Study On Function Of Silent Information Regulator2in Eimeria Tenella

Coccidiosis is one of the main forms of chicken diseases, which is caused by parasiticprotozoa and seriously does harm to the intensive chicken production. At present, thecontrol of coccidiosis mainly depends on drug, but increasingly strong drug resistancechallenges the control of coccidiosis. In fact, the new coccidial drug is an urgent need. Inrecent years, drug design based upon drug target becomes dominant method of drugmanufacture while the analysis of protein function, spatial structure through X-raydiffraction, and the mechanism of enzyme action is the basis of designing and developingnew drugs.Silent information regulator2(Sir2) proteins played an important role in cellbiological activities, such as chromatin silencing, gene regulation, metabolic regulation andantigenic variation. It was considered as a potential drug target. However, so far noresearch about Sir2has been reported in coccidia.The subject of the research was Eimeria tenella (E.tenella), which was most seriousdangerous to chicken in intensive chicken farming. Sir2gene sequence of Eimeria tenellawas obtained by gene annotation depended on comparative genomics and bioinformaticstechnology in present study. A potential EtSir2ORF was amplified and subcloned into thepET43a-EtSir2expression vector for prokaryotic expression. The enzymatic activity andkinetic analysis of EtSir2were measured by using Trypsin-coupled assay system.The data indicated that the full length of EtSir2ORF was909bp which encoded acytosolic protein composed of302amino acids. Amino acid sequences alignment of EtSir2with other Sir2s from several other model organisms by ClustalW2showed EtSir2shared24%～58%amino acid identities with other Sir2s. The EtSir2were phylogenetically mostclosely related to T. gondii and N. caninum, thereby demonstrating their high level ofgenealogical relatedness. Molecular mass of purified protein was about99ku. The resultswere in accordance with the reseacher’s expection.The results indicated that EtSir2was active with Km at41.7μM toward the NAD~+, Vmax=0.07349mmol/min/mg, and anapparent Km for Ac-peptide was51.2μM, Vmax=0.1093mmol/min/mg. For the enzymeinhibition studies, the results indited that lead compound nicotinamide (NAM) was ainhibitor against EtSir2, the IC50value for this inhibition was evaluated as355μM. Insummary, the present study successfully recombinant expressed and purified EtSir2, ourwork supplied the gap for Sir2of E.tenella, and the inhibitor NAM might be used as apotential lead compound in the discovery of anti-coccidian agents. The results of the studylaid the theoretical and experimental foundation for developing new anti-coccidia drug.