Construction Of CK1β-deficient In Toxoplasma Gondii

Toxoplasmosis is a epidemic zoonosis in worldwide caused by Toxoplasma gondii. Normal people are invisible infected with no obvious symptoms. The immune difecient patients like cancer, AIDS and organ transplants are more susceptible to infection with Toxoplasma gondii, causing systemic damage. Pregnant women who infected Toxoplasma gondii lead to fetal deformity and even death. Livestock which infected with Toxoplasma gondii caused epidemic outbreak and a large number deaths, affecting agricultural economic development. And yet there are no better clinical therapeutic drugs. Therefore, looking for potential drug targetsites become hotspot in the field of Toxoplasma gondii.Casein kinase is a type of serine / threonine protein kinase family which regulate cellular processes. Phosphorylation through a combination of the substrate. CK1 prefer to phosphorylate sbustrate, then complete in collaboration with other protein kinases in signal pathway process. It is divided into type I and II casein kinase according protein activity. Type I contains seven lsomers like CK1α, CK1β, CK1γ1, CK1γ2, CK1γ3, CK1δ and CK1ε. All of them are highly conserved in eukaryotic cells. Studies have shown that CK1β participate in physiological processes like Wnt signal transduction, membrane transport, cytoskeletal repair, DNA replication, RNA metabolism and relate to the development of tumor-related, it has become an important target for drug development.(1) TgCK1β prokaryotic expression Toxoplasma gondii CK1β cDNA was cloned by RT-PCR and constructing Prokaryotic expression vector TgCK1β-pET-22 b. Recombinant protein induced by IPTG, SDS-PAGE and western-blot analysis showed the molecular mass of the recombinant protein is 49 kDa. Recombinant protein purified by Ni-NTA, concentration is 1 mg/ml, purity reach more than 90%. The preparation of polyclonal antibodies in mice immunized with recombinant preteins. ELISA antibody titer reach 1:12800 using indirect immunofluorescence. IFA detect expression site in Toxoplasma gondii. The result confirmed CK1β mainly expressed in cell membrane of Toxoplasma gondii.(2) TgCK1β deletion strain constuction TgCK1β pairs flanking sequences were cloned into gene targeting vector pBluscript, while inserting resistance gene phle. Linearized plasmid introduced into the wild-type Toxoplama gondii using electric transfection. CK1β gene in Toxoplasma gondii target vector had been constructed, identified by PCR and Southern blot method; All of these lay the foundation for CK1β biological characteristics.