[go: up one dir, main page]

WO1995009923A1 - Procede de production de virus eukaryotiques recombines dans des bacteries - Google Patents

Procede de production de virus eukaryotiques recombines dans des bacteries Download PDF

Info

Publication number
WO1995009923A1
WO1995009923A1 PCT/US1993/009408 US9309408W WO9509923A1 WO 1995009923 A1 WO1995009923 A1 WO 1995009923A1 US 9309408 W US9309408 W US 9309408W WO 9509923 A1 WO9509923 A1 WO 9509923A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
bacmid
recited
composite
bacterial
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1993/009408
Other languages
English (en)
Inventor
Stephen C. Lee
Mark Steven Leusch
Verne A. Luckow
Peter O. Olins
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GD Searle LLC
Original Assignee
GD Searle LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by GD Searle LLC filed Critical GD Searle LLC
Priority to PCT/US1993/009408 priority Critical patent/WO1995009923A1/fr
Priority to AU53514/94A priority patent/AU5351494A/en
Publication of WO1995009923A1 publication Critical patent/WO1995009923A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention describes the production of eukaryotic virus shuttle vectors, and a novel method to produce recombinant virus shuttle vectors in bacteria.
  • the recombinant proteins are often expressed at high levels in cultured insect cells or infected larvae and are, in most cases functionally similar to their authentic counterparts (Luckow, 1991; Luckow and Summers, 1988; Maeda, 1989; Miller, 1988; Murhammer, 1991; O'Reilly et al., 1992).
  • AcNPV has a large (130 kb) circular double-stranded DNA (dsDNA) genome with multiple recognition sites for many restriction endonucleases, and as a result, recombinant baculoviruses are traditionally constructed in a two-stage process.
  • dsDNA circular double-stranded DNA
  • nonessential locus usually the polyhedrin gene.
  • This resultant plasmid DNA is called a transfer vector and is introduced into insect cells along with wild-type genomic viral DNA. About 1% of the resulting progeny are recombinant, with the foreign gene inserted into the genome of the parent virus by homologous recombination in vivo . The recombinant virus is purified to
  • plaque assays homogeneity by sequential plaque assays, and recombinant viruses containing the foreign gene inserted into the polyhedrin locus can be identified by an altered plaque morphology characterized by the absence of occluded virus in the nucleus of infected cells.
  • recombinant baculoviruses by standard transfection and plaque assay methods can take as long as four to six weeks and many methods to speed up the identification and purification of recombinant viruses have been tried in recent years. These methods include plaque lifts (Summers and Smith, 1987), serial limiting dilutions of virus (Fung et al., 1988) and cell affinity techniques (Farmer et al., 1989). Each of these methods require confirmation of the recombination event by visual screening of plaque morphology (O'Reilly et al., 1992), DNA dot blot hybridization (Luckow and Summers, 1988), immunoblotting (Capone, 1989), or amplification of specific segments of the baculovirus genome by polymerase chain reaction techniques
  • Co-expression vectors are transfer vectors that contain another gene, such as the lacZ gene, under the control of a second viral or insect promoter
  • viruses form blue plaques when the agarose overlay in a plaque assay contains X-gal, a chromogenic substrate for ⁇ -galactosidase .
  • blue plaques can be identified after 3-4 days, compared to 5-6 days for optimal representation of occlusion minus plaques, multiple plaque assays are still required to purify the virus. It is also possible to screen for colorless plaques in a background of blue plaques, if the parent virus contains the ⁇ -galactosidase gene at the same locus as the foreign gene in the transfer vector.
  • the fraction of recombinant progeny virus that result from homologous recombination between a transfer vector and a parent virus can be also be significantly improved from 0.1-1.0% to nearly 30% by using parent virus that is linearized at one or more unique sites near the target site for insertion of the foreign gene into the baculovirus genome (Kitts et al., 1990).
  • Linear viral DNA by itself is 15- to 150-fold less infectious than the circular viral DNA. A higher proportion of
  • recombinant viruses (80% or higher) can be achieved using linearized viral DNA (Hartig and Cardon, 1992;
  • Kitts, 1992; Kitts, 1992; Kitts et al., 1990 (marketed as BacPAK ⁇ , Clonetech; or as BaculoGold, Pharmingen) that is missing an essential portion of the baculovirus genome downstream from the polyhedrin gene.
  • Peakman et al., (1992) described the use of the Cre-lox sytem of bacteriophage P1 to perform cre-mediated site-specific recombination in vitro between a transfer vector and a modified parent virus that both contain the lox recombination sites. Up to 50% of the viral progeny are recombinant. Two disadvantages of this method are that there can be multiple insertions of the transfer vector into the parent virus, and that multiple plaque assays are still required to purify a recombinant virus.
  • the shuttle vector contains a yeast ARS sequence that permits autonomous replication in yeast, a CEN sequence that contains a mitotic centromere and ensures stable segregation of plasmid DNAs into daughter cells, and two selectable marker genes (URA3 and SUP4-o) downstream from the polyhedrin promoter (P polh ) in the order P Polh SUP4-o, ARS, URA3, and CEN.
  • the transfer vector contains the foreign gene flanked on the 5' end by baculovirus sequences and on the 3' end by the yeast ARS sequence.
  • Recombinant shuttle vectors which lack the SUP4-o gene can be selected in an appropriate yeast strain in the presence of a toxic amino acid analogue. Insect cells transfected with DNA isolated from selected yeast colonies produce virus and express the foreign gene under control of the polyhedrin promoter. Since all of the viral DNA isolated from yeast contains the foreign gene inserted into the baculovirus genome and there is no background of contaminating parent virus, the time-consuming steps of plaque purification are eliminated. With this method, it is possible to obtain stocks of recombinant virus within 10-12 days. Two drawbacks, however, are the relatively low
  • the present invention overcomes many of the
  • bacmid baculovirus shuttle vector
  • the present invention provides a method to produce recombinant eukaryotic viruses in bacterial cells.
  • the invention also relates to a composite shuttle vector, comprising:
  • nonessential locus of said viral DNA which is capable of driving the replication of said viral DNA in bacteria
  • preferential target site which includes heterologous DNA and a second bacterial genetic marker that is different than said first bacterial genetic marker.
  • Figure 1 Schematic outline for the generation of recombinant baculovirus shuttle vectors (bacmids) and site-specific transposon-mediated insertion of foreign genes into the baculovirus genome propagated in E. coli in which the donor plasmid and the helper are
  • FIG. 2 Flow chart for the construction of the bacmid transfer vectors pMON14271 and pMON14272. See text for details.
  • the light gray sections represent baculovirus sequences flanking the polyhedrin promoter in the 7327 bp AcNPV EcoRI fragment I.
  • the dark gray region represents the mini-F replicon derived as a
  • FIG. 3 Flow chart for the construction of the mini-Tn7 donor plasmids. See text for details. The left and right ends of Tn7 and the polyhedrin promoter are indicated by solid areas. The heavy and light dotted areas represent the ⁇ -glucuronidase gene and the SV40 poly(A) termination signals, respectively. The left diagonally-striped section represents a segment
  • Wide hatched regions (SL2nx and SL2 ⁇ b) represent synthetic
  • FIG. 5 SDS-PAGE of 35 S-methionine-labeled proteins expressed by traditional recombinant baculoviruses and composite bacmid vectors. All viral stocks were titered and SF21 cells were infected at a multiplicity of infection of 10. Cells were radiolabeled at 44.5 hours post-infection for 4 hours with 10 ⁇ Ci 35 S-methionine per 6 ⁇ 10 5 cells. The equivalent of 3.75 ⁇ 10 4 infected cells per lane were separated by electrophoresis on a 12% SDS-polyacrylamide gel. The gel was fixed, dried, and exposed to Kodak X-AR film ® for 76 hours at room temperature. The positions of Bio-Rad prestained molecular weight markers and expressed proteins are indicated.
  • VMON14272 Parent bacmid containing mini- F-Kan-IacZ ⁇ -mini-attTn7 in opposite orientation
  • pMON7124 Composite bacmid expressing ⁇ - glucuronidase. DNA originally transfected into insect cells also contained pMON7124 helper plasmid
  • VMON14221 Recombinant virus expressing ⁇ -glucuronidase constructed by classical method of homologous recombination in insect cells
  • VchMON14271 10 VchMON14271: :
  • bacmid A baculovirus shuttle vector capable of replication in bacteria and in susceptible insect cells
  • bacteria refers to any prokaryotic organism capable of supporting the function of the genetic elements described below.
  • the bacteria should support the replication of the low copy number replicon operationally linked to the baculovirus in the bacmid, most preferably mini-F.
  • the bacteria should support the replication of the donor plasmids, preferably moderate or high copy number plasmids or the host genome, most preferably either the bacteria chromosome, plasmids based on pMAK705, or plasmids based on pUC18.
  • the bacteria should support the replication of helper
  • the differentiable markers should confer the ability of cells possessing them to metabolize chromogenic substrates.
  • the differentiable markers should confer the ability of cells possessing them to metabolize chromogenic substrates.
  • baculovirus A member of the Baculoviridae family of viruses with covalently closed double- stranded DNA genome and which are
  • a plasmid containing a wild-type or altered transposon preferably a mini-Tn7 transposon, composed of the left and right arms of Tn7 flanking a cassette containing a genetic marker, a promoter, and the gene of interest.
  • the mini-transposon is on a pUC-based or pMAK705-based plasmid.
  • any replicating double-stranded DNA element such as the bacterial chromosome or a bacterial plasmid which carries a transposon capable of site-specific transposition into a bacmid.
  • the transposon contains a heterologous DNA and a genetic marker
  • locus A specific site or region of a DNA
  • mini-F A derivative of the 100 kb F plasmid which contains the RepFIA replicon, comprised of seven proteins including repE, and two DNA regions, oriS and incC , required for replication, maintenance, and regulation of mini-F replication.
  • mini-Tn7 A transposon derived from Tn7 which
  • a locus is non-essential if it is not required for an organisms replication as judged by the survival of that organism following disruption or deletion of that locus.
  • P polh A very late baculovirus promoter which is capable of promoting high level mRNA synthesis from any gene, preferably a heterologous DNA, placed under its
  • Plasmids are incompatible if they interact in such a way that they cannot be stably maintained in the same cell in the absence of selection for both plasmids.
  • Trans-acting elements are genes or DNA segments which exert their functions on another DNA segment independent of the trans-acting elements genetic linkage to that DNA segment.
  • transposon Any. mobile DNA element, including those which recognize specific DNA target sequences, which can be made to move to a new site by recombination or in sertion and does not require extensive DNA
  • bacmid recombinant baculovirus shuttle vector isolated from E. coli ; b, E. coli-derived bacmid; be, E. coli-derived composite bacmid; bch, mixture of E. coli-derived composite bacmid and helper plasmid; Bluo-gal,
  • halogenated indolyl-b-D-galactoside bp, base pair(s); Cam, chloramphenicol; cDNA, complementary DNA; ds, double-stranded; Gen, gentamicin; IPTG, isopropyl-b-D-thiogalactopyranoside; Kan, kanamycin; kb, 1000 bp; PCR, polymerase chain reaction; r, resistant or resistance; s, sensitive; SDS-PAGE, sodium dodecyl sulfate
  • spectinomycin/streptomycin Tet, tetracycline; Tn, transposon; tn ⁇ , transposition genes; ts, temperature-sensitive; U, units; v, insect cell-derived baculovirus; vc, insect cell-derived composite baculovirus; vch, mixture of insect cell-derived composite baculovirus and helper plasmid; X-gal, 5-bromo-3-chloro-indolyl- ⁇ -D-galactopyranoside; X-gluc (5-bromo-3-chloro-indolyl-b-D-glucopyranoside), : : , transposon insertion,
  • Bacmid can be constructed that will replicate in E. coli as a large plasmid and remain infectious when introduced into insect cells. Using bacteria or
  • E. coli as a host to propagate the shuttle vector gives us a wide variety of genetic tools to manipulate and analyze the structure of the baculovirus genome.
  • Recombinant virus (composite bacmid) DNA isolated from selected colonies is not mixed with parental, non-recombinant virus, eliminating the need for multiple rounds of plaque purification. As a result, this greatly reduces the time it takes to identify and purify a recombinant virus from 4-6 weeks (typical for conventional methods) to 7-10 days.
  • One of the greatest advantages of this method is that it permits the rapid and simultaneous isolation of multiple recombinant viruses, and is particularly suited for the expression of protein variants for structure/function studies.
  • a baculovirus transfer vector was first constructed that contains a bacterial replicon, a selectable marker, and a preferential target site for a site-specific transposon.
  • the baculovirus transfer vector contains a mini-F replicon (derived from the F' plasmid isolated from E.
  • coli strain DH5 ⁇ F'IQ which allows for autonomous replication and stable segregation of plasmids at a low copy number (Holloway and Low, 1987; Kline, 1985), a selectable kanamycin resistance marker derived from Tn903 (Oka et al., 1981; Taylor and Rose, 1988; Vieira and Messing, 1982), and attTn7, the target site for the bacterial transposon Tn7 (Craig, 1989; Berg et al., 1989). Unlike most of
  • Tn7 inserts at a high frequency into the single attTn7 site located on the E . coli chromosome and into DNA segments carrying attTn7 on a plasmid.
  • a mini-attTn7 is inserted into a DNA segment, also linked to the mini-F replicon and kanamycin resistance gene, which encodes the IacZ ⁇ peptide.
  • the insertion of the mini-attTn7 is such that it does not disturb the translational reading frame of the lacZa peptide.
  • the mini-F-Kan-IacZ ⁇ -mini-attTn7 sequences are inserted into a baculovirus transfer vector (derived from pVL1393) which lacks the baculovirus polyhedrin promoter and a portion of the polyhedrin coding sequences at the 5' end.
  • baculovirus transfer vector derived from pVL1393
  • Recombinant baculoviruses containing the mini-F-Kan-IacZ ⁇ -mini-attTn7 cassette are generated by
  • the transfecting susceptible cultured insect cells with this transfer vector and wild-type genomic baculovirus DNA are identified by their polyhedrin-minus phenotype in plaque assays and by DNA dot blot hybridization.
  • the baculovirus that is used is the Autographa californica nuclear polyhedrosis virus
  • baculovirus transfer vector is derived from AcNPV.
  • Susceptible host insect cells are derived from Spodoptera frugiperda (most preferably IPLB-SF21AE cells or its clonal isolate Sf9 cells), or from
  • Trichoplusia ni, Plutella xylostella, Manduca sexta, or Mamestra brassicae Calcium phosphate or lipofectin reagent is used to facilitate the transfection of the transfer vector and genomic viral DNA into susceptible insect cells.
  • Recombinant viral DNA containing the mini-F-Kan-IacZ ⁇ -mini-attTn7 cassette is isolated from infected insect cells and introduced into bacteria.
  • the bacterial strain used is E. coli DH10B.
  • the transformants, which replicate in bacteria under the control of the plasmid replicon are designated baculovirus shuttle vectors (bacmids). Bacmid DNAs transfected into susceptible host insect cell lines are infectious.
  • Donor replicons contain a transposon capable of site-specific transposition to its preferential target site present or in target bacmids.
  • the site-specific transposon is derived from Tn7 and the preferential target site is the mini-attTn7 located on a baculovirus shuttle vector.
  • the donor replicon is derived from pMON7117 which contains a deletion
  • mini-Tn7 derivative of Tn7 (mini - Tn7) (Barry, 1988).
  • the mini-Tn7 element on the donor plasmid is modified to contain a selectable drug resistance marker, a baculovirus promoter driving expression of a foreign gene, and a transcription termination poly(A) signal all flanked by the left and right ends of Tn7.
  • the selectable marker confers resistance to gentamicin
  • the baculovirus promoter is the AcNPV polyhedrin
  • plasmids we describe are small compared to traditional baculovirus transfer vectors and are easily manipulated. Easily manipulated to add or restrict genetic elements.
  • the efficiency of transposition of the mini-Tn7 element from the donor plasmid into the attachment site on the bacmid is high compared to generation of recombinants by homologous recombination. Insertions into the mini-attTn7 located in frame with a segment of DNA encoding the lacZa peptide on the bacmid prevent complementation between the ⁇ peptide produced by the bacmid and the acceptor polypeptide produced from a gene located on the chromosome of the bacteria. Therefore, transposon insertion events into the bacmid can be easily
  • Bacmid DNA can easily be isolated from E. coli and its structure analyzed by restriction endonuclease
  • helper functions or the donor segment might also be moved to the attTn7 on the chromosome to improve the efficiency of transposition, by reducing the number of open attTn7 sites in a cell which compete as target sites for transposition in a cell harboring a bacmid containing an attTn7 site.
  • E. coli strain DH10B (Grant et al., 1990) was used as the host for all bacterial plasmid manipulations.
  • E. coli strain DH5 ⁇ F'IQ (Jessee and Blodgett, 1988) was used as the source of F' plasmid DNA. Both strains were obtained from GIBCO/BRL (Grand Island, NY) as frozen competent cells.
  • Plasmids pBS2SKP (Alting-Mees and Short, 1989) and pBCSKP were obtained from Stratagene (La Jolla, CA). Plasmid pMAK705 (Hamilton et al., 1989) was obtained from Dr. Sidney Kushner (University of Georgia, Athens, GA). Plasmids pRAJ275 (Jefferson et al., 1986) was obtained from Clonetech (Palo Alto, CA). pSL301
  • pMON7124, and pMON7134 were obtained from Dr. Gerard Barry (Monsanto Agricultural Company, Chesterfield, MO) .
  • Plasmid pMON14007 was obtained from Dr. Verne Luckow (Monsanto Corporate Research, Chesterfield, MO) . All other plasmids were constructed specifically for this work.
  • Plasmids were transformed into frozen competent E. coli DH10B (Grant et al., 1990), obtained from
  • GIBCO/BRL using the procedures recommended by the manufacturer. Briefly, the frozen cells were thawed on ice and 33-100 ⁇ l of cells were incubated with 0.01-1.0 ⁇ g of plasmid DNA for 30-60 minutes. The cells were shocked by heating at 42oC for 45 seconds, diluted to 1.0 ml with antibiotic-free S.O.C. buffer (GIBCO/BRL), and grown at 37oC for 3 hours. A 0.1 ml sample of culture was spread on agar plates supplemented with the appropriate antibiotics. Colonies were purified by restreaking on the same selection plates prior to analysis of drug resistance phenotype and isolation of plasmid DNAs. Plasmids were also transformed into competent E. coli DH10B cells prepared by suspending early log phase cells in transformation and storage (TSS) buffer (Chung et al., 1989). TSS buffer,
  • Low-melting point agarose (GIBCO/BRL) was used to facilitate recovery of individual restriction fragments, when necessary. DNAs were separated on a 1% low-melting point agarose gel, stained with 2 ⁇ g/ml ethidium bromide for 15 minutes, and the products identified by
  • Sf9 cells (Summers and Smith, 1987), a clonal isolate of the IPLB-SF21-AE cell line (Vaughn et al., 1977) derived from the ovarian tissue of the fall armyworm, Spodoptera frugiperda , were used for the propagation of wild-type and recombinant baculoviruses.
  • the E2 strain Smith and Summers, 1978; Smith and Summers, 1979
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • IPL-41 medium (GIBCO/BRL) supplemented with 2.6 g/l tryptose phosphate broth (GIBCO/BRL) and 10% fetal bovine serum (J.RH. Biosciences) was used for the routine propagation of Sf9 cells.
  • Sf9 cells adapted for growth in Sf900 or Sf900-11 serum-free medium (GIBCO/BRL) were also used for some experiments. Cells were maintained as
  • Radiolabeling of infected cells with 35 S-methionine was performed as described by Luckow and Summers (1988).
  • Plasmid pMON14221 was constructed by replacing an NcoI/EcoRI fragment of
  • Recombinant viruses constructed using pMON14007 and pMON14221 transfer vectors are used as controls for comparing levels of expression of LTA 4 H and ⁇ -glucuronidase with composite bacmids. Recombinant viruses expressing ⁇ -glucuronidase were easily
  • pMON14102 was constructed by cloning a 1240 bp PstI fragment of pUC4-K (Taylor and Rose, 1988; Vieira and Messing, 1982) into the PstI site of pBluescript II SK(+) (Alting-Mees and Short, 1989).
  • F' plasmid DNA prepared from strain DH5a/(F'lac-pro)::Tn5 was digested with BamHI and EcoRI and ligated to an agarose gel-purified BamHI/EcoRI fragment from pMON14102 that confers resistance to kanamycin (Tn903).
  • the plasmid of one such transformant was designated pMON14181.
  • Plasmid pMON7134 was constructed by inserting a 523 bp HincII fragment of pEAL1 (Lichtenstein and Brenner, 1982) containing the attachment site for Tn7 (attTn7) into the HincII site of pEMBL9 (Dente et al., 1983).
  • a 112 bp mini-attTn7 sequence was amplified by polymerase chain reaction (PCR) from the plasmid pMON7134 using two primers AttRP-PR1 (5'- agatctgcaggaattcacataacaggaagaaaaatgc -3') [SEQ ID NO.1] and
  • AttSP-PR1 (5'- ggatccgtcgacagccgcgtaacctggcaaa -3') [SEQ ID NO.2],
  • PCR reactions were carried out using a DNA Thermal Cycler and GeneAmp PCR reagent kit (Perkin Elmer Cetus, Norwalk, CT). Thirty cycles were used to amplify the mini-attTn7. Each cycle consisted of three steps, denaturation of double stranded DNA (94oC, 1 min), annealing of oligonucleotide primers (50oC, 2 min), and polymerization of the complementary DNA strand (72°C, 3 min) . The amplified segment contains an 87 bp attTn7 (numbered -23 to +61 as described by Craig ( 1989 ) .
  • the 112 bp amplified fragment was digested with EcoRI and SalI and cloned into the EcoRI and SalI sites within the lacZ ⁇ region of the cloning vector pBCSKP to generate pMON14192 .
  • the EcoRI/SalI mini-attTn7 does not disrupt the reading frame of the lacZ ⁇ region of pBCSKP and has the E. coli glmS transcriptional terminator inserted in the opposite orientation from transcription directed by the Lac promoter, so colonies of E. coli strain DH10B harboring pMON14192 are blue on agar plates containing X-gal or Bluo-gal.
  • Plasmid pMON14192 was linearized with ScaI and used as a template for PCR in the presence of two new primers, l ⁇ cZA-PR1 (5'- tgatcattaattaagtcttcgaaccaatacgcaaaccgcctctccccgcgcg -3') [SEQ ID NO.3] and
  • reaction buffer contained 5% (v/v) DMSO to permit less stringent annealing.
  • Thirty cycles were used to amplify the mini-attTn7. Each cycle consisted of three steps, denaturation of double stranded DNA (94oC, 1 min), annealing of oligonucleotide primers (55oC, 2 min), and polymerization of the complementary DNA strand (72oC, 3 min).
  • the PCR primers were designed to amplify the entire lacZ ⁇ region of pMON 14192 or any pUC-based cloning vectors. Each primer contained a BbsI site
  • Primer lacZA-PR1 contains an EcoRI-compatible ('AATT,) site and primer lacZA-PR2 contains a SalI-compatible (TCGA,) site as part of the cleavage site (double underline above) flanking the BbsI recognition site (single underline above).
  • a DrdI site and a PacI site are also adjacent to the BbsI sites in lacZA-PR1 and lacZA-PR2, respectively.
  • the amplified 728 bp dsDNA fragment could therefore be cleaved with BbsI to generate EcoRI- and SalI-compatible sticky ends, even though there were internal EcoRI and Sall sites flanking the mini-attTn7 region towards the center of the fragment.
  • the 708 bp BbsI-cleaved PCR fragment was ligated to pMON14181 (mini-F-Kan) that was cleaved wit ⁇ E EcoRI and SalI and transformed into E. coli DH10B.
  • pMON14181 mini-F-Kan
  • pMON14231 mini-F-Kan-lacZa-mini- ⁇ ttTn7
  • Plasmid pMON14118 was constructed by digesting pVL1393 (Luckow, 1991; O'Reilly et al., 1992) with EcoRV and SmaI and recircularizing in the presence of T4 DNA ligase to remove the AcNPV polyhedrin promoter. Plasmid pMON14231 has two BamHI sites, one within the lacZa-mini-attTn7 region and the other at the junction between the mini-F and Kan genetic elements, so it was digested with a low concentration of BamHI to generate full-length linear molecules and ligated to the pMON14118 cleaved with BglII to generate pMON14271 and pMON14272.
  • Plasmids pMON14271 and pMON14272 differ only in the orientation of the mini-F-Kan-lacZa-mini-attTn7 cassette inserted into the pMON14118 transfer vector. Their structures were verified by digestion with BamHI, EcoRI, and XhoI. Upon transformation into E. coli DH10B, both plasmids confer resistance to ampicillin and kanamycin and have a Lac + phenotype on plates containing X-gal or Bluo-gal.
  • Both transfer vectors pMON14271 and pMON14272, were introduced into insect cells along with wild-type genomic AcNPV DNA using a calcium phospate-mediated transfection protocol (Summers and Smith, 1987). Putative recombinant viruses were identified by their occlusion minus phenotype under a stereo dissecting microscope and confirmed by DNA dot blot hybridization using 32 P-labeled pMON14181 DNA prepared by random priming (O'Reilly et al., 1992) as a probe to cell lysates (Summers and Smith, 1987) blotted 48 hr post infection onto nitrocellulose filter paper (Luckow and Summers, 1988).
  • vMON14271 and vMON14272 Three viruses for each construct were selected and purified free from wild-type parental virus by sequential plaque assays (Summers and Smith, 1987) and passage 1 stocks of each purified virus (vMON14271 and vMON14272) were prepared.
  • the prefix "v” is used to designate the source of viral stocks or viral DNA, in this case prepared from infected insect cells.
  • Genomic viral DNA was prepared from the infected cells used to generate the passage 1 stock of virus using the protocol described by Summers and Smith (Summers and Smith, 1987). Viral DNA constitutes approximately 25% of the total nucleic acid content of an infected cell nucleus very late in infection (>48 hr p.i.). Briefly, cells were lysed with lysis buffer (30 mM Tris-HCl, pH 8.0, 10 mM Mg acetate, and 1% Nonidet P-40), and the nuclei pelleted by centrifugation at 2000 rpm for 3 minutes.
  • lysis buffer (30 mM Tris-HCl, pH 8.0, 10 mM Mg acetate, and 1% Nonidet P-40
  • the nuclei were washed once in cold PBS and lysed with 4.5 ml extraction buffer (100 mM Tris-HCl, pH 8.0, 100 mM EDTA, 200 mM KCl). Approximately 200 ⁇ g of proteinase K was added and incubated at 50oC for 1 hour before adding 0.5 ml 10% Sarcosyl and incubating at 50oC overnight.
  • the DNA was purified by extracting once with buffer-saturated phenol and once with phenol/chloroform/isoamyl alcohol (25:24:1) before precipitating with ethanol.
  • Viral DNA was transformed into E. coli DH10B using frozen competent cells obtained from GIBCO/BRL.
  • Colonies on plates transformed with the viral DNAs vMON 14271 or vMON 14272 were kanamycin resistant and gave a Lac + (blue) phenotype in the presence of Bluo-gal or X-gal indicating complementation between the lacZa peptide expressed by the plasmid and the lacZ ⁇ M15 acceptor polypeptide expressed from the chromosome of E. coli DH10B.
  • the transformants were designated bMON14271 and bMON14272 to indicate their bacterial origin. Small amounts of pure bacmid DNA could be isolated from E. coli after alkaline lysis and purification over resin columns.
  • Bacmid DNA isolated from E. coli was introduced into insect cells using the calcium-phosphate transfection protocol (Summers and Smith, 1987). Three to five days post transfection the cells appeared swollen and detached easily from the plastic bottom of the flask like cells infected with viral DNA isolated originally from insect cells. Mock-infected cells attached tightly to the monolayer. Plaques produced by budded virus generated from transfections using E. coli-derived bacmid DNA were all occlusion minus (data not shown).
  • the replicon containing the element should be of small size, moderate or high copy number, and contain a drug resistance marker and a polylinker with unique restriction sites between the left (Tn7L) and right (Tn7R) arms of Tn7.
  • Plasmid pMON7104 (G. Barry, unpublished) is a derivative of pEMBL19P containing a 1258 bp AluI fragment encoding the gene (aacC1) for gentamicin acetyltransferase-3-I (AAC(3)-I) (Wohlleben et al., 1989).
  • the gentamicin resistance gene of pMON7104 was released by XbaI/PstI digestion and the resultant fragment was ligated to PstI/XbaI-digested pMON7117 (Barry, 1988), producing pMON14189.
  • SV40 poly-(A) transcription termination signal of pMON3327 (P. Hippenmeyer, unpublished) was released as a 244 bp fragment by BamHI/XbaI digestion and ligated to BamHI/XbaI-digested pMON14189, resulting in plasmid pMON14214.
  • pMON14214 was digested with NcoI and NotI and the restriction fragment sticky ends were removed by treatment with Mung-bean nuclease (Promega) using conditions described by the manufacturer. This fragment was recircularized by ligation, producing pMO ⁇ 14239.
  • pMON14239 was digested with BamHI and ligated to the synthetic double-stranded polylinker shown below (Boehringer Mannheim Biochemica),
  • Omega nuclease I-SceI recognizes the 18 bp sequence TAGGG,ATAA'CAGGGTAAT[SEQ ID NO.7] and generates a four bp 3' hydroxyl overhang (Colleaux et al., 1988).
  • Plasmid pMON14007 (Gierse et al., 1992) was digested with EcoRV and NotI and the fragment containing the AcNPV polyhedrin promotor and the human leukotriene A 4 hydrolase cDNA (hLTA 4 H) (Funk et al., 1987; Minami et al., 1987) was ligated to StuI/NotI-digested pSL301 (Brosius, 1989), producing plasmid pMO ⁇ 14209.
  • pMON14209 was digested with SpeI and NheI and the fragment containing the polyhedrin promoter and hLTA 4 H gene was ligated to XbaI-digested pMON 14255, resulting in plasmid
  • Plasmid pMON14327 was constructed by replacing the hLTA 4 H gene of pMON14314 with an NcoI/EcoRI fragment of pRAJ275 (Jefferson et al., 1986) which contains the coding sequences for the ⁇ -glucuronidase gene.
  • the plasmid pMON22300 is a derivative of the donor plasmid pMON14327 that has the cDNA for human myristoyl CoA:protein N-myristoyl transferase (Duronio et al., 1992) (hNMT) under the control of polyhedrin promoter.
  • the hNMT cDN A in this plasmid has a Pro to Leu mutation at amino acid position 127.
  • the resulting donor plasmids pMON14314, pMON14327, and pMON22300 therefore, have mini-Tn7 elements on a pUC-based plasmid containing a gentamicin resistance marker, the polyhedrin promoter driving expression of a foreign gene, a polylinker, an SV40 poly(A) transcriptional termination signal, and I-SceI site between the left and right arms of Tn7.
  • These donor molecules are incompatible with the helper plasmid, pMON7124.
  • This plasmid incompatibility can be used to eliminate the donor molecule after transposition to bacmid has occurred (See Example V).
  • the gentamicin resistance marker is used to select for transposition events to the target plasmid and the I-SceI site is used to facilitate the mapping of mini-Tn7 elements inserted into the genome of the target bacmids.
  • the donor molecules based on plasmid incompatability were sufficient to validate the concept of site-specific tansposition for this invention.
  • An alternative and more efficient method is the use of a temperature-sensitive donor plasmid.
  • the temperature-sensitive (ts) plasmid pMAK705 (Hamilton et al., 1989) containing a ts pSC101 origin of replication and ⁇ -galactosidase gene was digested sequentially with NruI and NdeI.
  • the ends were filled and dephosphorylated as described (Sambrook et al., 1989) and the 2.5 kb fragment containing the ts replicon and the ⁇ -galactosidase gene were isolated from 0.7% agarose using NA45 DEAE membrane according to the manufacturer's protocol with the following exception; after elution from the NA45 membrane in 300 ⁇ l high salt NET buffer, the fragment was concentrated using a Geneclean II kit into 15 ⁇ l sterile water.
  • pMON 14327 was linearized with EcoO109 and the ends filled. The linearized/filled pMON14327 was partially digested with 0.1 U AlwNI and immediately purified from enzyme using a Geneclean II kit.
  • the DNA was treated with 0.25 U Mung-bean nuclease as described in the manufacturers protocol.
  • the 5.2 kb fragment containing Tn7R, a gentamicin resistance gene, the AcNPV polyhedrin promoter driving a ⁇ -glucuronidase gene, an SV40 poly-(A) signal, and Tn7L was isolated from 0.7% agarose using NA45 DEAE membrane as described above. This fragment was mixed and ligated with the 2.5 kb NdeI/NruI fragment from pMAK705.
  • the resulting plasmid, pMON18127 was transformed into competent E. coli DH10B cells and outgrown at 30oC.
  • Cells were plated on LB agar medium containing 10 ⁇ g/ml gentamicin, 40 ⁇ g/ml IPTG and 200 ⁇ g/ml Bluo-gal and incubated at 30oC. Blue colonies were picked and purified at 30oC.
  • Verification of the ts phenotype was accomplished by diluting 12 independent isolates in 2 ml LB each and patching onto each of two plates of LB agar medium containing 10 ⁇ g/ml gentamicin, 40 ⁇ g/ml IPTG and 200 ⁇ g/ml Bluo-gal. One plate of each pair was incubated at 30oC the other at 44oC. Clones which gave rise to colonies on plates incubated at 30oC but not at 44oC were selected as ts (Hashimoto and Sekiguchi, 1976; Hashimoto-Gotoh and Sekiguchi, 1977). The structure of temperature-sensitive pMON18127 was confirmed by restriction analysis.
  • the mini-Tn7 element from pMON14327 was inserted into the chromosomal attTn7 site of E. coli DH10B.
  • the new strain of E. coli containing the mini-Tn7 element from pMON 14327 will be designated DH10B::Tn14327.
  • One hundred ⁇ l MAX Efficiency E. coli competent cells DH10B were transformed with 30 ng of helper plasmid pMON7124. Transformants were selected on LB agar medium containing 15 ⁇ g/ml tetracycline.
  • Transformants were selected on LB agar medium containing 10 ⁇ g/ml gentamicin and 15 ⁇ g/ml tetracycline and purified by streaking onto LB agar medium containing 10 ⁇ g/ml gentamicin. Isolated colonies were scored for ampicillin sensitivity by patching to LB agar medium containing 100 ⁇ g/ml ampicillin as described above. A gentamicin-resistant, ampicillin-sensitive colony was inoculated into 10 ml LB medium without antibiotic and grown overnight at 37oC. The overnight culture was then serial diluted to 10 -7 cells/ml and grown in LB overnight at 37oC. This entire outgrowth procedure was repeated a total of 4 times.
  • Cells from the fourth overnight were diluted in LB medium to 10 -4 , 10 -5 and 10 -6 cells/ml.
  • One hundred ⁇ l of each dilution was plated onto LB agar medium and grown overnight at 37oC.
  • Colonies from the 10 -5 and 10 -6 cells/ ⁇ l dilutions were replica plated onto LB agar medium containing 15 ⁇ g/ml tetracycline and grown overnight at 37oC.
  • Colonies from the master plate which did not grow as replicates on the medium containing 15 ⁇ g/ml tetracycline were streaked onto LB agar containing 10 mg/ml gentamicin and grown overnight at 37oC.
  • Transposition experiments were carried out by transforming a donor plasmid (pMON14314, pMON14327, or pMON22300) conferring ampicillin-and gentamicin-resistance into competent E. coli DH10B cells harboring the tetracycline-resistant helper plasmid pMON7124 and a kanamycin-resistant, lacZa+ bacmid (bMON1427 or bMON14272) and plating the cells out on LB agar plates containing kanamycin, tetracycline, gentamicin, X-gal, and IPTG.
  • Plasmid DNAs were purified from white kanamycin-, gentamicin-, and tetracycline-resistant, but ampicillin-sensitive colonies harboring the helper plasmid and the composite bacmid with the mini-Tn7 element inserted into the mini-attTn7 region of the lacZa region over QIAGEN resin columns. This mixture of plasmid DNAs was used to retransform E. coli DH10B, selecting for kanamycin and gentamicin resistance, and colonies were scored to confirm the absence of the tetracycline resistance marker present on the helper plasmid.
  • Calcium chloride competent cells were prepared from a culture of DH10B containing bacmid (bMON14272) and helper (pMON7124) grown in 2XYT medium containing 50 ⁇ g/ml kanamycin and 15 ⁇ g/ml tetracycline as described above.
  • bacmid bMON14272
  • helper pMON7124
  • 2XYT medium containing 50 ⁇ g/ml kanamycin and 15 ⁇ g/ml tetracycline as described above.
  • One hundred ⁇ l competent cells were mixed with 40 ng of the ts donor plasmid pMON18127, heat shocked at 42oC for 45 seconds, and outgrown in 1 ml S.O.C. medium at 30oC for 3.5 hours.
  • One hundred ⁇ l of cells were plated from undiluted or 10 -2 diluted outgrowth culture on prewarmed LB agar medium containing 50 ⁇ g/ml kanamycin, 10 ⁇ g/ml gentamicin, 15 ⁇ g/ml tetracycline, 40 ⁇ g/ml IPTG and 200 ⁇ g/ml bluo-gal and incubated overnight at 44oC. Between 77 and 88% of transformants were white (Lac-) kanamycin-resistant, gentamicin-resistant, and tetracycline-resistant and exhibited a single colony morphology.
  • Transformants both Lac- and Lac + , were purified by restreaking on selective media containing kanamycin, gentamicin and tetracycline.
  • Bacmid DNA was isolated from 3-5 ml overnight cultures using either a Magic mini prep kit or Qiawell-8 plasmid prep system. Insertion of the mini-Tn7 into the attTn7 site was verified by PCR using 2 different pairs of primers specific for both the mini-Tn7 element and sequences flanking the attTn7 site in the bacmid. PCR fragments of the expected sizes were observed only from composite bacmid (Lac-) isolates. Bacmid DNA isolated from non-recombinant (Lac + ) transformants gave the expected PCR product only when primer pairs were specific for the bacmid DNA alone.
  • coli DH10B::Tn14327 fifty ng of pMON7124 (helper) was transformed into 100 ml CaCl 2 competent DH10B::Tnl4327 containing the bacmid bMON14272.
  • cells were outgrown in 1 ml S.O.C. medium at 37oC for 3.5 hours.
  • One hundred ml of cells were plated from undiluted or 10 -2 diluted outgrowth culture on LB agar medium containing 50 mg/ml kanamycin, 10 mg/ml gentamicin, 15 mg/ml tetracycline, 40 mg/ml IPTG and 200 mg/ml bluo-gal and incubated overnight at 37oC.
  • DNAs from donor plasmids, the parent bacmids, and the composite bacmids isolated from E. coli and from insect cells were examined by digestion with BglII, EcoRI, I-SceI, NotI, PstI , Sse8387I, and XhoI and compared to the pattern generated by cleavage of wild-type Ac ⁇ PV D ⁇ A purified from budded virus.
  • Bacmid D ⁇ As isolated from E. coli and digested with BglII, PstI, or XhoI have the same or a similar restriction pattern as the corresponding viral D ⁇ A isolated originally from insect cells, indicating no gross structural differences between D ⁇ As isolated from the two sources.
  • the plasmid D ⁇ A was strikingly clean from contaminating E. coli chromosomal D ⁇ A compared to the crude viral D ⁇ A prepared from insect cells which was contaminated with insect chromosomal D ⁇ A.
  • the mini-F-Kan-lacZ ⁇ -mini- ⁇ ttTn7 cassette was inserted into the polyhedrin locus located in the Ac ⁇ PV restriction fragments BglII-C, PstI-D, and XhoI-D (data not shown).
  • the composite D ⁇ As had a single new insertion of the expected size and location in the mini-attTn7 as judged by the introduction of one or more restriction sites (EcoRI, I-SceI, NotI, Sse8387I) present in the mini-Tn7 donor cassette (data not shown).
  • the virus stock vchMON14271::Tn14327/pMON7124 was prepared by transfecting insect cells with a mixture of composite DNA and noninfectious pMON7124 helper plasmid DNA. No ⁇ -glucuronidase activity was detectable from uninfected cells or cells infected with wild-type AcNPV, or viruses expressing hLTA 4 H or hNMT (data not shown). These results indicated that the ⁇ -glucuronidase gene under the control of the polyhedrin promoter was expressed when the mini-Tn7 element from the donor plasmid was inserted into the mini-attTn7 site located in the bacmid.
  • Passage 2 stocks of viruses expressing ⁇ -glucuronidase, hLTA 4 hydrolase, and hNMT were prepared and titered.
  • the passage 2 stocks of virus were used to infect 6 ⁇ 10 5 SF21 cells/well in a 24 well plate at a multiplicity of infection of 10 virus particles per cell.
  • the cells were radiolabeled for 4 hours at 44.5 hours post infection with 35 S-methionine.
  • the cells were lysed and samples were separated by SDS-PAGE. An autoradiogram of the resulting gel is shown in Figure 5.
  • High levels of ⁇ -glucuronidase were produced by the control virus vMON14221 (lane 8), and by the composite viruses vcMON 14271::Tn14327 (lane 5), vcMON14272::Tn14327 (lane 7), and vchMON14271::Tn14327/pMON7124 (lane 6).
  • ⁇ -glucuronidase Slightly higher levels of ⁇ -glucuronidase were observed for vcMON14272::Tn14327 (lane 7) compared to vcMON14271::Tn14327 (lane 5) that might be attributed to the orientation of the mini-F-Kan-lacZa-mini-attTn7 cassette within the parent bacmids bMON14271 and bMON14272. Whether this effect will be seen for other heterologous genes inserted into these two bacmids is currently under investigation.
  • the expression of ⁇ -glucuronidase by the composite viruses is slightly less than that observed for vMON 14221 (lane 8), a recombinant virus constructed in a traditional manner by homologous recombination in insect cells.
  • At least three smaller species were also noted and are probably related to ⁇ -glucuronidase, since they are not present in wild-type AcNPV-infected (lane 2) or uninfected cells (lane 1) nor were they detected in cells infected with the parent viruses vMON14271 or vMON14272 (lanes 3 and 4).
  • High levels of human leukotriene A 4 hydrolase and human N-myristoyltransferase were expressed by the composite viruses vcMON14271::Tn14314 (lane 9) and vcMON14271::Tn22300 (lane 10).
  • the abundant expression of these heterologous genes demonstrates the general utility of the baculovirus shuttle vector technology to simply and rapidly generate recombinant baculoviruses.
  • helper functions or the donor casstte might also be moved to the attTn7 on the chromosome to improve the efficiency of transposition, by reducing the number of open attTn7 sites in a cell which compete as target sites for transposition in a cell harboring a shuttle vector containing an attTn7 site.
  • This invention is also directed to any substitution of analogous components. This includes, but is not restricted to, construction of bacterial-eukaryotic cells shuttle vectors using different eukaryotic viruses, use of bacteria other than E. coli as a host, use of replicons other than those specified to direct replication of the shuttle vector, the helper functions or the transposable element donor, use of selectable or differentiable genetic markers other than those specified, use of site-specific recombination elements other than those specified, and use of genetic elements for expression in eukaryotic cells other than those specified. It is intended that the scope of the present invention be determined by reference to the appended claims.
  • Capone J. 1989. Screening recombinant baculovirus plaques in situ with antibody probes.
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:2:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de production de baculovirus recombinés infectieux dans des bactéries. On a élaboré un nouveau vecteur navette de baculovirus (bacmid) contenant un réplicon bactérien à nombre de copies réduit, un marqueur dont la résistance aux médicaments peut être sélectionnée, ainsi qu'un site de fixation préféré pour un transposon bactérien spécifique de site, inséré dans un locus non essentiel du génome du baculovirus. Le vecteur navette peut se répliquer dans E. coli sous la forme d'un plasmide, son caractère hériditaire est stable, il est structurellement stable après de nombreuses générations de croissance. L'ADN de bacmid isolé à partir de E. coli est infectieux lorsqu'on l'introduit dans des cellules d'insectes lépidoptères sensibles. Des segemts d'ADN contenant un promoteur viral induisant l'expression d'un gène étranger dans des cellules d'insectes bordées par les extrémités gauche et droite du transposon spécifique de site puvent se transposer sur le site de fixation dans le bacmid propagé dans E. coli, lorsque des fonctions de transposition sont fournies in trans par un plasmide auxiliaire. Le gène étranger est exprimé lorsque le bacmide composite obtenu est introduit dans des cellules d'insectes.
PCT/US1993/009408 1993-10-07 1993-10-07 Procede de production de virus eukaryotiques recombines dans des bacteries Ceased WO1995009923A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1993/009408 WO1995009923A1 (fr) 1993-10-07 1993-10-07 Procede de production de virus eukaryotiques recombines dans des bacteries
AU53514/94A AU5351494A (en) 1993-10-07 1993-10-07 Method of producing recombinant eukaryotic viruses in bacteria

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1993/009408 WO1995009923A1 (fr) 1993-10-07 1993-10-07 Procede de production de virus eukaryotiques recombines dans des bacteries

Publications (1)

Publication Number Publication Date
WO1995009923A1 true WO1995009923A1 (fr) 1995-04-13

Family

ID=22237041

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/009408 Ceased WO1995009923A1 (fr) 1993-10-07 1993-10-07 Procede de production de virus eukaryotiques recombines dans des bacteries

Country Status (2)

Country Link
AU (1) AU5351494A (fr)
WO (1) WO1995009923A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044141A3 (fr) * 1997-03-27 1999-01-07 Univ British Columbia Vecteurs d'expression d'insectes
EP1144666B1 (fr) * 1999-08-18 2006-10-18 Oxford Brookes University Systeme d'expression du baculovirus
US7135337B2 (en) 1997-03-27 2006-11-14 Grigliatti Tom A Insect expression vectors
CN111808884A (zh) * 2020-07-23 2020-10-23 云舟生物科技（广州）有限公司 杆状病毒表达系统及其构建方法和应用
WO2023028440A3 (fr) * 2021-08-23 2023-05-04 Bioverativ Therapeutics Inc. Système d'expression de baculovirus
US12168776B2 (en) 2017-08-09 2024-12-17 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof
US12364774B2 (en) 2018-08-09 2025-07-22 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof for non-viral gene therapy

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LUCKOW, V.A. ET AL.: "Efficient generation of infectious recombinant baculoviruses by site-specific transposon mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli", JOURNAL OF VIROLOGY, vol. 67, no. 8, August 1993 (1993-08-01), pages 4566 - 4579 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044141A3 (fr) * 1997-03-27 1999-01-07 Univ British Columbia Vecteurs d'expression d'insectes
US7135337B2 (en) 1997-03-27 2006-11-14 Grigliatti Tom A Insect expression vectors
EP1144666B1 (fr) * 1999-08-18 2006-10-18 Oxford Brookes University Systeme d'expression du baculovirus
US7413732B1 (en) 1999-08-18 2008-08-19 Oxford Brookes University Baculovirus expression system
US8252278B2 (en) 1999-08-18 2012-08-28 Oxford Brookes University Baculovirus expression system
US12168776B2 (en) 2017-08-09 2024-12-17 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof
US12364774B2 (en) 2018-08-09 2025-07-22 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof for non-viral gene therapy
CN111808884A (zh) * 2020-07-23 2020-10-23 云舟生物科技（广州）有限公司 杆状病毒表达系统及其构建方法和应用
WO2023028440A3 (fr) * 2021-08-23 2023-05-04 Bioverativ Therapeutics Inc. Système d'expression de baculovirus

Also Published As

Publication number Publication date
AU5351494A (en) 1995-05-01

Similar Documents

Publication Publication Date Title
US5348886A (en) Method of producing recombinant eukaryotic viruses in bacteria
Luckow et al. Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli
US6090584A (en) Baculovirus artificial chromosomes and methods of use
US4745051A (en) Method for producing a recombinant baculovirus expression vector
Pennock et al. Strong and regulated expression of Escherichia coli β-galactosidase in insect cells with a baculovirus vector
Ho Je et al. A defective viral genome maintained in Escherichia coli for the generation of baculovirus expression vectors
US5244805A (en) Baculovirus expression vectors
DK172401B1 (da) Fremgangsmåder til fremstilling af rekombinante baculovirus-vektorer, vektorer fremstillet ved fremgangsmåderne, samt fremgangsmåde til fremstilling af heterologt protein under anvendelse af en baculovirus-vektor
EP0155476A1 (fr) Production de polypeptides dans des cellules d'insectes
JP3076833B2 (ja) 修飾されたバクロウイルス、その製造方法及び該バクロウイルスから得られる発現ベクター
US6485937B1 (en) System for rapid generation of recombinant baculovirus-based expression vectors for silkworm larvae
St Angelo et al. Two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells
CA2033070A1 (fr) Vecteurs d'expression du baculovirus
EP0397485A1 (fr) Vecteurs d'expression de baculovirus et leur utilisation pour l'expression de protéines étrangères dans des insectes ou cellules d'insectes
Yang et al. Control of baculovirus polyhedrin gene expression by very late factor 1
WO1995009923A1 (fr) Procede de production de virus eukaryotiques recombines dans des bacteries
EP0228036A2 (fr) Méthode de production de polypeptides sélectionnés dans des cellules d'insectes infectées par des virus et polypeptides ainsi isolés
Merrington et al. Manipulation of baculovirus vectors
Van Oers et al. Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of polyhedrin gene expression
CN102286534A (zh) 表达多外源基因的昆虫生物反应器及其构建方法和应用
Kitts Construction of baculovirus recombinants
US6106825A (en) Entomopoxvirus-vertebrate gene delivery vector and method
US6806064B2 (en) Baculovirus enhancer-like sequence
US5597689A (en) Nucleotide sequences permitting the replication and maintenance of a genetic information in animal cells
Xia et al. Adenine DNA methyltransferase M. CviRI expression accelerates apoptosis in baculovirus-infected insect cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU LV MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA