以人類HEK293細胞株苦味接受器TAS2R38及TAS2R43之穩定轉染株應用於苦味化合物的分析

Stable Expression of Human TAS2R38 and TAS2R43 in Human Embryonic Kidney 293 cells and Application in The Characterization of Bitter Compounds

馬瑞珊(Marisa Zakiya Ulfa)

指導教授 ： 鄭雪玲 ； -

國立屏東科技大學/農學院/生物科技系/碩士(2019年)

https://doi.org/10.6346/NPUST201900338

摘要

苦味接受器(TAS2R)存在於細胞的表面且可以偵測到多種不同的化合物。在人類的組織已經發現有25種不同亞型的苦味接受器，且能調節不同的生理功能，像是降血糖機制。但是苦味接受器進行異源表現時，經常無法表現於細胞膜上且表現量低，所以不利於進行單一亞型的研究。本研究將Lucy-human somatostatin receptor type 3 (Lucy-hsst3) 加在苦味接受器的N端以提升苦味接受器移至細胞膜。將Lucy-hsst3-TAS2R38-GFP (綠螢光蛋白) 轉染至HEK 293細胞，利用綠螢光蛋白證明重組後的TAS2R38位於細胞膜上。於是，於HEK 293細胞建立穩定表現Lucy-hsst3-TAS2R38-RFP或Lucy-hsst3-TAS2R43-RFP的轉染株。利用共軛焦顯微鏡證實TAS2R38及TAS2R43在各自的穩定轉染株是表現於細胞膜上。於是，以Gα-gustducin短暫轉染這些細胞株並以TAS2R38的激活劑PTC，TAS2R43的激活劑denatonium和quinine，或益生菌Bacillus amyloliquefaciens的胞外多醣 (EPS)刺激之效果發現，TAS2R38果然不被上述試劑活化，因為所選殖者為PTC味盲者的對偶基因而TAS2R43會被它的激活劑活化但不會被EPS活化。

關鍵字

苦味接受器 ； HEK 293 ； 穩定轉染 ； Exopolysaccharide

並列摘要

Bitter taste receptors (TAS2Rs) are cell surface receptors that are detected in a wide variety of compounds. They have been indicated to regulate multiple physiological reactions including the mechanisms of some hypoglycemic compounds. There are 25 isoforms of TAS2Rs in human tissues. The study of specific isoforms has been hampered because their expression are often low and they did not translocated to the cell membrane in heterologous expression systems. In this study, to improve the expression of TAS2Rs in heterologous cells, a new N-terminal tag Lucy-human somatostatin receptor type 3 (Lucy-hsst3) was added to TAS2Rs to improve membrane translocation. Transient transfection of HEK 293 cells by Lucy-hsst3-TAS2R38-GFP (green fluorescent protein) revealed that the recombinant TAS2R38 was translocated to the cell membrane. Thus, stable clones of HEK 293 expressing Lucy-hsst3-TAS2R38-RFP (red fluorescent protein) or Lucy-hsst3-TAS2R43-RFP were established. Confocal microscopy manifested that TAS2R38 and TAS2R43 were expressed in the cell membrane in the respective stable clones. Thus, the stable clones were transiently transfected by plasmid carrying Gɑ-gustducin and subjected to functional assays using TAS2R38 agonist PTC, TAS2R43 agonist denatonium and quinine, and the exopolysaccharide (EPS) from Bacillus amyloliquefaciens. Consequently, TAS2R38 was not activated by any of the agents because it is the non-tester allele. The TAS2R43 was activated by the agonist but not activated by EPS.

並列關鍵字

Bitter Taste Receptor ； HEK 293 ； Stable Transfection ； Exopolysaccharide

參考文獻

Bachmanov, A. A., & Beauchamp, G. K. (2007). Taste receptor genes. Annu Rev Nutr, 27, 389-414.

Behrens, M., & Meyerhof, W. (2011). Gustatory and extragustatory functions of mammalian taste receptors. Physiol Behav, 105(1), 4-13.

Behrens, M., & Meyerhof, W. (2006). Bitter taste receptors and human bitter taste perception. Cell Mol Life Sci, 63(13), 1501-1509. doi:10.1007/s00018-006-6113-8

Behrens, M., & Meyerhof, W. (2013). Bitter taste receptor research comes of age: from characterization to modulation of TAS2Rs. Semin Cell Dev Biol, 24(3), 215-221.

Breer, H., Eberle, J., Frick, C., Haid, D., & Widmayer, P. (2012). Gastrointestinal chemosensation: chemosensory cells in the alimentary tract. Histochem Cell Biol, 138(1), 13-24.