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⊿-EGFP-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⊿-EGFP-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.
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（A[ 1 098 -1300] ） by PCR and single restriction endonuelease 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.
出处
《上海师范大学学报（自然科学版）》
2012年第5期514-520,共7页
Journal of Shanghai Normal University(Natural Sciences)
基金
Foundation item:NSFC(31270883)
Science and Technology Commission of Shanghai Municipality(105405503400)
Innovation Program of Shanghai Municipal Education Commission(13ZZ103)
