Constitutive production of human interferons by mouse cells with bovine papillomavirus as a vector.

Abstract

Mouse C1271 cells were transformed with a chimeric plasmid consisting of bovine papillomavirus and human interferon (HuIFN) gene (either IFN-y or IFN-as) placed under the control of simian virus 40 early promoter. The transformed cells retained 30-50 copies each of the hybrid plasmid extrachromosomally and secreted a high level of HuIFNs constitutively up to 3-4 x 105 international units/ml.The secreted HuIFN-y had no detectable activity on mouse cells, whereas HuIFN-a5 was slightly active on mouse cells.Each IFN was neutralized either by anti-HuIFN-y or antiHuIFN-a antiserum. The partially purified M5S-labeled HuIFN-y produced by the transformed cells showed heterogeneous bands with apparent Mrs of 22,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. This value is similar to that of natural IFN-y and larger than the molecular weight calculated from the amino acid sequence, which suggests that HuIFN-y secreted by transformed mouse cells was glycosylated. The 35S-labeled IFN-a5, immunoprecipitated with anti-HuIFN-a antiserum from the supernatant of the transformed cells, had a molecular weight of mature protein. These results suggest that the bovine papillomavirus vector can be used to produce a large quantity of foreign proteins in mammalian cells.Interferons (IFNs) are a group of proteins that protect cells against viral infections and are classified currently into three types, IFN-a, IFN-P, and IFN-y, based on antigenic specificity, cellular origin, and physicochemical properties (1). Human (Hu) IFN-a comprises at least 13 subtypes encoded by the multigene family (2-5), whereas IFN-,B and IFN-y are each encoded by a single gene (6, 7). Well-characterized members of the IFN-a group demonstrate 80-95% amino acid sequence homology among each other (3), whereas there is only 29% homology between IFN-a and IFN-,B amino acid sequences (8). Even less homology was found between IFN-y amino acid sequence and other IFNs (9).In addition to antiviral activity, IFNs are known to exhibit various functions such as anticellular, antitumor, and immunoregulatory activities (10). To study those functions in more detail, large-scale production and purification of IFNs have been attempted (11,12 Fig.

The bovine papillomavirus genome and its uses as a eukaryotic vector

“…The second type of vector overcomes some of these disadvantages. They consist of: the 69 % transforming region of BPV; bacterial sequences, including an origin of replication and a selectable marker; a 'stimulatory segment' containing a foreign gene, which could overcome the inhibitory effect of the bacterial sequences; and the gene of interest [51,53,[61][62][63][64][65]. The third type of vector uses the whole BPV genome, bacterial sequence and the gene of interest [66][67][68].…”

“…These range from the bacterial proteins for chlor-1987 amphenicol acetyltransferase [80] and aminoglycoside phosphotransferase [44,71,72] to the human proteins growth hormone [76], tissue plasminogen activator [86] and a, /3 and y interferon [60,65,82]. They also include the viral glycoproteins hepatitis B surface antigen, HBSag [12,66,87,88], influenza virus haemagglutinin and cap-recognizing proteins [61,68] and type 1 human T-cell leukaemia virus small envelope protein [89].…”

“…All three interferons were correctly processed and secreted; interferon y was also glycosylated. All the interferons had antiviral activity and a5 and y were neutralized by antibodies raised to natural human interferons [65].…”

“…Removal of these bacterial sequences prior to transfection tends to restore the episomality of the vector and leads to less rearrangement provided that the linear plasmid is recircularized before transfection [76,80,81]. The lost enhancer function could to a certain extent, be restored by 'stimulatory segments' [51,53,63] and vectors containing these sequences tended to remain episomal [62,63,65]. There are however instances where this is not the case [64].…”

Interleukin 2 receptor beta chain expressed in an oligodendroglioma line binds interleukin 2 and delivers growth signal.

“…Although IL-2 has no detectable effect on the parental ONS-21 cells before and after cloning, the cloned cells were found to respond to IL-2 for proliferation when essentially following the general procedures (23). To construct pdKCRB, the Hha I fragment containing the entire coding region of the human IL-2R13 was inserted into BamHI-cleaved pdKCR containing the simian virus 40 early promoter (24). The resulting plasmid, pdKCRI3, was introduced together with the neomycin-resistance gene (pSTneoB; ref.…”