Molecular cloning, expression, and partial sequencing of the right 45 per cent of the Aleutian disease virus genome and a suspension method to determine reuse life of chemical disinfectants during clinical use

The right 45% of the genome of Aluetian disease virus, variant Gorham (ADV-G), was molecularly cloned into Bluescript plasmid. The result, pADV-1, contained a 2.16 kilobase pair ADV insert including a 430 bp fragment (HindIII site to the 5{dollar}prime{dollar} terminus) which has not been reported as having been cloned. A BamHI linker was added to the end of the ADV insert of pADV-1 to create pADV-2. The BamHI-excised ADV sequence from pADV-2 was cloned into each of the translation vectors pET-3a, pET-3b, and pET-3c. Since these translation vectors differ only in the reading frame of the BamHI site with respect to the translation start sequence, an evaluation of ADV protein production in all three reading frames was possible. It was found that ADV-specific protein was produced by all three recombinant plasmids (pADV-3a, pADV-3b, and pADV-3c). DNA sequence data confirmed that only one reading frame (corresponding to pADV-3a) was open. Reading frames corresponding to the other two plasmids (pADV-3b and pADV-3c) contained multiple termination signals. ADV-specific protein produced by these two plasmids was probably the result of internal initiation at one or three possible internal start sites identified by the DNA sequence data. This is supported by the size difference between ADV protein produced by pADV-3a (54,000) and that produced by pADV-3b or pADV-3c (44,000), as determined by Western blotting.