EP0647274A1 - Baculovirus recombine contenant un promoteur de cellules d'insecte - Google Patents

Baculovirus recombine contenant un promoteur de cellules d'insecte

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Publication number
EP0647274A1
EP0647274A1 EP93915581A EP93915581A EP0647274A1 EP 0647274 A1 EP0647274 A1 EP 0647274A1 EP 93915581 A EP93915581 A EP 93915581A EP 93915581 A EP93915581 A EP 93915581A EP 0647274 A1 EP0647274 A1 EP 0647274A1
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European Patent Office
Prior art keywords
insect
promoter
recombinant
expression cassette
gene
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EP93915581A
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German (de)
English (en)
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Kostas Iatrou
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University Technologies International Inc
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University Technologies International Inc
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Publication of EP0647274A1 publication Critical patent/EP0647274A1/fr
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/70—Enkephalins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • the present invention relates to a method of expressing heterologous proteins in insect cells using a recombinant baculovirus, comprising a structural gene for a heterologous protein functionally attached to an insect cellular promoter such that the protein is expressed in substantially all of the insect tissues into which the recombinant baculovirus was introduced.
  • the heterologous protein includes proteins toxic or harmful to the insect host or proteins whose unregulated expression will incapacitate the insect host.
  • the invention is also directed to expression cassettes, recombinant expression cassettes containing heterologous genes, transplacement fragments containing expression cassettes, transplacement fragments containing recombinant expression cassettes, vectors containing transplacement fragments and recombinant baculoviruses derived therefrom.
  • NDVs Nuclear polyhedrosis viruses
  • Baculoviridae whose virions are embedded into proteinaceous polyhedra in the nucleus of host cells.
  • SUBSTITUTE SHEET Baculoviruses provide alternatives to chemicals for controlling insect pests. Because most NPVs have a host range restricted to only a few closely-related species, they can be used without disrupting the balance of other insect and non-insect species (e.g., important predators) in the agricultural ecosystem. No baculovirus has been demonstrated to infect mammals, reptiles, birds, invertebrates such as earthworms or plants. To date, however, baculoviruses have met with only limited commercial success as control agents, due to difficulties with virus stability and, most importantly, slower speed of action than that achieved with chemical insecticides.
  • Certain baculoviruses specifically nuclear polyhedrosis viruses (NPVs) have a unique life cycle which involves the temporally regulated expression of two functionally and morphologically different viral forms, the budded form and the occluded form.
  • Nuclear polyhedrosis viruses produce large polyhedral occlusion bodies, which contain enveloped virus particles, within the nucleus of infected cells.
  • the occlusion body is composed of a matrix comprising a 29 Da protein known as polyhedrin. After the insect dies from infection, occlusion bodies containing virus are released from the dead larvae into the environment and spread the infection to other insects through contamination of the food supply.
  • occlusion bodies serve to protect the virus particles in the environment and also provide a means of delivering the virus particles to the primary site of infection in insects, the midgut epithelial cells.
  • the occlusion bodies When the occlusion bodies are ingested by the larvae, the alkaline pH of the midgut lumen of phytophagous lepidopteran larvae dissolves the paracrystalline matrix in which the virus particles are embedded, promoting infection.
  • Viral nucleocapsids are synthesized in the nucleus of the insect cell, move through the cytoplasm and bud from the plasma membrane of the cell resulting in the release of budded virus particles into the insect hemolymph.
  • the open circulatory system of the insect provides the virus with access to other tissues of the insect. Virtually all tissues within the host larvae are susceptible to infection by the budded virus.
  • Replication of the virus in other organs creates extensive tissue damage and eventually death.
  • the complete process can take 4-5 days in the laboratory, but may take more than a week in the field.
  • the synthesis of the budded and occluded forms of the virus is temporally regulated.
  • progeny budded viruses are released into the culture media beginning approximately 12 hour post infection (p.i.), and the release continues logarithmically through 22 hours p.i.
  • Occluded virus forms approximately at 20 hours p.i. and continues through 70 hours p.i. by which time approximately 70-100 polyhedral occlusions have formed in the nucleus.
  • This temporal regulation of viral development is reflected in the controlled transcription of specific viral genes.
  • Nuclear polyhedrosis virus genes are transcribed in a regulated cascade involving at least three phases of transcription: an early phase (0-6 hours p.i.) prior to viral DNA replication, a late phase (6-18 hours p.i.) involving DNA replication and budded virus formation, and the very late occlusion phase (1 8 through 70 hours p.i.).
  • baculovirus infection-mediated reduction of the population of insects occurs in the field only one to two weeks after application of wild-type baculovirus.
  • recombinant viruses have been generated which express non-viral proteins whose products are toxic to the infected insect under the control of viral or synthetic promoters.
  • the underlying premise for the creation of such recombinant viruses has been that the expression of the foreign protein in the larvae should inactivate or kill the larvae before they would normally succumb to viral infection.
  • Some recombinant viruses have been developed which employ the viral polyhedrin promoter (Merry weather et al., 1990, J. Gen. Virol. , 71 : 1 535-1 544; Tomalski and Miller, 1991 , Nature, 352:82-85; Maeda et al., 1991 , Virology, 184:777-780), the viral p10 promoter (Stewart et al., 1991 , Nature, 353:85-88; McCutchen et al., 1991 , Bio/Technology 9:848-852), or a synthetic promoter based on the previous two (Wang et al., 1991 , Gene, 100:131 -137) to express the desired foreign protein.
  • the viral polyhedrin promoter Maerry weather et al., 1990, J. Gen. Virol. , 71 : 1 535-1 544; Tomalski and Miller, 1991 , Nature, 352:82-85; Maeda et al
  • One of the key aspects of the development of recombinant baculoviruses as effective insecticides is the timing and site of expression of heterologous proteins following initial infection of the target insect.
  • the first cells to be infected are the columnar and regenerative cells of the midgut epithelium.
  • the generalized spread of the virus to other tissues of an infected larva through circulation does not occur until 36 hours after the virus is first observed in the gut epithelium.
  • heterologous genes under the control of the polyhedrin or p10 promoters in vivo may not occur until an even later time as there is some doubt as to the level of expression of genes under the control of the viral polyhedrin or p10 promoters in the epithelial cells of the midgut, the primary site of infection, as normal production of polyhedra is not observed in these cells (Granados and Lawler, 1981 , J. Virol., 108:297-308).
  • placing an insect incapacitating or toxic gene under the control of the polyhedrin or p10 promoter may offer modest advantages in the order of only 1 or 2 days in terms of accelerating insect death relative to an infection with a wild- type baculovirus.
  • the transcripts from this recombinant virus are expressed correctly only in the tissue in which this promoter is normally active, ovarian follicular cells of the insect, but not in any other tissues, for example fat body, other tissues of the abdomen such as muscles or ganglia or in non-expressing tissue culture cells such as Bm5 cells. Further, because of the presence of a thick basement membrane that completely surrounds each follicle, the later recombinant virus infects the follicle cells only in a limited fashion and only after a considerable time lag (e.g., 36- 48 hours) after in vitro inoculation (injection) of the insect with the virus.
  • a considerable time lag e.g. 36- 48 hours
  • the present invention is directed to the discovery that introduction into insect cells of recombinant baculoviruses containing DNA encoding for a heterologous protein functionally attached to an insect cellular promoter results in the expression of the protein in substantially all tissues of the insect into which the recombinant baculovirus is introduced commencing in the early stages of infection of those tissues.
  • this invention is directed towards a method of expressing heterologous proteins in insect cells comprising infecting insect cells with a recombinant baculovirus comprising a recombinant expression cassette, such cassette comprising a structural gene encoding a heterologous protein functionally attached to an insect cellular promoter wherein the insect cellular promoter is a promoter which is not naturally functionally attached to the structural gene, in such a manner that the heterologous protein is expressed in insect cells into which the baculovirus is introduced.
  • the heterologous protein may be a protein toxic to the insect host or a protein whose unregulated expression could incapacitate the insect host through unbalancing of an important physiological process.
  • this invention is also directed towards a method of achieving rapid inactivation of insect larvae by infecting the larvae with recombinant baculoviruses which contain a structural gene for a toxic or insect host incapacitating protein functionally attached to an insect cellular promoter wherein the insect cellular promoter is a promoter which is not naturally functionally attached to the structural gene, so that expression of the heterologous protein will occur in substantially all infected insect tissues in the early stages of infection of those tissues.
  • This invention is also directed to a method of expressing heterologous protein in insect cells comprising introducing into insect cells a recombinant expression cassette, such cassette comprising a structural gene encoding a heterologous protein functionally attached to the promoter of the cytoplasmic actin gene of B. mori.
  • This invention is also directed towards an expression cassette comprising an insect cellular promoter wherein insect cellular promoter is capable of expressing a heterologous gene functionally attached to the promoter when insect cells are infected with a recombinant baculovirus comprising the expression cassette and the heterologous gene and wherein the insect cellular promoter is a promoter not naturally functionally attached to the heterologous gene.
  • This invention is also directed to a recombinant expression cassette comprising a heterologous protein functionally linked to an insect cellular promoter, transplacement fragments comprising the expression cassettes and recombinant expression cassettes and vectors and recombinant baculoviruses derived therefrom.
  • FIGS. 1A-E Plasmid Vectors Used in the Generation of Recombinant Viruses
  • FIG. 1 A This is a schematic representation of the vector pBmA.
  • the 5' and 3' flanking sequences of the cytoplasmic actin gene are represented by the unfilled regions, and the transcribed sequences are indicated by the shaded region.
  • the line represents sequences from pBS/SK + containing the beta- lactamase (bla) gene which confers ampicillin resistance. All of the restriction enzyme sites shown in the multiple cloning site are unique in this plasmid.
  • FIG. 1 B This is a schematic representation of the vector pBmA.cat.
  • the region containing vertical lines represents the cat gene open-reading frame. All other designations are as in FIG. 1 A.
  • FIG. 1 C This is a schematic representation of the vector pBmp2s.
  • the region containing horizontal lines represents the deleted polyhedrin gene containing no promoter sequences. Black regions represent sequences flanking the polyhedrin gene in the BmNPV genome.
  • the line represents pUC9 DNA.
  • FIG. I D This is a schematic representation of the vector pBmp2s/A.cat.
  • the line represents DNA from pBS/SK + . All other designations are as described in FIGS. 1 A and 1 C.
  • FIG. 1 E This is a schematic representation of pBmp26.cat. The region containing horizontal lines represents the polyhedrin gene including all sequences necessary for strong promoter activity. All other designations are as described in FIG. 1 C.
  • FIGS. 2A-C Analysis of CAT Activity in Non-Occluded Virus
  • FIG. 2A Non-occluded virus from cells transfected with pBmNPV/A.cat or pBmNPV/P26.cat was separated from the medium as described in Example 2 and the level of CAT determined.
  • the upper spots (Ac-C) represent acetyl chloramphenicol, the products of the CAT reaction, while the lower spot in each assay (C) represents the substrate, chloramphenicol.
  • FIG. 2B Bm5 cells were assayed for CAT activity at the indicated times after addition of BmNPV/A.cat or BmNPV/P26.cat inoculum. Assays were performed using the amount of cell protein and length of incubation indicated and as described in Example 5.
  • FIG. 2C Bm5 cells were infected with the standard inoculum containing medium and virus (M + V), with non- occluded virus isolated from the inoculum (V), or mock infected ( — ). The cells were collected 1 hour after infection and were washed once (1 x) or five times (5x) with 1 ml PBS.
  • FIGS. 3A-C Time Course of CAT Activity in Infected Bm5 Cells
  • FIG. 3A Cells were infected with BmNPV/A.cat inoculum and CAT assays were performed at the indicated time post infection using 5 ⁇ g cell protein incubated for 1 hour.
  • FIG. 3B Cells were infected with BmNPV/P26.cat inoculum and assayed using 1 ⁇ g cell protein incubated for 1 hour at the indicated times post infection.
  • FIG. 3C Appropriate amounts of cell extract from cells infected with BmNPV/A.cat (actin) or BmNPV/P26.cat (polyhedrin) were assayed to quantitate CAT assays at each time point. The background CAT activity observed at 1 -2 hours p.i. was subtracted from each point. The values were plotted on a logarithmic scale.
  • FIG. 4 Analysis of CAT Activity in Infected Larvae
  • BmNPV/A.cat or BmNPV/P26.cat inoculum were injected with either BmNPV/A.cat or BmNPV/P26.cat inoculum and after 2 days were assayed for CAT activity in body wall (B), head (H), midgut (M), silk gland (S), and gonad (G) as described in Example 7. Tissues from three larvae were pooled for each assay.
  • FIGS. 5A-C Time Course of CAT Activity in Infected Larvae
  • FIG. 5 A CAT assays of body wall tissues from larvae injected with BmNPV/A.cat.
  • FIG. 5B CAT assays of body wall tissues from larvae injected with BmNPV/P26.cat.
  • FIG. 5C Appropriate amounts of body wall tissue from larvae injected with BmNPV/A.cat (solid circles) or BmNPV/P.26cat (open circles) were assayed for CAT activity, the background level of CAT activity observed at 2-4 hr. p.i. was subtracted and the values plotted on logarithmic scale.
  • FIGS. 6A and 6B Transfection of Cells with pBmA.cat
  • Tissue culture cells (Bm5 or Sf21 ) were transfected as described in Example 8. (Ac-C represents acetyl chloramphenicol; C represents the substrate chloramphenicol).
  • FIG. 6A CAT assays performed on the indicated amount of culture cells 2 days after cells were transfected with pBmA.cat.
  • FIG. 6B The amount of plasmid DNA present in the transfected cells was measured by dot-blot hybridization of the indicated amount of cell suspensions, using as a probe radioactively labelled pBS/SK + DNA, as described in Example 8.
  • the present invention generally relates to a method of expressing heterologous proteins in insect cells using a recombinant baculovirus comprising a structural gene for a heterologous protein functionally attached to an insect cellular promoter such that expression of the heterologous protein occurs in substantially all of the insect tissues into which the recombinant baculovirus was introduced.
  • the heterologous protein may be a heterologous protein which is toxic to the insect or any other heterologous protein whose unregulated expression could incapacitate the insect host through unbalancing of an important physiological process.
  • This invention is also related to expression cassettes containing insect cellular promoters, recombinant expression cassettes containing heterologous genes functionally attached to insect cellular promoters, transplacement fragments containing recombinant expression cassettes, vectors having transplacement fragments and recombinant baculoviruses derived therefrom.
  • baculovirus is used herein as an alternative to the term “nuclear polyhedrosis virus” or "NPV”. It encompasses viruses classified under subgroup A of the family of Baculoviridae.
  • expression cassette means a fragment of nucleic acid comprising an insect cellular promoter sequence, with or without a sequence containing mRNA polyadenylation signals, and one or more restriction enzyme sites located downstream from the promoter allowing insertion of heterologous gene sequences.
  • the expression cassette is capable of directing the expression of a heterologous protein when the structural gene encoding the heterologous protein is functionally attached to the insect cellular promoter by insertion into one of the restriction sites.
  • the recombinant expression cassette allows expression of the heterologous protein in an insect when the expression cassette containing the heterologous protein is introduced into the tissues of the insect by infection with recombinant baculovirus comprising the recombinant expression cassette.
  • the recombinant expression cassette allows expression at an early stage of infection and/or it allows expression in substantially all tissues of an insect.
  • recombinant expression cassette means an expression cassette further comprising a structural gene sequence encoding a heterologous protein inserted into a restriction enzyme site such that the structural gene sequence is functionally attached to the insect cellular promoter.
  • the heterologous protein is expressed under the control of the insect cellular promoter.
  • the recombinant expression cassette allows expression at an early stage of infection and/or it allows expression in substantially all tissues of an insect.
  • placement fragment means a DNA fragment which comprises: (1 ) an expression cassette sequence or a recombinant expression cassette sequence, and (2) a portion of a baculovirus genome that can sustain insertions of non-viral DNA fragments.
  • a portion of a baculovirus genome that can sustain insertions of non-viral DNA fragments means a portion of the genome into which non-viral DNA fragments can be inserted or which can be replaced with the non-viral DNA fragments without affecting viral infectivity, replication or assembly.
  • the sequence from the baculovirus genome comprises the 5' and 3' sequences of the polyhedrin gene of the B. mori baculovirus.
  • the portion of the baculovirus genome contains the polyhedrin gene and flanking sequences of the baculovirus AcNPV.
  • transplacement vector means nucleic acid which comprises: (1 ) a transplacement fragment, and (2) DNA sequences allowing replication and selection in E. coli.
  • the vector may be a plasmid or simply a linear DNA fragment.
  • a transplacement fragment or vector is used to produce recombinant baculoviruses through double recombination/cross ⁇ over events.
  • insect cells are transfected with the transplacement vector and DNA from wild-type baculovirus, a double cross-over event between the homologous portions of the baculovirus genome and the transplacement fragment will result in the replacement of a portion of the wild-type baculovirus sequence with the part of the transplacement fragment which contains the recombinant expression cassette.
  • the term "recombinant baculovirus” refers to a baculovirus whose genome comprises a recombinant expression cassette of the invention.
  • the recombinant baculovirus comprises B. mori nuclear polyhedrosis virus in which a section of the DNA sequence encoding the polyhedrin gene is replaced with a transplacement fragment comprising a heterologous structural gene functionally linked to an insect cellular promoter, preferably the promoter for the cytoplasmic actin gene of B. mori.
  • the recombinant baculovirus comprises A.
  • promoter means a DNA sequence which initiates and directs the transcription of a heterologous gene into an RNA transcript in cells.
  • insect cellular promoter is a promoter which will direct the expression of a heterologous structural gene when the gene is functionally linked to the insect cellular promoter in a recombinant expression cassette and the recombinant expression cassette is introduced into insect cells either by transfection of the cells directly or with a transplacement vector comprising the expression cassette and wild-type baculovirus DNA or by infection of the cells with a recombinant baculovirus comprising the expression cassette.
  • the insect cellular promoter allows expression at an early stage of infection and/or it allows expression in substantially all tissues of an insect.
  • insect cellular promoter is a promoter which does not normally direct the expression of the heterologous structural gene and is not naturally functionally attached to that structural gene.
  • the insect cellular promoter is not the promoter which normally directs the expression of the heterologous structural gene in the wild-type insect.
  • the insect cellular promoter is an insect cytoplasmic actin promoter; most preferably, the insect cellular promoter is the cytoplasmic actin promoter of B. mori.
  • the insect cellular promoter comprises the promoter for an insect ribosomal gene, tRNA gene, histone gene, or tubulin gene.
  • the heterologous gene it may be desirable to genetically modify the insect cellular promoter so as to make it capable of expressing the heterologous gene more efficiently in the cells of the insect brain, gut and/or muscle.
  • modification of the insect cellular promoter may be the addition of a tissue-specific "enhancer" element or some other DNA sequence to the expression cassette such that, while the promoter is expressed in substantially all tissues, it is over-expressed in certain tissues of the insect.
  • constitutive expression means that the promoter is expressed continuously in the insect cells. In the case of insect cells into which recombinant virus has been introduced, the promoter is expressed for at least 20 hours after introduction, more preferably for at least 30 hours after introduction and most preferably at least 60 hours after introduction.
  • the term "at an early stage of introduction” means production of the heterologous protein under the functional control of the insect cellular promoter occurs before expression would occur if the heterologous protein were functionally attached to a polyhedrin promoter or to other viral promoters that are functional after viral DNA replication.
  • expression occurs at an early stage of introduction where the heterologous protein is produced before about 1 0 hours, more preferably before about 8 hours, most preferably before about 5 hours post infection.
  • insect larvae are infected with recombinant virus containing the expression cassette, expression will occur at an early stage of introduction when it occurs before about 48 hours post infection, more preferably before 24 hours post infection and most preferably before 1 2 hours post infection.
  • substantially all insect tissues means that the heterologous protein is expressed in all insect tissues, more preferably the gut, brain, nervous system, fat body and muscle tissue, into which it is introduced by infection with a recombinant baculovirus comprising a structural gene coding for the heterologous protein functionally linked to an insect cellular promoter.
  • heterologous protein means that the structural gene encoding the heterologous protein is transcribed into mRNA and that the mRNA is further translated into protein.
  • the heterologous protein will be properly processed by the insect cell, although such processing may be in a tissue specific manner.
  • structural gene refers to those DNA sequences which, when functionally attached to a cellular promoter, will be transcribed and produce a heterologous protein in insect cells.
  • heterologous structural gene or “heterologous gene” is a structural gene which is not normally present in wild- type baculovirus genomes, but which may or may not be present in insect genomes.
  • the heterologous structural gene does not include the structural gene which is functionally attached to the insect cellular promoter in wild-type insect cells.
  • a heterologous structural gene is a structural gene which will be transcribed and will produce a protein when functionally attached to an insect cellular promoter in a recombinant expression cassette and thereafter introduced into cells of an insect either by infection of cells by a recombinant baculovirus containing the cassette or by transfection of cells with a transplacement fragment containing the cassette or with the recombinant expression cassette alone. While the CAT gene was used to characterize the expression of the heterologous protein under the control of the cellular promoter in the examples provided herein, it will be recognized that any heterologous structural gene meeting the above criteria may be used in the invention.
  • heterologous protein refers to a protein encoded by a heterologous structural gene and which is not normally expressed by the baculovirus, but which may be expressed by insect cells in a regulated manner.
  • the protein may be compatible or incompatible with the insect.
  • compatible heterologous proteins are chloramphenicol acetyltransferase, human alpha interferon (IFN- ⁇ ), insulin-like growth factor-ll (IGF-II), human interieukin 3, mouse interleukin 3, human and mouse interleukin 4, human T-lymphotropic virus (HTLV-1 ) p40 x , HTLV-1 env, human immunodeficiency virus (HIV- 1 ) gag, pol, sor, gp41 , and gp120, adenovirus E1 a, Japanese encephalitis virus env (N), bovine papillomavirus 1 (BPV1 ) E2, HPV6b E2, BPV1 E6, and human apolipoproteins A and E; ⁇ - galactosidase, hepatitis B surface antigen, HIV-1 env, HIV-1 gag, HTLV-1 p40 x , human IFN- ?, human interleukin 2, c
  • HA hemaggiutinin
  • LCMV lymphocytic choriomeningitis virus
  • Neurospora crassa activator protein polyomavirus T antigen
  • simian virus 40 (SV40) small t antigen SV40 large T antigen
  • Punta Toro phlebovirus N and Ns proteins simian rotavirus VP6, CD4 (T4), human erythropoietin, Hantaan virus structural protein, human epidermal growth factor (EGF) receptor, human insulin receptor, human B lymphotrophic virus 130-kd protein, hepatitis A virus VP1 , human tyrosine hydroxylase, human glucocerebrosidase, and mouse p53.
  • incompatible protein means either a toxic protein or an insect incapacitating protein of insect or non-insect origin whose unregulated expression could incapacitate the insect through unbalancing of an important physiological process.
  • An incompatible protein will enhance the inactivation of the insect by the baculovirus.
  • toxic gene means a heterologous DNA sequence which encodes for a heterologous protein product that inactivates the larvae or which results in extensive tissue damage to the larvae and eventually death.
  • Suitable toxic genes include genes that encode insect specific toxins or other gene products which, when inserted into the expression cassette improve the ability of the baculovirus to paralyze or kill the insect.
  • Such toxic genes include genes which encode for endotoxins from Bacillus thuringiensis.
  • Bacillus thuringiensis There are B. thuringiensis strains with activities against a wide range of insect species. Many strains produce a toxin that is active against lepidopteran larvae. Some strains produce toxins that affect dipteran or coleopteran larvae.
  • Suitable toxic genes include genes encoding: an insect specific neurotoxin, AalT, from the venom of the North African (Algerian) Scorpion, Androctonus australis Hector (Stewart et al., 1991 ); Tox-34, which encodes TxP-1 toxin associated with the mite Pyemotes tritici.
  • incapacitating protein means a protein whose unregulated expression could incapacitate the insect through unbalancing of an important physiological process.
  • incapacitating genes include: insect or non-insect neuropeptides such as FMRFamide-related peptides, enkephalin-related peptides, tachykinins, adipokinetic hormones, other myotropic peptides, such as proctolin, leucokinins, a tribekinins, drosulfakinins, locustakinin, locustamyotropins, leucopyrokinin, locustapyrokinin, leucosulfakinin, locustasulfakinin, allatostatins, allatotropin, alkaline phosphatases, collagenases, chitinases, juvenile hormone esterases and epoxide hydrolases, ecdysone and juvenile hormone receptors, eclosion hormone, prothoraci
  • introduction refers to either infection or transfection of insect cells.
  • infection refers to the invasion by pathogenic viral agents of cells where conditions are favorable for their replication and growth. Such invasion can be by placing the viral particles directly on the insect cell culture or by injection of the insect larvae with the recombinant virus.
  • the amount of recombinant virus injected into the larvae will be from 10 2 to 10 5 pfu of non-occluded virus/larvae.
  • larvae can be infected by the oral route using occlusion bodies carrying recombinant viruses.
  • the amount of occlusion bodies fed to the larvae is that amount which corresponds to the LD 50 for that species of baculovirus and insect host.
  • the LD 50 varies with each species of baculovirus and the age of the larvae.
  • the amount of occlusion bodies to be administered can readily determine the amount of occlusion bodies to be administered. Typically, the amount will vary from 10-10 6 occlusion bodies/insect. In those vectors in which the polyhedrin gene has been replaced by the expression cassette, the recombinant virus does not produce the polyhedrin protein, and occlusion body formation does not occur. However, co- infection of cell cultures with the budded virus forms of both recombinant and wild-type viruses provides occlusion bodies containing both recombinant and wild-type viruses and allows infection of insect larvae by the oral route. Alternatively, by constructing different transplacement vectors, it is possible to insert the recombinant expression cassette in other sites of the viral genome to generate occluded recombinant baculoviruses.
  • transfection refers to a technique for introducing purified nucleic acids into cells.
  • Calcium phosphate or other appropriate agents such as dextran sulfate are added to the DNA solution, and the solution is placed on the insect cells.
  • the insect cells will take up the DNA precipitated by the addition of calcium phosphate or other agents such as dextran sulphate to a DNA solution.
  • DNA can be introduced into the cells by electroporation.
  • the DNA is introduced into the cells by mixing the DNA solution with LipofectinTM (GIBCO BRL Canada, Burlington, Ontario) and adding the mixture to the cells.
  • transplacement fragment or transplacement vector containing the heterologous gene is constructed, host insect cells are transfected simultaneously with wild-type viral DNA and the transplacement fragment or vector DNA containing baculovirus DNA sequences homologous to the wild-type viral DNA.
  • the genetic recombination system of the host insect cell recombines the plasmid and viral DNAs. Double cross-over recombination events at homologous DNA sites results in the replacement of sequences of the viral genome with a portion of the transplacement fragment DNA, thereby inserting the recombinant expression cassette DNA containing the heterologous gene into the preferred site of the viral genome.
  • transplacement fragment or vector contains DNA sequences from the viral polyhedrin gene
  • a double recombination/cross-over event between the homologous viral sequences in the transplacement fragment and the wild-type baculovirus genome will result in the expression cassette replacing a portion of the polyhedrin gene of the wild-type genome.
  • recombinant viruses are selected by hybridization to heterologous gene probes and confirmed by restriction endonuciease and DNA sequence analysis identification techniques.
  • cells containing recombinant viruses with double cross- overs can be also identified visually because they do not contain viral occlusion bodies.
  • insect cells means insect cells from the insect species which are subject to baculovirus infection.
  • recombinant baculoviruses that effect an immediate incapacitation of pests by early expression of the incapacitating genes in the cells of the midgut would be advantageous. It has now been discovered that if a recombinant baculovirus contains a heterologous gene expressed under the control of a strong ubiquitous and constitutive insect cellular promoter, for example, the insect cytoplasmic actin promoter, the protein is expressed early in the baculovirus infection cycle and in substantially all infected tissues of the insect including the gut.
  • a strong ubiquitous and constitutive insect cellular promoter for example, the insect cytoplasmic actin promoter
  • recombinant nuclear polyhedrosis viruses with insect promoters have the additional advantage of a lack of potentially unstable sequence duplications of the viral genome. Therefore, if a heterologous gene whose product incapacitates the insect is placed under the control of an insect cellular promoter in a recombinant baculovirus, the gene product will be expressed in substantially all infected tissues of the insect at an early stage of infection and thus rapidly inactivate the insect. Furthermore, expression of compatible heterologous proteins may be placed under the control of an insect cellular promoter in a recombinant baculovirus for expression of the protein in infected insect tissue culture cells as a method of obtaining large amounts of the desired protein.
  • the present invention is directed, in part, to the discovery of an expression cassette which can be used to generate recombinant baculoviruses. that can express heterologous proteins under the control of an insect cellular promoter in substantially all tissues of the insect.
  • An expression cassette was constructed which contains an insect cellular promoter and a termination sequence.
  • the cassette also contains a polylinker sequence comprising a number of unique restriction sites into which the desired structural gene encoding a heterologous protein may be inserted.
  • the desired heterologous gene was inserted into the expression cassette such that the structural gene was functionally linked to the insect cellular promoter.
  • Another vector was also constructed which contains a portion of the baculovirus that can sustain insertions of non-viral DNA fragments.
  • the recombinant expression cassette containing the heterologous gene was excised from the first vector and inserted into a nucleotide site in the baculovirus sequence present on the other vector, thereby creating a transplacement vector.
  • the recombinant expression cassette and the transplacement vector were transfected separately into insect cells for expression of the heterologous protein.
  • the transplacement vector was also co-transfected into insect tissue culture cells with wild-type baculovirus DNA which is homologous to the portion of baculovirus DNA present on the transplacement vector.
  • Recombinant baculovirus genomes containing the expression cassette were generated by double cross-over events. Such recombinant viruses were used to infect insect cells for the expression of the heterologous protein.
  • an expression cassette was constructed comprising an insect cellular promoter and termination sequences.
  • the insect cellular promoter is able to direct the expression of a heterologous protein functionally attached to the insect cellular promoter when the expression cassette is introduced into insect cells.
  • an expression cassette was constructed to contain a multiple cloning site downstream of a mutationally inactivated translation initiation codon of the A 3 cytoplasmic actin gene of B. mori and upstream of the termination sequences of the cytoplasmic actin gene.
  • the expression cassette was present on the vector pBmA.
  • Other suitable promoters could include promoters from insect ribosomal genes, tRNA genes, histone genes or tubulin genes.
  • the gene or the cDNA sequence encoding the desired heterologous protein was inserted into one of the unique restriction enzyme sites present downstream from the insect cellular promoter in the expression cassette.
  • the desired heterologous protein is the chloramphenicol acetyltransferase structural gene sequence (CAT). This gene, cat, was inserted into the BamHI site of pBmA to create pBmA.cat.
  • CAT chloramphenicol acetyltransferase structural gene sequence
  • This gene, cat was inserted into the BamHI site of pBmA to create pBmA.cat.
  • a fragment containing the sequence for Heliothis virescens juvenile hormone esterase cDNA is inserted into the EcoRI site of the polylinker of plasmid pBmA.
  • a fragment containing the sequence for bovine preproenkephalin cDNA (Gubler and Hoffman, 1983, Gene, 25:263-269) is inserted between the EcoRI and BamHI sites of the polylinker of plasmid pBmA.
  • a fragment containing the gene for Drosophila FMRFamide-like peptide precursor (Schneider and Taghert, 1990, J. Biol. Chem., 265:6890-6895) is ligated into the EcoRI site of the polylinker of plasmid pBmA.
  • the genes for the neuropeptide proctolin, mouse tissue non-specific alkaline phosphatase, chicken protamine gene, Pyemotes tritici insectotoxin PxP-1 and Androctonus austra/is insectotoxin AalT will also be inserted into the expression cassette.
  • Another plasmid was constructed which contained a portion of a baculovirus genome which can sustain insertions of non-viral fragments without affecting viral infectivity, replication or assembly.
  • plasmid pBmp2s was constructed to contain a 10 kb Pst ⁇ fragment of BmNPV DNA including a partially deleted polyhedrin gene.
  • plasmid pAc.RI-l which contains the polyhedrin gene of AcNPV will be used.
  • Other portions of the genome of AcNPV that can similarly sustain insertions of foreign DNA and equivalent portions from other baculovirus genomes could also be used to construct transplacement vectors containing recombinant expression cassettes employing insect cellular promoters.
  • the recombinant expression cassette containing the insect cellular promoter and the desired heterologous structural gene was isolated from the first plasmid and inserted into a unique restriction enzyme site within the baculovirus sequences present on the second vector.
  • the A 3 cytoplasmic actin promoter-CAT structural gene recombinant expression cassette was excised with Sst ⁇ and inserted into the unique Sst ⁇ site in the deleted polyhedrin gene of plasmid pBmp2s to produce a transplacement vector (pBmp2s/A.cat).
  • FMRFamide-like peptide precursor and mouse tissue non-specific alkaline phosphatase, proctolin, the chicken protamine gene, Py emotes tritici insectotoxin TxP-1 and Androctonus australis insectotoxin AalT will be inserted into pBmp2s.
  • the expression cassettes described above can also be inserted into plasmid pAc.RI-l and into other vectors containing portions of the genomes of other baculoviruses that can sustain insertions of foreign DNA.
  • the resulting transplacement vector was used to generate recombinant viruses by transfection of cells with the vector DNA and wild-type BmNPV DNA.
  • the transplacement vector used was derived from pBmp2s
  • the resulting recombinant BmNPV virus was selected on the basis of its polyhedrin-minus phenotype and its structure confirmed by DNA hybridization and DNA sequencing. Replacement of the polyhedrin gene results in recombinant viruses which produce normal levels of budded virus but do not produce occlusion bodies. Such recombinant viruses replicate normally in insect cell cultures but are deficient in their ability to infect larvae by the oral infection route.
  • the lack of occlusion body formation in plaques produced by recombinant viruses carrying a foreign gene in place of the polyhedrin gene can be detected visually in the infected cells, and this provides a selectable phenotype for rapid recombinant virus selection.
  • the transplacement vector is derived from pAc.RI-l the recombinant AcNPV baculoviruses will be occluded into polyhedra.
  • an expression cassette containing an insect cellular promoter able to direct the expression of any desired heterologous protein such that when the recombinant expression cassette is introduced into insects by infection with a recombinant baculovirus containing the recombinant expression cassette the heterologous protein will be expressed in substantially ail of the insect tissues at an early stage in the infection. It is also obvious that the invention can be applied to any available baculoviruses and any insects that are subject to baculovirus infection.
  • BmNPV/A.cat A recombinant BmNPV (BmNPV/A.cat) containing the cat reporter gene under the control of the cytoplasmic actin promoter directed expression of CAT protein in both infected Bm5 cells and in all examined tissues of infected B. mori larvae. Placement of the cat gene under the control of the actin promoter resulted in faster induction of expression relative to that observed with the CAT gene under the control of the polyhedrin promoter both in infected Bm5 cells and in infected larvae. In the infected larvae, the constitutive actin promoter was active 24 hours earlier than the polyhedrin promoter. It is likely that the actin promoter is activated immediately upon entrance of the viral genome into the nuclei of infected cells.
  • Silkworm larvae were infected with BmNPV/A.cat by injection of non-occluded virus into the haemocoel, and CAT activity was observed in all tissues tested, including midgut. It was also determined that the actin-cat expression cassette is expressed in cells of S. frugiperda (Sf21 ) and C. fumiferana (Cf124) at levels comparable to those in Bm5 cells.
  • the expression cassette of this invention could be used for constructing recombinant baculoviruses for the control of a variety of insect species.
  • the vector, pBmA (FIG. 1 A), is a pBluescript (Stratagene) derivative of clone pA3-5500 which contains the A3 cytoplasmic actin gene of Bombyx mori (Mounier and PrudAppel, 1 986, Biochimie, 68:1053-1061 ).
  • Plasmid pBmA was constructed to contain 1 .5 kb of the A3 gene 5' flanking sequences and part of its first exon to position + 67 (relative to transcription initiation), a polylinker region derived from plasmid pBluescript (Stratagene) for insertion of foreign gene sequences, and an additional 1 .05 kb of the A3 gene sequences encompassing part of the third exon of the gene from position + 836 and adjacent 3' flanking sequences which contain signals required for RNA transcript polyadenylation.
  • This expression vector was constructed by (1 ) subcloning into plasmid Bluescript SK + (Stratagene) a 1 .5 kb Kpnl/Accl fragment of clone pA3-5500 containing the 5' flanking, 5' untranslated and coding sequences of the A3 gene up to position + 67 to generate plasmid pBmAp; (2) mutagenizing the ATG translation initiation codon present at position + 36 to + 38 of the actin coding sequence in plasmid pBmAp into AGG, AAG or ACG by the method of Kunkel (1985) P.N.A.S., 82:488-492 to generate plasmids pBmAp.AGG, pBmAp.AAG and pBmAp.ACG; (3) subcloning into plasmid pSP72 (Promega Corporation) a 1.05 kb Xhol/Sall fragment of clone pA3-5500, containing
  • the actin expression cassette pBmA (FIG. 1 A) was derived from plasmid pBmAo.AGG by digestion with Sail (complete digestion) and BamHI (partial digestion) to remove part of the polylinker sequence of plasmid pSP72 present at the 3' terminus of the actin insert, and religation with T4 DNA ligase. Translation initiation from any gene inserted in the pBmA actin expression cassette polylinker (FIG. 1 A) occurs from the first ATG triplet of the insert.
  • a 900 bp X ⁇ oll fragment containing the chloramphenicol acetyl transferase (CAT) open-reading frame was excised from pCARCAT-1 (Mitsialis et al., 1 987, P.N.A.S. , 84:7987-7991 ) by digestion of the DNA with the restriction enzyme XhoU.
  • This fragment includes the entire coding information of CAT, all of the 5' and most of the 3'-untranslated gene sequences, as well as 63 bp of the 5' untranslated region of the early transcription unit of SV40. It should be emphasized that the SV40 sequences are completely devoid of any promoter or enhancer elements. Therefore, the transcripts must depend on control elements contributed by the actin promoter sequences.
  • This fragment was inserted into the BamYW site of pBmA to create pBmA.cat (FIG. 1 B).
  • plasmid pBmp2 which contains a copy of the BmNPV polyhedrin gene, whose promoter and part of the coding sequences have been deleted, was created (latrou and Meidinger, 1989, Gene, 75:59-71 ).
  • plasmid Bmp/pP3 was constructed as described in latrou et al., 1985, J. Virol., 54:436-445. More specifically, the B.
  • Xba ⁇ linkers were ligated to the end-filled DNA in a reaction containing 66 mM Tris-HCI, pH 7.5, 66 ⁇ M ATP, 6.6 mM MgCI 2 , 10mM DTT, 0.125 ⁇ g/ml of Xbal linkers, 4 ⁇ glml of end-filled DNA and 300 units/ml T4 DNA ligase (Pharmacia LKB).
  • Linker- ligated DNA was purified by electroelution, restricted with Xbal, and circularized by incubating in the above reaction mix at a DNA concentration of 0.4 ⁇ g/ml with 30 units/ml T4 DNA ligase.
  • Transformation of circularized plasmid DNA into Escherichia coli HB101 resulted in the generation of a library of clones containing polyhedrin genes with segmental deletions of various lengths starting from the unique Xbal site of the gene.
  • Plasmid DNA from several clones was characterized by sequence analysis (Maxam and Gilbert, 1977, P.N.A.S. , 74:560-564), and plasmid pBmp2 which has the region from nt-90 to nt 339 of the polyhedrin gene deleted was selected as the recipient for insertion of the actin expression cassette.
  • Plasmid Pbmp2 was digested with Xbal and the Xbal site converted to an SstI site by the addition of linkers and religation.
  • the resulting plasmid was designated pBmp2s (FIG. 1 C).
  • the Sst fragment containing the A3 promoter-CAT structural gene DNA fragment was removed from Pbma.cat by digestion with Sst and then inserted into the SstI site of pBmp2s to create pBmp2s/A.cat (FIG. 1 D).
  • a recombinant virus, BmNPV/A.cat was obtained using this transfer vector as described in Example 2.
  • Plasmid pBmp26.cat contains a mutated polyhedrin gene promoter directing the expression of the cat structural gene. It has been constructed by subcloning the cat structural gene into plasmid pBmp26T. Plasmid pBmp26T is a derivative of plasmid pBmp26 which was initially selected from the library of clones generated by Bal 31 deletion mutagenesis of the polyhedrin gene described above for plasmid pBmp2, and shown by DNA sequence analysis to encompass a deletion of nucleotides + 27 to + 251 (relative to translation initiation) of the polyhedrin gene.
  • the polyhedrin gene translation initiation codon ATG was removed from plasmid pBmp26 containing the deleted polyhedrin gene, on a 2.0 kb Xhol/Xbal fragment and mutationally converted into ATT by the method of Kunkel (1985, P.N.A.S., 82:488-492).
  • the mutated 2.0 kb fragment was then cloned into Xhol/Xbal digested Bmp/pP14 to create pBmp26T.
  • Plasmid Bmp/pP14 was created in the same manner as Bmp/pP3 described above (latrou et al., 1985, J. Virol., 54:436-445) but contains the baculoviral sequences in the opposite orientation.
  • pBmp26T has a deletion from + 27 to + 146 with ATT instead of ATG.
  • Plasmid pBmp26T was then digested with Xbal, and the 900 bp Xholl CAT structural gene open-reading frame was inserted into the plasmid by blunt end ligation to create pBmp26.cat.
  • the polyhedrin promoter sequences are retained in this plasmid and direct the transcription of the cat structural gene.
  • Bm5 cells were plated in 10 cm 2 plates or microtiter wells at a density of 2x10 5 cells/cm 2 .
  • Transfection was accomplished by removing the culture medium, rinsing the cell monolayer with basal medium (no fetal calf serum) and adding 500 ⁇ l of transfection solution [30 ⁇ glml Lipofectin (GIBCO BRL Canada) in basal IPL-41 (JRH Biosciences, Inc.) containing 5 ⁇ g/ml of the transfer vector DNA and 0.2 ⁇ g/ml BmNPV DNA].
  • basal medium no fetal calf serum
  • the purified virus was propagated by infecting Bm5 cells grown in 25 cm 2 flasks at a density of 1 x 10 ⁇ cells/ml.
  • the medium from the infected cells, containing recombinant virus, was collected 4 to 7 days post infection and used as inoculum for subsequent experiments.
  • Bm5 cells to be infected with virus were seeded into 24-well microliter plates at a density of 2 x 10 5 cells (in 500 ⁇ l medium) per well. After 3 days, 100 ⁇ l of viral inoculum [10 ⁇ plaque forming units (pfu)] obtained by the method of Example 2, was added to each well. Time after infection was counted from the time that the virus was added to the medium. Cells were removed from the wells at the appropriate time by repeated pipeting.
  • Cells were pelleted from the medium at 3000 x g for 5 min., suspended in 1 ml PBS (10 mM KH 2 P0 4 , 2 mM NaH 2 P0 4 , 140 mM NaCI, 40 mM KCI) and repelleted. The cells were then resuspended in 200 ⁇ l of 0.25 M Tris-HCI, pH 7.8, freeze-thawed three times using dry ice to disrupt the cells and, after centrifugation, the supernatants retained for CAT assays.
  • PBS 10 mM KH 2 P0 4 , 2 mM NaH 2 P0 4 , 140 mM NaCI, 40 mM KCI
  • Silkworm larvae were reared on a diet of fresh mulberry leaves, and were injected with virus at the beginning of the 5th instar. After incubating the animals at 0°C for 1 hour, 1 0 ⁇ l of viral inoculum (10 5 pfu) obtained by the method of Example 2 was injected into the haemocoel using a 26 gauge needle. The infected animals were maintained as before and collected at the appropriate time for dissection.
  • Larvae were dissected in cold PBS, and the appropriate tissues were removed. Midguts were cut open longitudinally to allow removal of the gut contents, and all tissues were rinsed extensively with several changes of PBS.
  • the tissue samples were ground in 0.25 M Tris-HCI, pH 7.8 with a small pestle in a microcentrifuge tube, freeze-thawed and centrifuged as described above. All larval extracts were heated to 65 °C for 5 minutes to inactivate cellular deacetylase activities before use in CAT assays.
  • Assays for protein content and CAT activity of the extracts from transfected and infected cells and infected larvae were performed as follows.
  • cells (usually 2x10 5 to 2x10 ⁇ on plating day) were collected 24 hours post-transfection or 48 hours post-infection and rinsed with PBS (10 mM KH 2 P0 4 , 2 mM NaH 2 P0 4 , 140 mM NaCI, and 40 mM KCI). After pelleting at 3000 x g for 5 minutes, the cells were resuspended in 100 ⁇ l of 0.25 mM Tris-HCI, pH 7.8, and the suspension was freeze- thawed in dry ice 3 times to disrupt the cells.
  • Non-occluded virus was isolated from the medium by centrifugation at 50,000 rpm in a Beckman 100.2 rotor for 1 hour. After removal of the supernatant, the viral pellet was rinsed with 1 ml H 2 0, resuspended in 1 .6 ml H 2 0 and recentrifuged as before. The final pellet was again rinsed and resuspended in H 2 0.
  • Example 4 20 ⁇ l of isolated non-occiuded virus or medium containing NOV was assayed for 1 hour as described in Example 4. The results are shown in FIGS. 2A-C.
  • the upper spot (Ac-C) represents acetyl chloramphenicol, the products of the CAT reaction, while the lower spot in each assay (C) represents the substrate, chloramphenicol.
  • Medium collected from infected cells 5 days after infection and used as inoculum, was found to contain high levels of CAT activity (FIG. 2A, right).
  • NOV Non-occluded virus isolated from the medium was lysed by sonication and was also found to contain a significant amount of CAT activity even after extensive washing (FIG. 2A, left).
  • Bm5 cells were infected with BmNPV/A.cat or BmNPV/P26.cat inoculum obtained by the method of Example 2.
  • the cells were assayed for CAT activity by the method of Example 4.
  • Cells infected with either recombinant virus were found to contain background CAT activity 5 minutes after addition of the viral inoculum.
  • the level of CAT activity in these cells reached a plateau within 20 minutes (FIG. 2B), and this level was maintained for several hours.
  • Bm5 cells were infected with BmNPV/A.cat or
  • the cells were collected 1 hour after infection and were washed once (1 x) or five (5x) times with 1 ml PBS.
  • a portion of this CAT activity could be removed by washing the cells with PBS, but a significant fraction remained even after extensive washing.
  • none of the activity could be removed by washing (FIG. 2C). It was concluded, therefore, that this remaining activity represented CAT enzyme attached to or introduced into the cells during viral infection, and that this background value would have to be subtracted from all subsequent measurements in order to quantitate the amount of CAT enzyme present due to promoter activity during time course experiments.
  • Bm5 cells were infected with recombinant virus BmNPV/A.cat or BmNPV/P26.cat by the methods of Example 3, and cell extracts were assayed for CAT activity at various times after infection.
  • the CAT assays were performed using 5 ⁇ g cell protein incubated for 1 hour from cells infected with
  • CAT activity above the background level determined by the method of Example 5, was first detected in cells infected with BmNPV/A.cat at 5 hours post-infection (FIG. 3A), while in cells infected with BmNPV/P26.cat, activity above background was not observed until the 20 hour time point (FIG. 3B).
  • the cell extracts were then diluted appropriately to obtain quantitative CAT assays for all time points and the background values, determined by the method of Example 5 at 1 -2 hours post- infection, were subtracted from each point to generate the curves shown in FIG. 3C.
  • the actin promoter was transcriptionally active resulting in significant CAT activity above background level, while no activity could be detected from the polyhedrin promoter.
  • CAT activity derived from the polyhedrin promoter was detected at 20 hours p.i. At 20 hours p.i, the actin promoter was more active than the polyhedrin promoter, while at 30 hours p.i. CAT activity from the two promoters was roughly equal. Finally, after 30 hours p.i. expression from the polyhedrin promoter was higher than that from the actin promoter (by a factor of 3 at 50 hours p.i., the last point of the time course). Therefore, in Bm5 cells the actin promoter was active 1 5 hours earlier than the polyhedrin promoter. It is likely that the actin promoter is active immediately upon insertion of the viral genome into the cells.
  • Example 4 Fifth instar B. mori larvae were injected with recombinant virus BmNPV/A.cat or BmNPV/P26.cat by the method of Example 3, and various tissues were initially assayed for CAT activity 2 days post-infection by the method of Example 4 (FIG. 4). Tissues from three larvae were pooled for each assay. Larvae infected with either virus contained high levels of CAT activity in both the head and body wall, a lower level in the midgut, and still lower expression in the gonads. Although detectable, only very low levels of CAT activity could be seen in the silk glands.
  • larvae at the beginning of fifth instar were injected with the recombinant viruses BmNPV/A.cat or BmNPV/P26.cat by the method of Example 3, and body wall tissues were collected at different times post infection. The samples were then assayed for CAT activity by the method of Example 4 (FIGS. 5A-C).
  • Tissues from the injected larvae contained a low level of background CAT activity at early times post infection.
  • CAT activity above background was first observed in body wall tissues at 24 hours p.i. (FIG. 5A).
  • Larvae infected with BmNPV/P26.cat did not express any CAT activity above background levels until 48 hours p.i. (FIG. 5B).
  • FIG. 5C A quantitative analysis of the results obtained from all time points following subtraction of the background values is shown in FIG. 5C. Even at 48 hours p.i. the actin promoter was significantly more active than the polyhedrin promoter. At 60 hours p.i. the activity from the polyhedrin promoter was found to be higher than that from actin, but the difference in expression levels at that point was only six-fold.
  • plasmid pBmA.cat was transfected into tissue culture cells of both B. mori (Bm5) and Spodoptera frugiperda (Sf21 ) in a 24 well microliter plate. Wells were seeded with either 2x10 5 Bm5 cells or 4x10 5 Sf21 cells and the cells incubated in 200 ⁇ l transfection solution containing 5 ⁇ g/ml of pBmA.cat by the method of Example 2.
  • Transfected cells were collected, washed as above and suspended in 200 ⁇ l of PBS.
  • the cell suspensions (2 ⁇ l or 5 ⁇ l) were dot blotted onto Hybond N + membrane (Amersham, Canada Ltd.) and treated with 0.5 M NaOH followed by 0.5 Tris- HCI, pH 7.5.
  • Labeling of linearized pBS/SK + DNA with ⁇ - 32 P- dCTP and hybridization of this probe to the membrane was carried out at 65°C as described by Fotaki and latrou (1988) J. Mol. Biol. , 203:849-860.
  • the occluded form of BmNPV/A.cat is produced by infecting Bm5 cells simultaneously with the non-occluded forms of wild-type BmNPV and BmNPV/A.cat, as described previously (Price et al., 1989, P.N.A.S., 86:1453-1456) and collecting the resultant occlusion bodies.
  • These occlusion bodies contain both recombinant and wild-type virus and allow infection of insect larvae by the oral route.
  • the occlusion bodies are resuspended in distilled water, and the suspension is applied onto the diet (foliage or artificial diet) on which insects feed. In insect larvae infected orally with such occlusion bodies, CAT expression will occur in substantially all tissues at an early stage of introduction.
  • BmNPV/A.jhe juvenile hormone esterase
  • BmNPV/A.enk enkephalin-like peptides
  • proctolin BmNPV/A.pro
  • alkaline phosphatase BmNPV/A.aph
  • protamine BmNPV/A.cpr
  • FMRF- like peptides BmNPV/A.fmrf
  • insectotoxin TxP-l BmNPV/A.TxP- I
  • insectotoxin AalT insectotoxin AalT
  • Each of these recombinant baculoviruses incorporates a recombinant B. mori actin expression cassette containing a fragment of DNA encoding the corresponding heterologous protein, such that expression of the heterologous protein in insect cells infected with the recombinant baculovirus is directed by the B. mori actin promoter.
  • the method described in Example 1 is employed to generate the recombinant expression cassettes described below, and the latter are used in conjunction with plasmid pBmp2s (FIG. 1 C) to generate the corresponding transplacement vectors as described in Example 1 .
  • pBmA.jhe A 3.0 kb EcoRI fragment from plasmid 3hv16 containing the sequence for He/iothis virescens juvenile hormone esterase cDNA (Hanzlik et al., 1990, J. Biol. Chem., 264:12419-12425) is isolated by digestion of 3hv16 with EcoRI and inserted into the EcoRI site of the polylinker of plasmid pBmA (FIG. 1 A).
  • pBmA.enk A 1.2 kb Ncol/BamHI fragment excised from plasmid 921 containing the sequence for bovine preproenkephalin cDNA (Gubler and Hoffman, 1983, Gene, 25:263-269) is isolated by digestion of plasmid 921 with Ncol, and the 1 .2 kb fragment is inserted between the EcoRI and BamHI sites of the polylinker of plasmid pBmA.
  • sequences of the protruding 3' termini of the two synthetic fragments above allow their unidirectional insertion into the Bstxl site of pBmA.enk.
  • the resultant insertions are in-frame with the nucleotide sequences of preproenkephalin CDNA present in pBmA.enk such that translation of the mRNA synthesized under the control of the actin promoter yields fusion proteins which are longer than preproenkephalin by 1 5 or 14 amino acids, respectively, and contain the proctolin peptide sequence, RYLPT (amino acid residues 6-10 of SEQ ID NO:2 and amino acid residues 5-9 of SEQ ID N0:4).
  • pBmA.aph A 2.4 kb EcoRI fragment from plasmid p.1 .1 1 1 containing the structural gene for mouse tissue non ⁇ specific alkaline phosphastase (Hahnel and Schultz, 1989, Clin. Chim. Acta, 186:125-32) is isolated by digestion of p 1 .1 1 1 with EcoRI, and the 2.4 kb fragment is ligated into the EcoRI site of the polylinker of plasmid pBmA.
  • pBmA.fmrf A 1 .4 kb EcoRI fragment from plasmid pHS2A-3 containing the gene for Drosophila FMRFamide-like peptide precursor (Schneider and Taghert, 1990, J. Biol. Chem., 265:6890-6895) is isolated by digestion of pH52A-3 with EcoRI, and the 1 .4 kb fragment is ligated into the EcoRI site of the polylinker of plasmid pBmA.
  • pBmA.cpr A 0.45 kb Smal fragment from plasmid CPC4S4 containing the chicken protamine gene (Oliva and Dixon, 1989, J. Biol. Chem., 264:12472-12481 ) is isolated by digestion of CPC454 with Smal, and the 0.45 kb fragment is ligated into the Smal site of the polylinker of plasmid pBmA.
  • pBmA.TxP-1 A 0.94 kb EcoRI fragment from plasmid pTox-34 containing the sequence for Pyemotes tritici insectotoxin TxP-1 cDNA (Tomalski and Miller, 1991 , Nature, 352:82-85) is isolated by digestion of pTox-34 with EcoRI, and the 0.94 kb fragment is ligated into the EcoRI site of the polylinker of plasmid pBmA.
  • pBmA.AalT A 0.3 kb BamHI fragment from plasmid pTZ-AalT containing the sequence for Androctonus australis insectotoxin AalT cDNA (McCutchen et al., 1991 , Bio Technology, 9:848-852) is isolated by digestion with BamHI, and the 0.3 kb fragment is ligated into the BamHI site of the polylinker of plasmid pBmA.
  • Transplacement vectors pBmp2s/A.jhe, pBmp2s/A.enk, pBmp2s/A.aph and pBmp2s/A.fmrf, pBmp2s/A.pro, pBmp2s/A.cpr, pBmp2s/A.TxP-l and pBmp2s/A.AalT will be generated from the recombinant expression cassettes listed above, in conjunction with plasmid pBmp2s (FIG. 1 C), as described in Example 1 for transplacement vector pBmp2s/A.cat (FIG. I D).
  • Non-occluded recombinant baculoviruses BmNPV/A.jhe, BmNPV/A.enk, BmNPV/A.pro, BmNPV/A.aph, BmNPV/A.fmrf, BmNPV/A.cpr, BmNPV/A.TxP-l and
  • BmNPV/A.AalT will be generated by co-transfection of Bm5 cells with DNA from wild-type BmNPV and each of the above transplacement vectors, purified and amplified, as described in Example 2.
  • RNA extracted from tissues of infected larvae to 32 P-labelled probes derived from the heterologous genes; polymerase chain reaction (PCR) amplification of cDNA generated form the same RNA using appropriate primers derived from the known sequences of the heterologous genes; detection of the heterologous proteins themselves using available antibodies; and, in the cases of BmNPV/A.TxP-1 and BmNPV/A.AalT, also by monitoring the behavior of the infected larvae for paralytic symptoms.
  • PCR polymerase chain reaction
  • Example 1 1 - B. mori Recombinant Expression Cassettes Inserted into Autographa californica Nuclear Polyhedrosis Virus
  • transplacement vectors are created which can be used to generate recombinant Autographa californica Nuclear Polyhedrosis Viruses (AcNPVs) expressing the heterologous proteins listed in Example 10, under the control of the actin promoter of B. mori.
  • AcNPV Autographa californica Nuclear Polyhedrosis Viruses
  • Plasmid pAc.RI-l Smith et al., 1983, Mol. Cell. Biol., 3:21 56-2165, which contains the polyhedrin gene of AcNPV together with 4.0 kb of 5' and 2.1 kb of 3' flanking sequences, is used.
  • This plasmid is linearized with EcoRV which cleaves uniquely at a position located 40 bp upstream of the polyhedrin gene, and the SstI fragments of the recombinant expression cassettes 1-8 listed in Example 10 above, which contain the heterologous genes under the control of the actin promoter of B.
  • Acids Res., 15:10233-10248) have demonstrated that insertions of foreign DNA into this site of the genome of AcNPV leave all AcNPV functions unaffected, including that of the polyhedrin gene, thus permitting the generation of recombinant AcNPV baculoviruses which are occluded into polyhedra.
  • each plasmid containing a recombinant expression cassette as described in Example 10 is digested with SstI, the 3' protruding termini generated by SstI are removed by digestion with mung bean nuclease (New England Biolabs, Inc.), the SstI fragments containing the recombinant expression cassette are isolated by electroelution following electrophoresis in an agarose gel, and ligated into the EcoRV-digested plasmid pAc.RI-l with T4 DNA ligase, as described in Example 1 .
  • Recombinant baculoviruses AcNPV/A.jhe, AcNPV/A.aph, AcNPV/A.enk, AcNPV/A.pro, AcNPV/A.cpr, AcNPV/A.fmrf, AcNPV/A.TxP-l and AcNPV/A.AalT are generated and purified in Spodoptera frugiperda (Sf21 ) cells using the DNA of the transplacement vectors above and DNA from wild-type AcNPV, as described in Example 2.
  • Infection of Trichoplusia ni, B. mori or He/iothis virescens insect larvae with such recombinant baculoviruses by the oral route is accomplished by spreading aqueous suspensions of polyhedra prepared from infected Sf21 cells onto the diet (foliage or artificial diets) of these insects and allowing the larvae to feed on the occlusion body-containing diet.
  • expression of the heterologous proteins under the control of the actin promoter of B. mori will occur in essentially all insect tissues at an early stage of introduction.

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Abstract

Procédé pour exprimer des protéines hétérologues dans des cellules d'insecte, selon lequel on utilise un baculovirus recombiné, comprenant un gène structural pour une protéine hétérologue fixée fonctionnellement à un promoteur de cellules d'insecte. Dans ce procédé, le promoteur de cellules d'insecte est un promoteur qui n'est pas fixé fonctionnellement au gène structural, de sorte que la protéine hétérologue est exprimée dans les cellules d'insecte ayant le baculovirus recombiné. La protéine hétérologue comprend des protéine pouvant rendre l'insecte inoffensif. L'invention concerne également des cassettes d'expression comprenant un promoteur de cellules d'insecte capable de diriger l'expression d'une protéine hétérologue dans des tissus contenant la cassette d'expression, des cassettes d'expression recombinées contenant des protéines hétérologues, des fragments de transfert et des vecteurs contenant des cassettes d'expression recombinées, et les baculovirus qui en sont dérivés.
EP93915581A 1992-06-25 1993-06-25 Baculovirus recombine contenant un promoteur de cellules d'insecte Withdrawn EP0647274A1 (fr)

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CA2179394A1 (fr) * 1993-12-23 1995-06-29 Kostas Iatrou Procedes utilises pour exprimer des proteines dans des cellules d'insectes et procedes utilises pour eliminer des insectes
GB9405951D0 (en) * 1994-03-25 1994-05-11 Zeneca Ltd Biological control agents
FR2724393A1 (fr) * 1994-09-12 1996-03-15 Inst Nat Sante Rech Med Obtention d'un anticorps monoclonal recombinant humanise a partir d'un anticorps monoclonal murin, sa production en cellules d'insecte, et ses utilisations
US5843429A (en) * 1994-11-28 1998-12-01 The United States Of America As Represented By The Secretary Of Agriculture Gonad-specific virus which causes sterility in the corn earworm, Helicoverpa zea
EP0876497A1 (fr) 1995-12-22 1998-11-11 E.I. Du Pont De Nemours And Company Production de baculovirus de recombinaison
AU2789000A (en) * 1999-02-24 2000-09-14 University Technologies International Inc. Sequences for improving the efficiency of secretion of non-secreted proteins from mammalian and insect cells

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US5004687A (en) * 1985-05-21 1991-04-02 University Of Georgia Research Foundation, Inc. Insect virus vector with broadened host range

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