Cloning and In-Vitro Expression Analysis of the Cowpox Virus Red-Pock Gene
Robinson, Rachel A.
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Cowpox virus grown in developing chick embryos normally produces red, haemorrhagic pocks. However, up to 1% of these pocks can be white. The gene coding for pock redness has been located at 32 kilobas-pairs from the right-hand end of the cowpox virus genome. In this study the red-pock gene was cloned in two expression vectors: The first was pJG-300, which has a Lac Z gene and encodes hybrid proteins when the foreign DNA is inserted in the correct reading frame. These hybrid proteins can be detected by growing host cells on lactose minimal medium and can be isolated by affinity column chromatography. The second vector was pSP-64, which has a highly specific phage SP6 promoter followed by an extensive polylinker region. This vector was used in an in-vitro run-off transcription and 5 ' -capping reaction to yield functional mRNAs, which were then translated in-vitro in a rabbit reticulocyte system. It was found that the pJG-300 vector was difficult to use because its only unique restriction site was a Bam-H1 site. Bam-H1 proved to be an inefficient enzyme, so a new vector was created which carried a unique Eco-Rl site in its polylinker region. This study showed that the sp6 in-vitro expression system of Promega-Biotec promises to be a simple and efficient expression system, although the transcription reaction in this research was not successful