细菌荧光酶报告基因载体构建及在Hela细胞中的表达

采用ＰＣＲ点突变技术，构建含有细菌荧光酶融合ｌｕｘＡＢ报告基因的重组哺乳动物载体ｐｃＤＮＡ３－ｌｕｘＡＢ，脂质体ｌｉｐｏｆｅｃｔｉｎ转染和Ｇ４１８抗性筛选稳定转染的Ｈｅｌａ细胞。ＰＣＲ和酶切电泳证明构建的正确性；经稳定转染ｐｃＤＮＡ３－ｌｕｘＡＢ的Ｈｅｌａ细胞，用发光仪可测得较强的发光强度（最大值为４．１２ｍＶ／４０μｇ细胞总蛋白粗提物）。而亲本及ｐｃＤＮＡ３稳定转染的Ｈｅｌａ细胞则在本底值范围，ｐｃＤＮＡ３－ｌｕｘＡＢ稳定转染与亲本及ｐｃＤＮＡ３稳定转染的Ｈｅｌａ细胞在细胞形态和培养条件等方面无明显差异。本工作为细菌荧光酶作为报告基因在肿瘤细胞中的表达和应用建立了基础。

CONSTRUCTION OF THE EXPRESSION VECTOR BY THE FUSION OFBACTERIAL LUCIFERASE GENE AND ITS EXPRESSION IN HELA CELL

pcDAN3-luxAB was constructed by the fusion of bacterial luciferase gene (luxAB) into the mammalianexpression vector pcDNA3 by PCR and genetic engineering, by lipofectin and G418 selection it was stabllytransfected into Hela cell with Hela cell and the Hela cell transfected by pcDNA3 as controls. The correction ofboth the construct and the stable transfect were identified by PCR and Electrophoresis; considerable light(4.12mV / 40ug extracts of total cell protein) from the pcDNA3-luxAB transfected Hela cell was monitored byLuminometer while the values of the controls were in the range of background; the forms along with theculturing conditions of both the transfected and the untransfected Hela cells were indistinguishable.