| 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. |
| 关键词: 中性氨基酸转运蛋白 HA-SNAT3融合蛋白 构建和鉴定 |
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CHEN Chen1, ZHANG Zhou2
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1.College of Life and Environmental Sciences,Shanghai Normal University;2.College of Life Science and Biopharmacy,Shenyang Pharmaceutical University
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| 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 HA-SNAT3 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. |
| Key words: neutral amino acid transporter HA-SNAT3 fusion protein construction and characterization |
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