiently covering the gp64 protein.
- FIG. 14 is a diagram showing the production of pseudotype virus covered with the vesicular stomatitis virus G protein.
- FIG. 15 is a diagram showing the properties of pseudotyped viruses covered with the vesicular stomatitis virus G protein.
- FIG. 16 is a diagram showing gene transfer by pseudotyped virus covered with the vesicular stomatitis virus G protein.
- Figure 1 7 is the best mode for carrying out the c invention is a diagram illustrating a result of neutralization test of shea user de type Virus suffered vesicular mouth ⁇ quality Overview of construction of targeting vector
- PI inositol cell surface
- coat protein retains infectivity to insect cells, and the type of coat protein is limited. Only proteins with universal infectivity could be selected.
- the method for producing a baculovirus vector of the present invention comprises the steps of: codifying an insect cell with at least a plasmid containing a gene encoding a protein that can be expressed on the cell surface and any one of wild-type, mutant and recombinant baculovirus DNA.
- a pseudo-type baculovirus is produced, which comprises a transfection step of transfecting and which comprises at least a part of the DNA of the paculovirus and which is covered with a protein that can be expressed on the cell surface.
- pseudotyped baculovirus refers to the Paculovirus.
- a protein having a protein not derived from the viral DNA of the virus on the virus surface refers to a virus covered with a protein that is transiently expressed by a separately introduced plasmid.
- the term “transiently expressed” refers to expression without being integrated into viral DNA.
- the protein that can be expressed on the cell surface may be a protein that cannot infect insect cells or a protein that can infect insect cells.
- the term “protein capable of infecting insect cells” refers to a protein that, when expressed on a paculovirus envelope, enables the virus to enter insect cells. When this vector is used as it is for transfection into humans, etc., it is not necessary to infect insect cells.However, when this vector is used as an intermediate for the production of the final vector, it is propagated by infecting insect cells. To be able to infect insect cells.
- proteins that can infect insect cells include the gp64 protein, the viral envelope protein of Vircus, and the G protein of vesicular stomatitis virus.
- the protein that cannot be infected to insect cells is not particularly limited as long as it is a protein that can be expressed on the cell surface, but ligands / receptors corresponding to receptors or antigens specific to cells or tissues to which genes are to be introduced in vivo.
- a molecule capable of recognizing a cancer-specific antigen or a cell surface molecule different from a normal cell, a receptor molecule for an antigen specifically expressed in a virus-infected cell such as AIDS virus, and the like can be mentioned.
- insect cells to be transfected are not particularly limited, but Sf9 cells can be suitably used.
- the plasmid containing the gene encoding the protein that can be expressed on the cell surface is appropriately selected from known plasmids, and the gene encoding the desired protein is selected. It can be manufactured by purchasing. For example, pIB / g P 64 of the following examples, pA3Fb / gp64, and the like.
- the promoter in the plasmid is preferably a promoter that is well expressed in insect cells other than the gp64 promoter, and an actin promoter is particularly preferable.
- the baculovirus DNA may be any of wild-type, mutant and recombinant baculovirus virus DNA, but the baculovirus DNA is a recombinant baculovirus DNA, It is preferable that the DNA is a baculovirus virus that has undergone homologous recombination with a foreign gene and homologous recombination between the gp64 protein and a second foreign gene.
- the baculovirus DNA is preferably a recombinant baculovirus DNA, and is preferably a baculovirus DNA in which homologous recombination between a polyhedrin gene and a first foreign gene has been performed.
- Ekushiyon step simultaneously co-transfected Hue with a vector comprising a second foreign gene, it is preferable that the homologous recombination with the gp 6 4 protein and a second foreign gene takes place.
- the homologous recombination foreign gene is preferably either a transgene which is a target gene to be introduced into a target cell, or a marker gene which is used as an indicator for isolation or introduction. More preferably, the foreign gene is a transgene. It is preferable that the homologous and transformed foreign gene has a promoter that is well expressed in insect cells such as a polyhedron promoter. However, when the final vector is adapted to a host, the promoter of the introduced gene should be a host. Use a promoter that is expressed in.
- CAG promoter consisting of a chicken] 3-actin promoter and CMV enhancer
- the CMV promoter, the RSV promoter and the like are preferred.
- the foreign gene to be introduced is not particularly limited as long as it can be expressed by the promoter, but from the viewpoint of usefulness, defective genes involved in various genetic diseases, cytokins, neurotrophic factors, It is preferably a non-self antigen gene, a nucleotide sequence encoding a virus antigen, etc., a tumor suppressor gene, an antisense sequence such as Ras which is a cancer gene, or a suicide gene such as thymidine kinase.
- the method for producing a pseudotyped baculovirus of the present invention further comprises the step of, after the cotransfection step, adding to the cell in which a plasmid containing a gene encoding a protein that can be expressed on a cell surface is expressed by the cotransfection step.
- the method further comprises the step of infecting the produced insect with the infectious first pseudotyped paculovirus, amplifying the virus, and generating the second pseudotyped paculovirus. It is preferable in that it can be easily amplified.
- a method for producing a paculovirus vector includes a plasmid containing a gene encoding a protein that can be expressed on the cell surface, a recombinant baculovirus DNA lacking the gp64 gene, and a transgene.
- the gP 64 recombinant baculovirus DNA lacking a gene is preferably a recombinant Bakyurowi Luz DNA having two types of genes containing polyhedra promoter and marker downstream.
- a method for producing a paculovirus vector provides a method for producing a pac64 virus, in which a gp64 protein is coated on a particle surface, in an insect cell that transiently expresses a protein that can be expressed on the cell surface on the cell surface.
- the method is characterized by including at least a step of infecting the deficient paculovirus and germinating virus particles covered with a protein that can be expressed on the cell surface from the cell surface.
- insect cells that transiently express a protein that can be expressed on the cell surface are referred to as insect cells expressing the protein on a plasmid, and a plasmid encoding the protein.
- insect cells expressing the protein on a plasmid and a plasmid encoding the protein.
- the method for producing a baculovirus vector comprises, before the step, a step of producing a paclovirus deficient in the gp64 gene in which the gp64 protein is coated on the surface of a particle; an insect cell in which the gp64 protein is transiently expressed.
- the method may further comprise the step of transfecting viral DNA in which the gp64 gene has been deleted to germinate a baculovirus having the gp64 protein on the particle surface for only one generation.
- the baculovirus vector of the present invention is the baculovirus vector of the present invention. It is manufactured by one manufacturing method.
- a transfer vector incorporating the gene to be expressed, infectious virus DNA, and in the case of the present invention, a plasmid are simultaneously introduced into Sf9 cells (0.8 ⁇ 10 6 cells / 35 mm dish) at the same time. And dilute the culture supernatant on day 4 to form plaques on a 35 mm dish. Suspend it in 40 Om1 culture solution, vortex to elute the virus from agar, and centrifuge to collect the supernatant. This is inoculated transiently into Sf cells that have expressed the envelope protein, cultured again, and the supernatant is removed to obtain a white virus pellet. Plasmid is preferably introduced into Sf9 cells (1 ⁇ 10 6 cells / 35 mm) at about 6 ⁇ l of plasmid DNA 2 ⁇ transfection reagent (UniFector, Bridge, etc.).
- paculovirus infects mammalian cells and introduces the gene into the nucleus, but does not propagate in the cell. Accordingly, high expression in order to obtain the not high purified infectivity of the virus (1 X 1 0 9 - 1 0 pfu / ml) was prepared, and it is necessary to introduce a gene of high-copy.
- the Pakyurowirusu lacking the gp64 gene suffered the g P 6 4 protein on the particle surface was expressed transiently the plasmid with the desired protein This can be performed by infecting cells, and can be performed by, for example, the following known methods except that the plasmid is expressed.
- a purified virus of 0.2 ⁇ 10 10 pfu / m 1 can be obtained from a culture supernatant of 250 to 500 ml.
- the baculovirus vector according to another embodiment of the present invention has a baculovirus DNA lacking the gp64 gene, that the Shiyudotaipu virus suffered infected non protein insect cells characterized, for example, a gp 6 4 gene containing gp 6 4 promoter region, the region of the polyhedrin Yadenko each polyhedron promoter region
- the origin of the protein that cannot infect the insect cells is not limited thereto.
- Another baculovirus vector of the present invention is a pseudotyped virus in which the paculovirus DNA lacks the gp64 gene and has a protein capable of infecting insect cells, and the protein is the paculovirus.
- the baculovirus vector is a vector that transiently covers the gp64 protein without a transgene, such as AcA64 / GFP / LacZ * 64 (FIGS. 11 and 12) described below. Or a transgene such as AcA64 / GFP / CAGluc * 64 (Fig. 13), which transiently covers the gp64 protein, or a transgene.
- Te a protein that can be expressed on the cell surface other than g p64 protein, an infectious protein capable insect cells may be those which target one transiently.
- the pseudotyped paculovirus vector coated with a protein capable of infecting insect cells can be used for propagation and stock of the baculovirus vector in insect cells.
- the pseudotyped baculo-mouth virus vector which is coated with a protein that cannot infect insect cells, is used as a final vector for introducing a gene into any cell in vivo or in vitro, including humans. Can be used.
- the paculovirus vector When the paculovirus vector is administered to a living body, it is administered orally or parenterally (for example, intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal injection, rectal administration, transmucosal administration, Administration via the respiratory tract or into organs.
- parenterally for example, intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal injection, rectal administration, transmucosal administration, Administration via the respiratory tract or into organs.
- the dose and the number of doses can be appropriately adjusted according to the weight of the subject to be administered and the transgene.
- the gene transfer method of the present invention is characterized by introducing a gene into any of cells in a living body (excluding humans) and outside a living body using the baculovirus vector of the present invention.
- Gene transfer into mammalian cells can be performed in the same manner as a normal viral vector.
- a mammalian cell into which a gene is to be introduced is prepared on a plate or dish in a state of 60 to 80% confluent.
- the method of infection is the same as for general virus immunization.
- the culture supernatant is removed, and the recombinant virus is inoculated at an infectious titer (moi) of 10 to 100 per cell and adsorbed for 30 to 60 minutes.
- moi infectious titer
- the paculovirus may be inactivated by trapping in the serum, it is preferable to inactivate the fetal calf serum in the culture solution in advance.
- the baculovirus vector of the present invention a method for producing the same, and a method for introducing a gene using the same will be described in more detail with reference to Examples, but the present invention is not limited thereto.
- gp64 gene region Pakyu port virus (AcNPV) EcoRI- Smal fragment (nt 107, 325-1 12, 049 ) and incorporated into the same restriction enzyme site of pUC18, to produce a P UCgp641ocus.
- the transfer vector pAcGFP—CAGluc (Tani et al.), which incorporates the luciferase gene downstream of the GFP gene and the CAG promoter developed by Takasaki et al. (Niwa et al., 1986) downstream of the polyhedron promoter.
- the virus Ri we are deficient in g P 64 gene and its promoter region, in order to completely deficient in the envelope protein, do not show the infectious at all. Therefore, the gp64 protein is transiently overlaid on Ac ⁇ 64 / GFP / LacZ so that it can be infected only once.
- gp64 protein of a plasmid capable of expression in insect cells transiently insect Sf - 9 for intracellularly to produce a plasmid capable of expressing the gp64 protein
- Spel the gp64 gene from pUCg P 641ocus and Bglll in's a cut, built-in PUC18 cut the fragment with the same restriction enzyme, to prepare a pUCg P 64.
- Furthermore fragment excised with Hindlll and EcoRI containing the gp64 gene from pUCg P 64, built in pIB / V5-His were cleaved with the same enzyme (Invitrogen n), to prepare the expression vector pIB / gp64.
- P IB / V5- His Baculovirus Genes inserted downstream of the early gene promoter can be expressed efficiently in insect cells.
- IE promoter early gene promoter
- a similar expression vector using an actin promoter that is highly transcribed in insect cells was prepared.
- gp64-Fw Bgl: MAGATCTACCatggtaagcgctattgttt (SEQ ID NO: 1)
- gp64-Rv Sal
- TTGTCGACttaatat tgtctattacggttt SEQ ID NO: 2 were used.
- the amplified gp64 gene was cut with Bglll and Sail, and integrated into the BamHI and Sail sites of pA3Fb to produce pA3Fb / gp64.
- the above-described infectious virus DNA of AcLacZ, pUCA64 / GFP, and pIB / gp64 or pA3Fb / gp64 are co-transfected into Sf-9 cells.
- homologous recombination occurs between the viral DNA and pUCA64 / GFP in the nucleus of the insect cell, and the recombinant virus Ac ⁇ 64 / in which the gp64 gene region of the viral DNA has been replaced with the polyhedron motor and GFP gene.
- GFP / LacZ occurs.
- virus lacking the promoter region and gp64 genes since that is similar able to grow in normal insect cells co-transfected Hue transfected with the pIB / gp64 or PA3Fb / gp64 more transformer g p64 protein is provided to the cell surface, Despite deficiency in the gp64 gene, budding can occur while retaining infectivity by covering the gp64 protein.
- the recombinant virus can be distinguished from the parent AcLacZ by using the expression of GFP as an index. That is, the recombinant virus expresses both LacZ and GFP.
- the actual virus isolation was performed by homogenization and reintroduction of the culture supernatant 4 days after transfection and Sf-9 cells transfected with pIB / gp64 or pA3Fb / gp64 on the previous day using the usual plaque assay method.
- the plaques positive for both GFP and LacZ were collected and purified by repeating the plaque assay four times, replacing the gp64 gene region with the polyhedron promoter and GFP gene, and transiently causing the gp64 protein To obtain Ac ⁇ 64 / GFP / LacZ * 64 which retained infectivity.
- a firefly luciferase gene a red fluorescent protein (DsRed) gene
- a GFP gene a GFP gene
- the transferred transfer vector, pAcCAGluc, pAcCAGDsRed, and pAcCAGGFP were used. These vectors are designed to cause homologous recombination in the polyhedrin gene region. The production of pAcCAGluc has already been reported (Tani et al., 2001).
- pDsRed2-Nl (Clontech) was digested with Kpnl, and the DsRed gene was recovered and incorporated into pAcCAGMCS2 (Shoji et al., 1997) digested with the same restriction enzymes to produce pAcCAGDsRed.
- pIRES2-EGFP (Clontech) was digested with Ncol, the fragment containing GFP was blunted with klenow fragment, cut with Smal and Notl, and incorporated into pAcCAGMCS2 whose ends were smoothed with klenow fragment to produce pAcCAGGFP.
- Fig. 13 shows the procedure for producing a recombinant virus using pAcCAGluc.
- DNA was extracted from Ac ⁇ 64 / GFP / LacZ * 64, and cut at the unique Bsu36I site present in the LacZ gene to linearize the virus DNA.
- This linear viral DNA, pAcCAGluc, and pIB / gp64 or pA3Fb / gp64 were co-transfected into Sf-9 cells, and GFP-positive and LacZ-negative plaques were collected in the same manner.
- the black acetic acid was purified four times to prepare a recombinant baculovirus Ac ⁇ 64 / GFP / CAGluc * 64 having a luciferase gene.
- Fig. 14 Preparation of pseudotyped virus covered with vesicular stomatitis virus G protein
- the G protein gene of vesicular stomatitis virus was examined.
- pIB / V5- was a His and pA3Fb the G protein gene of vesicular stomatitis virus to produce a ⁇ off to pIB / VSVG ⁇ Pi P A3Fb / VSVG.
- the pseudotyped virus Ac ⁇ 64 / GFP / CAGluc * VSVG transiently covered with the vesicular stomatitis virus G protein is the pseudotyped virus Ac ⁇ 64 / GFP transiently covered with the gp64.
- / CAGluc * 64 it was found that various mammalian cells were efficiently infected.
- FIG. 16 shows that the infection titers (moi) per cell (moi) of Ac T64 / GFP / CAGluc * 64 and Ac 6464 / GFP / CAGluc * VSVG were inoculated to 293Tcell (manufactured by ATCC), respectively. Represents the result of inoculation.
- the culture supernatant was removed, the virus was inoculated, and the cells were adsorbed for 60 minutes.
- the cells are collected 24 to 48 hours after infection, and the value of luciferase gene expression is measured using a Bright-Glo lucit'erase Assay system (Promega).
- the virus vector and the antibody were mixed at the dilution concentration shown on the horizontal axis of the figure, pre-incubated for 1 hour at room temperature, and then infected with 293Tcel 1 cells as described above to measure expression. did.
- the concentration of the antibody in the virus vector is 1: 1.
- the vesicular stomatitis virus can be used without using the infectivity of the G protein to insect cells at all. It has been found that the baculovirus virus vector that has been covered can be obtained by multiplication. Therefore, it was demonstrated that the protein of the desired envelope can be freely applied regardless of whether the insect cell is infected. Also, according to this method, after infecting the cell into which the gene is to be introduced, the envelope protein is not expressed again, and a highly safe PacuMouth virus vector can be prepared.
- a paculovirus expressing two proteins on the particle surface construction of a paculovirus with two envelope proteins that define the host specificity of measles virus
- Measles is a highly contagious acute viral infection that kills 100,000 people each year, especially in developing countries.
- Measles virus has two envelope proteins, H and F, on the particle surface, and the H protein regulates the tissue specificity of the infection. That is, the measles virus strain (Edmonston strain) passaged in the laboratory can use CD46 or SLAM (Signaling lymphocyte activating molecule; CDwl50) as a receptor, whereas fresh isolates from the field only use SLAM as the receptor. It is clear that. Therefore, recombinant baculoviruses expressing H and F proteins from the Edmonston strain or fresh isolates on the particle surface were prepared, and the ability to reproduce the measles virus infection specificity with the paculovirus was examined. did.
- a plasmid incorporating the measles virus H and F genes derived from the Edmonston strain or a fresh isolate was cotransformed with AcA64 / GFP / CAGluc to obtain AcA64 / GFP. / CAGluc * MV-H / F was prepared.
- the specificity of the infection of these Paculoviruses was tested in a hamster-derived cell line (CH0 cells) expressing CD46 in SLAM. As a result, it was confirmed that the baculovirus virus vector covering the H and F proteins derived from the Edmonston strain infected SLAM-expressing CH0 cells and CD46-expressing CH0 cells.
- baculoviruses covered with H and F proteins derived from fresh isolates were able to transfer genes only to C0 cells expressing SLAM.
- Ac ⁇ 64 / GFP / CAGluc * VSVG a positive control virus, infected all CH0 cell lines
- Ac ⁇ 64 / GFP / CAGluc which lacked the gp64 protein of the negative control virus, showed no infectivity.
- This result indicates that two viral proteins can be displayed on the baculovirus particle surface while retaining biological activity. By presenting the two proteins on the particle surface, more accurate targeting will be possible.
- the viral envelope protein not only the viral envelope protein but also a single receptor antibody of the virus or a single-chain antibody against a cancer antigen can be displayed on the particle surface, and cells or cancer cells that are infected with the virus and express the envelope protein can be obtained. Only by introducing a suicide gene such as thymidine kinase and using it together with a prodrug, it becomes possible to eliminate only the target cells from the living body. In particular, AIDS virus chronically infects and highly expresses envelope proteins on the surface of infected cells.
- a novel paculovirus vector that can solve the above-mentioned various problems in the related art and can display a desired protein on the surface of a virus particle without having to show infectivity to insect cells, It is possible to provide a production method and a gene transfer method using the baculovirus vector.
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JP2004539540A JP4431039B2 (ja) | 2002-09-25 | 2003-09-25 | バキュロウイルスベクター、バキュロウイルスベクター製造方法及び遺伝子導入方法 |
AU2003266628A AU2003266628A1 (en) | 2002-09-25 | 2003-09-25 | Baculovirus vector, method of constructing baculovirus vector and gene transfer method |
EP03798509A EP1548119A4 (en) | 2002-09-25 | 2003-09-25 | BACULAR VIRUS VECTOR, METHOD OF CONSTRUCTING THE BACULAR VIRUS VECTOR AND GENTRANSFER METHOD |
US11/086,372 US7416890B2 (en) | 2002-09-25 | 2005-03-23 | Baculovirus vector, method of producing thereof and method of gene transfer |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2363463A1 (en) | 2006-02-09 | 2011-09-07 | Educational Foundation Jichi Medical University | Novel baculoviral vector |
US9327018B2 (en) | 2006-02-09 | 2016-05-03 | Educational Foundation Jichi Medical University | Recombinant baculovirus vaccine |
RU2588464C2 (ru) * | 2006-02-09 | 2016-06-27 | Эдьюкейшнл Фаундейшн Дзити Медикал Юниверсити | Бакуловирусный вектор и его применение |
CN114317608A (zh) * | 2020-12-28 | 2022-04-12 | 陕西杆粒生物科技有限公司 | 一种基因敲除型杆状病毒表达载体 |
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RU2491093C2 (ru) * | 2007-08-07 | 2013-08-27 | Эдьюкейшнл Фаундейшн Дзити Медикал Юниверсити | Бакуловирусные векторы с двойным промотором, включающим в себя промотор позвоночного и промотор бакуловируса, контролирующим иммуногенный слитый ген |
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DE19735593A1 (de) * | 1997-08-15 | 1999-02-18 | Hepavec Ag Fuer Gentherapie | Hüllprotein-modifizierter Baculovirus-Vektor für die Gentherapie |
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US6183993B1 (en) * | 1996-09-11 | 2001-02-06 | The General Hospital Corporation | Complement-resistant non-mammalian DNA viruses and uses thereof |
IL128769A0 (en) * | 1996-09-11 | 2000-01-31 | Gen Hospital Corp | Expression of an exogenous gene by us of a non-mammalian dna virus |
WO2002014527A2 (en) * | 2000-08-11 | 2002-02-21 | Boyce Thompson Institute For Plant Research, Inc. | Gp64-null baculoviruses pseudotyped with heterologous envelope proteins |
WO2002072853A1 (en) * | 2001-03-12 | 2002-09-19 | Ark Therapeutics Limited | Avidin-pseudotyped viral vectors and their use |
-
2003
- 2003-09-25 AU AU2003266628A patent/AU2003266628A1/en not_active Abandoned
- 2003-09-25 JP JP2004539540A patent/JP4431039B2/ja not_active Expired - Lifetime
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DE19735593A1 (de) * | 1997-08-15 | 1999-02-18 | Hepavec Ag Fuer Gentherapie | Hüllprotein-modifizierter Baculovirus-Vektor für die Gentherapie |
Non-Patent Citations (5)
Title |
---|
MANGOR, J. T. ET AL.: "A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein", J. VIROL, vol. 75, no. 6, 2001, pages 2544 - 2556, XP002209173 * |
OOMENS, A. G. ET AL.: "Requirement for GP64 to drive efficient budding of autographa californica multicapsid nucleopolyhedrovirus", VIROLOGY, vol. 254, no. 2, 1999, pages 297 - 314, XP004440038 * |
R. W. OLD ET AL., TRANSLATED BY MUTSO SEKIGUCHI ET AL.: "idenshi sosa no genri gensho 5th edition", 10 June 2000, BAIFUKAN CO., LTD., pages: 330 - 333, XP002977423 * |
See also references of EP1548119A4 * |
TANI, H. ET AL.: "Characterization of cell-surface determinants important for baculovirus infection", VIROLOGY, vol. 279, no. 1, 2001, pages 343 - 353, XP002973847 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2363463A1 (en) | 2006-02-09 | 2011-09-07 | Educational Foundation Jichi Medical University | Novel baculoviral vector |
EP2363462A1 (en) | 2006-02-09 | 2011-09-07 | Educational Foundation Jichi Medical University | Novel baculoviral vector |
US9023365B2 (en) | 2006-02-09 | 2015-05-05 | Educational Foundation Jichi Medical University | Recombinant baculovirus vaccine |
US9327018B2 (en) | 2006-02-09 | 2016-05-03 | Educational Foundation Jichi Medical University | Recombinant baculovirus vaccine |
US9333249B2 (en) | 2006-02-09 | 2016-05-10 | Educational Foundation Jichi Medical University | Recombinant baculovirus vaccine |
RU2588464C2 (ru) * | 2006-02-09 | 2016-06-27 | Эдьюкейшнл Фаундейшн Дзити Медикал Юниверсити | Бакуловирусный вектор и его применение |
CN114317608A (zh) * | 2020-12-28 | 2022-04-12 | 陕西杆粒生物科技有限公司 | 一种基因敲除型杆状病毒表达载体 |
CN114317608B (zh) * | 2020-12-28 | 2023-08-22 | 陕西杆粒生物科技有限公司 | 一种基因敲除型杆状病毒表达载体 |
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EP1548119A4 (en) | 2006-08-02 |
JP4431039B2 (ja) | 2010-03-10 |
JPWO2004029259A1 (ja) | 2006-01-26 |
AU2003266628A1 (en) | 2004-04-19 |
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