Cloning And Expression Profiling Of Sirtuin-like Histone Deacetylase 2 Genes(Et Sir2) During The Developmental Stages Of Eimeria Tenella(Phylum: Apicomplexa)

Currently, the chemotherapeutics/chemoprophylaxis for avian coccidiosis, which is accountered with the drug-resistance by Eimeria spp. and public concerns on drug residue in broiler products, has been yet applied in poultry production worldwidely. Although the anticoccidial live attenuated oocyst vaccines, a series of novel alternative immune prophylactic products for avian coccidiosis, has been limitedly used in some countries since 1990 s, because of its features such as intrinsic pathogenicity to chickens and spreading of parasite to environment. Therefore, the sustained control of coccidiosis in intensive poultry industry has been longing for newly powerful anticoccidials and/or vaccines for decades. Sir2 histone deacetylases(HDACs), members of NAD+-dependent histone deacetylase subfamily, play important roles in the chromatin remodeling, aging, cell metabolic pathway regulation, and other cellular processes, and have been demonstrated as potential drug-targets for tumor, allergy, autoimmune diseases, etc. in medicinal research. As revealed by the whole genome sequencing, apicomplexan parasites like as Toxoplasma gondii, Plasmodium falciparum and Eimeria spp. also possess two isoforms of Sir2 HDACs, named as Sir2 A and Sir2 B, but, unfortunately, the biochemical characterizations of these apicomplexan Sir2 were not profoundly investigated. In this thesis, we cloned and profiled the Sir2 A gene of Eimeria tenella(EtSir2) as well as its dynamic expression during its developmental stages, and achieved the following results:1. The full-length ORF sequence of EtSir2 A and partial coding sequence of EtSir2 B were cloned using RT-PCR. then the EtSir2 A sequences were analyzed by bioinformatics softwares. The EtSir2 A ORF was 909 bp in full-length, and encodes one protein of 302 amino acids. Bioinformatic analysis has shown that no signal peptide motif existed in EtSir2 A, which implies that this functional protein only localizes intracellular compartment. The recombinant EtSir2 A was also expressed using constructed vector pMAL-c2x-EtSir2 A in E. coli Transetta(DE3).2. The dynamic expression of EtSir2 A during E. tenella life cycle was evaluated by real-time quantitative PCR, and the results showed that the significantly differential transcription levels presented to different developmental stages. The unsporoulated oocyst stages had the highest transcription, while the second generation merozoites was exhibited with the lowest transcription level.