人硒蛋白SelK基因重组腺病毒表达载体的构建及鉴定

Objective: The recombinant adenovirus vector expressing human selenoprotein SelK was constructed and the efficient expression of SelK in human cell line was realized. Methods: AdEasy recombinant adenovirus vector system was used for the construction of the recombinant adenovirus vector expressing human selenoprotein SelK. The target fragment of human Selenoprotein SelK was amplified by PCR and inserted into adenoviral shuttle vector pShuttle-CMV, the constructed vector pShuttle-CMV-SelK-secis was linearized by restriction endonuclease Pme I and eletro-transformed into bacterium BJ5183 which had been transformed with plasmid AdEasy1 containing adenoviral backbone for homologous recombination. The positive clones were screened with selective media. The recombinant plasmid was linearized with endonuclease Pac I, introduced in AD293 cells by liposome-mediated transfection for packaging and subculture. And the recombinant vectors were amplified and purified. The virus titer was measured by TCID50 method. The overexpression of SelK in gastric cancer BGC-823 cells transfected by the vectors was identified by Western blot. Results: The recombinant adenovirus vector of SelK, pAdEasy-SelK, was successfully constructed and the virus titer reached 2 × 10 11pfu / ml. Human selenoprotein SelK was overexpressed in human cancer cells and identified by western blot. Conclusion: The recombinant adenovirus vector pAdEasy-SelK, which efficiently expressed human selenoprotein SelK, was constructed using AdEasy recombinant adenovirus vector system. And the constructed vectors make a foundation for the study of the molecular function of human selenoprotein SelK.