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WO1993017109A1 - Proteine de pointe du vib (2) - Google Patents

Proteine de pointe du vib (2) Download PDF

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Publication number
WO1993017109A1
WO1993017109A1 PCT/GB1993/000332 GB9300332W WO9317109A1 WO 1993017109 A1 WO1993017109 A1 WO 1993017109A1 GB 9300332 W GB9300332 W GB 9300332W WO 9317109 A1 WO9317109 A1 WO 9317109A1
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ser
leu
val
asn
thr
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PCT/GB1993/000332
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English (en)
Inventor
Michael Anthony Skinner
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BTG International Ltd
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British Technology Group Ltd
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Filing date
Publication date
Application filed by British Technology Group Ltd filed Critical British Technology Group Ltd
Priority to EP93904216A priority Critical patent/EP0627005A1/fr
Priority to JP5514621A priority patent/JPH07504814A/ja
Publication of WO1993017109A1 publication Critical patent/WO1993017109A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to the spike protein of infectious bronchitis virus (IBV) and to a recombinant DNA method of preparing it.
  • IBV infectious bronchitis virus
  • IBV is a virus which causes respiratory disease in the fowl, and is of particular importance in relation to poultry.
  • IBV is a virus of the type Coronaviridae. It has a single-stranded RNA genome, approximately 20 kb in length, of positive polarity, which specifies the production of three major structural proteins: nucleocapsid protein, membrane glycoprotein, and spike glycoprotein.
  • the spike glycoprotein is so called because it is present in the teardrop-shaped surface projections or spikes protruding from the lipid membrane of the virus.
  • the spike protein is believed likely to be responsible for immunogenicity of the virus, partly by analogy with the spike proteins of other coronavi ruses and partly by in vitro neutralisation experiments, see, for example, D. Cavanagh et al., Avian Pathology 13, 573-583 (1984).
  • spike protein is used to refer to the glycoproteinaceous material of the spike, it has been characterised by D. Cavanagh, Journal of General Virology 64, 1187-1191; 1787-1791; and 2577-2583 (1983) as comprising two or three copies each of two glycopolypeptides,
  • the polypeptide components of the glycopolypeptides S1 and S2 have been estimated after enzymatic removal of oligosaccharides to have a combined molecular weight of approximately 125,000 daltons. It appears that the spike protein is attached to the viral membrane by the
  • the protein comprises an extra cellular domain, a transmembrane domain and a cytoplasmic anchor domain.
  • European Patent Application Publication No. 218625A NRDC discloses the cloning of cDNA sequences coding for the spike protein precursor as well as sequences coding specifically for the S1 and S2 polypeptides.
  • Such a DNA molecule which codes for an IBV spike protein will hereinafter be referred to as "spike DNA" for brevity.
  • the disclosed spike DNA codes for the whol e spike protein, i.e. all 3 domains.
  • the present invention relates to a DNA molecule which codes substantially for a truncated IBV spike protein polypeptide.
  • the truncated IBV spike protein polypeptide produced as a result of the cloning and expression of a DNA molecule of the present invention is characterised in lacking the transmembrane domain and cytoplasmic anchor region of the native IBV spike protein.
  • a DNA molecule according to the invention is shown in the Sequence Listing (SEQ ID NO: 1). This DNA molecule was obtained as a result of research on the M41 strain of IBV, but it is expected that similarly truncated spike protein of cDNA of other IBV serotypes and strains such as Beaudette, M42, 6/82, Connecticut isolate A5968, Arkansas and Holland strains H120, H52, Ma5, D207, D212, D3128 and D3896, whether or not exhibiting a high degree of homology with M41, will express IBV spike protein.
  • DNA flanking sequences which may be, for example, cDNA to flanking sequences in the IBV RNA genome (other than transmembrane sequences) or may be foreign sequences derived from other genes, such as leader sequences that may assist in driving expression of the truncated polypeptide or may be a short sequence of plasmid DNA.
  • the DNA molecule should necessarily code for amino acids extending right up to the 5'- terminus or 3'-truncated end. It may be possible to obtain expression of the truncated spike protein lacking say, up to 5 or even 10 of the amino acids (30 nucleotides) at either end.
  • the invention also includes a vector containing the above defined DNA molecule, including a cloning vector such as a plasmid or phage or expression vector, preferably a pox virus vector, and a host containing the vector.
  • a cloning vector such as a plasmid or phage or expression vector, preferably a pox virus vector
  • Mammalian cells containing the above-defined DNA molecule are also included.
  • the invention includes isolated biosynthetic truncated spike protein polypeptide and its expression from mammalian cells.
  • Figures 1-17 show plasmid constructs of use in the preparation of DNA molecules of the present invention.
  • SEQ ID NO: 1 shows the complete nucleotide sequence of a cDNA molecule of the invention obtained from IBV genomic RNA M41 strain.
  • the IBV RNA of other strains is believed to be fairly similar to that of M41, and therefore oligonucleotides derived from DNA of the present invention can be used as primers for sequencing RNA of other serotypes thus enabling truncated cDNA for all or virtually all other serotypes to be prepared using methods described hereinafter.
  • those serotypes in which the entire IBV spike protein cDNA has a high degree of nucleotide sequence homology with IBV M41 strain are slightly preferred, as giving a wider choice of potential oligonucleotides.
  • the vectors included in the invention are cloning and expression vectors.
  • the DNA molecule of the present invention is conveniently multiplied by insertion in a prokaryotic vector, for example pBR322, and cloning in an appropriate host such as a bacterial host, especially E. coli. Alternatively, using appropriate different vectors it could be multiplied in (say) Bacillus species, or a yeast.
  • mammalian cells can be transfected by the calcium phosphate precipitation method or transformed by a viral vector.
  • Viral vectors include retroviruses and poxviruses such as fowl pox virus or vaccinia virus.
  • a DNA molecule of the present invention may be prepared by first obtaining full length IBV spike DNA in a suitable plasmid.
  • European Patent 218625A NRDC predicts the probable transmembrane domain of the spike protein and indicates the region of DNA coding for it.
  • a suitable endonuclease restriction site near the beginning of the DNA sequence coding for the transmembrane domain, can then be identified.
  • the IBV spike DNA may be cleaved and the truncated DNA molecule coding for the extracellular domain, introduced into a viral vector as described below.
  • the truncated IBV spike DNA can be introduced into the viral vector as follows.
  • the DNA is inserted into a plasmid containing an appropriate non-essential region of poxvirus DNA, such as the thymidine kinase gene of vaccinia virus or into any suitable non-essential region of fowlpox virus, e.g. as described in European Patent 353851A, so that the insert interrupts the NER sequence.
  • a poxvirus promoter e.g. the vaccinia virus p7.5K promoter, which is usable in vaccinia virus or avipoxviruses, or a fowlpox virus promoter as described in our prior patent applications publication Nos.
  • WO89/03879 is also introduced into the NER sequence in such a position that it will operate on the inserted truncated spike DNA sequence.
  • a "marker" gene with its own promoter e.g. the lac Z gene will be inserted along with the sequence coding for the truncated spike protein.
  • leader sequence is the region between the TAATTATT of the promoter sequence and the ATG initiation codon of the gene.
  • leader sequences could be derived from: (i) part or all of the sequences found downstream of other poxviral promoters e.g. the vaccinia virus p7.5 promoter (ii) part or all of the leader sequences from foreign genes that have been shown to be well expressed in cells infected by the appropriate recombinant poxviruses or (iii) synthetic sequences shown to promote efficient translation in poxvirus-infected cells.
  • the replacement of appropriate sequences can be accomplished using PCR cloning or by inserting synthetic oligonucleotides.
  • the choice of leader sequence to be used and the method of insertion is well within the ability of skilled man.
  • the Example 2 hereinafter illustrates how the procedure could be performed.
  • the invention therefore further relates to a vector wherein containing part or all of a sequence found downstream of a poxvirus promoter, not being the poxvirus promoter of use in the vector, between the promoter and the IBV DNA Molecule.
  • the preferred poxvirus is fowlpox virus. It may be that the inserted truncated IBV DNA contains a sequence, which, in the fowl pox vector, leads to premature termination of transcription. In this case, the truncated spike DNA would have to be modified slightly by one or two nucleotides, thereby to allow transcription to proceed along the full length of the gene.
  • the vector can be introduced into any appropriate host by any method known in recombinant DNA technology.
  • Hosts include E. coli. Bacillus spp, animal cells such as avian or mammalian cells and yeasts.
  • the method of introduction can be transformed by a plasmid or cosmi d vector, or infection by a phage or viral vector etc. as known in recombinant DNA technology.
  • Example 2 of European Patent Application Publication No. 218625 describes the preparation of cDNA coding for the spike protein precursor of IBV strain M41. It describes therein the preparation of plasmids pMB276 and pMB250 containing the entire M41 spike protein cDNA sequence.
  • An initial step in the preparation of a DNA molecule encoding a truncated IBV spike protein was to join pMB276 and pMB250 to produce a full length clone of the IBV M41 spike gene.
  • Plasmids pMB276 and pMB250 were digested with Ndel (20 units) in 50mM tris-HCl pH 8.0, 10mM MgCl 2 , 50mM NaCl, final volume 20 ⁇ l. The digested DNA was then phenol-extracted with an equal volume of TE-saturated phenol, ether extracted twice with an equal volume of water-saturated ether, then ethanol- precipitated. The precipitated DNA was resuspended in 15 ⁇ l water.
  • Transformant colonies were grown in L broth plus tetracycline and DNA was isolated therefrom using a standard procedure described by Holmes and Quigley (1981), Analytical Biochemistry 114: 193-197. Following digestion of the isolated DNA with Ndel and agarose gel electrophoresis, it was apparent that, of 48 clones screened, one (no. 17) had inherited the desired fragments from the parental plasmids, viz. a fragment of circa 6kbp from pMB276, Fig. 1 and a fragment of about 4kbp from pMB 250, Fig. 2.
  • the desired recombinant plasmid would also have a fragment, following Pstl digestion, equivalent to the length of pBR322 (Pi ⁇ l sites flank the M41 spike cDNA).
  • Analysis of clone 17 showed that it did not have a pBR322-sized Pstl fragment, indicating that the two Ndel fragments had ligated together in the wrong relative orientation.
  • Clone 17 DNA was therefore digested with Ndel and religated (using procedures described above) to allow isolation of recombinants with the two Ndel fragments in the correct orientation.
  • Analysis of Pstl-digested DNA from a number of clones showed that about 50% had religated to give the correct orientation.
  • One of these clones was saved, as pMB374, Fig, 3.
  • the IBV M41 spike protein gene was cut out of pMB374 by digestion of the plasmid with Tthlll 1, see Fig. 3, in 10mM tris-Hcl pH 7.4, 10mM MgCl 2 , 50mM NaCl, 10mM (J-mercaptoethanol, at 65°C in a final volume of 20 ⁇ l.
  • the DNA was made blunt-ended by the addition of 0.025mM dATP, dCTP, dGTP, dTTP and 5 units of Klenow polymerase, followed by incubation for In at room temperature.
  • the digestion products were electrophoresed on an agarose gel using standard procedures as described by Maniatis et al., (1982) in "Molecular cloning: a laboratory manual” (Cold Spring Harbor Laboratory) and a 5kb fragment, containing the spike gene was purified using "Geneclean II” (Bio 101) as per supplier's instructions.
  • the purified DNA was then cloned into the Smal site of pGS20 (from Dr. G. L. Smith, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, as described in Mackett, Smith & Moss (1984), J. Virol. 49, 857-864) to make pGSS2, see Fig. 4.
  • pGSS2 was digested with Styl, the DNA was made blunt-ended with Klenow polymerase and the 1.95kb fragment (2430-4384) was recovered and purified. This fragment was ligated into pUC19 digested with Smal. Recombinants carrying the inserted fragment were isolated and the orientation of the inserted fragment was checked by digestion of their plasmid DNA with Mlul and BamHl . The required recombinant had a small Mlul /BamHl fragment of 480 bp (and not 1480bp) and was given the title pUC/M41Sty, Fig. 5 (note that ligation of the blunt-ended Styl fragment into the Smal site restores the Styl sites but not the Smal site).
  • Plasmids pGSS2, Fig. 4, and pUC/M41Sty, Fig. 5, were both digested with BamHl and Afl2 and fragments of 2.2kb and 3.8kb, respectively, were recovered. The purified fragments were ligated together and recombinants were isolated. The required recombinant (titled pUC/M41 Barn-Sty), Fig. 6, had the 0.85kb BamHl /Afl2 fragment of pUC/M41Sty replaced by a 2.2kb fragment from pGSS2, Fig. 6.
  • a DNA fragment containing the fowlpoxvirus 4b promoter driving a lacZ reporter gene was cut out of plasmid pNM4b30 (see the relevant fowlpox virus promoter patent specification (WO89/03879), page 35, Table 2) using EcoRl and Nrul.
  • the fragment was end-repaired and was then blunt-end ligated into the end-repaired Bgl2 site of a plasmid containing part of the terminal BamHl fragment of fowlpoxvirus (pB3ME, described in Boursnell et al., 1990, J. Gen. Virol. 71, 621-628) to create plasmid pEFL10.
  • the vaccinia virus p7.5 promoter was then introduced, on a 300bp EcoRl (end-repaired) DNA fragment from pGS20 (see above), into the Scal site of pEFL10.
  • a recombinant with the p7.5 promoter in such an orientation that transcription from it is initiated in the opposite direction to that from the fowlpoxvirus 4b promoter, identified by restriction analysis using BamHl. was titled pEFL29.
  • Chick embryo fibroblasts (CEFs), at 80% confluence, were infected with the Duphar "Poxine" strain of fowlpoxvirus at a multiplicity of infection (m.o.i.) of 1.
  • pEFS17 DNA (lO ⁇ g per 25cm 2 flask) was introduced to the cells using the 'Lipofectin' method (BRL) under manufacturer's instructions.
  • BTL 'Lipofectin' method
  • Five days post-infection when there was complete cytopathic effect, the cells were harvested.
  • Virus released from the cells by freeze/thawing three times, was used at various dilutions to infect CEFs which were then overlaid with agarose to allow plaques to form.
  • plaques were visible the plates were overlaid with X-gal agarose. Two days later, blue plaques were picked and virus was released by freeze/thawing. The virus was titrated again, overlaid with X-gal agarose and blue plaques were picked again. This procedure was repeated three more times. Finally two plaques (fpl74P ⁇ 1111 and fpl74P ⁇ 1121) were chosen for further characterisation.
  • CEFs were infected with the fpEFS17 recombinant viruses (or with a control 'poxine'/lacZ recombinant virus or mock-infected) at a m.o.i. of 10.
  • tissue culture medium was replaced with methionine-free medium to 'starve' the cells (i.e. to deplete the cells of their intracellular methionine pool) for lh.
  • the cells were then labelled with
  • the protein-A/Sepharose was washed thrice with RIPA buffer, then resuspended in SDS-PAGE sample buffer and boiled for 3 min. The samples were then applied to a 5-10% gradient SDS-PAGE gel and electrophoresed. The gel was fixed and exposed by fluorography.
  • the Example below describes the replacement of the untranslated IBV spike sequences with sequences derived from part of the leader downstream of the p7.5 promoter, by cloning synthetic oligonucleotides between the BamHl site in the leader and a Spel site near the 5' end of the IBV spike coding sequence. The complete leader is then cloned upstream of the truncated IBV spike gene from pEFS 17 to give pEFS 20.
  • the 83 base pair BamHI-Spel fragment (SEQ ID NO 3) in pEFS17 is replaced with a synthetic leader based on p7.5 (SEQ ID NO 4) using the oligonucleotides MAS-H7 and MAS-H8 (SEQ ID 5 and 6 respectively).
  • Plasmid pGSS2 (Fig. 4) was digested with BamHl (1059) and Spel (3358), and fragments of 10kb and 2.2kb (Fig. 9 were recovered.
  • BamHl (1059) and Spel (3358)
  • fragments of 10kb and 2.2kb (Fig. 9 were recovered.
  • To anneal synthetic oligonucleotides MAS-H7 and MAS-H8, 50 pmol of each were mixed in 10 ⁇ l water. They were then boiled for 3 minutes and allowed to cool slowly to room temperature. The annealed oligonucleotide duplex (0.2 to 5 pmol) was then ligated to the 10 kb BamHI-Spel fragment from pGSS2.
  • the required recombinant, pGSS3 (Fig. 10), had retained the BamHl and Spel sites but had deleted a 2.2 kb Spel fragment relative to pGSS2.
  • Plasmid pGSS4 was digested with BamHl and EcoRl, repaired with Klenow polymerase then a 4.9 kb fragment (Fig. 13) was recovered and ligated into pEFL29 (Fig. 7) digested with Smal.
  • Plasmids pEFS17 and pEFS19 were digested with Ncol and Bglll then 3 kb (Fig. 15) and 11.8 kb (Fig. 16) fragments, respectively, were recovered and ligated together.
  • the required recombinant pEFS20 (Fig. 17) was checked by digestion with Kpnl,
  • Recombinant fowlpox viruses were derived, using pEFS20, and analysed as described above in Example l.III for pEFS17.
  • ORGANISM Infectious bronchitis virus
  • GGT TTT CTT AAG GAC CTT GCG TGT GCT CGT GAA TAT AAT GGT TTG CTT
  • ORGANISM Infectious bronchitis virus
  • ORGANISM Infectious bronchitis virus

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Abstract

L'invention concerne une molécule d'ADN qui code pour un polypeptide tronqué de la protéine de pointe du virus infectieux de la bronchite (VIB); ce polypeptide tronqué de la protéine de pointe du VIB est caractérisé par l'absence d'un domaine transmembrane et d'un domaine de fixation cytoplasmique que l'on trouve dans la protéine de pointe naturelle du VIB.
PCT/GB1993/000332 1992-02-19 1993-02-17 Proteine de pointe du vib (2) Ceased WO1993017109A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP93904216A EP0627005A1 (fr) 1992-02-19 1993-02-17 Proteine de pointe du vib (2)
JP5514621A JPH07504814A (ja) 1992-02-19 1993-02-17 Ｉｂｖスパイクタンパク質（２）

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9203509.6 1992-02-19
GB929203509A GB9203509D0 (en) 1992-02-19 1992-02-19 Ibv spike protein(2)

Publications (1)

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WO1993017109A1 true WO1993017109A1 (fr) 1993-09-02

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PCT/GB1993/000332 Ceased WO1993017109A1 (fr) 1992-02-19 1993-02-17 Proteine de pointe du vib (2)

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EP (1) EP0627005A1 (fr)
JP (1) JPH07504814A (fr)
AU (1) AU3508493A (fr)
CA (1) CA2117468A1 (fr)
GB (2) GB9203509D0 (fr)
WO (1) WO1993017109A1 (fr)
ZA (1) ZA931191B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109985235A (zh) * 2019-01-29 2019-07-09 苏州世诺生物技术有限公司 鸡传染性支气管炎基因工程亚单位疫苗

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100064A (en) * 1984-04-06 2000-08-08 Chiron Corporation Secreted viral proteins useful for vaccines and diagnostics
DE69233270T2 (de) * 1991-04-25 2004-09-30 Akzo Nobel N.V. Subeinheits-Impfstoff gegen Hundecoronavirus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005806A1 (fr) * 1985-03-29 1986-10-09 National Research Development Corporation Proteine "pointe" du virus de la bronchite infectieuse
EP0221609A1 (fr) * 1985-10-31 1987-05-13 Duphar International Research B.V Protéines et peptides ayant une activité antigénique et vaccins du virus de bronchite infectieuse (IBV)
NL8700953A (nl) * 1987-04-22 1988-11-16 Duphar Int Res Nieuwe antigeen werkzame eiwitten en peptiden, en infectieuze bronchitis virus (ibv)-vaccins.
EP0423869A1 (fr) * 1989-10-20 1991-04-24 Akzo Nobel N.V. Vaccin contre le virus de la bronchite infectieuse (IBV)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005806A1 (fr) * 1985-03-29 1986-10-09 National Research Development Corporation Proteine "pointe" du virus de la bronchite infectieuse
EP0221609A1 (fr) * 1985-10-31 1987-05-13 Duphar International Research B.V Protéines et peptides ayant une activité antigénique et vaccins du virus de bronchite infectieuse (IBV)
NL8700953A (nl) * 1987-04-22 1988-11-16 Duphar Int Res Nieuwe antigeen werkzame eiwitten en peptiden, en infectieuze bronchitis virus (ibv)-vaccins.
EP0423869A1 (fr) * 1989-10-20 1991-04-24 Akzo Nobel N.V. Vaccin contre le virus de la bronchite infectieuse (IBV)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOLECULAR IMMUNOLOGY vol. 26, no. 1, January 1989, OXFORD, UK; pages 7 - 15 J.A. LENSTRA ET AL. 'Antigenicity of the peplomer protein of infectious bronchitis virus' *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109985235A (zh) * 2019-01-29 2019-07-09 苏州世诺生物技术有限公司 鸡传染性支气管炎基因工程亚单位疫苗

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GB9303123D0 (en) 1993-03-31
CA2117468A1 (fr) 1993-09-02
JPH07504814A (ja) 1995-06-01
ZA931191B (en) 1994-08-19
EP0627005A1 (fr) 1994-12-07
GB2264497A (en) 1993-09-01
AU3508493A (en) 1993-09-13
GB9203509D0 (en) 1992-04-08

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