Chemistry: molecular biology and microbiology – Vector – per se
Patent
1992-01-17
1994-07-26
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Vector, per se
536 241, 536 2372, 4352351, 935 6, C12N 1511, C12N 1586, C12N 1539, C12N 701
Patent
active
053326768
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is in the field of recombinant DNA technology and relates to avipox promoters useful for the expression of foreign DNA inserted into a fowlpox virus vector.
2. Description of the Prior Art
Poxviruses are large viruses with a complex morphology containing linear double-stranded DNA genomes. They are among the few groups of DNA viruses that replicate within the cytoplasm of the cell. They are subclassified into six genera: orthopoxviruses, avipoxviruses, capripoxviruses, leporipoxviruses, parapoxviruses and entomopoxviruses. Vaccinia virus (VV), an orthopoxvirus, is the most widely studied of the poxviruses, and is the subject of U.S. Pat. No. 4,603,112 (Paoletti et al.,). Fowlpox virus (FPV) is an avipoxvirus or arian poxvirus.
Recent advances in recombinant DNA technology have allowed VV to be used as a vector to carry and express foreign genes. Foreign DNA is introduced into the VV genome by a process of homologous recombination. Homologous recombination involves essentially (1) pre-selecting a length of the VV genome in some region which does not impair the replication and normal functioning of the virus (hereinafter called a "non-essential region"), (2) making a construct comprising a VV promoter and a length of foreign DNA within a copy of the non-essential region (NER) so that the foreign DNA is under the control of the promoter and so that the promoter-foreign DNA combination is flanked by extensive sequences of non-essential region of VV DNA, (3) co-infecting appropriate tissue culture cells with the VV and with the construct and (4) selecting cells containing VV in which the pre-selected length has been recombined in vivo so that it is replaced in the genome by the construct DNA. The recombinant VV expresses the foreign gene in vivo, stimulating the immunity to the protein in an appropriate host. The procedure has considerable potential for use in vaccination.
More recently, similar technology has been applied to fowlpox virus (FPV). Although VV promoters have been used successfully in laboratory constructs of FPV, it is undesirable to incorporate elements of such VV, an orthopoxvirus which has a wide host range recombinant vaccine, for fear of recombination events which could pose a health risk. There is therefore a need to develop FPV promoters for use in recombinant FPV. Certain FPV promoters have been described in UK Patent Application Publication No. 2211504A or PCT Application WO/89/03879 (NRDC). However, each promoter has its own peculiar characteristics of strength and timing of promotion. A choice of promoters is therefore very highly desirable.
One of the major proteins of VV is the 4a core protein, DNA coding for which has been sequenced by E. Van Meir and R. Wittek, Archives of Virology 102, 19-27 (1988). The mRNA for such a protein might be strongly promoted if it exists in FPV. The task of locating and cloning new FPV promoters is made more difficult because only very limited data have been published about the DNA sequence of the FPV genome. A greater amount of the VV genome has been sequenced, but the FPV genome is much larger than that of VV. Estimates have put it at from 240 to 360 kbp compared with 186 kbp in VV. There is no publicly available library of FPV DNA. Homologies and heterologies between a few parts of the FPV and VV genomes are known.
SUMMARY OF THE INVENTION
It has now been found that FPV does have a counterpart to the 4a protein of VV and that it is preceded by a strong promoter.
The science of promoters of poxvirus DNA is at present poorly understood. It is known that certain regions to the 5' or "upstream" end of a gene serve to assist in transcribing genomic DNA into messenger RNA by binding the RNA polymerase involved in the transcription so that the mRNA which contains the start codon of the gene can be transcribed. Such upstream regions are referred to as the "promoter". It is often not possible a priori to say for certain which nucleotides of the upstream sequence are essential and which a
REFERENCES:
Binns, M. M. et al. 1987 Nucleic Acids Res. vol. 15 pp. 6563-6573.
Binns, M. M. et al. 1989 Virology vol. 170 pp. 288-291.
Van Meir, E. et al. 1988, Arch. Virol vol. 102 pp. 19-27.
Boyle, D. B. et al. 1988, Virus Research vol. 10 pp. 343-356.
Binns Matthew M.
Boursnell Michael E. G.
Campbell Joan I. A.
British Technology Group Limited
Mosher Mary E.
Schwartz Richard A.
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