Cloning and expression of uvrA gene of Corynebacterium pseudotuberculosis to value its role in NER pathway

Abstract

Caseous lymphadenitis is a infection that affects sheep and goats and caused by Corynebacterium pseudotuberculosis. DNA repair, the target of this study, is essential for survival of any organism, to prevent potential deleterious effects. The objective of this study was to characterize uvrA gene from C.psudotuberculosis using in silico and in vitro techniques. In silico analyzes were performed to check the physical and chemical parameters, search for conserved domains and modeling of 3D structure using bioinformatics programs. The analysis shows that the protein has a molecular weight of about 101.5 kDa and a conserved domain of ABC transporters family and ABC excinuclease. The amplification of uvrA gene was carried out using a Long Range PCR Kit and the insert was cloned into the TOPO PCR 2.1 vector. E. coli strain DH5α was electroporated with recombinant plasmid and after that these were digested with restriction enzymes BamH1 and Xho1 for subsequent cloning into the expression vector pProEX HTB. The constructions were confirmed by sequencing. Financial support: CNPq and FAPEMIG