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文昌鱼中一个2A肽介导的多基因表达载体构建
Authors
华俊豪
李光
王义权
Publication date
28 January 2018
Publisher
Abstract
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.国家自然科学基金(31372188,31471986,31672246
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Last time updated on 10/06/2020