【摘要】 钠离子依赖的中性氨基酸转运蛋白SNAT3是SLC38基因家族N系统的成员之一,主要在脑部和视网膜的星形胶质细胞中表达,负责转运谷氨酰胺,在脑部和肝脏的谷氨酸-谷氨酰胺循环中发挥重要作用.为了方便检测SNAT3在细胞膜上的表达和定位,本研究采用PCR扩增和酶切连接的方法将HA标签连接到质粒pBK-CMVΔ(1098-1300)-SNAT3中大鼠SNAT3的N端,构建了真核生物表达载体pBK-CMVΔ-SNAT3.用脂质体转染法将该表达载体瞬时转染进人胚肾细胞(HEK293T cells),通过Western blotting检测HA-SNAT3融合蛋白的表达.结果表明,HA-SNAT3能够正确在细胞膜上表达.pBK-CMVΔ-SNAT3表达载体的成功构建对于进一步研究SNAT3的结构和功能提供了有效方法.更多还原

【Abstract】 Sodium- coupled neutral amino acid transporter 3( SNAT3) is a member of SLC38 gene family which primarily expresses in astrocytes in the brain and retina. It transports L- glutamine predominantly and plays an important role in glutamate-glutamine cycle in the brain and liver. In order to easily detect the expression and localization of SNAT3 on the membrane,we constructed a eukaryotic expression plasmid with an HA tag at the N-terminus of rat SNAT3 in the mammalian expression vector pBK-CMVΔ( 1098- 1300) by PCR and double restriction endonuclease digestion techniques.After transiently transfected the plasmid into human embryonic kidney cells( HEK293T),expression of HA-SNAT3 fusion protein was detected by western boltting. The result suggested that the HASNAT3 fusion protein could normally express on the membrane. The plasmid pBK-CMVΔ( 1098-1300)-HA-SNAT3 will provide a useful tool for the investigation of structure and function of SNAT3 in the future.更多还原