EP0482061A1 - Adn codant pour une sequence de signaux polypeptidiques dans le virus vaccinal - Google Patents

Adn codant pour une sequence de signaux polypeptidiques dans le virus vaccinal

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Publication number
EP0482061A1
EP0482061A1 EP90910856A EP90910856A EP0482061A1 EP 0482061 A1 EP0482061 A1 EP 0482061A1 EP 90910856 A EP90910856 A EP 90910856A EP 90910856 A EP90910856 A EP 90910856A EP 0482061 A1 EP0482061 A1 EP 0482061A1
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Prior art keywords
sequence
dna
gene
poxvirus
promoter
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EP90910856A
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German (de)
English (en)
Inventor
Dairena Frances Gaffney
Arvind Hirabhai Patel
Nigel Dennis Stow
John Herbert Subak-Sharpe
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BTG International Ltd
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BTG International Ltd
National Research Development Corp UK
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Publication of EP0482061A1 publication Critical patent/EP0482061A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C12N2710/24122—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
    • 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

Definitions

  • This invention is in the recombinant DNA field and relates to DNA coding for the secretion signal sequence of a polypeptide expressed by a strain of vaccinia virus.
  • vaccinia virus has been used as a vector for Introducing a foreign gene Into mammalian cells.
  • the foreign gene 1s Introduced into the virus 1n a "non-essential region" within Its genome in which 1t does not Interfere with essential functions of virus, so that the virus 1s capable of at least limited, If not full, replication 1n the mammalian host.
  • Transcription of the foreign gene requires a vaccinia virus promoter, which could be obtained by excising the relevant DNA from elsewhere 1n the genome, to be co-inserted upstream of the foreign gene within the non-essential region.
  • VV vaccinia virus
  • P7.5K The vaccinia virus promoter most widely used is the "7.5K” promoter (P7.5K). It promotes the transcription of a gene (7.5 gene) which encodes a protein of relative molecular mass of about 7.5 kiloDaltons.
  • the gene and Its promoter have been partially sequenced by S. Venkatesan £i -, Cell 25, 805-813 (1981).
  • the inventors' research was founded on the discovery that the 35kD polypeptide is secreted from the Lister strain, which does grow well and on the hope that the Lister strain of VV might contain a powerful promoter for the 35kD polypeptide. Such a promoter might be useful for linking to a heterologous gene to enhance expression of that gene In VV recombinants, i.e. to Improve on the 7.5K promoter.
  • a signal sequence is one which enables the polypeptide to pass through the cell membrane of the infected cell and enter the culture medium. During secretion, the signal sequence 1s cleaved, resulting 1n production of mature protein in the culture medium. In the present instance a putative signal sequence was discovered initially by sequencing the 35K gene and comparing its sequence with that of the 7.5K gene.
  • the first twelve codons of the open reading frame for the gene are the same for the two viruses, but thereafter they diverge.
  • the putative signal sequence of the 35K gene comprises the first 17 codons of the open reading frame, for the 35 gene.
  • the first 20 codons of this ORF are shown below: ATG AAA CAA TAT ATC GTC CTG GCA TGC ATG Met Lys Gin Tyr He Val Leu Ala Cys Met TGC CTG GCG GCA GCT GCT ATG CCT GCC AGT Cys Leu Ala Ala Ala Ala Met Pro Ala Ser (See also SEQUENCE ID NO: 1 which differs only in that the underlining 1s omitted and numbering is added).
  • the underlined portion represents bases which are not present 1n the corresponding DNA of the WR strain.
  • the 35K gene (a term used herein to denote the gene from which the 35kD protein 1s translated) codes differently from the 7.5K gene, not only in that 1t contains these additional bases, but also in that 1t is frameshifted immediately downstream of the additional sequence.
  • the references herein to a relative molecular mass of 35kD for the mature protein are labels of convenience and not to be construed as denoting that this mass is necessarily accurate. A precise r.m.m. is calculable from the amino acid sequence shown hereinafter.
  • the present invention provides a DNA molecule which is suitable for use in the manufacture of a cassette, ultimately for insertion into the genome of a poxvirus, said DNA molecule encoding the following amino add sequence (SEQUENCE ID NO: 2): Met Lys Gin Tyr lie Val Leu Ala Cys Met Cys Leu Ala Ala Ala Ala Met Pro Ala Ser.
  • This sequence which might further contain one, two, three of four additional amino acids (following on from the C-terminal end of SEQUENCE ID NO: 2), i.e. Leu, possibly Gin, possibly another Gin and possibly thereafter Ser, is herein referred to as "the signal sequence DNA”.
  • signal sequence is used notwithstanding that it 1s probable that only the first 17 codons are required 1n secretion of the 35kD protein, at least 3 additional codons encoding amino adds of the mature 35kD protein being considered essential for the use of the signal sequence in a recomblnant vector.
  • the signal sequence DNA can be varied by appropriate substitutions of amino acids, particularly in the hydrophobic region from lie onwards by hydrophobic substitutes (Gly, Ala, Val, Leu, lie, Cys, Met, Phe, Tyr, Trp, Pro, His and sometimes Lys). The permissible extent of such substitution can be determined by experiment.
  • the cassette will preferably include a promoter, desirably a strong promoter, for example the 7.5K promoter or the promoter which precedes the 35K gene. It will preferably also include a multiple cloning site downstream of the signal sequence, so that a foreign gene can easily be Inserted.
  • a promoter desirably a strong promoter, for example the 7.5K promoter or the promoter which precedes the 35K gene. It will preferably also include a multiple cloning site downstream of the signal sequence, so that a foreign gene can easily be Inserted.
  • the foreign gene to be introduced Into the mammalian cells can be in principle any which is foreign with respect to the poxvirus with which the cells are to be infected.
  • the cassette conveniently also contains, therefore, the foreign gene in frame with the signal sequence DNA.
  • a preferred cassette therefore comprises the following elements linked together so that transcription can occur:- [promoter (such as VV 7.5K)3-["signal sequence DNA"3-[optional1y further downstream sequence from the 35 gene, e.g. DNA encoding SEQUENCE ID NO: 3, but bearing in mind that DNA encoding the first 4 amino acids of SEQUENCE ID NO: 3 could be part of the signal sequence DNA and would not then be repeated here..-[foreign gene or a multiple cloning site for insertion of the foreign gene]-[a viral transcription termination signal].
  • the invention includes a vector, such as a bacterial or yeast plasmid, carrying the cassette DNA together with flanking poxvirus sequence at each end thereof (from the infecting poxvirus).
  • recombination vector This is referred to herein as a "recombination vector", meaning a vector which is ultimately destined for use in the homologous recombination process.
  • the flanking poxvirus sequence in the recombination vector interchanges with the corresponding sequence in the "parent" strain of the infecting poxvirus, whereby the cassette DNA becomes inserted into that poxvirus.
  • three separate plasmids of this type are provided with multiple cloning sites for insertion of the foreign gene into whichever of the three possible reading frames will put it in frame with the signal sequence.
  • the invention includes a recombinant poxvirus resulting from homologous recombination and therefore containing the cassette DNA, animal cells infected with the recombinant poxvirus, the foreign protein obtained directly or indirectly from recombinant infected cells by in vitro culture, and, where national patent law permits, a method of vaccination which comprises administering the recombinant virus to an animal subject.
  • Figure 1 1s a restriction map of part of the vaccinia virus (Lister strain) genome, showing the location of the 35 gene;
  • Figure- 2 is a plasmid diagram showing schematically the construction of a preferred cassette for use in preparing a recombination vector of the invention;
  • the invention is of interest in relation to poxvirus vectors generically.
  • the poxvirus family have sufficient similarity to allow the VV 7.5K promoter to be used 1n fowlpox, capripox cowpox, and dovepox.
  • the invention is hereinafter described with reference to VV, it will be appreciated that the promoter and signal sequence combination is likely to be useful in aiding gene expression and product secretion in any other poxvirus in which the 7.5 promoter is functional for transcription of its geno ic DNA.
  • the signal sequence of the invention will be used in association with the 35K promoter DNA or its 7.5K counterpart which precedes it. It is known from Cochran a ⁇ . il-. J- Virol 54, 30-37, ( 1985 ) that the 137 bp of VV DNA immediately upstream from the ATG start codon includes both the early and late promotion functions of the 7.5K promoter in WR strain. Of these 137 bp, the last 18 (counting backwards) were not present * 1n the constructs described by Cochran £ ⁇ si. , and are therefore presumably non-essential.
  • the 35K promoter 1n the Lister strain 1s almost Identical, having only two base substitution within the 85 bp length upstream of the putative mRNA start site. It 1s highly likely that the 137-18 115 bp length could be shortened, if desired, and various deletions or changes made in the promoter sequence, as may be determined by experiment, e.g. deletion mapping, synthesis of ollgonucleotides, site-directed mutagenesis etc. as well known in the art. All such variant sequences are within the scope of the term "7.5K promoter" (P7.5K) or "35K promoter" (P35K) as used herein.
  • the signal sequence could be used in poxviruses in conjunction with other poxvirus promoters or Tn other viruses together with their respective promoters, e.g. in herpes simplex virus.
  • the promoter need not be a strong one, as the signal sequence will ensure secretion irrespective of promoter strength.
  • any 17-codon DNA coding for the signal sequence of the precursor polypeptide is coupled with DNA coding for at least the three N-terminal amino acids, preferably at least the five N-terminal amino acids, of the mature 35kD protein, and followed by the desired foreign gene (exogeneous with respect to the poxvirus). This will normally ensure that the foreign, protein is secreted by the host cells.
  • the protein secreted will be the foreign protein fused at its N-terminus to amino acids from the N-term1nal portion of the mature 35kD protein.
  • the desirable number of amino acids from the N-terminus of the mature 35kD protein Is a matter for experiment, but expression of a fusion polypeptide has been obtained when using a site in the 22nd or 42nd codon of the reading frame (including the signal sequence) for fusion of the foreign gene.
  • the invention includes any method of insertion, e.g. by creating a cassette containing the promoter and signal sequence DNA or by introducing the signal sequence DNA separately. Indeed, site-directed mutagenesis could be used to alter existing DNA.
  • the plasmid "pi" (arbitrary designation) contains non-essential region left-hand sequence, promoter, signal sequence, a multiple cloning site having restriction sites W, X, Y and Z (four are shown, but the term "multiple" means two or more) and non-essential region right-hand sequence.
  • X is a restriction site which enables the sequence X-X from plasmid "p2" to be inserted in the cloning site, resulting in the recombination plasmid "p3".
  • NER non-essential region
  • the NER is of the same poxvirus, and, if possible, the same strain as 1s Intended for the expression of the foreign gene.
  • some NERs might be substantially homologous in several poxviruses, in which case the NER need not be of the homologous virus. Precise homology of the NER of the recombination vector with that of the Infecting poxvirus, i.e. that in which recombination 1s to take place, is not necessarily required.
  • Suitable NERs are for vaccinia virus the TK gene or 35K gene (this being non- essential and within the terminal inverted repeat region) and for fowlpox virus (FPV) a non-essential region within the inverted terminal repeat (ITR) as described in NRDC's UK Patent Application Publication No. 2220941A (or the equivalent PCT Application Publication No. W089/12684, but the genomic location of the NER 1s not critical. Where it is within a ITR two copies of the foreign gene enter the poxvirus genome.
  • homologous recombination does not require a NER, but can take place within an essential gene so long as the promoter signal sequence and foreign DNA are inserted into a non-essential region.
  • the NER can Itself be flanked by a region which is in an essential gene.
  • the cassette contains at least 1000 bp of flanking sequence, i.e. the NER sequence and optional further flanking sequence, at each end thereof.
  • the preferred construct conveniently comprises:
  • Such a construct Is cloned Into a suitable vector such as a bacterial plasmid vector, to produce the required recombination vector.
  • Sequences (1) and ( 2) are not necessarily distinct, nor are (6) and (7).
  • the termination signal might be important for the production of mature mRNA.
  • Recombination to produce the recombinant VV or other poxvirus can take place by any method known in VV recombinant technology and essentially comprises introducing an appropriate strain of poxvirus into animal cells in vitro and also introducing the recombination vector of the invention into those cells. Any animal cells infectable by VV will suffice, e.g. rat, rabbit, chicken or even human.
  • the principal use contemplated for the invention is in the in vitro s y nthesis of proteins, using vaccinia virus or another virus as a vector for the introduction into animal cells of foreign DNA to be expressed as protein. It 1s, however, also possible that incorporation of a signal sequence might help in the in vivo presentation of the foreign product in an animal which is to be vaccinated with a recombinant poxvirus, e.g. poultry with fowlpox virus.
  • the foreign protein thus produced will probably require enzymatic treatment to remove any amino adds of the mature 35kD VV protein fused to the foreign protein sequence, and/or to remove glycosylation. Any conventional such treatment can be used.
  • the Invention includes the foreign protein whether directly or indirectly obtained from the animal cells. The following Example illustrates the invention.
  • the strategy used to map the gene encoding the 35kD polypeptide Involved several stages: (i) Initially the 35kD polypeptide was purified and used to develop an antiserum which was capable of recognizing the product of the 35K gene amongst the polypeptides synthesized in vitro from infected cell mRNA.
  • Rabbit kidney . (RK) cells were infected with the virus and 2.5 h after virus addition washed extensively with serum-free phosphate buffered saline (PBS).
  • PBS serum-free phosphate buffered saline
  • the proteins secreted into PBS during the next 3-4 h were collected and subjected to DEAE cellulose ion exchange chromatography on a column of Whatman DE52.
  • a preparation of the 35kD protein was obtained which contained only small amounts of contaminating proteins.
  • the DEAE cellulose column-purified preparation was used to immunise rabbits, and an antiserum obtained which was able to precipitate the 35kD protein secreted.
  • Figure 1 shows the location of the 35K gene on the vaccinia virus genome.
  • the boxed regions indicate the inverted terminal repeat sequences and the positions of the terminal Hindlll (H) fragments B and G are shown.
  • the expanded regions show the two copies of the 2.2 kb BamHI (Ba) plus Sail (S) fragment which arrested translation of the 35K gene (arrowed). 'Ss' indicates S_s_ ⁇ I sites.
  • AACTGATCAC TAATTCCAAA CCCACCCACT TTTTATAGTA AGTTTTTCAC CCATAAATAA 1440 .-* mRNA TAAATACAAT AATTAATTTC TCGTAAAAGT AGAAAATATA TTCTAATTTA TTGCACGGTA 1500
  • GGT TCA ACG ATC GTC GAT ACA AAA TGT GTC AAG AAT CTA GAG TTT AGC 2179 Gly Ser Thr He Val Asp Thr Lys Cys Val Lys Asn Leu Glu Phe Ser 180 185 190 195
  • the published sequence for strain WR corresponds to residues 1267-1605 and 2405-2570.
  • the counterpart in the above sequence of the 137 bp upstream of the start codon identified by Cochran e_£ Si-. lG£- £i£. as containing the W 7.5K promoter in WR strain would be from 1407 to 1543 (immediately preceding the start codon at 1544).
  • the longest open reading frame was 235 amino acids and extended beyond the Sail site at its C terminus.
  • the N-terminal end of the predicted polypeptide encodes a hydrophobic region of 17 amino adds which functions as a signal sequence. Immediately beyond this the predicted amino add sequence is (SEQUENCE ID NO: 5):
  • This sequence corresponds to the limited amino add sequence for the N-terminus of DE52 column-purified mature 35kD protein (Unknown-Ala-Unknown-Leu-Gin-Gin) which had previously been obtained for us, in confidence, by J.E. Fothergill and B. Dunbar of the University of Aberdeen, making us confident that the sequenced gene encodes the secreted 35kD protein.
  • the remainder of the 35K gene (downstream of the Sail site) was determined by sequencing an overlapping cloned DNA fragment and the complete gene encodes a further 23 amino acids.
  • the sequence of the 2.2 kb BamHI plus Sail fragment is shown together with approximately 300 residues downstream of the Sail site.
  • the amino a ids encoded by the 35K open reading frame are indicated.
  • the 19 residues missing from strain WR DNA are underlined (1580-1598 inclusive) and the asterisked proline indicates the position (amino acid number 1) of the N-terminus of the mature secreted 35kD protein (I.e. following cleavage of the signal sequence).
  • Also shown are the mapped positions of the 5' and 3' ends of the strain WR 7.5K mRNA (Venkatesan e_i &1-, loc. cit.)
  • the underlined restriction enzyme recognition sites are those for Clal. BamHI and Sail (see Example 2).
  • Example 2 This Example relates to the testing of the promoter and signal sequences.
  • Figure 3 shows a linearised map of the plasmids used in the construction of cassette DNA comprising promoter, signal sequence (omitted in a comparative construct) and foreign gene.
  • the BamHI-Sall sub-fragment of a terminal Hindlll fragment was cloned in the widely available plasmid pUC19.
  • the resulting plasmid, which has most of the coding sequence for the 35kD protein, was designated pG62.
  • PG620 was constructed from these two plasmids and carries the BamHI to Xbal fragment which includes the entire 35K gene.
  • the Clal fragments coding for all except the first 42 N-terminal " amino adds of the 35kD precursor polypeptide were deleted as follows.
  • pG62 was cleaved with Hindlll and partially with Clal.-
  • a DNA fragment carrying the pUC19 and the vaccinia DNA sequence starting from the BamHI to the second Clal site was isolated from an agarose gel by electroelution and ligated to the Clal and Hindlll fragment of the plasmid p48-15 forming pD35.
  • a Bglll linker was introduced in the Clal site downstream from the 35K promoter in pD35 forming pD35-L.
  • the vaccinia DNA sequence in pD35-L was then subcloned on an EcoRI to Sail fragment into pAT153 cleaved with the same enzymes.
  • the resulting plasmid pD351 carries the DNA sequence left of the 35K gene [35K(D], the 35K promoter (P35K) and the coding sequence for 42 N-term1nal amino adds (42 aa of the precursor polypeptide, including 17 aa signal sequence) followed by a unique Bglll site and the DNA sequence right of the 35K gene [35K(R)].
  • lacZ gene and the £al genes, each on a BamHI fragment, were cloned separately into the Bglll site of pD351 to form plasmids pD352 and pD351/CAT respectively.
  • These constructs have the above marker genes linked in frame to DNA encoding the N-terminal 42 amino adds of the 35K precursor polypeptide and contain the 35K promoter.
  • a vector for introducing the lacZ and the £ai gene immediately downstream from the ATG translation initiation codon of the 35K gene was constructed. Such a vector lacks the signal sequence but contains the promoter linked to the foreign gene.
  • pD351 was cleaved with Bglll, the ends filled in and partially digested with Seal.
  • the DNA fragment carrying the 35K(R) sequence starting from Bglll/filled 1n end, pAT153 and the 35K(L) sequence ending at Sea site 18bp upstream from the ATG start codon was Isolated from an agarose gel by electroelutlon and 11gated to a linker (SEQUENCE ID NO: 6):- ACTCAATCAA TAGCAATTAT GGATCC to form pD356. This allowed us to generate a BamHI site immediately downstream from the ATG codon and also to delete the N-terminal coding sequence of the 35K gene in pD351. The sequence upstream from the ATG codon remained unchanged.
  • the BamHI DNA fragments encoding the lacZ and . ⁇ g ⁇ genes were inserted separately to form pD357 and pD356/CAT respectively. These constructs have the first ATG codon of the 35K gene linked in frame to the marker genes.
  • the EcoRI and fiamHI fragment of pD356 carrying 35K(R) sequence and pAT 153 was 11gated to the EcoRI and Sphl fragment of pG62 carrying the 35K(L), the 35K promoter plus part of the coding sequence (first 7 amino adds) of the precursor polypeptide 1n the presence of an oligonucleotide linker with Sphl and BamHI sticky ends carrying the coding sequence downstream from the Sph site (amino adds -10-5, followed by the BamHI site) to form pD358.
  • TK(L) and TK(R) refer to segments of vaccinia DNA which include the left and right portion of the TK gene.
  • pSC8 has the lacZ gene under the control of the UK W late promoter flanked on both sides by vaccinia TK sequences; S. Chakrabarti e_ ⁇ aL Mol . and Cell. Biol. 5. 3403-3409 (1985 ) ], to construct pSC8/351.
  • This plasmid has the lacZ. gene fused to DNA encoding the N-terminal 42 amino adds of the 35K precursor polypeptide and under the control of the 35K promoter.
  • the UK promoter is upstream from the 35K promoter.
  • Calcium phosphate-precipitated plasmids pD357 and pV327 (containing the lacZ gene linked to the 35K promoter but no signal sequence and inserted Into the 35K and TK loci, respectively) were transfected Into CV-1 cells infected with wild type vaccinia virus strain Lister. Recombinant viruses (blue plaques) were isolated as reported previously [Mackett fil il., J- Virol. 42, 857-864 (1984); Chakrabartl fit l- , Mol . Cell. Blol. 5, 3403-3409 (1985)].
  • Recombinant viruses containing the lacZ gene under the control of the 35K promoter either 1n the 35K locus or TK gene (V357 and V327, respectively) efficiently expressed beta-galactosidase activity. As expected, most of the activity was located intracellularly. Southern blot analysis confirmed that the lacZ gene had been inserted into the 35K locus in both inverted terminal repeats of the V357 genome.
  • Plasmids pV328, pV331 pV325 and pD351-CAT were used to construct the recombinant viruses V328, V331 , V325 and V351-CAT respectively, as described above.
  • the £& ⁇ gene was Inserted Into the TK locus of strain Lister in viruses V328, V331 and V325 and into both copies of the 35K gene 1n V351-CAT.
  • V328, V331 , V325 and V351-CAT respectively contain sequences encoding 0, 22, 42 and 42 amino adds of the precursor polypeptide linked in frame to the 5' end of the . ⁇ ai gene.
  • the £a ⁇ gene was specifically mutated so as to remove the one potential N-linked glycosylation site Asn-Gln-Thr at amino acid positions 34-36.
  • the amino acids Asn-34 and Thr-36 were substituted with Gin and Val respectively as follows.
  • the BamHI fragment carrying the .cat gene was subcloned into vector pTZ19U (Pharmacia), and a single stranded DNA template form of this plasmid, pTZ19U-CAT, was propagated in £. coll strain TGI with the phage M13K07 as helper [Mead fii si. , Protein Engineering 1, 67-76 (1986)].
  • An oligonucleotide SEQUENCE ID NO: 7
  • the mutant £at gene on the BamHI fragment of pTZ19U-CAT14 was subsequently cloned in the correct orientation into recombination vectors pV326 and pV329 to form plasmids pV328-l and pV331-10 respectively.
  • An approximately 270 bp Bell and Bglll fragment of pD351 specifying the 35K promoter and the coding sequences for the N-terminal 42 amino acids was inserted together with the BamHI mutant _ai fragment from pTZ19U-CAT14 Into the TK locus of pV321 to form plasmid pV325-ll.
  • the plasmids pV328-l, pV331-10 and pV325-ll are identical to pV328, pV331 and pV325 respectively except for the two amino acids substitutions disrupting the potential N-linked glycosylation site.
  • the mutant £a constructs were Introduced into the TK locus of the strain Lister genome by homologous recombination, and the recombinant viruses V328-1 , V331-10 and V325-11 were isolated as described above. Analysis of the CAT activity produced by viruses carrying mutated cat genes
  • the N-terminal 22 and 42 amino acids of the precursor polypeptide are both able to function as signal sequences and efficiently direct secretion of proteins to which they are attached.
  • the hybrid proteins because they route the hybrid proteins through the secretory pathway, one potential problem 1s that proteins that are not normally glycosylated may become so if they possess glycosylation sites.
  • the results show that there is a possibility of circumventing this problem either by using tunicamycin or by removing the glycosylation sites by site-specific mutagenesis of the gene encoding the protein to be secreted.
  • GGT TCA ACG ATC GTC GAT ACA AAA TGT GTC AAG AAT CTA GAG TTT AGC 2179 Gly Ser Thr He Val Asp Thr Lys Cys Val Lys Asn Leu Glu Phe Ser 180 185 190 195 GTT CGT ATC GGA GAC ATG TGC AAG GAA TCA TCT GAA CTT GAG GTC AAG 2227 Val Arg He Gly Asp Met Cys Lys Glu Ser Ser Glu Leu Glu Val Lys 200 205 210

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'ADN à séquence de signaux encode la séquence aminoacide (ID. Séquence n°1) ou une variante légèrement modifiée de ladite séquence aminoacide. Il est destiné à être intégré dans des constructions d'ADN recombinant d'un poxvirus et d'un gène étranger afin de faciliter la sécrétion du produit d'expression du gène étranger.
EP90910856A 1989-07-11 1990-07-10 Adn codant pour une sequence de signaux polypeptidiques dans le virus vaccinal Ceased EP0482061A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB898915870A GB8915870D0 (en) 1989-07-11 1989-07-11 Dna coding for a polypeptide signal sequence in vaccinia virus
GB8915870 1989-07-11

Publications (1)

Publication Number Publication Date
EP0482061A1 true EP0482061A1 (fr) 1992-04-29

Family

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Family Applications (2)

Application Number Title Priority Date Filing Date
EP90910856A Ceased EP0482061A1 (fr) 1989-07-11 1990-07-10 Adn codant pour une sequence de signaux polypeptidiques dans le virus vaccinal
EP90307513A Withdrawn EP0408301A1 (fr) 1989-07-11 1990-07-10 DNA codant pour une séquence signal polypeptidique dans vaccinia

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP90307513A Withdrawn EP0408301A1 (fr) 1989-07-11 1990-07-10 DNA codant pour une séquence signal polypeptidique dans vaccinia

Country Status (6)

Country Link
EP (2) EP0482061A1 (fr)
JP (1) JPH05504253A (fr)
AU (1) AU633334B2 (fr)
CA (1) CA2063389A1 (fr)
GB (2) GB8915870D0 (fr)
WO (1) WO1991000911A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0862449A4 (fr) * 1995-09-29 1998-11-11 Immunex Corp Inhibiteur de la chemokine
DE69738977D1 (de) * 1996-03-29 2008-10-23 Zeon Corp Neues fusionsprotein, dafuer kodierendes gen, rekoerwendung

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2583429B1 (fr) * 1985-06-18 1989-11-03 Transgene Sa Vecteur d'expression de l'interferon u dans les cellules de mammifere, procede pour sa mise en oeuvre et produit obtenu et composition pharmaceutique contenant l'interferon u
DE68919846T2 (de) * 1988-02-12 1995-07-06 Commw Scient Ind Res Org Poxvirus-vektoren.
CA2001001A1 (fr) * 1988-10-21 1990-04-21 Matthew M. Binns Promoteur du virus de l'acne molluscum
NL1004764C2 (nl) 1996-12-12 1998-06-15 Aluminium Verkoop Zuid B V Arminrichting voor een uitvalscherm en uitvalscherm voorzien van dergelijke arminrichtingen.

Non-Patent Citations (1)

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Title
See references of WO9100911A1 *

Also Published As

Publication number Publication date
GB9015168D0 (en) 1990-08-29
CA2063389A1 (fr) 1991-01-12
EP0408301A1 (fr) 1991-01-16
AU633334B2 (en) 1993-01-28
GB2233655B (en) 1993-04-21
GB2233655A (en) 1991-01-16
AU5955590A (en) 1991-02-06
JPH05504253A (ja) 1993-07-08
GB8915870D0 (en) 1989-08-31
WO1991000911A1 (fr) 1991-01-24

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