钠离子依赖的中性氨基酸转运蛋白SNAT2- HA融合蛋白的构建及鉴定

摘要

Sodium coupled Neutral Amino acid Transporter 2 ( SNAT2 ) , widespread expressed in the mammalian tissues, plays an important role in the pathways of glutamate glutamine cycle and gluconeogenesis. To determine easily SNAT2 expression and location in the membrane,we construct an eukaryotic expression plasmid with HA at the C terminus of SNAT2 in pBK CMVA( 1098 -1300) by molecular biological methods. After transiently transfected into human embryonic kidney cells (HEK293T) ,correctly expression and location of SNAT2 HA fusion proteins are observed in cell membranes by western blot. The plasmid pBK CMVA (1098 - 1300) SNAT2 HA provides a good tool for studying structure and function of SNAT2 in the future.%钠离子依赖的中性氨基酸转运蛋白SNAT2在哺乳动物组织中广泛表达,具有转运中性氨基酸的功能,在谷氨酸-谷氨酰胺循环、肝脏糖质新生等生物通路中发挥重要作用.为了方便测定SNAT2在细胞膜上的表达和定位,本研究采用PCR扩增和酶切连接将HA标签蛋白与SNAT2的C末端连接,构建了真核生物表达载体pBK - CMV△( 1098 - 1300) - SNAT2 - HA表达载体.用脂质体转染法将该表达载体瞬时转染到HEK293T细胞中,通过Western blot法检测到SNAT2 - HA融合蛋白在细胞膜上的正确表达和定位.pBK - CMV△( 1098 - 1300) -SNAT2 - HA表达载体的成功构建,为今后对SNAT2的结构和功能的研究提供了有效方法.