KR101055305B1 - Method for producing a virus containing two or more target genes simultaneously - Google Patents

Abstract

The present invention provides two or more recombinant plasmids obtained by recombining two or more target genes into separate herpes simplex virus amplicon plasmids, together with fHSVΔpacΔ27 0+ and pEBHICP27. It provides a method for producing a virus containing two or more target genes at the same time, comprising the step of co-transduction and packaging.

Herpes Simplex Virus Amplicon Plasmid

Description

Method for producing viruses co-packaged with two or more target genes}

The present invention relates to a method for producing a virus containing two or more target genes simultaneously, and more particularly, by recombining two or more target genes into a separate herpes simplex virus amplicon plasmid. The present invention relates to a method for producing a virus containing two or more target genes simultaneously, the method comprising co-transduction and packaging of two or more recombinant plasmids obtained into recipient cells.

Herpes simplex virus amplicon plasmids (HSV amplicon plasmids) are propagated in bacteria and at least the viral DNA elements necessary for DNA replication origin (oriS) and virion DNA cleavage / packaging ( pac ). (Spaete, RR, and Frenkel, N. (1982) .The herpes simplex virus amplicon: a new eukaryotic defective-virus cloning-amplifying vector. Cell 30, 295-304. ). Unlike other viral vectors of limited size, the HSV amplicon virus vector can package up to 150kb of DNA into a viral capsid, which has a large transgene capacity and allows the production of viral proteins inside the plasmid. The absence of the gene ("gutted vector") has the advantage of not toxic to the host cell. It also has the ability to deliver genes to different types of cells.

In recent years, HSV amplicon plasmid vector can be packaged in the virus capsid without defective Herpes simplex or adeno helper-virus, which is a little contaminated during the production of herpes simplex virus. Raefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y., Wild, P., and Geller, AI (1996). virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.Journal of virology 70, 7190-7197; Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, XO, and Chiocca, EA (2001 Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.Mol Ther 3, 591-601; Saeki, Y., Ichikawa, T., Saeki, A., Chiocca, EA, Tobler, K., Ackermann, M., Breakefield, XO, and Fraefel, C. (1998) .Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors.Human gene therapy 9, 2787-2794; Stavropoulos, T.A., and Strathdee, C.A. (1998). An enhanced packaging system for helper-dependent herpes simplex virus vectors. Journal of virology 72, 7137-7143.

Meanwhile, when introducing two or more gene expression units into one virus (ie, a viral particle) using a conventional viral vector system, two or more genes within one HSV amplicon plasmid Expression units expressing genes (either a cassette or a cassette in which two genes are expressed by one promoter using IRES using two or more expression cassettes of promoter-1 gene-polyA) should be separately introduced into the virus. . However, in this case, when the size of the plasmid, i.e., the expression unit, increases, the ligation becomes difficult, and production is difficult, and when the genes are transferred in various combinations, the gene structure of each combination must be separately produced according to the gene combinations. have.

The present inventors conducted various studies to develop a method for simultaneously delivering two or more genes to a single cell using the HSV amplicon plasmid vector. As a result, when two or more amplicon plasmids expressing each target gene are mixed and co-transfected into the permissive cell line with helper BAC DNA and pEBHICP27 plasmid, they are surprisingly surprising. It was found that target genes present on each other amplicon plasmid can be packaged in one virus at the same time. This can not be explained by the existing theory that even if two or more amplicon plasmids are packaged to produce a virus, only one type of amplicon plasmid is concatenated by a rolling-circle mechanism (see FIG. 2). It is a new discovery.

Accordingly, the present invention provides a method for easily preparing a virus comprising simultaneously packaging amplicon plasmids containing each target gene into recipient cells, i.e., simultaneously containing two or more target genes. For the purpose of

According to one aspect of the present invention, two or more recombinant plasmids obtained by recombining two or more target genes into a separate herpes simplex virus amplicon plasmid, respectively, include fHSVΔpacΔ27 0+ and Provided is a method for the convenient production of a virus containing two or more target genes simultaneously, comprising co-transduction and packaging of the recipient cells with pEBHICP27.

The herpes simplex virus amplicon plasmid may have a nucleotide sequence of SEQ ID NO: 1, the target genes may be two or more reverse differentiation inducing genes, for example two or more selected from the genes of SEQ ID NOs: 2-7 Can be.

According to the present invention, in packaging the recombinant plasmid recombinant the target gene with fHSVΔpacΔ27 0+ and pEBHICP27 by co-transduction into a recipient cell, when the recombinant plasmid is two or more species, that is, two or more recombinant plasmids When packaging was performed in the presence, it was found that the target genes contained in each recombinant plasmid were simultaneously introduced into the virus. This suggests that even if a virus is produced by packaging two or more amplicon plasmids, only one amplicon plasmid is introduced into the viral capsid by a rolling-circle mechanism mechanism (see FIG. 2). Given that, it is very surprising. Therefore, the virus production method of the present invention can be usefully used for virus production in which genes encoding two or more kinds of reverse differentiation inducing factors are introduced.

The present invention accommodates two or more recombinant plasmids obtained by recombining two or more target genes into separate herpes simplex virus amplicon plasmids together with fHSVΔpacΔ27 0+ and pEBHICP27. Provided is a method of preparing a herpes simplex virus amplicon virus simultaneously containing two or more target genes, the method comprising co-transduction and packaging into cells.

The preparation method according to the present invention provides a method for preparing a herpes simplex virus comprising two or more target genes simultaneously, Saeki et al. The developed helper virus-free system is used (see FIG. 1). In this system, the entire HSV-1 genome, except for the pac (packaging) signal, is placed in a bacterial artificial chromosome (BAC) (fHSVΔpac △ 27 0+), and the ICP27 gene is removed from the BAC DNA for safety. It is provided as plasmid pEBHICP27. Co-transfection of these three plasmids into permissive cell lines, such as vero 2-2 cells, results in the gene products necessary for the production of HSV particles produced from BAC DNA and pEBHICP27 plasmids. Due to the structure of the HSV particles, the amplicon DNA is concatenated by the rolling circle mechanism to fill the capsid until the amplicon DNA reaches 150 kb, which is the capacity of the HSV capsid. (See Figure 2). The production method of the present invention is described in Saeki et al. In a non-helper virus system such as two or more amplicon DNAs, that is, two or more kinds of recombination obtained by recombining two or more target genes into a separate Herpes simplex virus amplicon plasmid, respectively. Co-transduction of the plasmids is based on new findings that viruses are produced that contain two or more target genes simultaneously.

In the production method of the present invention, the target gene is not particularly limited, and includes all genes to be introduced into a host cell through a virus as a medium. Preferably, genes encoding reprogramming-inducing factors involved in reprogramming of differentiated cells are included. The reverse differentiation inducing factor may be a gene encoding the previously known reverse differentiation factor, that is, Sox2, Oct3 / 4, Nanog, Klf4, Lin28, or Myc, each having a nucleotide sequence of SEQ ID NOs: 2-7. Those skilled in the art will recognize that various factors known or known to be involved in inducing differentiation, as well as Sox2, Oct3 / 4, Nanog, Klf4, Lin28, c-Myc described above, can be applied without limitation to the preparation method according to the present invention. will be.

The recombinant plasmid may be obtained by recombining two or more target genes into a separate herpes simplex virus amplicon plasmid, preferably a herpes simplex virus amplicon plasmid having a nucleotide sequence of SEQ ID NO: 1. The recombination may be performed by cloning the target gene by conventional genetic engineering methods using appropriate restriction enzymes such as NheI, NotI, BamHI, BglII, XbaI and EcoRI.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, the scope of the present invention is not limited to these examples.

Example

As a model gene, a gene encoding GFP (green fluorescence protein) (SEQ ID NO: 8) and a gene encoding DsRed2 (red fluorescence protein) (SEQ ID NO: 9) were selected.

PHGCX, a herpes simplex amplicon plasmid (Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, XO, and Chiocca, EA (2001) .Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27 -deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.In Mol Ther 3, 591-601, the gene encoding GFP is already attached after the herpes simplex virus immediate early promoter (IE4 / 5 promoter). As a recombinant plasmid expressing GFP.

pHGCX was cut out of GFP by double treatment of NruI and NotI restriction enzymes, and vector was prepared by CIP (calf intestinal alkaline phosphatase) treatment. The DsRed2 gene on pDsRed2 (Invitrogen) was then ligated with a vector cut and digested with SmaI and NotI restriction enzymes to encode DsRed2 followed by the herpes simplex virus immediate early promoter (IE4 / 5 promoter). Located recombinant plasmid (pHSVamp-DsRed2) was constructed.

Saeki, Y. et al. (Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, XO, and Chiocca, EA (2001). system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.Mol Ther 3, 591-601, modified to produce Vero 2-2 cells: African green monkey kidney packaging was performed co-transformed into cells; from Dr. Rozanne Sandri-Goldin, University of California, Irvine, CA, USA. That is, in a 60 mm cell culture container, pHGCX (0.116 ug), pHSVamp-DsRed2 (1.392 ug), pEBHICP27 (0.07 ug), and helper-BAC DNA (fHSVΔPACΔ27 0+; 0.698 ug) were mixed and FugeneHD 8: 2 After mixing together with Opti-MEM after 15 minutes at room temperature was evenly dropped on Vero 2-2 cells. After incubating the cells for 4 hours at 37 ° C, DMEM supplemented with 3 ml of 6% FBS was added and allowed to produce HSV amplicon virus for 60 hours. Cell scrapers were used to scrape the cells and freeze and thaw three times in a dry ice / ethanol bath and a water bath at 37 ° C., allowing the HSV amplicon viruses in the cells to be released into the medium. The culture was centrifuged at 2000 x g to obtain supernatant except cell precipitate. 2-3 days after the virus obtained in the medium was infected with G16-9 cells (human glioblastoma; Glioblastoma; Gli36 derived cell line tnat express HSV-1 VP 16 constitutuvely; widely available from many labs) The number of cells expressing fluorescent proteins (GFP and DsRed2) was then measured. The result is shown in FIG. 3. As can be seen in Figure 3, it can be seen that about 80% of cells infected with the virus prepared by the production method according to the present invention commonly express GFP and DsRed2 in one cell.

Comparative example

The recombinant plasmid expressing GFP and the recombinant plasmid expressing DsRed2 obtained in Example 1 were separately packaged in the same manner as in Example 1, and the obtained viruses were mixed and infected with G16-9 cells to fluorescein proteins (GFP and DsRed2) expression was measured. The result is shown in FIG. 4. As can be seen in Figure 4, only about 2.6% of cells can be seen that the common expression of GFP and DsRed2 in one cell.

FIG. 1 is a schematic diagram illustrating a process for preparing an amplicon vector through a virus-free helper system using fHSVΔpacΔ27 0+ and pEBHICP27 system.

FIG. 2 is a schematic diagram illustrating a process in which an amplicon vector is produced by concatenating multiple copies of an amplicon plasmid concatenate by a rolling circle mechanism as a conventional packaging method.

FIG. 3 shows that GFP-9 and DsRed2 are infected with G16-9 cells by infecting G16-9 cells with a virus obtained by co-transfection of two different amplicon plasmids with units expressing GFP and DsRed2, respectively, together with BAC DNA and pEBHICP27. Indicates the result of measuring the proportion of cells co-expressing in total cells (* P = 0.00042; # P = 0.00242).

FIG. 4 shows that a virus is separately prepared from an amplicon plasmid containing GFP and DsRed2, mixed with a virus expressing GFP and a virus expressing DsRed2, and infected with G16-9 cells to express GFP and / or DsRed2. The result of measuring the ratio of cells is shown (* P = 0.00013; #P = 0.02).

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Method for producing viruses co-packaged with two or more target          genes <130> PN0181 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 6723 <212> DNA <213> herpes simplex virus 7 <400> 1 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 60 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 120 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 180 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata 240 cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg gtaccgagct 300 cggatccact agtccagtgt ggtggaattc tgcagatatc cagcacagtg gcggccgctc 360 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag 420 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 480 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 540 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcgttaat 600 taaaattcca gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt caaaaataat 660 aataaccggg caggccatgt ctgcccgtat ttcgcgtaag gaaatccatt atgtactatt 720 taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt tttatcatgg 780 gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc agcaaaagtg 840 atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa ccgctgtttg 900 gtctgctttc tgacaaactc ggaacttgtt tattgcagct tataatggtt acaaataaag 960 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttg 1020 tccaaactca tcaatgtatc ttatcatgtc tggatctctg acctgagatt ggcggcactg 1080 aggtagagat gcccgaaccc ccccgaggga gcgcgggacg cgccggggag ggctggggcc 1140 ggggagggct ggggccgggg agggctgggg ccggggaggg ctggggccgg ggagggctgg 1200 ggccggggag ggctggggcc ggggagggct ggggctgggg agggctgggg ctggggaggg 1260 ggcggtggtg tgtagcagga gcggtgtgtt gcgccggggt acgtctggag gagcgggagg 1320 tgcgcggtga cgtgtggatg aggaacagga gttgttgcgc ggtgagttgt cgctgtgagt 1380 tgtgttgttg ggcaggtgtg gtggatgacg tgacgtgtga cgtgcggagt gcgccgtgct 1440 ctgttggttt cacctgtggc agcccgggcc ccccgcgggc gcgcgcgcgc gcaaaaaagg 1500 cgggcggcgg tccgggcggc gtgcgcgcgc gcggcgggcg tggggggcgg ggccgcggga 1560 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga 1620 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga 1680 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga 1740 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ccagacgccg aaaacgggcc 1800 ccccccaaaa cacacccccc gggggtcgcg cgcggccctt taaagcggtg gcggcgggca 1860 gcccgggccc cccgcggccg agactagcga gttagacagg caagcactac tcgcctctgc 1920 acgcacatgc ttgcctgtca aactctacca ccccggcacg ctctctgtct ccatggcccg 1980 ccgccgccgc catcgcggcc cccgccgccc ccggccgccc gggcccacgg gcgccgtccc 2040 aaccgcacag tcccaggtaa cgagctcgaa ttaattcttg aagacgaaag ggcctcgtga 2100 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagggt cgacccaggt 2160 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 2220 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 2280 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 2340 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 2400 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 2460 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 2520 ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 2580 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 2640 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 2700 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 2760 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 2820 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 2880 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 2940 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 3000 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 3060 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 3120 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 3180 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 3240 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 3300 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 3360 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 3420 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 3480 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 3540 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 3600 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 3660 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 3720 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 3780 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 3840 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 3900 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 3960 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 4020 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 4080 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 4140 ataggtcgac ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 4200 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 4260 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4320 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 4380 cagccggcca ttatgcacga ccccgccccg acgccggcac gccgggggcc cgtggccgcg 4440 gcccgttggt cgaacccccg gccccgccca tccgcgccat ctgccatggg cggggcgcga 4500 gggcgggtgg gtccgcgccc cgccccgcat ggcatctcat taccgcccga tccggcggtt 4560 tccgcttccg ttccgcatgc taacgaggaa cgggcagggg gcggggcccg ggccccgact 4620 tcccggttcg gcggtaatga gatacgagcc ccgcgcgccc gttggccgtc cccgggcccc 4680 ccggtcccgc ccgccggacg ccgggaccaa cgggacggcg ggcggcccaa gggccgcccg 4740 ccttgccgcc cccccattgg ccggcgggcg ggaccgcccc aagggggcgg ggccgccggg 4800 taaaagaagt gagaacgcga agcgttcgca cttcgtccca atatatatat attattaggg 4860 cgaagtgcga gcactggcgc cgtgcccgac tccgcgccgg ccccgggggc gggcccgggc 4920 ggcggggggc gggtctctcc ggcgcacata aaggcccggc gcgaccgacg cccgcagacg 4980 gcgccggcca cgaacgacgg gagcggctgc ggagcacgcg gaccgggagc gggagtcgca 5040 gagggccgtc ggagcggacg gcgtcggcat cgcgacgccc cggctcggga tcgggatcgc 5100 atcggaaagg gacacgcgga cgcggggggg aaagacccgc ccaccccacc cacgaaacac 5160 aggggacgca ccccgggggc ctccgacgac agaaacccac cggtccgcct tttttgcacg 5220 ggtaagcacc ttgggtgggc ggaggagggg gggacgcggg ggtggaggag gggggacgcg 5280 ggggcggagg aggggggacg cgggggcgga ggagggggga cgcgggggcg gaggaggggg 5340 gacgcggggg cggaggaggg ggctcacccg cgttcgtgcc ttcccgcagg aggaacgtcc 5400 tcgtcgataa gctgatccat cgccaccatg gtgagcaagg gcgaggagct gttcaccggg 5460 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 5520 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 5580 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 5640 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 5700 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 5760 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 5820 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 5880 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 5940 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 6000 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 6060 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 6120 ctcggcatgg acgagctgta caagtaaagc ggccaacttg tttattgcag cttataatgg 6180 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 6240 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg gtttgaagat 6300 cttccgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 6360 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 6420 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 6480 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggactatt 6540 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 6600 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 6660 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 6720 ttt 6723 <210> 2 <211> 954 <212> DNA <213> Homo sapiens <400> 2 atgtacaaca tgatggagac ggagctgaag ccgccgggcc cgcagcaaac ttcggggggc 60 ggcggcggca actccaccgc ggcggcggcc ggcggcaacc agaaaaacag cccggaccgc 120 gtcaagcggc ccatgaatgc cttcatggtg tggtcccgcg ggcagcggcg caagatggcc 180 caggagaacc ccaagatgca caactcggag atcagcaagc gcctgggcgc cgagtggaaa 240 cttttgtcgg agacggagaa gcggccgttc atcgacgagg ctaagcggct gcgagcgctg 300 cacatgaagg agcacccgga ttataaatac cggccccggc ggaaaaccaa gacgctcatg 360 aagaaggata agtacacgct gcccggcggg ctgctggccc ccggcggcaa tagcatggcg 420 agcggggtcg gggtgggcgc cggcctgggc gcgggcgtga accagcgcat ggacagttac 480 gcgcacatga acggctggag caacggcagc tacagcatga tgcaggacca gctgggctac 540 ccgcagcacc cgggcctcaa tgcgcacggc gcagcgcaga tgcagcccat gcaccgctac 600 gacgtgagcg ccctgcagta caactccatg accagctcgc agacctacat gaacggctcg 660 cccacctaca gcatgtccta ctcgcagcag ggcacccctg gcatggctct tggctccatg 720 ggttcggtgg tcaagtccga ggccagctcc agcccccctg tggttacctc ttcctcccac 780 tccagggcgc cctgccaggc cggggacctc cgggacatga tcagcatgta tctccccggc 840 gccgaggtgc cggaacccgc cgcccccagc agacttcaca tgtcccagca ctaccagagc 900 ggcccggtgc ccggcacggc cattaacggc acactgcccc tctcacacat gtga 954 <210> 3 <211> 1083 <212> DNA <213> Homo sapiens <400> 3 atggcgggac acctggcttc ggatttcgcc ttctcgcccc ctccaggtgg tggaggtgat 60 gggccagggg ggccggagcc gggctgggtt gatcctcgga cctggctaag cttccaaggc 120 cctcctggag ggccaggaat cgggccgggg gttgggccag gctctgaggt gtgggggatt 180 cccccatgcc ccccgccgta tgagttctgt ggggggatgg cgtactgtgg gccccaggtt 240 ggagtggggc tagtgcccca aggcggcttg gagacctctc agcctgaggg cgaagcagga 300 gtcggggtgg agagcaactc cgatggggcc tccccggagc cctgcaccgt cacccctggt 360 gccgtgaagc tggagaagga gaagctggag caaaacccgg aggagtccca ggacatcaaa 420 gctctgcaga aagaactcga gcaatttgcc aagctcctga agcagaagag gatcaccctg 480 ggatatacac aggccgatgt ggggctcacc ctgggggttc tatttgggaa ggtattcagc 540 caaacgacca tctgccgctt tgaggctctg cagcttagct tcaagaacat gtgtaagctg 600 cggcccttgc tgcagaagtg ggtggaggaa gctgacaaca atgaaaatct tcaggagata 660 tgcaaagcag aaaccctcgt gcaggcccga aagagaaagc gaaccagtat cgagaaccga 720 gtgagaggca acctggagaa tttgttcctg cagtgcccga aacccacact gcagcagatc 780 agccacatcg cccagcagct tgggctcgag aaggatgtgg tccgagtgtg gttctgtaac 840 cggcgccaga agggcaagcg atcaagcagc gactatgcac aacgagagga ttttgaggct 900 gctgggtctc ctttctcagg gggaccagtg tcctttcctc tggccccagg gccccatttt 960 ggtaccccag gctatgggag ccctcacttc actgcactgt actcctcggt ccctttccct 1020 gagggggaag cctttccccc tgtctccgtc accactctgg gctctcccat gcattcaaac 1080 tga 1083 <210> 4 <211> 918 <212> DNA <213> Homo sapiens <400> 4 atgagtgtgg atccagcttg tccccaaagc ttgccttgct ttgaagcatc cgactgtaaa 60 gaatcttcac ctatgcctgt gatttgtggg cctgaagaaa actatccatc cttgcaaatg 120 tcttctgctg agatgcctca cacggagact gtctctcctc ttccttcctc catggatctg 180 cttattcagg acagccctga ttcttccacc agtcccaaag gcaaacaacc cacttctgca 240 gagaagagtg tcgcaaaaaa ggaagacaag gtcccggtca agaaacagaa gaccagaact 300 gtgttctctt ccacccagct gtgtgtactc aatgatagat ttcagagaca gaaatacctc 360 agcctccagc agatgcaaga actctccaac atcctgaacc tcagctacaa acaggtgaag 420 acctggttcc agaaccagag aatgaaatct aagaggtggc agaaaaacaa ctggccgaag 480 aatagcaatg gtgtgacgca gaaggcctca gcacctacct accccagcct ttactcttcc 540 taccaccagg gatgcctggt gaacccgact gggaaccttc caatgtggag caaccagacc 600 tggaacaatt caacctggag caaccagacc cagaacatcc agtcctggag caaccactcc 660 tggaacactc agacctggtg cacccaatcc tggaacaatc aggcctggaa cagtcccttc 720 tataactgtg gagaggaatc tctgcagtcc tgcatgcagt tccagccaaa ttctcctgcc 780 agtgacttgg aggctgcctt ggaagctgct ggggaaggcc ttaatgtaat acagcagacc 840 actaggtatt ttagtactcc acaaaccatg gatttattcc taaactactc catgaacatg 900 caacctgaag acgtgtga 918 <210> 5 <211> 1440 <212> DNA <213> Homo sapiens <400> 5 atgaggcagc cacctggcga gtctgacatg gctgtcagcg acgcgctgct cccatctttc 60 tccacgttcg cgtctggccc ggcgggaagg gagaagacac tgcgtcaagc aggtgccccg 120 aataaccgct ggcgggagga gctctcccac atgaagcgac ttcccccagt gcttcccggc 180 cgcccctatg acctggcggc ggcgaccgtg gccacagacc tggagagcgg cggagccggt 240 gcggcttgcg gcggtagcaa cctggcgccc ctacctcgga gagagaccga ggagttcaac 300 gatctcctgg acctggactt tattctctcc aattcgctga cccatcctcc ggagtcagtg 360 gccgccaccg tgtcctcgtc agcgtcagcc tcctcttcgt cgtcgccgtc gagcagcggc 420 cctgccagcg cgccctccac ctgcagcttc acctatccga tccgggccgg gaacgacccg 480 ggcgtggcgc cgggcggcac gggcggaggc ctcctctatg gcagggagtc cgctccccct 540 ccgacggctc ccttcaacct ggcggacatc aacgacgtga gcccctcggg cggcttcgtg 600 gccgagctcc tgcggccaga attggacccg gtgtacattc cgccgcagca gccgcagccg 660 ccaggtggcg ggctgatggg caagttcgtg ctgaaggcgt cgctgagcgc ccctggcagc 720 gagtacggca gcccgtcggt catcagcgtc agcaaaggca gccctgacgg cagccacccg 780 gtggtggtgg cgccctacaa cggcgggccg ccgcgcacgt gccccaagat caagcaggag 840 gcggtctctt cgtgcaccca cttgggcgct ggaccccctc tcagcaatgg ccaccggccg 900 gctgcacacg acttccccct ggggcggcag ctccccagca ggactacccc gaccctgggt 960 cttgaggaag tgctgagcag cagggactgt caccctgccc tgccgcttcc tcccggcttc 1020 catccccacc cggggcccaa ttacccatcc ttcctgcccg atcagatgca gccgcaagtc 1080 ccgccgctcc attaccaaga gctcatgcca cccggttcct gcatgccaga ggagcccaag 1140 ccaaagaggg gaagacgatc gtggccccgg aaaaggaccg ccacccacac ttgtgattac 1200 gcgggctgcg gcaaaaccta cacaaagagt tcccatctca aggcacacct gcgaacccac 1260 acaggtgaga aaccttacca ctgtgactgg gacggctgtg gatggaaatt cgcccgctca 1320 gatgaactga ccaggcacta ccgtaaacac acggggcacc gcccgttcca gtgccaaaaa 1380 tgcgaccgag cattttccag gtcggaccac ctcgccttac acatgaagag gcatttttaa 1440                                                                         1440 <210> 6 <211> 630 <212> DNA <213> Homo sapiens <400> 6 atgggctccg tgtccaacca gcagtttgca ggtggctgcg ccaaggcggc agaagaggcg 60 cccgaggagg cgccggagga cgcggcccgg gcggcggacg agcctcagct gctgcacggt 120 gcgggcatct gtaagtggtt caacgtgcgc atggggttcg gcttcctgtc catgaccgcc 180 cgcgccgggg tcgcgctcga ccccccagtg gatgtctttg tgcaccagag taagctgcac 240 atggaagggt tccggagctt gaaggagggt gaggcagtgg agttcacctt taagaagtca 300 gccaagggtc tggaatccat ccgtgtcacc ggacctggtg gagtattctg tattgggagt 360 gagaggcggc caaaaggaaa gagcatgcag aagcgcagat caaaaggaga caggtgctac 420 aactgtggag gtctagatca tcatgccaag gaatgcaagc tgccacccca gcccaagaag 480 tgccacttct gccagagcat cagccatatg gtagcctcat gtccgctgaa ggcccagcag 540 ggccctagtg cacagggaaa gccaacctac tttcgagagg aagaagaaga aatccacagc 600 cctaccctgc tcccggaggc acagaattga 630 <210> 7 <211> 1365 <212> DNA <213> Homo sapiens <400> 7 ctggattttt ttcgggtagt ggaaaaccag cagcctcccg cgacgatgcc cctcaacgtt 60 agcttcacca acaggaacta tgacctcgac tacgactcgg tgcagccgta tttctactgc 120 gacgaggagg agaacttcta ccagcagcag cagcagagcg agctgcagcc cccggcgccc 180 agcgaggata tctggaagaa attcgagctg ctgcccaccc cgcccctgtc ccctagccgc 240 cgctccgggc tctgctcgcc ctcctacgtt gcggtcacac ccttctccct tcggggagac 300 aacgacggcg gtggcgggag cttctccacg gccgaccagc tggagatggt gaccgagctg 360 ctgggaggag acatggtgaa ccagagtttc atctgcgacc cggacgacga gaccttcatc 420 aaaaacatca tcatccagga ctgtatgtgg agcggcttct cggccgccgc caagctcgtc 480 tcagagaagc tggcctccta ccaggctgcg cgcaaagaca gcggcagccc gaaccccgcc 540 cgcggccaca gcgtctgctc cacctccagc ttgtacctgc aggatctgag cgccgccgcc 600 tcagagtgca tcgacccctc ggtggtcttc ccctaccctc tcaacgacag cagctcgccc 660 aagtcctgcg cctcgcaaga ctccagcgcc ttctctccgt cctcggattc tctgctctcc 720 tcgacggagt cctccccgca gggcagcccc gagcccctgg tgctccatga ggagacaccg 780 cccaccacca gcagcgactc tgaggaggaa caagaagatg aggaagaaat cgatgttgtt 840 tctgtggaaa agaggcaggc tcctggcaaa aggtcagagt ctggatcacc ttctgctgga 900 ggccacagca aacctcctca cagcccactg gtcctcaaga ggtgccacgt ctccacacat 960 cagcacaact acgcagcgcc tccctccact cggaaggact atcctgctgc caagagggtc 1020 aagttggaca gtgtcagagt cctgagacag atcagcaaca accgaaaatg caccagcccc 1080 aggtcctcgg acaccgagga gaatgtcaag aggcgaacac acaacgtctt ggagcgccag 1140 aggaggaacg agctaaaacg gagctttttt gccctgcgtg accagatccc ggagttggaa 1200 aacaatgaaa aggcccccaa ggtagttatc cttaaaaaag ccacagcata catcctgtcc 1260 gtccaagcag aggagcaaaa gctcatttct gaagaggact tgttgcggaa acgacgagaa 1320 cagttgaaac acaaacttga acagctacgg aactcttgtg cgtaa 1365 <210> 8 <211> 749 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 8 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtactaatga 720 ccccgtaatt gattactatt aataactaa 749 <210> 9 <211> 678 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequence <400> 9 atggcctcct ccgagaacgt catcaccgag ttcatgcgct tcaaggtgcg catggagggc 60 accgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120 cacaacaccg tgaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180 ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240 gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300 gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 360 aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 420 atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 480 acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540 tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 600 atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 660 caccacctgt tcctgtag 678  

Claims (4)

Two or more recombinant plasmids obtained by recombining two or more target genes into a separate herpes simplex virus amplicon plasmid, respectively, were co--received together with fHSVΔpacΔ27 0+ and pEBHICP27 to recipient cells. A method for producing a virus containing two or more target genes simultaneously, comprising the steps of transducing and packaging. The method according to claim 1, wherein the herpes simplex virus amplicon plasmid has a nucleotide sequence of SEQ ID NO: 1. The method according to claim 1, wherein the target gene is a gene encoding at least two differentiation induction factors selected from the genes of SEQ ID NOs: 2 to 7. delete

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