【摘要】 2A肽(P2A)介导的多基因表达载体具有高裂解活性且上、下游基因等摩尔表达等优点,已广泛应用于动物转基因研究.文昌鱼(amphioxus)作为一种新兴的模式动物,尚无应用这种表达载体的报道,为此在pXT7转录系统基础上构建一个P2A介导的多基因表达载体.将体外转录的P2A介导的mRNA注入文昌鱼卵细胞并受精后,经激光共聚焦显微镜和蛋白质免疫印迹(Western blot)检测表明,该mRNA在文昌鱼胚胎中能够高效地翻译和剪切,并且在信号肽的作用下eGFP蛋白定位于细胞核中,而mCherry蛋白定位于细胞膜上,上、下游蛋白间的剪切效率达到91%;进而构建了由文昌鱼热激蛋白基因启动子(BbHsp70)启动,并由P2A介导的多基因表达载体,实验证明其在热诱导和上、下游蛋白剪切方面均达到了预期效果.更多还原

【Abstract】 The multicistronic expression vector mediated by 2A peptide( P2A) possesses advantages such as high cleavage efficiency,equimolar amounts of upstream and downstream gene expression,so that it has been widely applied to animal transgenic research.However,up to date,no multicistronic expression vector is available for the application in amphioxus,a promising new model animal for evolutionary development study,therefore we constructed a multicistronic expression vector harboring P2A based on pXT7 transcription system. After microinjecting mRNA transcribed in vitro into amphioxus eggs and fertilization,we observed the embryos under a laser confocal microscope and analyzed protein expression using Western blot assay. The results showed that the mRNA was translated and the P2A mediated protein was cleaved effectively in amphioxus embryos.Moreover,the eGFP protein was targeted to nucleus and mCherry protein to cell membrane under the role of corresponding signal peptides.The separation efficiency of the two proteins was about 91% .Furthermore,we constructed a heatshock protein gene promoter( BbHsp70) mediated P2A multicistronic expression vector,and the experiments showed the efficiency of gene expression and protein separation achieved the desired results.更多还原