Selenium Speeds Reactions

Background: Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV2) VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation). Results: A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion: It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli) can interrupt the downstream work. Background Potato virus X (PVX), a potexvirus, is a filamentous rodshaped virus which contains a single plus-sense RNA molecule. The RNA is capped, polyadenylated, and encodes five open reading frames (ORF)[1]. PVX was designed to express foreign genes[2]. Its coat promoter was duplicated in the vector to drive the foreign ORF transcription. The PVX vector has been used to express foreign genes in many host plants[3], in translation studies[4], and for investigating gene silencing[5]. In the current study, the PVX vector was used to express the gp53 gene of BVDV Singer strain[6] and VP1 of CPV-2[7] for vaccine development in a plant vector. The construction of the PVX vector required the new plasmid to be amplified in E. coli before inoculating plants, which would be necessary to construct a plant virus vectored viral genes for a vaccine for these two viruses. In molecular cloning, plasmids need to be amplified in E. coli to produce enough plasmid DNA for molecular manipulation. The foreign gene in the expression vectors is not expressed until it is in a eukaryotic host cell; thus, it was surprising to find, when the plasmid was amplified in E. coli, the foreign gene was expressed and it killed the bacPublished: 5 March 2007 BMC Molecular Biology 2007, 8:17 doi:10.1186/1471-2199-8-17 Received: 2 October 2006 Accepted: 5 March 2007 This article is available from: http://www.biomedcentral.com/1471-2199/8/17 © 2007 Guo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.