EP0431038A1 - Herstellung von nichtstrukturellen proteinen des bluetongue-virus unter verwendung eines baculovirus-expressionsvektors - Google Patents

Herstellung von nichtstrukturellen proteinen des bluetongue-virus unter verwendung eines baculovirus-expressionsvektors

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Publication number
EP0431038A1
EP0431038A1 EP89909839A EP89909839A EP0431038A1 EP 0431038 A1 EP0431038 A1 EP 0431038A1 EP 89909839 A EP89909839 A EP 89909839A EP 89909839 A EP89909839 A EP 89909839A EP 0431038 A1 EP0431038 A1 EP 0431038A1
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btv
protein
polypeptide
cells
infected
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French (fr)
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Polly Roy
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to a process for producing bluetongue virus proteins.
  • Bluetongue virus is the prototype virus of the Qvbi ⁇ iTus genus (Reoviridae family) . It is vectored to vertebrates by Culicoides species and causes disease in certain ruminants, notably sheep.
  • the genome of BTV consists of 10 unique double-stranded (ds) RNA molecules, each believed to code for a single polypeptide product. (Gorman et al. , 1981; Sanger and Mertens, 1983).
  • the ten dsRNA species are contained in an inner core structure that contains five types of proteins, two that are major (VP3 and VP7) and three that are minor components (VPl, VP4 and VP6) .
  • the core is surrounded by an outer capsid consisting of two major proteins, VP2 and VP5, to give a complete virion particle with a diameter of approximately 69 nm.
  • non-structural proteins In addition to the above-mentioned structural proteins, three non-structural proteins (NSI, NS2 and NS3) appear in BTV infected cells. Their function in the replication or morphogenesis of BTV is not known. Two viral specific entities, tubules and granular inclusion bodies, are routinely obserbed in BTV infected cells (Lecatsas, 1968) . These morphological structures are attached to the intermediate filament component of the cell's cytoskeleton (Eaton, B.T. et al. , 1988) and are presumed to be involved in the virus assembly process.
  • tubular structures are composed entirely of one type of polypeptide, namely the 64,000 Dalton (64 kD) NSI protein, which is the gene product of BTV dsRNA middle size segment No. 6 (M6) .
  • 64,000 Dalton (64 kD) NSI protein which is the gene product of BTV dsRNA middle size segment No. 6 (M6) .
  • M6 BTV dsRNA middle size segment No. 6
  • a process for producing a polypeptide comprising a bluetongue virus non-structural protein in antigenic form which comprises infecting susceptible insects or cultured insect cells with an expression vector having a DNA segment coding for said polypeptide.
  • An example of such a polypeptide is the NSI protein of bluetongue virus.
  • insects and cultured insect cells are capable of producing bluetongue virus non- structural proteins in morphological forms which resemble structures observed in bluetongue-virus infected mammalian, cells.
  • BTV-NSI when produced in accordance with the invention in insect cells has been found to "self-assemble" into tubules which are similar to tubules observed in bluetongue virus infected sheep cells.
  • the expression vectors used in the method of the invention may comprise a recombinant baculovirus having a DNA segment coding for a polypeptide comprising a bluetongue virus non-structural protein.
  • Such recombinant baculoviruses may include promoter systems native to naturally occurring baculoviruses, for example the so-called “polyhedrin” promoter, or they may include other promoter systems capable of directing expression of polypeptide in transformed insect or cultured insect cells.
  • Especially suitable cultured insect cells are those of Spodoptera f_vugipe ⁇ da.
  • antigenic form as used herein to refer to a protein is meant a protein which is capable of exhibiting an antigenic property of a native bluetongue virus protein, e.g. the capability of binding to an antibody to said protein.
  • BTV serotype 10 (BTV-10) NSI gene product using an expression system based on recombinant baculoviruses is illustrated by the following Example.
  • the expressed protein has been shown to be similar in size and antigenic properties to the authentic BTV NSI protein. It reacts with BTV ' antibody and forms numerous tubular structures in the cytoplasm of the infected insect cells. The tubular structures have been purified to homogeneity from infected cell extracts. Further the expressed polypeptide has been used to identify antibodies to five United States BTV serotypes in infected sheep sera, indicating the potential of the expressed protein as a group reactive antigen in the diagnosis of BTV infections.
  • Example the BTV-10 M6 gene product is expressed in an insect baculovirus expression vector derived from AcNV.
  • A. Viruses and cells United States prototype BTV-10 was plaque cloned using monolayers of BHK-21 cells. The viral dsRNA was purified as described by Yamaguchi et al. (1988) and the 10 individual RNA segments separated and isolated as described previously (Purdy et al. , 1984). AcNPV and recombinant baculovirus stocks were grown and assayed in confluent monolayers of S. fvugipe ⁇ da cells in medium containing 10 fetal calf serum according to the procedures described by Brown and Faulkner (1977) • Occasionally virus stocks were made using spinner cultures of these insect cells.
  • B DNA cloning of the BTV10-M6 RNA.
  • Polyadenylation of BTV-10 dsRNA and synthesis of cDNA copies of the polyadenylated M6 RNA using an oligo(dT) 1 _ 1 o primer were undertaken as described previously (Purdy et al. , 1984).
  • the RNA templates were removed by treatment with 0.5 M K0H and double-stranded DNA generated by self-annealing. The products were repaired using the Klenow large fragment of DNA polymerase, followed by 3' tailing with dC and annealing to Pstl-cut, dG-tailed, pBR322 plasmid DNA (Maniatis et al. , 1982).
  • RNA gel electrophoresis blotting and hybridization.
  • Purified BTV-10 RNA was resolved on an agarose gel, blotted onto a Genescreen membrane (New England Nuclear, Boston MA) and hybridized to nick- translated cloned DNA by procedures described previously (Purdy et al. , 1984).
  • Plasmid DNA manipulations were carried out following the procedures described by Maniatis and associates (Maniatis et al. , 1982) . Restriction enzymes, T4 DNA ligase and the Klenow large fragment of DNA polymerase, were purchased from New England Biolabs, Inc. (Beverly, MA) . Calf intestine alkaline phosphatase was obtained from Boehringer Mannheim (FGR) .
  • S. fvugipevda cells were transfected with mixtures of infectious AcNPV DNA and DNA obtained from plasmid pAcBTV 10-6. Recombinant viruses were obtained as described previously (Inumaru and Roy, 1987) . One of the derived recombinant viruses was designated AcBTV 10-6.
  • glycerol, 5% ⁇ -mercaptoethanol, 62.5 mM Tris-HCl, 0.01 bromophenol blue, pH 6.8) were added to each sample and the mixture heated at 100°C for 10 min.
  • Proteins were analysed by electrophoresis in a 10-30$ linear gradient polyacrylamide gel in the presence of SDS (SDS-PAGE) as described by Laemmli (1970) . After electrophoresis, the gel was stained with 0.25% Coomassie brilliant blue.
  • the membrane was soaked for 1 hr at room temperature in anti-rabbit IgG-goat IgG-alkaline phosphatase conjugate (Sigma Chemical Co.), also diluted in blocking buffer. After further washing with PBST, bound antibodies were detected by incubating using Fast BB salt and a-Naphthyl phosphate (Sigma Chemical Co.) as substrate.
  • PBST PBS
  • a solid phase indirect micro-ELISA was used to demonstrate the reactivity of recombinant AcBTV 10-6 antigen with various polyclonal BTV antisera.
  • S. fs gipexda cells infected 2 hr previously with recombinant AcBTV 10-6 virus (see above) were collected and subjected to freezing and thawing followed by low speed centrifugation to remove cellular debris.
  • the supernatant was diluted (1:10- to 1:10,000-fold) with sodium carbonate buffer (15 mM Na-CO-, 36 mM NaHC0_, pH 9-6)-
  • sodium carbonate buffer 15 mM Na-CO-, 36 mM NaHC0_, pH 9-6)
  • a 96-well polyvinylchloride microplate (Flow Laboratories) was coated overnight at 4xC with 50fl of the diluted antigen.
  • the plate was washed three times between each step of the following protocol by flooding the wells of the plate with PBST buffer.
  • the antigen-coated microplate was washed and blocked with blocking solution for 3 hr at room temperature.
  • BTV antisera were diluted (1:100- to 1:12,800-fold) in blocking solution and 50 ⁇ l of diluted sera were added to each well.
  • Cells were infected with the AcBTV 10-6 recombinant baculovirus and the infected cells harvested 4 or 5 days post-infection. The cells were recovered, washed twice with PBS and resuspended in 10 mM Tris-HCl (pH 7-4) and disrupted by sonication. The resulting cell extract was loaded oh a 10% to 50 (wt/v) sucrose gradient in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4) and centrifuged at 40,000 rpm for 3 hr using an SW4l rotor. After centrifugation, the gradient was fractionated and a portion of each fraction was subjected to gel electrophoresis. The peak fractions containing NSI protein were pooled and pelleted by centrifugation for 2 hr at 40,000 rpm. The pellet was resuspended in 10 mM Tris HC1 buffer (pH 7.4).
  • a plasmid (pBTV10-6) containing the complete BTV-10 M6 sequence was constructed from two overlapping clones as described above (Fig. 2) .
  • the restriction enzyme M ⁇ el was used to isolate the entire coding region of the M6 DNA (including 1 bases upstream from the ATG initiation codon and 59 bases downstream from the TAG stop codon) and the DNA used to prepare a recombinant baculovirus transfer vector.
  • the 60 kD protein was NSI
  • a sample of the AcBTV 10-6 infected cell extract was electrophoresed, transferred onto a Durapore membrane and subjected to Western analyses using anti-BTV-10 sera as described in Methods.
  • the 60 kD protein (lane c) was identified by the alkaline phosphatase conjugate detection procedure.
  • tubules of various lengths were evident. In cross-section, they exhibited a diameter of ca 60 nm. Many of the tubules appeared to contain ribosome-like particles (Fig. 5. R arrowheads). Both the structures and arrangement of the tubules were comparable to the tubular structures reported by others in BTV infected BHK-21 cells (Huismans and Els, 1979). As expected, polyhedra were only evident in the AcNPV infected cells (Fig. 5. P arrowheads).
  • Fibrous structures were observed both in AcNPV and in the recombinant virus infected cells (Fig. 5) • In the latter, the tubules appeared to be aligned along the edges of bundles of the fibrous material (Fig. 5c and d) . Both the tubules and the fiberous structures were randomly oriented with respect to one another. In some cells bundles of fibers were seen in both the nucleus and the cytoplasm (Fig. 5e, f) , with tubules mostly, but not exclusively associated with the cytoplasmic fibers. Fewer tubules were seen in the nuclei (Fig. f)• Whether these tubules were formed in the nucleus, or occurred there because the cell was in the terminal stage of infection, is not known.
  • Recombinant virus infected cell extracts were absorbed to microtiter plates (representing some 50 ng of NSI protein per well) and incubated with either polyclonal BTV-2, or BTV-10, or BTV-11, or BTV-13, or BTV-17 sheep antisera, or with normal sheep serum as a control.
  • the derived antigen-antibody complexes were detected by incubating with anti-sheep-alkaline phosphatase conjugates followed by the addition of an enzyme substrate.
  • Fig. 7 all five BTV antisera reacted with the recombinant antigen in proportion to the end point titer of the antisera. No reaction was detected with the normal sheep serum. No reactivity was obtained when each of the BTV antisera was tested with AcNPVinfected cell extracts.
  • NSI is the major viral protein synthesized in BTV-infected cells and is a major constituent of the tubules which are observed in such cells. (Huismans and Els, 1979). It has now unexpectedly been found that baculovirus expressed NSI protein also form tubules in insect cells. The two overlapping clones (#14 and 39) were used to construct a plasmid containing the complete gene (pBTV 10-6) . A recombinant baculovirus was prepared with the NSI gene sequences under the control of the AcNPV polyhedrin promoter. The data herein shows that the BTV NSI protein is expressed to a high level in insect cells infected with this recombinant baculovirus.
  • NSI, NS2 and NS3 can be used as group specific probes to detect the presence of bluetongue virus (all 24 serotypes); Ritter and Roy, 1988.
  • BTV 10 NSI antigen can be used to detect antibodies to the US strains (BTV-2, BTV-10, BTV-11, BTV-14 and BTV-17) ; Urakawa and Roy, 1988.
  • a culture of AcNPV-BTV-10 has been deposited at the European Collection of Animal Cell Cultures (ECACC) on 10th August 1989 under Accession No. 89081017.
  • Fig. 1 Strategy used to determine the sequences of cDNA clones of the BTV- 10 M6 gene. The distance and directions in which individual strands of two overlapping clones were sequenced are shown by the solid arrows. Restriction enzyme sites used are as follows: Ma, Mael; Hd, Hind l; R, HinfI; D, DdeI; Pv,PuuI; Ms, spI.
  • FIG. 2 Schematic diagram of the construction of the transfer vector pAcBTVlO-6.
  • a pBR332-based plasmid (pBTV 10-6) containing the entire coding region of the BTV-10 M6 DNA was constructed from two overlapping partial clones as described in Methods.
  • the complete clone (pBTV10-6) was used to construct the transfer vector (pAcBTVlO-6) as described in Methods.
  • the sequence of the 5' insertion site was determined by the method of Maxam and Gilbert (18) using a Hin ⁇ HL restriction fragment (BTV DNA residue #296).
  • Fig. 3 Hydropathic plot and distribution of cvsteine residues for the predicted M6 gene product of BTV-10.
  • the plot involves a span setting of 21 amino acids.
  • Fig.4 Expression of NSl protein bv recombinant baculoviruses derived from the pAcBTV10-6 transfer vector.
  • S. frugiperda cells were infected with recombinant (c) or wild type AcNPV (b). Proteins were recovered at 72 hr post-infection and an aliquot of each sample was resolved by gel electrophoresis. Uninfected cells were treated similarly (a). The resolved protein bands were either detected by staining with Coomassie brilliant blue (A), or blotted onto nitrocellulose membranes and detected immunolog ⁇ cally with a ti BTV-10 serum (B) as described in Methods. The positions of NSI and the AcNPV polyhedrin protein (P) are indicated. Molecular weights (in kD) are indicated on the left.
  • FIG. 5 Electron micrographs of NSl-derived tubules produced by S. frugiperda cells infected with recombinant viruses.
  • S. frugiperda cells infected with AcNPV (panel a), or a recombinant virus (panels b-f) were fixed with 2% glutaraldehyde 72 hr post-infection and processed for electron microscopy as described in Methods.
  • P polyhedrin
  • V virus particles
  • M mitochondria
  • R ribosomes
  • F fibrous structure
  • T tubules
  • N nuclear membrane.
  • FIG. 6 Three-dimensional arrangement of the tubules in the infected S. frugiperda cells.
  • A A series of representative virus (A-H) of the single arrays in a thin section of the tubules were photographed at 10° intervals of specimen tilt.
  • B A schematic three-dimensional interpretation of the structure are represented (a, b and c).
  • Fig. 7 Reaction of bluetongue antibodies to recombinant baculovirus derived antigen using indirect ELISA: Recombinant AcBTV 10-6 infected cell extracts were adsorbed to the solid phase and examined using 1:100 to 1:12800 dilutions of sheep anti-BTV-2 serum ( ⁇ e), or anti-BTV- 10 serum (o o), or anti-BTV-11 serum ( ⁇ A), or anti-BTV-13 serum ( ⁇ ⁇ ), or anti-BTV-17 serum (G ⁇ ), or with normal sheep serum (gg U).
  • Fig. 8 SDS-PAGE of purified NSI protein stained with Coomassie brilliant blue: AcBTV10-6 recombinant baculovirus infected cells were disrupted and tubules were recovered by sedimenting in sucrose gradient (10-50% w/v) centrifugation as described in Methods. The peak fractions containing NSl were pooled and subjected to SDS-PAGE analysis. The proteins recovered from unpurif ⁇ ed recombinant virus infected cells (1) are compared with NSl protein purified through sucrose gradient centrifugation (2). - /S

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EP89909839A 1988-08-16 1989-08-15 Herstellung von nichtstrukturellen proteinen des bluetongue-virus unter verwendung eines baculovirus-expressionsvektors Withdrawn EP0431038A1 (de)

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GB888819453A GB8819453D0 (en) 1988-08-16 1988-08-16 Production of bluetongue virus non-structural proteins using baculovirus expression vector
GB8819453 1988-08-16

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