Analyzing Intracellular Midbody Localization In Kinesin Moter Protein KIF14 And The Preparation Of CENP-E Antibody

Kinesins are superfamily of molecular motors that travel along microtubule tracks driven by ATP hydrolysis to fulfill their roles in various cellular processes.Therefore, kinesin molecules play a important role in cell proliferation,division and transferring, they also participate in the directional transport of intracellular organelles and biomacromolecule and maintainning cytoskeletal structure. Kinesin molecules KIF14 (Kinesin Family member 14, KIF14) is crucial for the forming of midbody and cell division in telephase. When using siRNA to interference KIF14, cellds could not form complete midbody and unable to make the final cut and eventually form a multicore cell.This thesis consists of two parts, the first part of the paper mainly explores the KIF14. We use innunofluorescence staining to clarify its intracellular location of endogenous KIF14 and use siRNA to knock down KIF14 to confirmed that KIF14 is not only involved in cytokinesis, but also involved in chromosome alignment. At the same time, we use synchronized cell as sample to run western blot and find obvious upshift in late mitotic cells, which suggest that KIF14 regulate cell mitosis through posttranslational modification in late mitosis. To study the domain of Kif14 that determines its midbody localization in telophase in eukaryotic cells, the green fluorescent protein (GFP) expression plasmids for different Kifl4 domains were constructed and transfect into HeLa cells respectively. We found N-terminal extension of KIF14 determines its localization in midbody in telophase, which lay the foundation for clarifying the mechanism of cutting in the midbody.The second part mainly discusses the preparation of polyclonal antibody about CENP-E. Using molecular cloning technique to construct prokaryotic expression plasm id of pHis-CENPEC410 and then His-CENPEC410 fusion protein was induced by IPTG and the fusion protein was purified by affinity chromatography using Ni-NTA beads. Using the purified protein as antigen to immune New Zealand white rabbits to produce specific polyclonal antibody of CENP-E. The antibodies serum were detected by immunoblotting and Co-Immunoprecipitation, the purified antibodies were detected by immunofluorescene staining. The results of immunoblotting and Co-Immunoprecipitation demonstrated that the antibody serum is effective and the purified antibody can be applied to Immunofluorescene. CENP-E polyclonal antibody with high specificity and sensitivity was obtained, which lay the foundation for the follow-up study of CENP-E protein.