Direct molecular cloning of foreign genes into poxviruses and methods for the preparation of recombinant proteins
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United States of America Patent
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Jul 24, 2001
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N/A
app pub date -
Dec 19, 1994
filing date -
Aug 26, 1991
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Expired
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Abstract
A method is disclosed for producing a modified eukaryotic cytoplasmic DNA virus by direct molecular cloning of a modified DNA molecule comprising a modified cytoplasmic DNA virus genome. The inventive method comprises the steps of (I) modifying under extracellular conditions a DNA molecule comprising a first cytoplasmic DNA virus genome to produce a modified DNA molecule comprising the modified cytoplasmic DNA virus genome; (II) introducing the modified DNA molecule into a first host cell which packages the modified DNA molecule into infectious virions; and (III) recovering from the host cell virions comprised of the modified viral genome. The host cell is infected with a helper virus which is expressed to package the modified viral genome into infectious virions. Examples of packaging a modified poxvirus genome by a helper poxvirus of the same or different genus are described. Also disclosed are novel poxvirus vectors for direct molecular cloning of open reading frames into a restriction enzyme cleavage site that is unique in the vector. In one model poxvirus vector, the open reading frame is transcribed by a promoter located in the vector DNA upstream of a multiple cloning site comprised of several unique cleavage sites.
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