摘要
Na+偶联的中性氨基酸转运蛋白SNAT1是一种膜蛋白,属于转运蛋白第38家族(SLC38),在中枢神经系统(CNS)中起到重要的生理学作用.为了更容易地检测SNAT1在细胞膜上的表达,以pBK-CMVΔ-SNAT1为模板,用PCR,双酶切和T4连接酶连接的方法在SNAT1的C端加上了一个HA标签蛋白,构建了一个新质粒pBK-CMVΔ-SNAT1-HA.用该质粒瞬时转染人胚胎肾细胞(HEK293T),转染36 h以后,融合蛋白SNAT1-HA可以用HA抗体检测.Western blot结果显示在约54 kDa处有一条特异性条带,与预期分子量大小一致.利用重组质粒pBK-CMVΔ-SNAT1-HA可以更方便地检测到SNAT1在细胞膜上的表达与定位,为进一步研究SNAT1结构与功能奠定了基础.
The sodium-coupled neutral amino acid transporter 1 （SNAT1） is a membrane protein which belongs to the solute carrier 38 family （ SLC38 ） , it plays an important physiological role in the central neural system （CNS）. In order to determine easily SNAT1 expression in the membrance of cells, we construct an eukaryotic expression plasmid with HA tag at the C terminus of SNAT1 on the base of pBK - CMVA - SNAT1 by PCR, double enzyme digestion and ligation. Plasmids of pBK - CMVA -SNAT1 -HA were transiently transfected into human embryonic kidney cell （HEK293T）. After transfection 36 h, the fusion proteins of SNAT1 - HA were determined using HA antibody by western blotting. The result showed that there was a specific band at around 54 kDa, which is consistent to the predicted molecular weight. It is more convenient to detect the expression of SNAT1 in the cell membrane using the recombinant plasmid of pBK - CMVA - SNAT1 - HA, which will facilitate further structural and functional studies of the neutral amino acid transporter SNAT1.
出处
《上海师范大学学报（自然科学版）》
2011年第5期515-521,共7页
Journal of Shanghai Normal University(Natural Sciences)
基金
NSFC(30870560)
Science and Technology Commission of Shanghai Municipality(105405503400)
The Innovation Program of Shanghai Municipal Education Commission(09ZZ139)
Shanghai Normal University(DZL808 and DRL804)
Shanghai Leading Academic Discipline(S30406)
