A simplified system for generating recombinant E3-deleted canine adenovirus-2
| Autor: | Jiasen Liu, Zuo Yu, Bo-tao Li, Liandong Qu, Dongchun Guo, Qian Jiang, Chuan-song Quan |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Genetic Vectors
Green Fluorescent Proteins Restriction Mapping Genome Viral Adenoviruses Canine Biology Transfection Genome Madin Darby Canine Kidney Cells law.invention Dogs Plasmid Cytopathogenic Effect Viral Shuttle vector law Animals Molecular Biology Gene Sequence Deletion Recombination Genetic Adenovirus genome Virology Molecular biology Subcloning Genetic Techniques cardiovascular system Recombinant DNA Expression cassette |
| Zdroj: | Plasmid. 77:1-6 |
| ISSN: | 0147-619X |
| DOI: | 10.1016/j.plasmid.2014.10.005 |
| Popis: | Canine adenovirus type 2 (CAV-2) has been used extensively as a vector for studying gene therapy and vaccine applications. We describe a simple strategy for generating a replication-competent recombinant CAV-2 using a backbone vector and a shuttle vector. The backbone plasmid containing the full-length CAV-2 genome was constructed by homologous recombination in Escherichia coli strain BJ5183. The shuttle plasmid, which has a deletion of 1478 bp in the nonessential E3 viral genome region, was generated by subcloning a fusion fragment containing the flanking sequences of the CAV-2 E3 region and expression cassette sequences from pcDNA3.1(+) into modified pUC18. To determine system effectiveness, a gene for enhanced green fluorescent protein (EGFP) was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique NruI and SalI sites. Transfection of Madin-Darby canine kidney (MDCK) cells with the recombinant adenovirus genome containing the EGFP expression cassette resulted in infectious viral particles. This strategy provides a solid foundation for developing candidate vaccines using CAV-2 as a delivery vector. |
| Databáze: | OpenAIRE |
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