WO1992014818A2 - Systeme d'expression d'entomopoxvirus comprenant des sequences de spheroïdine ou de thymidine-kinase - Google Patents

Systeme d'expression d'entomopoxvirus comprenant des sequences de spheroïdine ou de thymidine-kinase Download PDF

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WO1992014818A2
WO1992014818A2 PCT/US1992/000855 US9200855W WO9214818A2 WO 1992014818 A2 WO1992014818 A2 WO 1992014818A2 US 9200855 W US9200855 W US 9200855W WO 9214818 A2 WO9214818 A2 WO 9214818A2
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sequence
asn
ile
gene
leu
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PCT/US1992/000855
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WO1992014818A3 (fr
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Richard W. Moyer
Richard L. Hall
Michael E. Gruidl
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University Of Florida
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Priority to MX9200697A priority Critical patent/MX9200697A/es
Priority to AU16634/92A priority patent/AU663709B2/en
Priority to JP4508743A priority patent/JPH06506594A/ja
Priority to ZA921163A priority patent/ZA921163B/xx
Priority to IL100983A priority patent/IL100983A0/xx
Priority to IE051592A priority patent/IE920515A1/en
Priority to NZ241662A priority patent/NZ241662A/en
Priority to CN92101985A priority patent/CN1065293A/zh
Publication of WO1992014818A2 publication Critical patent/WO1992014818A2/fr
Publication of WO1992014818A3 publication Critical patent/WO1992014818A3/fr
Priority to US09/370,861 priority patent/US6410221B1/en

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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C12N9/1211—Thymidine kinase (2.7.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates generally to the field of recombinantly-produced proteins and specifically to novel, recombinant Entomopoxvirus proteins, protein regulatory sequences and their uses in expressing heterologous genes in transformed hosts.
  • Poxviruses are taxonomically classified into the family Chordopoxvirinae, whose members infect vertebrate hosts, e.g., the Orthopoxvirus vaccinia, or into the family Entomopoxvirinae. Very little is known about members of the Entomopoxvirinae family other than the insect host range of individual members.
  • One species of Entomopoxvirus (EPV) is the Amsacta moorei Entomopoxvirus (AmEPV), which was first isolated from larvae of the red hairy caterpillar Amsacta moorei
  • AmEPV is the type species of genus B of EPVs and is one of three known EPVs which will replicate in cultured insect cells [R. R. Granados et al, "Replication of Amsacta moorei Entomopoxvirus and Autographa
  • AmEPV is one of the few insect poxviruses which can replicate in insect cell culture; AmEPV is unable to replicate in vertebrate cell lines.
  • the AmEPV doublestranded DNA genome is about 225 kb unusually A+T rich (18.5% G+C) [W. H. R. Langridge et al, Virology, 76:616620 (1977)].
  • Recently, a series of restriction maps for AmEPV were published [R. L. Hall et al. Arch. Virol., 110:77-90 (1990)]. No DNA homology to vaccinia has been detected [W. H. Langridge, J. Invertebr. Pathol., 42:77- 82 (1983); W. H. Langridge, J. Invertebr. Pathol., 43:41- 46 (1984)].
  • the viral replication cycle of AmEPV resembles that of other poxviruses except for the appearance of occluded virus late in infection.
  • AmEPV once a cell is infected, both occluded and extracellular virus particles are generated.
  • the mature occlusion body particle which is responsible for environmentally protecting the virion during infection, consists of virus embedded within a crystalline matrix consisting primarily of a single protein, spheroidin.
  • Spheroidin the major structural protein of AmEPV, has been reported to be 110 kDa in molecular weight and to consist of a high
  • eukaryotic viral vectors are either tumorigenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with resultant gene products and accidental infections. Further, in some eukaryotic host-viral vector systems, the gene product itself exhibits antiviral activity, thereby decreasing the yield of that protein.
  • Vaccinia virus has recently been developed as a eukaryotic cloning and expression vector [M. Mackett et al, DNA Cloning, Vol. II, ed. D. M. Glover, pp. 191-212, Oxford: IRL Press (1985); D. Panicali et al, Proc. Natl. Acad. Sci. USA. 8j3: 5364-5368 (1982)]. Numerous viral antigens have been expressed using vaccinia virus vectors [E. Paoletti et al, Proc. Natl. Acad. Sci. USA. 81:193-197 (1984); A. Piccine et al, BioEssays, 5:248-252
  • poxviruses have several advantageous features as vaccine vectors. These include the ability of poxvirus-based vaccines to stimulate both cell-mediated and humoral immunity, minimal cost to mass produce vaccine and the stability of the lyophilized vaccine without refrigeration, ease of administration under non-sterile condition, and the ability to insert at least 25,000 base pairs of foreign DNA into an infectious recombinant, thereby permitting the simultaneous expression of many antigens from one recombinant.
  • the invention provides an
  • Entomopoxvirus polynucleotide sequence free from other viral sequences with which it is associated in nature, which comprises a sequence encoding the Entomopoxvirus spheroidin gene and/or its regulatory sequences, an allelic variant, an analog or a fragment thereof.
  • the spheroidin DNA sequence is isolated from the Amsacta moorei Entomopoxvirus and is illustrated in Fig. 2 [SEQ ID NO:1].
  • Another aspect of the invention is the
  • polynucleotide sequence encoding the Entomopoxvirus spheroidin promoter or an allelic variant, analog or fragment thereof.
  • the spheroidin promoter sequence is characterized by the ability to direct the expression of a heterologous gene to which the sequence or fragment is operably linked in a selected host cell.
  • the present invention provides a recombinant polynucleotide sequence comprising a sequence encoding the Entomopoxvirus spheroidin protein and/or its regulatory sequences, an allelic variant, analog or fragment thereof, linked to a second
  • heterologous gene in a selected host cell.
  • Another embodiment provides the sequence encoding the spheroidin protein linked to the heterologous gene in a manner permitting expression of a fusion protein.
  • Still another embodiment provides the heterologous gene inserted into a site in the spheroidin gene so that the heterologous gene is flanked on both termini by spheroidin sequences.
  • the invention provides an Entomopoxvirus polynucleotide sequence free from other viral sequences with which it is associated in nature, comprising a sequence encoding the Entomopoxvirus
  • tk thymidine kinase
  • sequences an allelic variant, an analog or a fragment thereof.
  • the sequence originates from the Amsacta moorei Entomopoxvirus and is illustrated in Fig. 3 [SEQ ID NO: 8].
  • sequence encodes the Entomopoxvirus tk promoter, allelic variant or a fragment thereof.
  • the tk promoter sequence is
  • a further aspect of the invention provides a recombinant polynucleotide sequence described above encoding the Entomopoxvirus tk gene and/or its regulatory sequences, an allelic variant, or a fragment thereof, linked to a heterologous gene.
  • This polynucleotide sequence provides the tk promoter sequence operably linked to the heterologous gene to direct the expression of the heterologous gene in a selected host cell.
  • Another embodiment provides the sequence encoding the tk protein linked to the heterologous gene in a manner permitting expression of a fusion protein.
  • Still another embodiment provides the heterologous gene
  • Another aspect of the invention is an
  • Entomopoxvirus spheroidin polypeptide a fragment thereof, or an analog thereof, optionally fused to a heterologous protein or peptide.
  • polynucleotide molecules which comprise one or more of the polynucleotide sequences described above.
  • This molecule may be an expression vector or shuttle vector.
  • the molecule may also contain viral sequences originating from a virus other than the
  • Entomopoxvirus which contributed the spheroidin or tk polynucleotide sequence, e.g., vaccinia.
  • the present invention provides a recombinant virus comprising a polynucleotide sequence as described above. Also provided are host cells infected with one or more of the described
  • the present invention also provides a method for producing a selected polypeptide comprising culturing a selected host cell infected with a recombinant virus, as described above, and recovering said polypeptide from the culture medium.
  • the invention provides a method for screening recombinant host cells for insertion of heterologous genes comprising infecting the cells with a recombinant virus containing a polynucleotide molecule comprising the selected heterologous gene sequence linked to an incomplete spheroidin or tk polynucleotide sequence or inserted into and interrupting the coding sequences thereof so that the heterologous gene is flanked at each termini by an Entomopoxvirus spheroidin or tk
  • the absence of occlusion bodies formed by the expression of the spheroidin protein in the spheroidin containing cell indicates the integration of the heterologous gene.
  • methotrexate or a nucleotide analogue of methotrexate, formed by the integration of the inactive thymidine kinase sequence indicates the insertion of the
  • Fig. 1 is a physical map of AmEPV illustrating restriction fragments thereof and showing the spheroidin gene just to the right of site #29 in the Hindlll-G fragment.
  • Fig. 2 provides the AmEPV DNA sequence of the Amsacta moorei Entomopoxvirus spheroidin gene
  • flanking sequences [SEQ ID NO:1], the deduced amino acid sequences of the spheroidin protein [SEQ ID NO: 6], and five additional open reading frames (ORFs) .
  • Fig. 3 provides the DNA sequence of the Amsacta moorei Entomopoxvirus thymidine kinase (tk) gene and flanking sequences [SEQ ID NO: 8], the deduced amino acid sequences of the tk protein [SEQ ID NO: 11], and two additional ORFs.
  • tk Amsacta moorei Entomopoxvirus thymidine kinase
  • Fig. 4 provides the nucleotide sequences of the synthetic oligonucleotides designated RM58 [SEQ ID NO: RM58]
  • Fig. 5 is a schematic map of an AmEPV fragment illustrating the orientation of the spheroidin ORF on the physical map and indicating homologies.
  • the present invention provides novel
  • Entomopoxvirus EDV
  • Recombinant polynucleotide vectors containing the sequences, recombinant viruses containing the sequences, and host cells infected with the recombinant viruses are also disclosed herein. These compositions are useful in methods of the invention for the expression of heterologous genes and production of selected proteins in both insect and mammalian host cells.
  • Novel polynucleotide sequences of the invention encode the EPV spheroidin gene and/or its flanking sequences, including sequences which provide regulatory signals for the expression of the gene.
  • the invention also provides novel polynucleotide sequences encoding the EPV thymidine kinase (tk) gene and/or its flanking
  • RNA or DNA sequences More preferably, the polynucleotide sequences of this
  • inventions are DNA sequences.
  • spheroidin and tk polynucleotide sequences obtained from the Amsacta moorei Entomopoxvirus (AmEPV). While this is the presently preferred species for practice of the methods and compositions of this invention, it is anticipated that, utilizing the techniques described herein, substantially homologous sequences may be
  • the AmEPV spheroidin DNA sequence is reported in Fig. 2 as spanning nucleotides # 1 through 6768 [SEQ ID NO:1]. Within this sequence, the spheroidin gene coding sequence spans nucleotides #3080 to #6091 [SEQ ID NO:21]. A fragment which is likely to contain the promoter
  • sequences spans nucleotide #2781-3199 [SEQ ID NO:22]. Other regions of that sequence have also been identified as putative coding regions for other structural or regulatory genes associated with spheroidin. These other fragments of interest include the following sequences: nucleotide # 1472 through 2151 [SEQ ID NO: 23] encoding the G2R ORF [SEQ ID NO:3]; nucleotide #2502 through 2987 [SEQ ID NO: 24] encoding the G4R ORF [SEQ ID NO: 5]; and the following sequences transcribed left to right on Fig.
  • the AmEPV ORF G4R [SEQ ID NO: 5] which is immediately upstream of the spheroidin gene has
  • HM3 ORF capripoxvirus HM3 ORF
  • a homolog of the HM3 ORF is found in vaccinia virus just upstream of a truncated version of the cowpox virus ATI gene. Therefore, the microenvironments in this region are similar in the two viruses.
  • Two other ORFs relate to counterparts in vaccinia virus. These ORFs include the 17 ORF of the vaccinia virus HindIII-I fragment (17) [J. F. C. Schmitt et al, J. Virol..
  • oligonucleotide probes Transcription of the spheroidin gene is inhibited by cycloheximide, suggesting it is a late gene. Consistent with this prediction are the observations that spheroidin transcripts were initiated within a TAATG motif (See Fig. 2, nucleotide #3077- 3082) and that there was a 5' poly(A) sequence, both characteristic of late transcripts.
  • AmEPV tk DNA sequence including flanking and regulatory sequence, is reported in Fig. 3, as
  • the tk gene coding sequence spans nucleotides # 234 to 782 [SEQ ID NO: 28] (transcribed right to left on Fig. 3) .
  • Another fragment of interest may include nucleotides #783 through #851 [SEQ ID NO: 29] of that sequence or fragments thereof.
  • a fragment likely to contain the promoter regions spans nucleotide #750 - 890 [SEQ ID NO:30].
  • Other regions of that sequence have also been identified as putative coding regions for other structural or regulatory genes associated with tk. These other fragments of interest include the following
  • the location of the AmEPV tk gene maps in the EcoRI-Q fragment near the left end of the physical map of the AmEPV genome (Fig. 1) [see, also, R. L. Hall et al, Arch. Virol.. 110:77-90 (1990), incorporated by reference herein]. Because of the orientation of the gene within the AmEPV genome, transcription of the gene is likely to occur toward the terminus. There are believed to be similar tk genes, or variations thereof, in other
  • polynucleotide sequences when used with reference to the invention can include the entire EPV spheroidin or tk genes with regulatory sequences flanking the coding sequences.
  • the illustrated AmEPV sequences are also encompassed by that term.
  • fragments of the coding sequences with flanking regulatory sequences are also encompasses.
  • the definition also encompasses the regulatory sequences only, e.g., the promoter sequences, transcription sites, termination sequences, and other regulatory sequences.
  • Sequences of the invention may also include all or portions of the spheroidin or tk genes linked in frame to a heterologous gene sequence.
  • polynucleotide sequences of the invention may include sequences of the spheroidin or tk genes into which have been inserted a foreign or heterologous gene sequence, so that the EPV sequences flank the heterologous gene sequence.
  • Polynucleotide sequences of this invention also include sequences which are capable of hybridizing to the sequences of Figs. 2 and 3, under stringent conditions, which sequences retain the same biological or regulatory activities as those of the figures. Also sequences capable of hybridizing to the sequences of Figs. 2 and 3 under non-stringent conditions may fall within this definition providing that the biological or regulatory characteristics of the sequences of Figs. 2 and 3, respectively, are retained. Examples of stringent and non-stringent conditions of hybridization are
  • polynucleotide sequences of this invention also include allelic variations (naturally- occurring base changes in the EPV species population which may or may not result in an amino acid change) of DNA sequences encoding the spheroidin or tk protein sequences or DNA sequences encoding the other ORFs or regulatory sequences illustrated in Figs. 2 and 3.
  • DNA sequences which encode spheroidin or tk proteins of the invention which differ in codon sequence due to the degeneracies of the genetic code or variations in the DNA sequences which are caused by point mutations or by induced modifications to enhance a
  • sequence data in Figs. 2 or 3 as well as the denoted characteristics of spheroidin or thymidine kinase, it is within the skill of the art to obtain other DNA sequences encoding these polypeptides.
  • the structural gene may be manipulated by varying individual nucleotides, while retaining the correct amino acid(s), or varying the nucleotides, so as to modify the amino acids, without loss of enzymatic activity.
  • Nucleotides may be substituted, inserted, or deleted by known techniques, including, for example, in vitro mutagenesis and primer repair.
  • the structural gene may be truncated at its 3'- terminus and/or its 5'-terminus while retaining its biological activity. It may also be desirable to ligate a portion of the polypeptide sequence to a heterologous coding sequence, and thus to create a fusion peptide.
  • polynucleotide sequences of the present invention may be prepared synthetically or can be derived from viral RNA or from available cDNA-containing plasmids by chemical and genetic engineering techniques or
  • AmEPV proteins, spheroidin, thymidine kinase and their respective regulatory sequences, as described herein, may be encoded by polynucleotide sequences that differ in sequence from the sequences of Figs. 2 and 3 due to natural allelic or species
  • polypeptides also refer to any of the naturally occurring sequences and various analogs, e.g., processed or
  • truncated sequences or fragments including the mature spheroidin or tk polypeptides and mutant or modified polypeptides or fragments that retain the same biological activity and preferably have a homology to Fig. 2 or 3, respectively, of at least 80%, more preferably 90%, and most preferably 95%.
  • EPV spheroidin proteins encoded by the EPV spheroidin and tk polynucleotide sequences. Putative amino acid sequences of the two EPV proteins as well as additional putative proteins encoded by the ORFs of these sequences which are identified in Figs. 2 and 3, respectively.
  • EPV spheroidin has no significant amino acid homology to any previously reported protein, including the polyhedrin protein of baculovirus. Both spheroidin and tk are nonessential proteins, which makes them desirable as sites for insertion of exogenous DNA.
  • AmEPV tk amino acid sequence Comparison of the AmEPV tk amino acid sequence with other tk genes reveals that the AmEPV tk gene is not highly related to any of the vertebrate poxvirus tk genes (43.4 to 45.7%). The relatedness of the vertebrate tk proteins to AmEPV is still lower (39.3 to 41.0%), while African Swine Fever (ASF) showed the least homology of all the tk proteins tested (31.4%). Although ASF has many similarities to poxviruses, and both ASF and AmEPV infect vertebrate hosts, the tk genes indicate little commonality and/or indication of common origin stemming from invertebrate hosts.
  • the spheroidin and thymidine kinase polypeptide sequences may include isolated naturally-occurring spheroidin or tk amino acid sequences identified herein or deliberately modified sequences which maintain the biological or regulatory functions of the AmEPV
  • polypeptides respectively identified in Figs. 2 and 3. Therefore, provided that the biological activities of these polypeptides are retained in whole or part despite such modifications, this invention encompasses the use of all amino acid sequences disclosed herein as well as analogs thereof retaining spheroidin or tk biological activity. Typically, such analogs differ by only 1, 2, 3, or 4 codon changes. Similarly, proteins or functions encoded by the other spheroidin or tk ORFs may include sequences containing minor amino acid modifications but which retain their regulatory or other biological
  • polypeptides with minor amino acid variations from the natural amino acid sequence of Entomopoxvirus spheroidin or thymidine kinase in particular, conservative amino acid replacements.
  • Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families:
  • acidic aspartate, glutamate
  • basic lysine, arginine, histidine
  • non-polar alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • uncharged polar glycine
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides,
  • polypeptides are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with
  • proteins or polypeptides of the present invention may be expressed in host cells and purified from the cells or media by conventional means [Sambrook et al. Molecular Cloning. A Laboratory Manual, 2d edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)].
  • This invention also relates to novel viral recombinant polynucleotide molecules or vectors, which permit the expression of heterologous genes in a selected host cell.
  • a polynucleotide vector of the invention comprises the polynucleotide sequence encoding all or a portion of the spheroidin or tk gene, the RNA polymerase from a selected poxvirus, and the polynucleotide sequence encoding a desired heterologous gene.
  • the sequence includes the regulatory region, and most
  • the promoter region, of either the EMV spheroidin or tk gene preferably, the promoter region, of either the EMV spheroidin or tk gene.
  • the source of the polymerase is not limited to EMV; rather, any poxvirus RNA polymerase may be utilized.
  • the viral vectors may contain other viral elements contributed by another poxvirus, either vertebrate or invertebrate, with the only EPV sequences being provided by the presence of the EPV spheroidin or tk gene sequences, or fragments thereof.
  • Numerous conventional expression viral vectors and expression systems are known in the art. Particularly desirable vectors systems are those of vertebrate or invertebrate poxviruses.
  • the Entomopoxvirus spheroidin and tk gene regulatory sequences may be used in other virus vector systems which contain a poxvirus RNA polymerase to
  • expression systems in general, and the components thereof, including expression vectors and transformed host cells, are within the art. See, generally, methods described in standard texts, such as Sambrook et al, supra.
  • the present invention is therefore not limited to any particular viral expression system or vector into which a polynucleotide sequence of this invention may be inserted, provided that the vector or system contains a poxvirus RNA polymerase.
  • the vectors of the invention provide a helper independent vector system, that is, the presence or absence of a functional spheroidin or tk gene in a poxvirus contributing elements to the vector, e.g. , contributing the RNA polymerase, does not affect the usefulness of the resulting recombinant viral vector. Because both spheroidin and tk are non-essential genes, the viral vectors of this invention do not require the presence of any other viral proteins, which in helperdependent systems are contributed by additional viruses to coinfect the selected host cell.
  • heterologous gene include insect and mammalian cells.
  • the viral vector comprises the EPV spheroidin or tk gene sequences of the invention inserted into any member of the family Entomopoxvirinae, e.g., EPVs of any species
  • the host cell will be limited to cells of insects normally infected by EPVs.
  • the viral vector comprises the EPV spheroidin or tk gene sequences of the invention inserted into a vertebrate poxvirus, such as vaccinia or swinepox
  • the host cells may be selected from among the mammalian species normally infected by the wild-type vertebrate poxvirus.
  • such mammalian cells may include human cells, rodent cells and primate cells, all known and available to one of skill in the art. According to one aspect of the subject
  • vectors of the present invention may utilize a fragment of the polynucleotide sequence of EPV spheroidin, particularly the promoter and ancillary regulatory sequences which are responsible for the naturally high levels of expression of the gene.
  • spheroidin sequences may be found within the sequence of Fig. 2 [SEQ ID NO:1], more particularly within the region of nucleotides # 2781 through 3199 [SEQ ID NO:22]. Smaller fragments within that region may also provide useful regulatory sequences. The desired
  • spheroidin promoter sequence can be utilized to produce large quantities of a desired protein by placing it in operative association with a selected heterologous gene in viral expression vectors capable of functioning in either a vertebrate or invertebrate host cell.
  • regulatory sequence and a selected protein gene, such that the regulatory sequence is capable of directing the replication and expression of the protein in the
  • the resulting protein expressed in the host cell may be a fusion protein consisting of all or a portion of the spheroidin protein and the heterologous protein.
  • the heterologous protein may be produced alone.
  • SEQ ID NO: 8 may be employed in the construction of an expression vector to drive expression of a heterologous protein, or a fusion protein, in a selected known expression system.
  • tk regulatory sequences are desirably obtained from the sequence of Fig. 3 [SEQ ID NO: 8], particularly in the fragment occurring between nucleotide #750 through 890 [SEQ ID NO: 30]. Smaller fragments within that region may also provide useful regulatory sequences.
  • an advantage of the use of the novel EPV spheroidin or tk promoter sequences of this invention is that these regulatory sequences are capable of operating in a vertebrate poxvirus (e.g., vaccinia)-mammalian cell expression vector system.
  • vaccinia a vertebrate poxvirus
  • the strong spheroidin promoter can be incorporated into the vaccinia virus system through homologous recombination.
  • the promoter for the EPV spheroidin gene can be utilized directly in the vaccinia or swinepox virus expression vector.
  • the spheroidin or tk polynucleotide sequence may be isolated and purified from a selected
  • Entomopoxvirus e.g., AmEPV
  • restriction endonuclease enzymes to produce a fragment comprising all or part of the spheroidin or tk gene.
  • a fragment may be chemically synthesized.
  • the desired AmEPV sequences may be obtained from bacterial cultures
  • This plasmid contains the 4.51 kb BglII fragment AmEPV DNA sequence inserted into a BamHI site in the conventional vector pUC9.
  • the plasmid pRH7 was constructed by digesting AmEPV genomic DNA, obtained as described in Example 1, with Bsp1286I, and the resulting fragments with HaeII. T4 DNA polymerase is employed to blunt end the AmEPV DNA and the fragment containing the spheroidin gene is ligated to the large fragment of a Smal digested pUC9 fragment. This fragment contains the entire spheroidin open reading frame and some flanking sequence, included within the nucleotide sequence
  • Example 8 The regulatory sequences of the tk gene as well as the structural gene is described below in the Example 8. It was constructed by inserting the EcoRI-Q fragment of AmEPV into the conventional vector pUC18.
  • Bacterial cultures containing plasmids pRH512, pMEG tk-1, and pRH7 have been deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA. The deposited cultures are as follows: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
  • the plasmids can be obtained from the deposited bacterial cultures by use of standard procedures, for example, using cleared lysate-isopycnic density gradient procedures, and the like.
  • the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the
  • electrophorese DNA fragments tail and anneal plasmid and insert DNA, ligate DNA, transform cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.
  • spheroidin gene is the closest site to genetically engineer a usable insertion sequence for cloning.
  • restriction sites closer to the initiating Met of the spheroidin gene must be deliberately inserted.
  • Methods for the insertion of restriction sites are known to those of skill in the art and include, the use of an intermediate shuttle vector, e.g., by cloning the EPV sequence into the site of an appropriate cloning vehicle. It will be recognized by those skilled in the art that any suitable cloning vehicle may be utilized provided that the spheroidin or tk gene and flanking viral DNA may be functionally incorporated.
  • a spheroidin shuttle vector may be constructed to include elements of the spheroidin structural gene, a cloning site located or introduced in the gene to enable the selected heterologous gene to be properly inserted into the viral genome adjacent to, and under the control of, the spheroidin promoter, and flanking viral DNA linked to either side of the spheroidin gene to
  • flanking viral DNA facilitates insertion of the spheroidin-foreign geneflanking sequence into another expression vector.
  • the presence of flanking viral DNA also facilitates insertion of the spheroidin-foreign geneflanking sequence into another expression vector.
  • the shuttle vectors may thereafter be modified for insertion of a selected gene by deleting some or all of the sequences encoding for spheroidin or tk synthesis near the respective transcriptional start sites.
  • Examples of such sites in spheroidin are nucleotides #3077 and 3080 and in tk includes nucleotide #809.
  • oligonucleotide linker sequences may be inserted at the site of the deletion.
  • a polynucleotide linker sequence which may be either a natural or synthetic
  • oligonucleotide may be inserted at the site of the deletion to allow the coupling of DNA segments at that site.
  • One such linker sequence may provide an
  • this linker sequence may encode, if desired, a polypeptide which is selectively cleavable or digestible by conventional chemical or enzymatic methods.
  • the selected cleavage site may be an enzymatic cleavage site, including sites for cleavage by a proteolytic enzyme, such as
  • enterokinase factor Xa
  • trypsin collegenase
  • the cleavage site in the linker may be a site capable of being cleaved upon exposure to a selected chemical, e.g. cyanogen bromide or
  • the cleavage site if inserted into a linker useful in the sequences of this invention, does not limit this invention. Any desired cleavage site, of which many are known in the art, may be used for this purpose.
  • the linker sequence may encode one or a series of restriction sites. It will be recognized by those skilled in the art who have the benefit of this disclosure that linker sequences bearing an appropriate restriction site need not be inserted in place of all or a portion of the spheroidin structural sequence, and that it would be possible to insert a linker in locations in the
  • Entomopoxvirus genome such that both the sequence coding for the selected polypeptide and the spheroidin
  • sequence coding for the selected polypeptide could be inserted into the tk gene in place of all or a portion of the tk structural sequence and under the transcriptional control of the tk promoter.
  • PCR Polymerase chain reaction
  • the polynucleotide sequence may be used as a shuttle vector to transfer a selected heterologous gene to a selected virus.
  • the polynucleotide sequence encoding the EMV spheroidin gene or EMV tk gene, or a fragment thereof is linked to a heterologous gene.
  • the polynucleotide sequence further contains a flanking region on either side of the
  • Such a flanking region may be derived from EPV, or
  • the target virus may be complementary to the target virus.
  • a selected heterologous gene into a vaccinia virus to create a recombinant virus, one would utilize flanking regions complementary to the targeted vaccinia virus.
  • the heterologous gene is inserted within the EPV spheroidin or tk gene, so that the selected EPV
  • this cassette may be used for transfer into a wild type EPV having homologous sequences to the flanking sequences.
  • the insertion or linkage of the foreign gene into the tk or spheroidin sequences of the present invention or the linkage of flanking sequences foreign to the spheroidin or tk genes may be accomplished as
  • the vectors of the subject invention may use cDNA clones of foreign genes, because poxvirus genes contain no introns, presumably as a consequence of a totally cytoplasmic site of infection.
  • any selected gene may be inserted into the vector at an available restriction site to produce a recombinant shuttle vector.
  • Virtually any gene of interest could be inserted into the vectors described herein in order to obtain high expression of the desired protein.
  • Restriction sites in the fragment may thereafter be removed so as to produce a preferred spheroidin or tk shuttle vector, having one or more cleavage or cloning sites located in the 3' direction downstream from the spheroidin promoter sequence.
  • a vector of this invention may comprise a heterologous gene which is inserted into all or a portion of the EMV spheroidin or tk protein encoding sequence to interrupt the protein's natural processing.
  • a vector of this invention may comprise a heterologous gene which is inserted into all or a portion of the EMV spheroidin or tk protein encoding sequence to interrupt the protein's natural processing.
  • the vector is transferred to another virus which contains a wild-type spheroidin or tk gene,
  • the Entomopoxvirus spheroidin gene (Fig. 2 SEQ ID NO:1) and/or the tk gene (Fig. 3 SEQ ID NO: 8) can be used as the location for the insertion of exogenous or
  • a shuttle vector so constructed may be useful as a marker for research and production techniques for identifying the presence of successfully integrated heterologous genes into the selected expression system.
  • the tk gene is a particularly desirable site for insertion of a selected heterologous gene. Unlike spheroidin, tk is produced early in infection and in lesser quantities. Additionally, many poxviruses possess tk genes which may be sufficiently homologous to the EPV tk to provide easy recombination. For example, in
  • the vaccinia tk gene is a common insertion site.
  • this gene is particularly desirable for construction of a shuttle vector to shuttle selected genes directly between vector systems. More
  • a foreign gene may be desirably inserted into the EPV tk gene sequence between nucleotide #460 and #560 (See Fig. 3).
  • homologous recombination The homologous recombination techniques used to insert the genes of interest into the viruses of the invention are well known to those skilled in the art.
  • the shuttle vectors when co-infected into host cells with a wild-type virus, transfer the cassette containing the selected gene into the virus by homologous recombination, thereby creating recombinant virus vectors.
  • Expression of a selected gene is accomplished by infecting susceptible host insect cells with the recombinant viral vector of this invention in an
  • An EPV expression vector is propagated in insect cells or insects through
  • infectious vectors can be used to produce the selected gene in suitable insect cells, thus facilitating the efficient expression of the selected DNA sequence in the infected cell.
  • EPV spheroidin gene (or tk gene) - heterologous gene fragment is inserted into a vertebrate poxvirus by the same methods as described above, the recombinant virus may be used to infect mammalian cells and produce the heterologous protein in the mammalian cells.
  • a gene inserted into the tk site of a vaccinia virus system could be transferred directly to the tk locus of an Entomopoxvirus vector of the subject invention or vice versa.
  • This shuttling could be accomplished, for example, using homologous
  • insertion of a selected gene into the spheroidin gene or tk gene in a viral vector permits the gene to be shuttled into other viruses having homologous spheroidin or tk sequences, respectively.
  • the following description illustrates an exemplary vector of this invention, employing the gene coding for human ⁇ -interferon (IFN- ⁇ ) synthesis as the heterologous gene.
  • IFN- ⁇ human ⁇ -interferon
  • a DNA fragment containing the IFN- ⁇ gene is prepared conventionally with restriction enzyme digested or blunt ended termini and cloned into a
  • the hybrid gene may comprise the spheroidin promoter, the IFN- ⁇ protein coding sequences, and sequences encoding a portion of the polypeptide sequence of the spheroidin protein, provided all such coding sequences are not deleted from the particular shuttle vector utilized.
  • the resulting shuttle vector contains the AmEPV spheroidin gene sequence coupled to the IFN- ⁇ gene.
  • the hybrid spheroidin-IFN- ⁇ gene of the recombinant shuttle vector is thereafter transferred into the genome of an appropriate Entomopoxvirus, such as the preferred
  • Entomopoxvirus AmEPV to produce a recombinant viral expression vector capable of expressing the gene encoding for ⁇ -interferon in a host insect cell.
  • Transfer of the hybrid gene to a wild-type virus is accomplished by processes which are well known to those skilled in the art.
  • appropriate insect cells may be
  • infected cells are then transfected with the shuttle vector of the subject invention. These procedures are described, for example, in DNA Cloning: A Practical
  • caterpillars and cultured gypsy moth cells can be used.
  • the hybrid gene is transferred to the wild-type AmEPV by homologous recombination between the
  • a mixture is produced comprising wild-type, nonrecombinant EPVs and recombinant EPVs capable of expressing the IFN- ⁇ gene.
  • transfection is the preferred process for transfer of the hybrid gene into the EPV genome, it will be understood by those skilled in the art that other procedures may be suitably utilized so as to effect the insertion of the gene into the EPV genome and that recombination may be accomplished between the recombinant shuttle vector and other strains of EPV (or other
  • the preferred recombinant AmEPV expression vector comprising a hybrid spheroidin-IFN- ⁇ gene
  • the preferred, but by no means only, method of selection is by screening for plaques formed by host insect cells infected with viruses that do not produce viral occlusions. Selection may be performed in this manner because recombinant EPV viruses which contain the spheroidin or tk protein coding
  • sequences interrupted by the heterologous gene are defective in the production of viral occlusions due to the insertional inactivation of the spheroidin gene.
  • the selection procedure may involve the use of the ⁇ -galactosidase gene to facilitate color selection.
  • This procedure involves the incorporation of the E. coli ⁇ -galactosidase gene into the shuttle vector and is well known to those skilled in the art. This technique may be of particular value if the exogenous DNA is inserted into the tk gene so that the spheroidin gene is still expressed. It will be recognized by those skilled in the art that alternative selection procedures may also be utilized in accordance with the present invention.
  • the DNA from a recombinant virus is thereafter purified and may be analyzed with
  • EPV viral vectors of the present invention are not oncogenic or tumorigenic in mammals. Also, the vectors and methods provided by the present invention are characterized by several advantages over known vectors and vector systems.
  • EPV viral vectors of the present invention are not oncogenic or tumorigenic in mammals. Also, the vectors and methods provided by the present invention are not oncogenic or tumorigenic in mammals. Also, the vectors and methods provided by the present invention are not oncogenic or tumorigenic in mammals. Also, the
  • Entomopoxvirus (AmEPV) gene expressions are similar to those of vaccinia. Therefore, it is possible to transfer the strongly expressed spheroidin gene, or the thymidine kinase gene, as an expression cassette, not only in insect cells, but for use in vertebrate poxviruses such as vaccinia and swinepox virus.
  • exogenous DNA which can be packaged into a virus is not anticipated to be encountered when using the novel EPV vectors and methods of the subject invention.
  • Still another advantage lies in the expression power of the EPV spheroidin regulatory sequences, which when in operative association with a heterologous gene in a vector of this invention, should produce high levels of heterologous protein expression in the selected host cell.
  • EPV vectors of this invention and methods for employing them to express selected heterologous proteins in insect or mammalian cells, as described above, are characterized by the advantage of replication in insect cells, which avoids the use of mammalian viruses, thereby decreasing the possibility of
  • the expression system of this invention is also a helper independent virus expression vector system. These two characteristics are shared by known baculovirus
  • the EPV expression vector system (EEVS) using the vectors of this invention has some important distinguishing features compared to the baculovirus expression systems (BEVS).
  • BEVS baculovirus expression systems
  • Virus family Poxviridae Baculoviridae
  • Polyhedrin is not found in mammalian systems.
  • the present invention also provides a method for screening recombinant host cells for insertion of heterologous genes is provided by use of the recombinant viral polynucleotide molecules of this invention.
  • the heterologous gene may be linked to the spheroidin or tk regulatory sequences in the absence of the complete coding sequences, or it may be inserted into the
  • recombinant vector is cultured under conditions suitable for expression of the heterologous protein, either unfused or as a fusion protein with a portion of the spheroidin sequence.
  • the absence of occlusion bodies which would ordinarily be formed by the expression of the intact spheroidin protein indicates the integration of the heterologous gene.
  • the viral vector similarly contained either incomplete or interrupted EPV tk encoding sequence, the absence of thymidine kinase function (e.g., resistance to methotrexate or an analogue thereof) formed by the
  • integration of the inactive thymidine kinase sequence indicates the insertion of the heterologous gene.
  • a parent virus is deleted of part of its tk or spheroidin gene, and is thereafter mixed with a viral vector containing intact tk or
  • recombinants would express the methotrexate resistance or produce occlusion bodies, respectively, thus indicating integration of the active tk or spheroidin genes and the foreign gene.
  • Another embodiment of the present invention involves using novel EPV expression systems of the subject invention for insect control. Control of insect pests can be accomplished by employing the vectors and methods of the invention as described above.
  • a gene coding for an selected insect toxin may be inserted into the viral expression vector under the control of the spheroidin or tk regulatory sequences or within either of the two genes for purposes of
  • the resulting EPV vector containing the toxin gene is applied to the target pest or its surroundings.
  • the viral vector will infect the target pest, and large quantities of the toxin will be produced, thus resulting in the control of the pest.
  • Particularly large quantities of the toxin protein can be produced if the regulatory sequences of the Entomopoxvirus spheroidin gene are used to express the toxin.
  • the spheroidin gene can be left intact and the toxin gene inserted into a different
  • Entomopoxvirus gene such as the tk gene.
  • the toxin will be produced by the system and then effectively coated or encapsulated by the natural viral production of spheroidin.
  • the subject invention pertains to the use of novel regulatory elements from Entomopoxvirus to
  • polynucleotide sequences of the invention can also be used with viral vaccines, e.g., known vaccinia virus vaccines, to enhance the production of vaccinia virus.
  • EPV spheroidin promoter sequences into known viral vectors which are used to express selected proteins in a mammalian host in vivo may enable the powerful spheroidin promoter to increase expression of the protein in the viral vaccine.
  • This aspect of the invention provides a significant improvement over other expression systems, including the baculovirus expression system (BEVS).
  • the AmEPV inoculum for cell culturing was from an AmEPV-infected, freeze-dried E. acrea larva stored at -70°C [R. L. Hall et al, Arch. Virol.. 110:77-90
  • the larva was crushed and macerated in 5 ml of EX-CELL 400 (with penicillin and streptomycin but without fetal bovine serum) to which 0.003 g of cysteine-HCl had been added to prevent melanization.
  • the debris was pelleted at 200 ⁇ g for 5 minutes, and the supernatant was passed through a 0.45- ⁇ m-pore-size filter.
  • the gypsy moth cells were infected with AmEPV by addition of the inoculum to a preconfluent monolayer of cells (about 0.1 to 1 PFU per cell), with occasional agitation of the dish during the first day. Infected cells were harvested 5 to 6 days postinfection.
  • AmEPV DNA was prepared from the infected cells by one of two methods.
  • the first method involved in situ digestion of infected cells embedded within agarose plugs, after which the released cellular and viral DNAs were separated by pulsed-field electrophoresis [Bio-Rad CHEF-II-DR system].
  • IPLB-LD-652 cells were infected with first-cell-culture-passage AmEPV. Infected cells were harvested 6 days postinfection by centrifugation at 200 ⁇ g for 5 minutes, rinsed, and resuspended in modified Hank's phosphate-buffered saline (PBS), which contained 15 g of glucose per liter, but no Ca 2+ or Mg 2+ .
  • PBS Hank's phosphate-buffered saline
  • 1% SeaPlaque GTG agarose prepared in modified Hank's PBS and equilibrated at 37°C
  • infected cells were mixed 1:1 with infected cells to yield 5 ⁇ 10 6 cells per ml in 0.5% agarose.
  • Digestion to release DNA was done by gentle shaking of the inserts in 1% Sarkosyl-0.5 M EDTA-1 mg of proteinase K per ml at 50°C for 2 days [C. L. Smith et al. Methods Enzymol.. 151:461-489 (1987)].
  • the CHEF-II- DR parameters for DNA separation were 180 V, a pulse ratio of 1, 50 initial and 90 second final pulse times, and a run time of 20 to 25 hours at 4°C.
  • the separating gel was 1% SeaKem GTG agarose in 0.5x TBE buffer
  • the second method of viral DNA preparation used the extracellular virus found in the infected-cell- culture supernatant.
  • the supernatant from 10-day- postinfection cell cultures was clarified by
  • Viral pellets were resuspended in 6 ml of 1x TE. DNase I and RNase A (10 and 20 ⁇ g/ml final
  • restriction endonuclease enzymes e.g., Bam HI, EcoRI, HindIII, PstI and Xhol. generating the various fragments which appear on the physical map of Fig. 1.
  • reference to each restriction fragment will refer to the enzyme and the applicable letter, e.g., BamHI-A through BamHI-E, EcoRI-A through EcoRI-S, etc.
  • EXAMPLE 2 ISOLATION OF THE SPHEROIDIN GENE
  • OBs occlusion bodies
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • spheroidin for protein microsequencing was deionized with AG501X8 resin [Bio-Rad, Richmond, CA] . The gels were polymerized overnight at 4°C.
  • 2x Laemmli sample buffer consisting of 125 mM Tris-HCl (pH 6.8), 4% SDS (w/v), 10% ⁇ -mercaptoethanol (v/v), and 20% glycerol (v/v) was used.
  • OB suspension samples were diluted 1:1 with 2x Laemmli sample buffer and boiled for 5 minutes. Several lanes of an OB protein preparation were separated electrophoretically.
  • the spheroidin protein (113 kDa) was the predominant protein of the purified OBs.
  • Spheroidin within SDS-polyacrylamide gels was tested for glycosylation by periodic acid-Schiff staining [R. M. Zacharius et al. Anal. Biochem.. 30:149-152 (1969)].
  • PVDF Immobilon polyvinylidene difluoride
  • Biosystems gas-phase sequencer Microsequencing of the intact protein was unsuccessful, presumably because the N terminus of the protein was blocked.
  • Cyanogen bromide cleavage was performed on samples of spheroidin eluted from the PVDF membrane to generate internal peptide fragments for sequencing.
  • the 8 and 9 kDa polypeptides represented overlapping partial CNBr cleavage products which together yielded the longest continuous amino acid sequence: Met-Ala-(Asn or Arg)-Asp-Leu-Val-Ser-Leu-Leu- Phe-Met-(Asn or Arg)-(?)-Tyr-Val-(Asn?)-Ile-Glu-Ile-Asn- Glu-Ala-Val-(?)-(Glu?) [SEQ ID NO: 34].
  • the amino acid sequence obtained from the 6.2 kDa fragment was Met-Lys- Ile-Thr-Ser-Ser-Thr-Glu-Val-Asp-Pro-Glu-Tyr-Val-(Thr or Ile)-Ser-(Asn?) [SEQ ID NO:35].
  • a partial sequence for the 15 kDa fragment was also obtained: (Asn?)-Ala-Leu-Phe-(Phe?)(Asn?)-Val-Phe [SEQ ID NO:36].
  • the question marks in the above sequences indicated undetermined or unconfirmed amino acids. All sequences were ultimately located within the spheroidin gene sequence.
  • a BglII AmEPV DNA library was prepared by digesting the genomic AmEPV DNA with BglII according to manufacturer's instructions. Plasmid pUC9 [GIBCO;
  • restriction enzyme digestions of the genomic DNA was a 4.4 BglII fragment and an EcoRI-D fragment.
  • sequence derived from the 6.2 kDa CNBr fragment was used to design a degenerate oligonucleotide for use as a hybridization probe to locate the spheroidin gene in a clone.
  • the nucleotide sequence of this probe called RM58 [SEQ ID NO: 12] was GA5GT7GA6CC7GA5TA6GT, where 5
  • the peptide sequence of the probe was: Glu-Val-Asp-Pro-Glu-Tyr-Val [SEQ ID NO: 37].
  • the DNA probe was radiolabeled either with [ ⁇ - 3 2 P]dCTP by the random oligonucleotide extension method [A. P. Feinberg et al. Anal. Biochem.. 132:6-13 (1983)] or with [ ⁇ - 32 P]ATP and T4 polynucleotide kinase [Sambrook et al, supra1. These same procedures were used for all other oligonucleotide probes described below. Both types of probes were purified by passage through spun columns of Sephadex G-50.
  • Hybridization with the oligonucleotide probe was done at 37 or 45°C with BLOTTO [Sambrook et al, supra] and was followed by two washes at room temperature with 0.3 M NaCl-0.06 M Tris (pH 8) -2 mM EDTA for 5 minutes.
  • the RM58 probe [SEQ ID NO: 12] hybridized to the 4.4 kb BglII fragment and the EcoRI-D fragment of AmEPV DNA [See Fig. 1] .
  • a plasmid produced by the shotgun cloning, recombinant pRH512 (a BglII 4.56 kb fragment inserted into the BamHI site of pUC9 which contains about 1.5 kb of the 5' end of the spheroidin gene) was also identified by this hybridization with the RM58
  • the 4.51 kb pRH512 BglII insert was isolated, radiolabeled as described above, and hybridized back to various AmEPV genomic digests as follows.
  • the DNA-DNA hybridization was done at 65°C with BLOTTO [Sambrook et al, supra] and was followed by two washes at room
  • the 4.51 kb BglII insert of pRH512 was thereafter sequenced by two procedures.
  • One is the double-stranded plasmid sequencing method [M. Hattori et al. Anal. Biochem.. 152:232-238 (1986)] performed with "miniprep" [Sambrook et al, supra] DNA and 1 pmol of universal, reverse, or custom-designed oligonucleotide primer in each sequencing reaction.
  • Nested exonuclease II deletions [S. Henikoff, Methods Enzvmol.. 155:156-165 (1987) ] were used to sequence plasmid pRH512 according to this method. Deletions were made from the universal primer end.
  • the DNA was cut with EcoRI. filled in with ⁇ -thiophosphate dNTPs [S. D. Putney et al, Proc. Natl. Acad. Sci. USA. 78:7350-7354 (1981) ] by use of the Klenow fragment of E. coli DNA polymerase, cut with Smal, and treated with exonuclease III. Samples were removed every 30 seconds, re-ligated. and used to transform E. coli SURE cells by electroporation. Sequencing reactions were carried out with the universal primer.
  • a second sequencing method was performed using a combination of M12 shotgun sequencing with standard and universal and reverse M13 primers into M13 phage to permit single-stranded sequencing as follows. Plasmid pRH512 was sonicated to produce random fragments,
  • Plaque lifts were screened with a radiolabeled probe prepared from the 4.5 kb insert found in pRH512 to identify appropriate clones for shotgun single stranded sequencing [see, e.g., Sambrook et al, supra].
  • Dral AmEPV DNA library was prepared by digesting genomic DNA with Dral. These Dral fragments were shotgun cloned into Smal-digested, phosphatase-treated vector M13mpl9. Preparations of M13 virus and DNA were made by standard procedures [J. Sambrook et al, supra]. Ligation and heat shock transformation
  • the standard PCR primers used for this reaction were RM92 [SEQ ID NO: 15] (GCCTGGTTGGGTAACACCTC) and RM118 [SEQ ID NO: 16] (CTGCTAGATTATCTACTCCG) .
  • This sequencing revealed that there was a single HindIII site at base 931 and that the 2 ' end of the spheroidin open reading frame (ORF) was truncated (Fig. 2).
  • PCR inverse polymerase chain reaction
  • TTTCAAATTAACTGGCAACC was GGGATGGATTTTAGATTGCG.
  • the resulting 2.2 kb inverse PCR product was digested with Clal. and a 1.7 kb fragment was gel
  • the 1.7 kb PCR fragment was sequenced with RM83 as a primer. Additional PCR primers were made to the new sequence as it was identified.
  • the sequencing process employed Sequenase, 5 pmol of each primer, and 10 to 50 ng of template. Prior to being sequenced, the PCR products were chloroform extracted and purified on spun columns [Sambrook et al, supra] of Sephacryl S-400. The DNA sequence was assembled and aligned, and consensus sequence was produced [R. Staden, Nucleic Acids Res..
  • the relevant Clal sites of the 1.7 kb PCR fragment are at positions 3485 and 6165. This fragment was radiolabeled and used as a probe to locate additional clones, i.e., pRH827 (307 bp), pRH85 (1.88 kb), and pRH87 (1.88 kb) from the BglII fragment library. Plasmids pRH85 and pRH87 were sequenced using the same nested exonuclease II deletions and sequencing procedure, as described above for pRH512. Sequencing of the inverse PCR products with custom-designed primers confirmed that plasmids pRH85 and pRH87 represented the same 1.88 kb
  • Fig. 1 The orientation of the spheroidin ORF on the physical map is shown in Fig. 1. It is interesting to note that the 1.7 kb inverse PCR fragment only hybridized to the AmEPV HindIII-G fragment.
  • the amino acid sequence derived from the 8 and 9 kDa overlapping CnBr-generated polypeptides is found from nucleotide positions 4883 to 4957 [SEQ ID NO: 38]. That derived from the 6.2 kDa polypeptide is found from nucleotides 3962 to 4012 [SEQ ID NO: 39], and that derived from the 15 kDa polypeptide is found from nucleotides 4628 to 4651 [SEQ ID NO: 40].
  • concentration was about 0.1 to 1 PFU per cell.
  • the dishes were occasionally agitated during a 3 hour
  • RNA from the infected cells was isolated by the guanidinium thiocyanate-cesium chloride procedure [J. M. Chirgwin et al. Biochemistry. 18:5294-5299 (1979)].
  • Primer extension reactions were carried out with primer RM165 [SEQ ID NO: 17], a 35-base
  • the primer was end labeled with [ ⁇ - 32 P]ATP and T4 polynucleotide kinase and purified on a "spun column" [Sambrook et al, supra] .
  • 40 ⁇ g of total infected-cell RNA and 10 6 cpm of radiolabeled primer were coprecipitated with ethanol.
  • the pellet was resuspended in 25 ⁇ l of hybridization buffer [80%
  • RNA-primer hybrids were ethanol precipitated, resuspended, and used for five individual reactions. Each reaction contained 8 ⁇ g of total infected-cell RNA, 50 mM Tris-HCl, (pH 8.3), 50 mM KC1, 10 mM dithiothreitol, 10 mM MgCl 2 , 4 U of avian myeloblastosis virus reverse transcriptase (Life
  • dNTP deoxynucleoside triphosphate
  • ddNTP dideoxynucleoside triphosphate
  • dNTP/ddNTP ratios were 4:1, 5:1, 5:1, and 2:1, for the C, T, A, and G reactions, respectively.
  • the reactions were carried out at 42°C for 30 minutes.
  • chase buffer (4 ⁇ l of 5 mM dNTP mixture and 1 ⁇ l of 20-U/ ⁇ l reverse transcriptase) was added to each reaction mixture, which was then incubated for an additional 30 minutes at 42°C. Reaction products were separated on a sequencing gel (8%
  • transcription of the gene initiates within the TAAATG element of the proposed late promoter element.
  • the spheroidin ORF (G5R) was initially identified by sequencing back through the RM58
  • TTTT TNT early gene termination signals include three TTTT TNT early gene termination signals and TAAATG, which presumably represents a late transcription start signal used to initiate transcription and translation of the spheroidin gene.
  • TAAATG a late transcription start signal used to initiate transcription and translation of the spheroidin gene.
  • Several adjacent translation termination codons are also present within the 92 bp upstream of the spheroidin ORF.
  • ORF G4R [SEQ ID NO:24] showed homology to ORF HM3 of capripoxvirus. In vaccinia virus, the ORF HM3 homolog was found very near the site of an incomplete ATI gene.
  • RM03 and RM04 Two oligonucleotides, RM03 and RM04, based on different but strongly conserved regions of the tk genes of several poxviruses and vertebrates [C. Upton et al, J. Virol.. .60:920-927 (1986); D. B. Boyle et al. Virology, 156:355-365 (1987)] were prepared by the methods referred to above. RM03 was the 32-fold
  • RM04 [SEQ ID NO: 19] was (GGNCCCATGTT(C/T)TCNGG with 32-fold degeneracy and
  • oligonucleotide probes RMO3 and RMO4 to a Southern blot of the EcoRI-digested EPV DNA.
  • EcoRI-Q EcoRI-Q
  • pMEGtk-1 One such clone was called pMEGtk-1.
  • the recombinant clones containing the EcoRI-Q fragment oriented in both directions relative to the pUC18 vector sequences were used for sequencing. Sequential nested deletions were generated by the method of Henikoff, cited above, as described for pRH512. These clones were used for sequencing the entire EcoRI-Q fragment.
  • RM129 is a non-degenerate oligonucleotide
  • ORF Q2 potentially encodes for a protein of 182 amino acids (21.2 kDa) [SEQ ID NO:10].
  • ORF Q3 potentially encodes a polypeptide of at least 68 amino acids but is incomplete and is transcribed in the opposite direction from ORF Q2.
  • ORF Ql [SEQ ID NO: 31] potentially encodes a small peptide of 66 amino acids (7.75 kDa) [SEQ ID NO:9].
  • TTTTTAT potential poxvirus early transcription termination signal sequence
  • the deduced amino acid sequence for the tk encoded by the ORF Q2 of the EcoRI-Q fragment can be compared to the tk genes for the poxviruses swine pox [W. M. Schnitzlein et al, Virol.. 181:727-732 (1991); J. A. Feller et al, Virol . , 183 : 578-585 (1991) ] ; fowlpox [Boyle et al., supra; M. M. Binns et al, J. Gen. Virol..
  • monkeypox J. J. Esposito et al, Virol.. 135:561-567 (1984)]; capripoxvirus [P. D. Gershon et al, J. Gen.
  • the AmEPV tk gene was tested functionally by cloning the gene into a vaccinia virus strain tk mutant, as follows.
  • the plasmid was transfected by Lipofectin
  • the cells were either rat tk, human 143 tk, or CV-1 cell lines onto which the vaccinia virus VSC8 was propagated. These cells were maintained in Eagle's Minimal Essential Medium with Earle's salts [Massung et al, Virol.. 180:347-354 (1991) incorporated by reference herein].
  • VSC8 vaccinia strain [Dr. Bernard Moss] contains the ⁇ -galactosidase gene driven by the vaccinia P 11 promoter (P 11 -Lac Z cassette) inserted into the viral tk gene. While VSC8 contains an inactive tk gene due to the insertion of the ⁇ -galactosidase, portions of the vaccinia tk sequence remain. VSC8 is thus tk- and, upon staining with X-Gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside), will form blue plaques (ß-galactosidase positive).
  • X-Gal 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside
  • Lipofectin solution (20 ⁇ g of Lipofectin in 50 ⁇ l of dH 2 O) was added to 10 ⁇ g plasmid DNA (pHGN3.1/AmEPV EcoRI-Q) in 50 ⁇ l of dH 2 O and incubated for 15 minutes at room temperature. After a 2 hour period of viral
  • the monolayers were washed three times with serum-free OptiMEM. Three milliliters of serum-free OptiMEM was then added to each 60 mm dish. The Lipofectin/DNA mixture was slowly added dropwise with gentle swirling and incubated an additional 12 to 18 hours at 37°C. Fetal bovine serum was then added (10% final) and the infected cells were harvested at 48 hours postinfection.
  • Recombinant viruses containing the EcoRI -Q fragment inserted into the hemagglutinin (HA) gene of vaccinia, were identified by hybridization of AmEPV
  • the radiolabeled product was then hybridized to an EcoRI digest of AmEPV DNA. If orientation of the gene is such that the tk ORF reads toward the end of the genome, hybridization would be expected to the EcoRI-E fragment; whereas if the gene is read toward the center of the genome, hybridization would be expected to the
  • the results indicate hybridization not only to the EcoRI-E fragment, but also to the EcoRI-A fragment. These results infer that the orientation of the tk gene is with reading toward the left end of the genome.
  • Hybridization of the run-off extension product also to the EcoRI-A fragment is consistent with the presence of an inverted terminal repetition, common in poxviruses, with identical sequences residing in both the EcoRI-A and the EcoRI-E fragments.
  • the optimal growth temperature for AmEPV in the laboratory is 28°C, whereas that of the vertebrate poxviruses is 37°C.
  • the recombinant virus was capable of growing at 37°C in the presence of methotrexate [Sigma], indicative of a tk + phenotype.
  • the subject invention encompasses recombinant polynucleotide sequences, plasmids, vectors, and transformed hosts which are equivalent to those which are specifically exemplified herein in that the
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Amsacta moorei entomopoxvirus
  • TAACATATTT TTTATTAAAA TGAATAAAAT ATATATTGTT ATTGTCAATA TTTTATATCA 2228 TTTTACAGTC TTATTTTTTT TTTTTGCTTT TAGGTATAAT TTTACCTTCT AAACGTTTAT 2288
  • CTCCCCAAAC ATCTACAGTA GATGGTTTAT TAGATTCTGT GTTATACACA TCTGCTGGAT 2348
  • TATAATATCA ATCATAATTT TTATATATAT TTTATCTAAA AGGACTTTTT ATTTTTTATA 3064
  • AAC GAC GAA AAT AAA ATT ATA TTG GAA GAA ATT GAA GCA GAA TAT AGA 4267 Asn Asp Glu Asn Lys Ile Ile Leu Glu Glu Ile Glu Ala Glu Tyr Arg
  • AAA GTA CCC AAA AAT TTA AGA CTT TGG GGA TGG ATT TTA GAT TGC GAT 4459 Lys Val Pro Lys Asn Leu Arg Leu Trp Gly Trp Ile Leu Asp Cys Asp
  • AAA CAA CAT TAT ACT AAT GTA ATT ATA TTA GAG TAC GCA AAT ACA TAT 4603 Lys Gin His Tyr Thr Asn Val Ile Ile Leu Glu Tyr Ala Asn Thr Tyr
  • GCT ATT AAA GTA ATT AAT GAT TTA TTA TTA ATT AAC GGA
  • AAG ACT CTA CCA AAT GAA AAG TAT GGT GGT GTT GAT AAG AAA TTT AAA 5131 Lys Thr Leu Pro Asn Glu Lys Tyr Gly Gly Val Asp Lys Lys Phe Lys
  • AGA GGT CTT TTA TAT GGT CCT GAA TAT GTA CAT CAC AGA TAT CAA GAA 5755 Arg Gly Leu Leu Tyr Gly Pro Glu Tyr Val His His His Arg Tyr Gin Glu
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Amsacta moorei entemopoxvirus
  • ATTTTATCAC AAAATTGTTC TAAATCATTT TCTTCAAAAA ATTGACACTC ATCTATGCCA 540
  • AAATTATCAA A ATG GAT TTA CTA AAT TCT GAT ATA ATT TTA ATA AAT ATT 890
  • AGC AAT ATA AGT AAT ATT ATA CTA CCA CAT TCT ATA GAA TTT TTA AAT 1178 Ser Asn Ile Ser Asn Ile Ile Leu Pro His Ser Ile Glu Phe Leu Asn

Abstract

L'invention se rapporte à de nouvelles séquences polynucléotidiques d'entomopoxvirus (EPV) qui ne sont pas associées à d'autres séquences virales avec lesquelles elles sont naturellement associées, à des vecteurs polynucléotidiques de recombinaison contenant ces séquences, à des virus de recombinaison contenant aussi ces séquences, et à des cellules hôtes infectées par ces virus de recombinaison, ainsi qu'à des procédés d'utilisation de ces séquences pour l'expression de protéines hétérologues à la fois dans des cellules hôtes d'insectes et dans celles des mammifères.
PCT/US1992/000855 1991-02-19 1992-02-12 Systeme d'expression d'entomopoxvirus comprenant des sequences de spheroïdine ou de thymidine-kinase WO1992014818A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
MX9200697A MX9200697A (es) 1991-02-19 1992-02-12 Sistema de expresion de entomopoxvirus.
AU16634/92A AU663709B2 (en) 1991-02-19 1992-02-12 Entomopoxvirus expression system comprising spheroidin or thymidine-kinase sequences
JP4508743A JPH06506594A (ja) 1991-02-19 1992-02-12 新規なエントモボックスウイルス発現系
ZA921163A ZA921163B (en) 1991-02-19 1992-02-18 Novel entomopoxvirus expression system
IL100983A IL100983A0 (en) 1991-02-19 1992-02-18 Entomopoxvirus expression system
IE051592A IE920515A1 (en) 1991-02-19 1992-02-18 Novel Entomopoxvirus Expression System
NZ241662A NZ241662A (en) 1991-02-19 1992-02-19 Entomopoxvirus spheroidin, thymidine kinase and fragments, recombinant polynucleotides
CN92101985A CN1065293A (zh) 1991-02-19 1992-02-19 新的昆虫痘病毒表达系统
US09/370,861 US6410221B1 (en) 1991-02-19 1999-08-09 Entomopoxvirus expression system

Applications Claiming Priority (4)

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US65758491A 1991-02-19 1991-02-19
US657,584 1991-02-19
US82768592A 1992-01-30 1992-01-30
US827,685 1992-01-30

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US07/991,867 Continuation-In-Part US5476781A (en) 1991-02-19 1992-12-07 Entomopoxvirus spheroidin gene sequences

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IE (1) IE920515A1 (fr)
IL (1) IL100983A0 (fr)
MX (1) MX9200697A (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013812A2 (fr) * 1992-12-07 1994-06-23 University Of Florida Nouveaux genes de l'entomopoxvirus, nouvelles proteines et leurs procedes d'utilisation
WO1998050571A1 (fr) * 1997-05-07 1998-11-12 University Of Florida Vecteur d'administration de gene a base d'entomopoxvirus pour vertebres
US6130074A (en) * 1992-06-01 2000-10-10 American Cyanamid Company Five Giralda Farms Recombinant insect virus with reduced capacity for host-to-host transmission in the environment and methods to produce said virus

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL106038A0 (en) * 1992-06-16 1993-10-20 Commw Scient Ind Res Org Recombinant entomopoxvirus

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0397560A2 (fr) * 1989-05-08 1990-11-14 Kai-Chung Leonard Yuen ADN de la spéroidine et vecteurs d'expression d'entomopoxvirus recombinants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0397560A2 (fr) * 1989-05-08 1990-11-14 Kai-Chung Leonard Yuen ADN de la spéroidine et vecteurs d'expression d'entomopoxvirus recombinants

Non-Patent Citations (1)

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Title
Virology, vol. 175, no. 2, April 1990, Academic Press, Inc., L. Yuen et al.: "Identification and sequencing of the spheroidin gene of choristoneura biennis entomopoxvirus", pages 427-433, see the whole article *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6130074A (en) * 1992-06-01 2000-10-10 American Cyanamid Company Five Giralda Farms Recombinant insect virus with reduced capacity for host-to-host transmission in the environment and methods to produce said virus
WO1994013812A2 (fr) * 1992-12-07 1994-06-23 University Of Florida Nouveaux genes de l'entomopoxvirus, nouvelles proteines et leurs procedes d'utilisation
WO1994013812A3 (fr) * 1992-12-07 1994-11-10 Univ Florida Nouveaux genes de l'entomopoxvirus, nouvelles proteines et leurs procedes d'utilisation
WO1998050571A1 (fr) * 1997-05-07 1998-11-12 University Of Florida Vecteur d'administration de gene a base d'entomopoxvirus pour vertebres
US6106825A (en) * 1997-05-07 2000-08-22 University Of Florida Entomopoxvirus-vertebrate gene delivery vector and method

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CA2103550A1 (fr) 1992-08-20
NZ241662A (en) 1995-03-28
MX9200697A (es) 1993-03-01
WO1992014818A3 (fr) 1992-12-10
AU663709B2 (en) 1995-10-19
IE920515A1 (en) 1992-08-26
ZA921163B (en) 1992-12-30
YU16292A (sh) 1994-06-24
EP0573613A1 (fr) 1993-12-15
IL100983A0 (en) 1992-11-15
CN1065293A (zh) 1992-10-14
JPH06506594A (ja) 1994-07-28

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