Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1999-12-13
2003-07-29
Mertz, Prema (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C435S069520, C435S071100, C435S071200, C435S471000, C435S325000, C435S320100, C435S252300, C435S254110
Reexamination Certificate
active
06600020
ABSTRACT:
TECHNICAL FIELD
The present invention relates to recombinant vectors, recombinant viruses, transformants, canine interleukin 18, canine interleukin 1&bgr; converting enzyme and interleukin 18 production methods, where the objective is to mass produce by genetic manipulation.techniques canine interleukin 18 the protein primary structure of which is derived from canine genetic information and to thus make pharmaceuticals for animal use (antitumour drugs/anti-allergy drugs/antiviral drugs/vaccine adjuvants).
TECHNICAL BACKGROUND
Interleukin 12 (hereinafter abbreviated to IL12), which shows immune control action, is a cytokine which possesses actions comprising interferon &ggr; inducing activity and physiological activity in regard to, for example, activating natural killer cells and type 1 helper T cells (reference 1) and, in particular, as a result of a powerful activating action in respect of cellular immunity, it is regarded as having very good prospects as an antitumour drug and anti-allergy drug candidate (references 2 and 3). Interleukin 18 (hereinafter abbreviated to IL18) has also been recently cloned as a cytokine showing the same kind of activity as IL12 (reference 4), and it has been reported that due to synergistic action with IL12 the activities thereof are further increased (reference 5).
Following mouse IL18 (reference 4), human IL18 cDNA has also been cloned (reference 6) by genetic manipulation techniques, and mass production using these gene recombination techniques has been investigated. However, IL18 does not have a signal sequence required for secretion from within cells. Hence, in order to be secreted from within cells in the active form, processing of an IL18 precursor protein by means of an interleukin 1&bgr; converting enzyme (hereinafter abbreviated to ICE) is necessary and so, when the IL18 gene is introduced on its own into animal cells, it is not expressed as the active form of IL18 (reference 7) and therefore the efficient mass production of recombinant form IL18 using cells has been difficult.
Development as remedies for tumours, allergies and viral diseases, etc, may be expected by means of mass production using IL18 gene recombination techniques.
In pets, particularly dogs, in common with humans, there are known many tumours such as mammary gland tumours, allergic dermatitis, and numerous viral diseases such as parvovirus infection and distemper infection, and the development of remedies for these is desired.
The cloning of canine IL18 has not yet been reported. Hence, if canine IL18 could be cloned, it is possible that it could form a novel canine remedy.
Again, if it were possible to readily mass produce IL18 in cells using gene recombination techniques, then there could be expected to emerge L18 applications as, for example, human and animal antitumour drugs, anti-allergy drugs and antiviral drugs, etc.
Against this background, with the objective of cloning canine IL18 cDNA and large-scale expression of the IL18 gene, and based on an original concept, the present inventors have succeeded in cloning the gene coding for canine IL18 from canine cDNA, and furthermore they have produced a recombinant baculovirus containing DNA coding for an IL18 precursor protein, and it has been discovered that by infecting insect cells or larvae with this, surprisingly, the active form of IL18 is produced without ICE treatment, and it has been further discovered that by producing a gene in which there is added the gene coding for the signal sequence in front of the gene coding for the active form of canine IL18, the level of production of the active form of IL18 is further enhanced. Furthermore, the gene coding for canine ICE has been successfully cloned from canine cDNA and a recombinant baculovirus containing at the same time DNA coding for a canine IL18 precursor protein and DNA coding for canine ICE has been produced, and canine IL18 has been successfully mass produced by infecting insect cells or larvae, and hence a method for producing IL18 simply and on a large scale has been established. The present invention has been perfected based on these discoveries.
DISCLOSURE OF THE INVENTION
Specifically, the present invention relates to canine interleukin 18 having at least one of the abilities selected from an ability to act on canine leukocytes and induce antiviral active factors and factors which enhance class II MHC expression on canine tumour cells; an ability to promote the proliferation of canine lymphocytes; an ability to enhance Fas ligand expression on canine lymphocytes and canine tumour cells; an ability to obstruct and destroy canine tumour cells; an ability to bring about a reduction in size of tumours occurring in the bodies of dogs; and an ability to activate canine leukocytes and suppress canine allergies.
Furthermore, the present invention offers recombinant vectors which bring about the production of IL18
, Escherichia coli
transformants possessing these recombinant vectors, recombinant baculoviruses which bring about the production of IL18 in insect cells or larvae, IL18 obtained therefrom, and also an IL18 production method. Moreover, the present invention also offers a gene coding for canine IL18, canine ICE and a gene coding for canine ICE. Furthermore, it offers a canine immune disease remedy containing IL18.
OPTIMUM FORM FOR PRACTISING THE INVENTION
A recombinant vector in which there has been inserted DNA coding for canine IL18 of the present invention can be produced, for example, as follows. After extracting poly (A) RNA from canine cells, cDNA synthesis is carried out and, using primers based on the gene sequences coding for mouse or human IL18, polymerase chain reactions (hereinafter abbreviated to PCR) are conducted.
Furthermore, from the synthesized cDNA a phage library is produced and, by carrying out plaque hybridization with the gene fragments obtained by PCR, full length canine IL18 cDNA can be cloned. Full length canine ICE cDNA can be cloned similarly.
As methods for obtaining RNA from canine organs and the like, there are the usual methods such as, for example, those employing polysome isolation, sucrose density gradient centrifugation and electrophoresis. The extraction of the RNA from the aforesaid canine organs and canine cells can be carried out by selection of a suitable method from amongst the guanidine thiocyanate-cesium chloride method where guanidine thiocyanate treatment is carried out followed by CsCl density gradient centrifugation (reference 8), the method of phenol extraction following treatment with a surfactant in the presence of a ribonuclease inhibitor, using a vanadium complex (reference 9), the guanidine thiocyanate-hot phenol method, the guanidine thiocyanate-guanidine hydrochloride method, the guanidine thiocyanate-phenol chloroform method, and the method where treatment with guanidine thiocyanate is carried out followed by treatment with lithium chloride, and RNA precipitation effected.
From canine organs and, for example, mitogen-stimulated canine monocytes and lymphocytes, mRNA is isolated by the usual methods, for example the lithium chloride/urea method, the guanidine isothiocyanate method and the oligo dT cellulose column method, etc, and cDNA is synthesized from the mRNA obtained by the usual methods, for example by the method of Gubler et al. (reference 10) or the method of H. Okayama et al. (reference 11). To synthesize cDNA from the mRNA obtained, basically besides using avian myeloblastosis viral (AMV) or other such reverse transcriptase, there may be combined methods where DNA polymerase or the like is employed using partial primers. The use of commercial synthesis or cloning kits is convenient.
Using this cDNA as a template, PCR is carried out using primers based on mouse or human base sequences and furthermore, after ligating the synthesized cDNA to a &ggr; phage vector, packaging is carried out by mixing in vitro with &ggr; phage coat protein, etc, and the
E. coli
which constitutes the host is infected with these created phage particles. In such circumstances, &ggr; phage-infected
E.
Mertz Prema
Nixon & Vanderhye P.C.
Toray Industries Inc.
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