| 摘要: |
| SLC38基因家族编码的Na+偶联的中性氨基酸转运蛋白3(SNAT3)是System N中的一员.它在大脑谷氨酸-谷氨酰胺循环以及肝细胞质膜的谷氨酰胺转运等生理过程中发挥着重要作用.为了方便检测SNAT3在细胞膜上的表达与定位,本研究采用PCR扩增和酶切连接技术将增强型绿色荧光蛋白(EGFP)与SNAT3的 N末端连接,通过菌液PCR、酶切和DNA测序验证得到pBK-CMV(Δ［1098-1300］)-EGFP-SNAT3重组真核表达质粒.用脂质体转染法将构建好的质粒瞬时转染进入人体胚肾细胞(HEK293T cells),利用激光扫描共聚焦显微镜和Western blot技术检测EGFP-SNAT3融合蛋白的表达和亚细胞定位.结果表明,EGFP-SNAT3重组质粒在细胞中正确表达并定位于细胞膜上.EGFP-SNAT3重组质粒的成功构建将有助于日后进一步研究SNAT3的结构和功能. |
| 关键词: 增强型绿色荧光蛋白 EGFP-SNAT3融合蛋白 质粒构建 表达鉴定 |
| DOI： |
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| 基金项目:国家自然基金资助项目(31270883);上海市教育委员会科研创新项目(13ZZ103) |
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| Expression and identification of rats glutamine transporters EGFP-SNAT3 fusion protein |
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LI Min1, MIN Weiyong1, ZHANG Zhou2
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1.Collegeof Life and Environmental Sciences,Shanghai Normal University;2.College of Life Science and Biopharmacy,Shenyang Pharmaceutical University
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| Abstract: |
| The sodium-coupled neutral amino acid transporter 3(SNAT3) encoded by the SLC38 gene family is a member of the System N.It plays an important role in Glutamate-Glutamine shuttle in the brain and glutamine transportation in the membrane of liver cells.In order to detect expression and localization of SNAT3 on the membrane conveniently,in this study,an enhanced green fluorescent protein(EGFP) sequence was constructed to the N terminus of SNAT3 by PCR amplification,enzyme digestion and ligation.After the recombinant eukaryotic expression plasmid of pBK-CMV(Δ［1098-1300］)-EGFP-SNAT3 was transiently transfected into HEK293T cells mediated by liposomal for 36 hours,expression and localization of EGFP-SNAT3 fusion protein were detected by laser scanning confocal microscope and Western blot.The results showed that EGFP-SNAT3 expressed and localized correctly on the membrane.The recombinant plasmid of EGFP-SNAT3 constructed successfully may contribute to the further study of structure and function of SNAT3 in the future. |
| Key words: enhanced green fluorescent protein EGFP-SNAT3 fusion protein plasmid construction expression and identification |
