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pBK-CMV⊿-EGFP-SNAT2质粒载体构建及其表达鉴定
李 洋, 张 舟
上海师范大学
摘要:
SNAT2是SLC38基因家族编码的Na+偶联中性氨基酸转运蛋白中属于system A的成员之一.它在谷氨酸-谷氨酰胺循环、肝脏糖质新生等生物通路中发挥重要作用.增强型绿色荧光蛋白EGFP的连接对于SNAT2在细胞表达中的定位以及检测都有很大的帮助.采用PCR扩增和酶切技术将EGFP连接到质粒pBK-CMV⊿-SNAT2中SNAT2的N端.通过酶切后琼脂糖凝胶电泳和测序验证得到pBK-CMV⊿-SNAT2质粒载体,将构建好的质粒瞬时转染进人胚肾细胞(HEK293 cells)用Western blot和激光共聚焦电子显微镜检测EGFP SNAT2的表达与亚细胞定位.结果表明:EGFP SNAT2在细胞中正确表达并定位于细胞膜上.pBK-CMV⊿-SNAT2质粒载体的构建对于进一步研究SNAT2的结构和功能具有重要意义.
关键词:  SNAT2  EGFP  构建  表达
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Construction and identification of EGFP SNAT2 fusion protein in mammalian expression vector
LI Yang, ZHANG Zhou
College of Life and Environment Sciences,Shanghai Normal Universit
Abstract:
SNAT2 is the second member of the sodium coupled neutral amino acid transporters (SNATs) of the SLC38 gene family.It plays an important role in Glutamate glutamine cycle in the brain and gluconeogenesis in the liver.In order to detect expression and localization of SNAT2 on the membrane conveniently,the C terminus of enhanced green fluorescence protein (EGFP) was linked to the N terminus of SNAT2 in the mammalian expression vector pBK-CMV(Δ[1 098-1300])by PCR and single restriction endonuclease digestion techniques.After the plasmid pBK-CMV-EGFP-SNAT2 was transiently transfected into HEK293T cells for 36 hours,the expression and localization of EGFP SNAT2 fusion protein were detected by western blot and laser scanning confocal microscope (LSCM).The result showed that EGFP SNAT2 expressed and localized on the membrane normally.The construction of the expression vector of pBK-CMV-EGFP-SNAT2 will be of great benefit to investigation of structure and function of SNAT2 in the future.
Key words:  SNAT2  EGFP  construction  expression