Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1990-10-09
1993-11-02
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
536 241, 435235, 4353201, 935 6, 935 8, 935 36, 424 89, C12N 1510, C12N 1511, C12N 1586
Patent
active
052582949
DESCRIPTION:
BRIEF SUMMARY
This invention relates to recombinant vectors and their construction, and in one aspect this invention relates to recombinant fowlpox virus vectors and use thereof.
Poxviruses are large DNA viruses which replicate within the cytoplasm of infected cells. The vaccinia virus is the most widely studied member of the poxvirus family and has been widely used to express a number of foreign genes inserted into the vaccinia virus genome. In recent years, recognition of non-essential regions of the vaccinia virus genome, followed by mapping and nucleotide sequence determination, has made it possible to insert and express a wide variety of foreign genes through the medium of recombinant vaccinia viruses. Such recombinant vaccinia viruses have the potential to deliver vaccine antigens to a variety of animal species. However, for faithful expression of an inserted gene, it is important to position the gene near unique viral promoter regions.
A recognised problem resides in a risk of cross species infection and therefore the spread of disease from one animal species to another through the widespread use of vaccinia virus in animals. Thus, the construction of recombinants based on host specific poxviruses is desirable, e.g. fowlpox virus for poultry or ORF for sheep vaccines. Although promoter sequences have been described for a number of early and late genes of vaccinia viruses, little is known about promoter regions in other pox viruses. In order to construct host specific live viral vaccines, it is of fundamental importance that the molecular biology of those viruses, and in particular the biology of transcriptional control elements or promoter region(s) in the pox virus be understood. Therefore, it is necessary to characterise and isolate these regions before utilising them in suitable recombinant vectors. Such vectors show great potential for the manufacture of viral vaccines and the like.
Accordingly, it is an object of the present invention to overcome, or at least alleviate, one or more of the deficiencies and/or difficulties related to the prior art.
Accordingly, in a first aspect of the present invention there is provided a gene sequence including a first promoter for the expression of a major early fowlpox virus protein.
In a preferred aspect of the present invention, the gene sequence further includes a second promoter for the expression of a late fowlpox virus protein in opposite orientation to said first promoter.
The gene sequence may be approximately 40 base pairs in length, consisting of two promoter elements in opposite orientation to each other.
As stated above, this promoter sequence, in its preferred form, is characterised in that it has a major early function and a late function in opposite orientation to the early function. The early function of the promoter compares well with the strongest known promoter of vaccinia virus (PL11). Because of the strength of this promoter and its unique bidirectional nature, it has important applications in the construction of both vaccinia (VV) and FPV vectors and other recombinant poxviruses for the delivery of foreign antigens. Its small and defined size makes it a versatile DNA element which may express two foreign genes of interest simultaneously in a pox virus expression system as described below. Since its early function compares well in strength with PL11, a VV promoter widely used for expression of foreign genes during late stages of virus growth cycle, it may be a promoter of choice when high levels of expression is required during early stages of virus growth. Its promoter strength is 3-4 times greater than the VV P7.5 promoter which has been widely used, to date, to express genes in recombinant VV, FPV and other poxviruses (for a review see Moss and Flexner, 1987). The gene sequence may be particularly useful to induce cytotoxic T-lymphocyte (CTL) response to an expressed antigen since late promoter expressed gene products appear not be recognised by CTL (Coupar et al, 1986). Being a FPV native promoter, it may be a promoter of choice in developing FPV b
REFERENCES:
Hanggi, M. et al., 1986, Embo Journal vol. 5 pp. 1071-1076.
Esposito, J. J. et al., 1989, Advances in Veterinary Science and Comparative Medicine, vol. 33, pp. 195-247.
Onuma et al., Am. J. Vet. Res., 45:112-1218 (1984).
S. Berger et al., Guide to Molecular Cloning Techniques: Methods in Enzymology, 152:113-129 (1987).
Boyle, D. B. et al., 1986, J. Gen. Virol., vol. 67, pp. 1591-1600.
Boyle, D. B. et al., 1988, Virus Research, vol. 10, pp. 343-356.
Tsao, H. et al., 1988, J. Virol., vol. 62, pp. 4832-4834.
Tomley, F. et al., 1988, J. Gen. Virol., vol. 69, pp. 1025-1040.
Boyle Bernard D.
Kumar Sharad
Commonwealth Scientific and Industrial Research Organisation
Mosher Mary E.
Schwartz Richard A.
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