稳定表达甜味受体蛋白T1R2/T1R3的HEK293细胞系的建立

Establishment of HEK293 Cell Line Stably Expressing Sweet Preceptor Protein T1R2/T1R3

以小鼠舌组织为对象,提取总mRNA,并以此为模板,使用自行设计的引物通过RT-PCR扩增Gα15、T1R2和T1R3目的片段。构建重组质粒pEGFP-C1-Gα15、pDsRed1-N1-T1R2、pcDNATM6.2/N-YFP-DEST-T1R3。以脂质体介导的方法转染HEK293细胞,经抗性筛选后,通过极限稀释法获得稳定表达T1R2/T1R3的HEK293细胞系,最后通过RT-PCR,荧光显微镜及Western blot方法在基因及蛋白质水平上对建立的稳定表达细胞系进行鉴定。基因及蛋白质水平上的结果均表明,目的基因Gα15、T1R2/T1R3成功导入HEK293细胞中,并且稳定表达。该细胞系的建立为细胞水平上甜味机理的体外研究(如甜味识别热动力学等)提供了稳定的细胞来源。

It is to establish HEK293 cell line which could stably express sweet taste receptor protein T1R2/T1R3.Firstly,extract total mRNA from mouse tongue tissue,then amplify Gα15,T1R2 and T1R3 target gene fragment by RT-PCR with self-designed primers and above total mRNA template.Then,establish the recombinant plasmid pEGFP-C1-Gα15,pDsRed1-N1-T1R2,pcDNATM6.2/N-YFP-DEST-T1R3 and introduce them into the HEK293 cells by liposome.After resistance screening,the HEK293 cell line with the ability of stable expressing T1R2/T1R3 was obtained in a limit dilution method.Finally,the stable cell line with sweet receptor protein T1R2/ T1R3 was identified by RT-PCR,fluorescence microscopy and Western blot.The results in gene and protein level show that Gα15、T1R2/T1R3 are successfully introduced into HEK293 cell line and are stably expressed.The establishment of HEK293 cell line provides a stable cell source for the study of sweetness mechanism in vitro（such as sweet recognition thermodynamics,etc.） at cell