Production and purification of non replicative canine adenovirus type 2 derived vectors
2013
Szelechowski, Marion | Bergeron, Corinne | Gonzalez-Dunia, Daniel | Klonjkowski, Bernard | Centre de Physiopathologie de Toulouse-Purpan (INSERM U563 - CNRS UMR1037) ; Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Institut Claudius Regaud (ICR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de lutte contre le cancer (CLCC)-Centre National de la Recherche Scientifique (CNRS) | Virologie UMR1161 (VIRO) ; École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES) | INSERM; CNRS; Fondation pour la Recherche Medicale; Agence Nationale pour la Recherche [ANR-10-Blanc-1322]; European Commission; FRM (programme "Equipe FRM"); UE [245266] | European Project: 245266,EC:FP7:KBBE,FP7-KBBE-2009-3,ORBIVAC(2010)
International audience
اظهر المزيد [+] اقل [-]إنجليزي. Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 10(10) infectious particles per ml and can be directly administrated in vivo.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
المعلومات البيبليوغرافية
تم تزويد هذا السجل من قبل Institut national de la recherche agronomique