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A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

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Author: Maassen, C.B.M.
Institution: TNO Preventie en Gezondheid
Source:Journal of Immunological Methods, 1, 223, 131-136
Identifier: 234929
doi: doi:10.1016/S0022-1759(98)00206-3
Keywords: Biology · EAE · Electroporation · Lactic acid bacteria · Mucosal tolerance · Oral vaccination · Plasmid extraction · Administration, Oral · Bacterial Proteins · Deoxyribonuclease BamHI · Deoxyribonucleases, Type II Site-Specific · DNA, Bacterial · Genetic Engineering · Immune Tolerance · Immunodominant Epitopes · Lactobacillus · Lactobacillus casei · Plasmids · Recombination, Genetic · Transformation, Genetic · Bacteria (microorganisms) · lactobacilli casei · Lactobacillus casei · Posibacteria


Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated from Lactobacillus casei to transform other Lactobacillus strains. The isolation of plasmid DNA from Gram-positive lactobacilli is complicated by the resilience of the peptidoglycan layer. Here a rapid, safe and efficient method is described that combines enzymatic breakdown of the cell wall and purification of the plasmid by commercially available DNA-binding columns. For the lysis-resistant L. casei strain, this method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency. Chemicals/CAS: Bacterial Proteins; Deoxyribonuclease BamHI, EC 3.1.21.-; Deoxyribonucleases, Type II Site-Specific, EC; DNA, Bacterial; endodeoxyribonuclease PvuI, EC 3.1.21.-; Immunodominant Epitopes