Human Cyclin E prokaryotic expression vector and expression of.docVIP

 PAGE \* MERGEFORMAT 10 Human Cyclin E prokaryotic expression vector and expression of Authors: Li Shan, Pan Quan, Yun-Long Li, Ye Xin [Keywords:] Cyclin, E;, gene cloning;, prokaryotic expression [Abstract] Objective: To construct prokaryotic expression of Cyclin E gene vector and expressed in E.coli BL21 and purified. Methods: PCR amplification of human Cyclin E gene fragment, and cloned into the prokaryotic expression vector pET32a () in the recombinant plasmid pET32a () Cyclin E. The restriction endonuclease EcoR I and Xho I double restriction enzyme digestion and sequencing, the transformation of E.coli BL21, after IPTG induced expression of histidine fusion protein. Results: The total length of approximately 1200 bp of the human Cyclin E gene fragments. To construct the recombinant plasmid pET32a () Cyclin E, after transforming E.coli BL21 by IPTG induction, express the relative molecular mass (Mr) of about 60,000 of the fusion protein. After SDSPAGE, Western blot analysis showed that induced expression of the protein Cyclin E. Conclusion: The successfully constructed the expression vector pET32a () Cyclin E, and expressed a recombinant protein HisCyclin E, for further screening in vivo and in vitro with the combination of Cyclin E and CDK2 protein, laid the foundation for the new. [Keywords:] Cyclin E; gene cloning; prokaryotic expression Cell cycle is a continuous dynamic, multi-stage, multi-factor involved in the regulation of the precise and orderly process can be divided into five periods: the G0 phase, G1 phase, S phase, G2 phase, M phase, with G1 / S restriction point and G2 / M restriction points 2. In the mammalian cell cycle operation, there are many enzymes involved in, in which the core is a group of enzymes as a catalytic subunit of the cyclin-dependent kinase (CDK) and a group of proteins as a regulatory subunit of the cycle (Cyclins) composed of different dimeric complexes [1], formed after a combination of both cell cycle regulator