【摘要】 乳腺生物反应器具有广阔的开发前景,而提高目的基因的表达量是该领域一个重要的研究课题。因此,选用强的非特异性启动子pCAG,而不是乳腺特异性启动子来实现高效表达;通过Cre/loxP系统和山羊β-乳球蛋白启动区(pBLG)表达Cre重组酶来实现载体的自删除以达到乳腺特异表达的目的。方法:构建含PolyA终止信号的Cre乳腺特异表达元件PolyA-pBLG-cre,插至强启动子pCAG驱动的报告基因lacZ表达载体中,构建成乳腺特异表达载体pCBCZ(pCAG-loxPPolyA-pBLG-cre-loxP-lacZ)。转染细胞实验结果:PCR鉴定确认pCBCZ载体在小鼠乳腺上皮细胞(HC11)中发生Cre-loxP同源重组。X-Gal染色表明载体能驱动lacZ在HC11细胞中高效表达β-半乳糖苷酶,而在NIH 3T3细胞中仅少量表达。结论:构建的pCBCZ载体能高效驱动外源基因在乳腺细胞中表达,且具有较好的乳腺特异性,为研发乳腺生物反应器表达载体提供新的方法。更多还原

【Abstract】 Mammary gland bioreactor is considered as a promising approach for generating pharmaceutical proteins. Yet,the low-level expression of the transgene is one of the bottlenecks in this technique. Here,a strong and constitutively expressing promoter instead of a mammary gland specific but weak promoter was used to improve transgene expression; meanwhile,a Cre /loxP system driven by a β-lactoglobulin( BLG) gene promoter was used to restrict the strong expression in mammary gland. Methods: Construct the mammary gland-specific expression unit( Polyadenylation signal-pBLG-cre),this unit was then flanked by a pCAG strong promoter and a lacZ reporter. Thus,a mammary gland-restricted expression vector named pCBCZ( pCAG-loxP-PolyA-pBLG-creloxP-lacZ) was constructed. The in vitro experiment results: Cre-loxP recombination was detected in the mouse epithelial( HC11) cells transfected with pCBCZ vector. X-Gal staining experiment showed that the vector can strongly drive β-galactosidase expression in HC11 cells,but weak in NIH 3T3 cells. These data indicate that the pCBCZ vector can effectively drive gene expression at a mammary gland-restricted manner,which provide a new strategy for the generation of an effective mammary gland bioreactor.更多还原