| The quality and efficacy of a protein can be superior when expressed in mammalian cells, which have the capacity for proper protein folding, assembly, and post-translational modification, versus other hosts such as bacteria, plants and yeast. However, the low expression level of foreign gene is the main problem faced with the biopharmaceutical academy and industry. The expression levels of introduced genes in mammalian cells are primarily determined by the cellular DNA at the site of integration and the regulatory elements of expression in the cassette of introduced genes. So this study focused on the optimization of the expressional regulatory elements in the cassette of interesting gene and the attenuation of selecting gene (Neo gene), which guarantees the foreign gene being integrated into transcriptional hotspots of the chromosome.Firstly, a series of expression vectors containing diverse combinations of expressional regulatory elements such as hCMV, hEF-1 α promoter, hCMV Enhancer, hEF-1 α 1st intron, translational enhancer H213 and V163 were constructed. And then the expression efficiency of these regulatory elements were, based on the assay of k2tPA (a mutant of tPA) activity by monitoring fibrin clearance, valuated by the use of the targeted integration system. The results showed that the two new hybrid regulatory elements hCMV+H213 and hEF-1 α + V163, in comparison with the only hCMV promoter, increased the expression efficiency by nearly 60% and 40%, respectively.Secondly, several modes of attenuation of Neo gene such as wild and mutant type of Neo gene, weak promoter lack of enhancer, frame-shift ATG, changed Kozak sequence and an intron in which Neo gene was placed were applied to construct expression vectors aiming at targeting mammalian integration sites that supported high levels of expression. These vectors were valuated, by the use of the random integration system, with the rates of forming colonies and expression levels of k2tPA in the mixed...
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