Molecular genetic construction of vaccine strains of...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/74 (2006.01) C07K 14/285 (2006.01) C12N 1/21 (2006.01) C12N 9/10 (2006.01) C12N 9/22 (2006.01) C12P 19/34 (2006.01) A61K 39/00 (2006.01)

Patent

CA 2253809

Tools for genetically engineering Pasteurellaceae are provided. Replication- conditional plasmids which are useful for the Pasteurellaceae have been isolated and characterized. The plasmids can be utilized for delivery of DNA segments into the Pasteurellaceae in situations where control of extrachromosomal replication desired, such as in achieving allelic exchange or site-directed mutagenesis. A restriction endonuclease, HsoI, was isolated from a bovine lung isolate of Haemophilus somnus. The enzyme was found to be a true isoschizomer of HinPI, a commercially available enzyme originally isolated from Haemophilus influenzae Pl. Commercially available HhaI methyl transferase was found to protect against cleavage by both enzymes. Methylation of foreign plasmid DNA was found to enhance transformation of Haemophilus somnus in excess of four orders of magnitude.

L'invention propose des instruments de génie génétique applicables aux Pasteurellaceae. Des plasmides à réplication conditionnelle, utiles aux Pasteurellaceae, ont été isolés et caractérisés. Les plasmides peuvent être utilisés pour l'apport de segments d'ADN à l'intérieur de Pasteurellaceae lorsqu'il est souhaitable de pouvoir régler la réplication à l'extérieur des chromosomes, par exemple, lors d'un échange allélique ou d'une mutagénèse dirigée. On a isolé une endonucléase de restriction, HsoI, d'un isolat d'Haemophilus somnus dans un poumon de bovidé. Il s'est avéré que l'enzyme représente un isoschisomère authentique de HinPI, qui est une enzyme disponible dans le commerce, initialement isolée d'Haemophilus influenzae PI. On a également constaté que la méthyle transférase HhaI, disponible dans le commerce, assure une protection contre le clivage par les deux enzymes. On a découvert que la méthylation de l'ADN d'un plasmide étranger stimule la transformation d'Haemophilus somnus en la multipliant par quatre, voire plus.

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