WO1989007140A1 - Systeme d'expression par genes (en particulier pour la proteine de rotavirus vp7) employant un peptide de signal etranger et eventuellement une sequence d'ancrage transmembrane - Google Patents

Systeme d'expression par genes (en particulier pour la proteine de rotavirus vp7) employant un peptide de signal etranger et eventuellement une sequence d'ancrage transmembrane Download PDF

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WO1989007140A1
WO1989007140A1 PCT/AU1989/000038 AU8900038W WO8907140A1 WO 1989007140 A1 WO1989007140 A1 WO 1989007140A1 AU 8900038 W AU8900038 W AU 8900038W WO 8907140 A1 WO8907140 A1 WO 8907140A1
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protein
gene
cell
signal peptide
rotavirus
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PCT/AU1989/000038
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Gerald Wayne Both
Peter Lawrence Whitfeld
Sally Clare Stirzaker
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Commonwealth Scientific And Industrial Research Or
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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
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/033—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • 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
    • C12N2710/24011—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
    • 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

  • the present invention relates to novel genes for the expression of proteins and in particular novel genes expressing antigens of rotaviruses and more particularly to VF7 antigens having altered amino acid sequences.
  • Rotaviruses are a major cause of diarrheal disease. Worldwide they account for some 140 million cases of illness annually with an associated one million deaths (Robbins & Freeman Sci. Am. 259:126 1988). Approximately 50% of hospitalized cases of diarrheal illness for children in the 6-24 months age group in the U.S.A., Japan and Australia are rotavirus induced (Kapikian & Channock 1985) pp 863-906 in "Virology” B N Fields (Ed) Raven Press, New York.
  • VP7 is the major viral protein against which neutralizing antibodies are directed, it is a prime candidate for the development of a rotavirus vaccine affording protection through a single viral protein.
  • the present inventors have investigated the virus on a molecular level with a view to developing a rotavirus vaccine based on recombinant DNA technology. It was earlier found by cloning and sequencing rotavirus dsRNA genome segments, that segment 9 is the one which codes for VP7 protein in the Simian rotavirus SAIL The equivalent genes describing the VP7 proteins for a human strain S2 and a bovine strain NCDV have also been cloned and sequenced (Both et al P.N.A.S. .80:3091-3095, 1983; Gunn et al, J. Virology 54:791-797, 985).
  • VP7 proteins as deduced from the gene sequences reveals certain features which are conserved, notably two regions of hydrophobic amino acids HI and H2 near the amino terminus. These are involved in directing newly synthesized VP7 to its correct location in the cell for virus assembly. Virus particles which are partially assembled in the cell cytoplasm migrate to the membrane of the rough endoplasmis reticulum (ER) . The immature particles then bud through the membrane becoming transiently enveloped as they do so. The VP7 protein may be acquired at this time or later when the enveloping membrane is lost. The VP7 protein is therefore unusual in that it is retained in the ER for virus assembly. Most other viral glycoproteins are transported to the cell surface membrane or directed to other organelles in the cell. In fact there is no evidence for transport of VP7 beyond the ER of the infected cell.
  • ER endoplasmis reticulum
  • the site at which the signal peptide is cleaved from the precursor protein to yield mature VP7 was also determined using genetic engineering and protein sequencing techniques (Stirzaker et al. J. Cell Biol. 105: 2897-2903, 1987). It is now clear that glutamine residue 51 (numbered according to the codons in the open reading frame of the VP7 gene) is the N-terminal residue of mature VP7. It is predicted (Von Heijne Nuc. Ac. Res 14: 4683-4699 1988) that this cleavage site is conserved in all serotypes of VP7 whose structure has been determined (Gorziglia, et al. J. Gen. Virol. 62:2445-2454, 1986).
  • the H2VP7 signal peptide (comprising residues 30-50 of the open reading frame) (Fig. 1) was replaced with one consisting of the N-terminal 16 amino acids of the influenza haemagglutinin (HA) (HAQVP7, Fig. 1A) , an integral membrane protein which is directed to the cell surface (Gething and Sambrook Nature 193: 620-625, 1981).
  • HA influenza haemagglutinin
  • Fig. 1A an integral membrane protein which is directed to the cell surface
  • the HA signal peptide was then fused to phe47 of VP7 to conserve the usual cleavage site of VP7(HAFVP7 Fig. 1A) .
  • This precursor was correctly processed in vitro to yield a molecule indistinguishable from the wild-type (Fig. 1C and Stirzaker, et al. J. Gen. Virol. 67:245-2454, 1987).
  • the fate of the protein in this case was remarkably different.
  • VP7 produced from the wild-type precursor remained intracellular (Fig. 2A)
  • that derived from the hybrid precursor was rapidly secreted from the cell and according to its increased size was modified with complex carbohydrate (Fig. 2B) .
  • the present invention therefore provides a gene coding for a secreted VP7 protein with an N-terminus indistinguishable from that derived from the wild-type VP7 precursor protein.
  • the only known modification to secreted VP7 is due to the addition of complex, endoglycosidase H-resistant carbohydrate attached at asn 69.
  • the present invention consists in a gene including a sequence coding for a protein naturally retained in the endoplasmic reticulum and a sequence coding for a foreign signal peptide derived from a gene coding for a protein which is naturally transported beyond the endoplasmic reticulum, the gene sequence for the signal peptide being so fused to the gene sequence for the protein that cleavage of the protein from the signal peptide takes place at a site such that the antigenic characteristics and/or biological properties of the cleaved protein are the same as those of the naturally occurring protein.
  • the present invention consists in a vector, cell or organism carrying the gene according to the present invention.
  • the present invention consists in a process for the preparation of the protein comprising causing the gene to be expressed in a culture medium and recovering the protein from the culture medium, and in the protein so expressed.
  • the protein may be any protein naturally retained in the ER including Rotavirus VP7 protein, glucose regulated proteins GRP78 (also known as BiP) and GRP94, protein disulphide isomerase, HMG CoA Reductase and adenovirus E19 protein. It is presently preferred, however, that the protein is Rotavirus VP7 protein.
  • the term "protein naturally retained in the ER” is taken to mean proteins which are, in organisms in which they naturally occur, so retained. The term includes also derivatives of such proteins which correctly fold to the extent that they will still be transported out of the endoplasmic reticulum.
  • the signal peptide may be derived from any suitable protein that is naturally transported beyond the endoplasmic reticulum. These signal peptides include those from influenza haemagglutinin, from yeast invertase and from growth hormone, with the signal peptide from influenza haemagglutinin being preferred.
  • the nucleotide sequence of the gene should be such that the signal peptide is fused to the protein in a way which ensures that the cellular process of cleavage of the protein from the signal peptide occurs at the correct site on the protein i.e. at the point at which the protein would be cleaved from its natural signal peptide. If need be a suitable linker group may be included in the gene to ensure that the protein when processed has its natural N-terminal sequence.
  • the present inventors have made a further surprising discovery. They have found that proteins which are naturally resident in the cytoplasm, including the organelles present therein, may be correctly expressed and bound in the membrane of a cell if the gene for the protein is appropriately fused with a suitable signal peptide and a suitable transme brane anchor domain.
  • the present invention consists in a gene including:-
  • the protein naturally retained in the cytoplasm may be from an organelle or a component of an organelle.
  • the anchor domain used in preferred embodiments of this aspect of the present invention may be derived from haemagglutinin, the VSV glycoprotein G, the IgG i munoglobulin protein, the histocompatability antigen HLA-2A or from another C-terminally-anchored membrane protein. However, it is most preferred that the anchor domain is derived from haemagglutinin.
  • the signal peptide-anchor domain is derived from a type II membrane protein such as influenza neuraminidase, respiratory syncytial virus G protein or asialoglycoprotein, and most preferably influenza neuraminidase.
  • the present invention consists in a gene including:-
  • sequence coding for a combined signal peptide-transmembrane anchor domain the sequence coding for the signal peptide anchor domain being derived from a gene coding for a protein which is normally transported beyond the endoplasmic reticulum, the sequence coding for the combined signal peptide-transmembrane anchor domain being fused in-frame to the sequence coding for the N-terminal region of the protein so that the gene will upon expression in a eukaryotic cell give rise to a correctly processed, appropriately folded, membrane bound version of the protein naturally exported from the cell.
  • the signal peptide-anchor domain is derived from a type II membrane protein such as influenza neuraminidase, respiratory syncytial virus G protein or asialogylcoprotein. At present it is preferred that the signal peptide-anchor domain is derived from influenza neuraminidase.
  • the present invention consists in a vector, cell or organism carrying either of the genes defined above.
  • the present invention consists in a process for the preparation of the protein comprising the steps of causing the gene to be expressed and recovering the cells or the membrane therefrom to which the protein is bound.
  • the invention still further consists in such a protein when bound to the surface of a eukaryotic cell.
  • the invention still further consists in antigenic preparations containing such proteins and to antigenic preparations comprising suitable viral vectors containing these genes, with adenovirus and vaccinia virus being preferred vectors.
  • the present invention further consists in a gene which, upon expression in a eukaryotic cell, gives rise to a correctly processed, membrane-bound version of VP7 which is no longer located on the ER but is transported to and displayed on the cell surface.
  • Figure 1(A) shows the construction of VP7 genes with altered signal peptides.
  • Bold type and lines indicate VP7 sequences: other sequences are derived from influenza haemagglutinin (CHO) Endo H-sensitive carbohydrate: (CHO***) Endo H-resistant carbohydrate. Arrows indicate cleavage sites.
  • B and C Partial N-terminal sequence of 35 S-methionine-labelled HAQVP7 and HAFVP7 after treatment of the protein with puroglutamate aminopeptidase (+) . Radioactivity released after each cycle of Edman degradation was determined by liquid scintillation counting.
  • Figure 2 shows the cellular location and transport of VP7 produced from precursor H2VP7 (panel A) or HAFVP7
  • Figure 3 shows the structure of HAFVP7A. Amino acid residues in italics are derived from haemagglutinin. Val 326 of VP7 is replaced by Ser and Ala is derived from the synthetic oligonucleotide. The arrow shows the cleavage site.
  • FIG. 4 cellular location and transport of secreted variant HAFVP7 (tracks 1,2,5,6) or C-terminally anchored variant HAFVP7A (tracks 3,4,7,8).
  • VP7 recovered from transfected COS cells by immunoprecipitation was digested with Endo H as indicated (+) .
  • Track 9 contains standard marker proteins of 30,46,60 and 92.5 kd.
  • FIGS 5 Figure 5 sensitivity of proteins expressed in COS cells to digestion with endo glycosidase F.
  • COS cells were transfected with HAFVP7 (tracks 1,2) or HAFVPYA (tracks 3,4) and intact cells were digested with endo F.
  • FIG. 6 screening of vaccinia virus plaques with radiolabelled antibodies.
  • Cells were infected with vaccinia virus strain WR or recombinants W-VP7 or W-HAFVP7A (Cell-surface variant) . Plaques were incubated with normal rabbit serum (NRS), mouse anti vaccinia (M-c -W) or rabbit anti SA11 (R-cC-SAll) serum incubated with iodine 125-labelled protein A. Construction of novel VP7 genes
  • VP7 genes were constructed using procedures similar to those described in "Molecular Cloning: A Laboratory Manual", Maniatis et al. (1982) Cold Spring Harbor Press.
  • the SA11 VP7 gene was previously cloned into the Xhol site of the SV40-based expression vector pJC119 to create plasmid pHC9 (Poruchynsky, et al. J. Cell. Biol. 101:2199-2209, 1985).
  • the gene encoding the VP7 precursor carrying the HA signal peptide i.e. HAFVP7 (Fig. 1A) was constructed in two stages as follows. Oligonucleotides of 65 -and 58 bases encoding the first sixteen amino acids i.e. the signal peptide of the HA from the influenza strain A/NT/60/68/29C and the first 12 residues i.e.
  • VP7 amino acids 51-61 of VP7 plus a linking ser residue were synthesized using an Applied Biosystems Model 380A DNA synthesizer. These oligonucleotides were complementary for 15 bases at their 3' ends. The synthetic oligonucleotides were phosphorylated, annealed and elongated using Klenow DNA polymerase to make them double stranded. The fragment was cut with Xhol and Ncol to generate 5' Xhol and 3' Ncol-compatible ends then ligated with a 4.3kb EcoRI-XhoI fragment and a 3.7 b EcoRI-NcoI fragment prepared from pJC9 to recreate the SV40 expression vector carrying the modified gene (HAQVP7) (Fig. 1A) .
  • HAQVP7 modified gene
  • the modified gene was also excised from this plasmid using Xhol and subcloned into the Sail site of the Bluescript KS M13+ vectpr (Stratagene) then transcribed into RNA in vitro.
  • the RNA was translated in rabbit reticulocyte lysates in the presence of canine pancreatic icrosomes under which conditions the processed VP7 protein was produced. N-terminal analysis of this species (Stirzaker & Both, (1989) Cell, In press) showed that processing had not occurred at the expected site i.e. at glutamine 51, but four residues further downstream (Fig. IB).
  • the gene was further modified in an effort to obtain correct cleavage of the HA signal peptide from VP7.
  • Single stranded template DNA was prepared for gene HAQVP7 using the helper phage M13K07 as described by Stratagene.
  • An oligonucleotide was synthesized to insert 12 nucleotides coding for the amino acids phe-leu-arg-ala preceding the N-terminal glutamine residue of VP7. This mutation was constructed using the techniques and reagents provided in the Biorad mutagenesis kit, except that DNA was transformed into E.coli strain MV1190 and ampicillin resistant colonies were selected.
  • This Bluescript plasmid was digested with Xhol and Ncol to prepare the 5'-terminal fragment encoding the HA signal peptide now fused to the phe residue 47 of VP7.
  • the HAFVP7 gene (Fig. 1A) in the SV40 vector was again constructed by three fragment ligation as described above. This gene could have been constructed in a single step if the signal peptide cleavage site could have been accurately predicted.
  • the cleavage site of HAFVP7 translated in vitro was checked by N-terminal sequencing (Stirzaker et. al. 1989 Cell, In Press) and confirmed as correct . (Fig. 1C) .
  • the gene HAFVP7 was further modified to add a C-terminal transmembrane anchor domain as follows.
  • the 3' terminal BamHI fragment of the VP7 gene in pJC9 was subcloned into M13mpl0 and single stranded template DNA was prepared.
  • An oligonucleotide complementary to bases 1014-1035 of the gene was synthesized with mismatched bases to introduce a Bgll site at codons 325/326 of the VP7 gene.
  • the oligonucleotide was elongated with Klenow DNA polymerase in the presence of DNA ligase to form double-stranded DNA which was transformed into E.coli strain JM101. Mutants were selected by hybridization with the radiolabelled oligonucleotide at a temperature near its Tm.
  • a 528bp 5'HgaI-3' Xhol fragment was prepared from mutated, double-stranded M13 DNA and ligated with 4.83kb EcoRI-Hgal and a 4.3kb EcoRI-XhoI fragments from pJC9 to produce the gene VP7Bgl in the SV40 vector.
  • the C-terminal membrane anchor domain of influenza haemagglutinin was prepared as follows. Oligonucleotides of 77 and 74 bases which were complementary for 13 residues at their 3' ends were synthesized, phosphorylated, annealed and elongated with Klenow DNA polymerase to make a 130bp double stranded fragment. This was digested with BamHI to produce terminii compatible with the Bglll site which had been introduced into VP7Bgl as described above.
  • VP7Bgl was cut with Bglll (a unique site) and the membrane anchor fragment was incorporated into the plasmid by ligation.
  • the plasmid was recut with Bglll to eliminate molecules which had closed without acquiring the fragment and the DNA was transformed into E.coli RR1. Colonies carrying the transmembrane anchor fragment were identified by hybridization using one of the radiolabelled synthetic oligonucleotides as a probe. This plasmid was called VP7A.
  • gene HAFVP7A in the SV40 vector was produced by three fragment ligation using the 5'Xhol-Ncol fragment from gene HAFVP7, the 4.3kb EcoRI-XhoI from pJC9 and the 3.8kb EcoRI-NcoI fragment from VP7A. Expression of VP7 Genes in COS Cells
  • Wild-type and modified VP7 genes in the SV40 vector pJC119 were introduced into COS cells by electroporation (Chu et al. Nucleic acids Res. .15:1311-1326, 1987) using a Biorad Gene Pulser at a capacitance of 250uF with a pulse of 0.3Kv. Cells were allowed to recover at room temperature for 10-15 min. then gently resuspended in 3ml of DMEM with 10% foetal calf serum and plated in a 60 mm dish. The following day the medium was changed to remove
  • HAFVP7A The gene encoding the HAFVP7 precursor (Fig. 1) was fused with a C-terminal transmembrane anchor domain also derived from the influenza haemagglutinin gene.
  • the DNA coding of this HA segment was spliced, inframe, to the VP7 gene at penultimate codon 325 and the stop codon of the HA segment was used to terminate protein synthesis (Fig. 3). This construction is called HAFVP7A.
  • endoglycosidase F a protein which removes both simple and complex carbohydrate from proteins.
  • Intracellular VP7 produced in cells transfected with HAFVP7 was insensitive to endo F indicating that the cells remained intact during digestion (Fig. 5, tracks 1 & 2) .
  • putative cell-surface expressed VP7 derived from HAFVP7A was sensitive to endo F (Fig. 5, tracks 3,4), indicating its accessibility and confirming its cell-surface location. It may also be possible to achieve cell-surface expression of other derivatives of VP7.
  • the present inventors have found that attaching the HA C-terminal anchor domain to the penultimate residue of the secreted variant deletion 47-61 (Poruchynsky et al, 1985) also results in surface presentation of the antigen. Similar results could be expected for related deletion mutations 43-61 and 42-61. However, for none of these variants is it known whether signal peptide processing occurs and the immunological effectiveness of these variant proteins is largely uncharacterized. It may also be possible to replace the HA segments with segments of equivalent function from other similarly transported proteins. However, in a number of different constructions that were tried, the hybrid VP7 protein was not transported to the ER.
  • a recombinant vaccinia virus carrying the gene for the cell-surface expressed VP7 was also constructed.
  • Recombinant virus plaques expressing either cell-surface Vp7 or the wild-type protein were screened using a radiolabelled antibody. Plaques expressing the modified gene gave a strong signal compared with those carrying the wild-type gene which gave a weak signal (Fig. 6), further confirming the surface location of the modified antigen.

Abstract

La présente invention se rapporte à de nouveaux gènes servant à l'expression de protéines. Ces gènes permettent l'expression de protéines qui sont normalement retenues à l'intérieur d'une cellule, afin qu'elles soient transportées hors de la cellule ou qu'elles se lient à la membrane cellulaire de la cellule. La présente invention se rapporte en outre à de nouveaux gènes servant à l'expression de protéines qui sont naturellement transportées hors de la cellule ou liées à la membrane de la cellule. Les nouveaux gènes de la présente invention sont particulièrement utiles dans l'expression d'antigènes de rotavirus et notamment de la protéine de rotavirus VP7 qui est normalement retenue dans le réticulum endoplasmique de la cellule. En permettant l'expression de protéines, telles que la protéine de rotavirus VP7, sur la membrane de la cellule ou sous une forme transportée hors de la cellule, on obtient un procédé avantageux qui permet de produire cet antigène pour son utilisation dans des vaccins.
PCT/AU1989/000038 1988-02-05 1989-02-03 Systeme d'expression par genes (en particulier pour la proteine de rotavirus vp7) employant un peptide de signal etranger et eventuellement une sequence d'ancrage transmembrane WO1989007140A1 (fr)

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WO1992020805A1 (fr) * 1991-05-13 1992-11-26 Pierre Fabre Medicament Adn recombine codant pour un peptide signal, polypeptide d'interaction et sequence d'ancrage de membrane
WO1994001567A1 (fr) * 1992-07-08 1994-01-20 Unilever N.V. Procede pour immobiliser des enzymes sur la paroi cellulaire d'une cellule microbienne en produisant une proteine de fusion
WO1995020660A2 (fr) * 1994-01-27 1995-08-03 University Of Massachusetts Medical Center Immunisation par inoculation d'une unite de transcription d'adn
US5643578A (en) * 1992-03-23 1997-07-01 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
WO1998024912A2 (fr) * 1996-12-04 1998-06-11 Heska Corporation Vaccin contre la peste obtenu par recombinaison
EP1019435A1 (fr) * 1996-01-29 2000-07-19 Georgetown University Amplification de la reponse d'une proteine de recombinaison exprimee
WO2001032894A1 (fr) * 1999-11-04 2001-05-10 Zymogenetics, Inc. Presentation de surface cellulaire de proteines par des cellules hotes recombinantes
EP1293571A2 (fr) * 1992-07-08 2003-03-19 Unilever N.V. Procédé pour l'immobilisation d'enzymes sur la paroi cellulaire d'une cellule microbienne eucaryote en produisant une protéine de fusion
US6686168B1 (en) 1999-11-04 2004-02-03 Zymogenetics, Inc. Cell surface display of proteins by recombinant host cells
US20110142869A1 (en) * 2008-08-04 2011-06-16 Ira Berkower Membrane proximal region of hiv gp41 anchored to the lipid layer of a virus-like particle vaccine
US8252551B2 (en) 2003-12-23 2012-08-28 Novozymes Biopharma Dk A/S 2-micron family plasmid and use thereof
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EP0509841A3 (en) * 1991-04-18 1993-08-18 Tonen Corporation Co-expression system of protein disulfide isomerase gene and useful polypeptide gene and process for producing the polypeptide using its system
EP0509841A2 (fr) * 1991-04-18 1992-10-21 Tonen Corporation Système de co-expression d'un gène de la protéine disolfure isomerase et d'un gène d'un polypeptide utile et procédé de production de ce polypeptide en utilisant un tel système
WO1992020805A1 (fr) * 1991-05-13 1992-11-26 Pierre Fabre Medicament Adn recombine codant pour un peptide signal, polypeptide d'interaction et sequence d'ancrage de membrane
US6841381B1 (en) 1992-03-23 2005-01-11 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
US5643578A (en) * 1992-03-23 1997-07-01 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
US7850956B2 (en) 1992-03-23 2010-12-14 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
EP1293571A3 (fr) * 1992-07-08 2004-02-18 Unilever N.V. Procédé pour l'immobilisation d'enzymes sur la paroi cellulaire d'une cellule microbienne eucaryote en produisant une protéine de fusion
AU685057B2 (en) * 1992-07-08 1998-01-15 Unilever Plc Process for immobilizing enzymes to the cell wall of a microbial cell by producing a fusion protein
US6027910A (en) * 1992-07-08 2000-02-22 Unilever Patent Holdings B.V. Process for immobilizing enzymes to the cell wall of a microbial cell by producing a fusion protein
WO1994001567A1 (fr) * 1992-07-08 1994-01-20 Unilever N.V. Procede pour immobiliser des enzymes sur la paroi cellulaire d'une cellule microbienne en produisant une proteine de fusion
EP1293571A2 (fr) * 1992-07-08 2003-03-19 Unilever N.V. Procédé pour l'immobilisation d'enzymes sur la paroi cellulaire d'une cellule microbienne eucaryote en produisant une protéine de fusion
WO1995020660A2 (fr) * 1994-01-27 1995-08-03 University Of Massachusetts Medical Center Immunisation par inoculation d'une unite de transcription d'adn
WO1995020660A3 (fr) * 1994-01-27 1995-12-21 Univ Massachusetts Medical Immunisation par inoculation d'une unite de transcription d'adn
EP1019435A1 (fr) * 1996-01-29 2000-07-19 Georgetown University Amplification de la reponse d'une proteine de recombinaison exprimee
EP1019435A4 (fr) * 1996-01-29 2001-12-12 Univ Georgetown Amplification de la reponse d'une proteine de recombinaison exprimee
WO1998024912A3 (fr) * 1996-12-04 1998-09-11 Heska Corp Vaccin contre la peste obtenu par recombinaison
WO1998024912A2 (fr) * 1996-12-04 1998-06-11 Heska Corporation Vaccin contre la peste obtenu par recombinaison
US6686168B1 (en) 1999-11-04 2004-02-03 Zymogenetics, Inc. Cell surface display of proteins by recombinant host cells
WO2001032894A1 (fr) * 1999-11-04 2001-05-10 Zymogenetics, Inc. Presentation de surface cellulaire de proteines par des cellules hotes recombinantes
US8252551B2 (en) 2003-12-23 2012-08-28 Novozymes Biopharma Dk A/S 2-micron family plasmid and use thereof
US8969064B2 (en) 2003-12-23 2015-03-03 Novozymes Biopharma Dk A/S Gene expression technique
US9057061B2 (en) 2003-12-23 2015-06-16 Novozymes Biopharma Dk A/S Gene expression technique
US20110142869A1 (en) * 2008-08-04 2011-06-16 Ira Berkower Membrane proximal region of hiv gp41 anchored to the lipid layer of a virus-like particle vaccine
US9005631B2 (en) * 2008-08-04 2015-04-14 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Membrane proximal region of HIV gp41 anchored to the lipid layer of a virus-like particle vaccine
US9486518B2 (en) 2008-08-04 2016-11-08 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Membrane proximal region of HIV GP41 anchored to the lipid layer of a virus-like particle vaccine

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AU626288B2 (en) 1992-07-30
EP0398944A4 (en) 1991-09-11
AU3045389A (en) 1989-08-25
EP0398944A1 (fr) 1990-11-28

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