【摘要】 用PCR技术获得NIa-pro、NIa-vpg、NIb基因片段,将这些基因分别克隆到pBD-GAL4载体中,构建酵母双杂交系统的诱饵载体pBD-GAL4-NIa-pro、pBD-GAL4-NIa-vpg和pBD-GAL4-NIb。测序正确后,将重组质粒导入YRG-2酵母菌株,检测其表达产物对酵母细胞有无毒性及对报告基因有无激活作用。结果表明,获得了正确的NIa-pro、NIa-vpg、NIb基因片段,并成功克隆到pBD-GAL4诱饵载体中,且转化有诱饵载体的YRG-2在SD/-Trp营养缺陷平板上生长良好,说明表达产物对酵母细胞无毒性,对报告基因也无自激活作用,为下一步利用酵母双杂交系统检测与NIa-pro、NIa-vpg、NIb蛋白相互作用的蛋白奠定基础。更多还原

【Abstract】 NIa-pro, NIa-vpg and NIb genes were amplified by PCR and then fused with pBD-GAL4. After confirmation with sequence analysis, the plasmid was transformed into the yeast cell YRG-2, and its toxicity and transcriptional activation was tested by color assay. As a result, the NIa-pro, NIa-vpg and NIb were successfully amplified and cloned into pBD-GAL4. The YRG-2 transformed with bait plasmids grew well on SD/-trp plate, without toxicity and autonomous activation effect. The bait vectors, pBD-GAL4-NIa-pro, pBD-GAL4-NIa-vpg and pBD-GAL4-Nib, constructed were expressed correctly, and could not activate the transcription of reporter gene alone in yeast two-hybrid system.更多还原