Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Patent
1994-12-16
1998-06-09
Guzo, David
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
424 936, 435 691, 4352351, 4353201, 536 2372, 536 241, A01N 6300, C12P 2100, C12N 1586, C07H 2104
Patent
active
057629242
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/AU93/00284, filed Jun. 15, 1993.
This invention relates to the production of recombinant entomopoxviruses (EPV's), particularly recombinant Heliothis armigera entomopoxviruses (HaEPV's), capable of expressing heterologous DNA sequences. Particular applications of the invention include the use of the recombinant entomopoxviruses as biological insecticides and in the production of desired, biologically-active proteins, polypeptides and peptides in cell culture.
Entomopoxviruses are large, double-stranded DNA viruses of insects, and have to date been described from species of caterpillars, beetles and locusts (Goodwin et al., 1991). The economic importance of these insect groups has led to serious consideration of EPV's as potential biological control agents, and investigation by others has documented various characteristics which support their use in this capacity. For example, while the collective EPV host range is broad, covering the important insect groups, individual EPV isolates generally have a narrow host range, allowing potentially high levels of control specificity. Additionally, vertebrates (and vertebrate cell cultures) exposed to large amounts of infectious EPV have shown no sign of infection or other discernible ill-effect (Buckner & Cunningham, 1972; Langridge, 1973).
These factors confer significant potential for the use of EPV's as insect control agents. Unfortunately however, most EPV's exhibit low levels of pathogenicity. This trait, which has prevented serious attempts to develop the viruses as major commercial insecticides, may be overcome, conceivably, by the production of recombinant entomopoxviruses capable of expressing heterologous DNA sequences encoding agents toxic or otherwise deleterious to insects.
Recombinant entomopoxviruses also hold great potential for the production of homogenous and biologically active proteins, polypeptides and peptides. Such products are presently generated either from recombinant bacteria or through the use of expression vectors in eukaryotic cells. The first method is technically more simple, but suffers from the drawback that many proteins of eukaryote origin are not correctly processed in bacteria. Without correct processing many proteins are biologically inactive, and thus of little use or value. On the other hand, production from expression vectors in vertebrate cells is traditionally more expensive, with smaller yields of protein. Some eukaryotic expression vectors (e.g. baculoviruses) also cause lysis of the host cell.
In contrast to the baculoviruses, EPV's do not necessarily cause lysis of the infected cell, thereby offering potential for long term persistent infection of large scale cell culture. This should permit enhanced production efficiency of proteins, especially of those which are secreted from the host cell, since collection of the product (via periodic removal of cell culture medium) will not require destruction of the cells.
Thus it is an object of the present invention to provide recombinant entomopoxviruses suitable for use as biological insecticides and/or as expression vectors for the production of desired proteins, polypeptides and peptides in cell culture systems. To achieve this object it is necessary that non-essential regions are identified in the genomes of EPV's. The identification of such regions would provide sites which could be utilised for development of an EPV viral vector, with heterologous DNA inserted into the non-essential region of the EPV genome by any of the methods known in the art (but most conveniently, homologous recombination), optionally with deletion of the non-essential region or portion thereof prior to insertion of the foreign DNA.
The spheroidin protein of EPV is a major component of the occlusion body (spheroid). Since occlusion is only required for horizontal virus transmission, and not infection, the single spheroidin gene driven by a strong promoter provides a very attractive site for the insertion of heterologous sequences into the EPV genome. The sequence of th
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Dall David James
Fernon Carol Anne
Sriskantha Alagacone
Commonwealth Scientific and Industrial Research Organisation
Guzo David
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