家蚕S期激酶相关蛋白的克隆表达和定位研究

Skp (S-phase kinase associated protein) was a kind of F-box protein which participated in the ubiquitination. A cDNA sequence containing three Skp1 conservative domains was cloned in a Bombyx mori cDNA library, and designated as BmSkp since it encoded an S phase kinase-associated protein of silkworm. This gene was inserted into a vector pET-28a with both EcoR I and Xho I restriction sites, and then expressed in E. coli BL21 (DE3). The recombinant protein was determined to be 26 kD by the SDS-PAGE analysis. The target protein was purified by affinity chromatography and used to immune a male New Zealand rabbit for the production of anti-serum. The polyclonal antibody was generated and the titer was 1:128,000 measuring by ELISA and was determined for specificity by Western blot after purified by affinity chromatography using immobilized protein A. The polyclonal antibody was generated and the titer was 1:21800 measuring by ELISA and was determined for specificity by Western blot after purified by affinity chromatography using immobilized protein A. The total proteins and RNA from four developmental stages and eight different tissues of silkworm were extracted for real-time PCR and Western blot, in order to analysis the transcription and expression level of BmSkp. Subcellular localization of BmSkp was shown to be mainly present in cytoplasm of the BmN cells. These researches may lay a foundation for the study of BmSkp gene function.