Establishment Of The Stable Sweet Receptor Protein T1R2/T1R3-Expressed Cell Strain

Taste is one important part of everyone's daily life, especially important for sweet, which can indicate the presence of nutrients, and enhance the flavor and taste of food, influence the choice of food intake. Carbohydrate sweeteners with intrinsic fine flavor and high energy are more popular. But excessive intake of theses sweeterners may be lead to obesity and obesity-related diseases. Consequently, it is crucial to develop new low-calorie or non-caloric sweeteners for the development of food industy. In order to meet the growing demand of these sweeteners, many scientists are making efforts to interpret the mechanism of sweet perception from different aspects, which will facilitate to the development of novel sweeteners. The mainly methods include calcium imaging and electrophysiology for the study of the sweet perception mechanism at the cell level, while our lab use isothermal titration calorimetry(ITC) as a study tool to explore the thermodynamic properties of interaction between sweeteners and sweet receptors and desired to elaborate the sweet recognition mechanism in a new view. Previous studies in our lab, an effective method to separate and purify taste bud cells from mouse tongue have been set up, meanwhile one kind of cell lines-NCI-H716was found, which express the sweet receptor protein T1R2/T1R3and substitute taste cell. The ITC experimental conditions suitable for cell system were and the interaction thermodynamic properties between different concentrations of the same kind of sweetener, the same concentration of the different sweeteners and the cell were analysed. Above study results demonstrated that ITC experiment can be used as a new means to study the physical and chemical mechanisms of interaction between receptor and ligand molecules by the thermodynamic parameters at the cell level. However, the stable thermodynamic data can not be obtained using the taste cells or the NCI-H716cells, whch may result from the stable and purity of cells and cell protein. Accordingly, it is crucial for the study of thermodynamic mechanism for sweet perception to establish a cell line which posseses stable and simple protein components. Therefore, stable cell strain expressing sweet receptor protein T1R2/T1R3and Ga15was established with the method of heterologouse expression technology which have been widely used in other field and sweet mechanism at the cell level. The establishment of this stable cell strain will be helpful for our study and provide methodological foundation for e a large number of other researches on the basis of the cell. The results of this study are as follows:(1) Obtain of Ga15, T1R2and T1R3gene and establishment of eukaryotic expression vector. According to Gal5(NM₀10304.3), T1R2(NM₀31873.1) and T1R3(NM₀31872) reference sequence published in the NCBI, primers were designed by Primer5.0, which were used to amplify target gene. The gene encoding Ga15, T1R2and T1R3were successful obtained by the designed primers and mRNA from the C57BL/J6. Then the eukaryotic expression vectors were established by the genes encoding Ga15, T1R2and T1R3were inserted into the pEGFP-Cl, pDsRed1-N1, pcDNATM6.2plasmids repectively. The restriction enzyme digestion results showed that the targe genes have been accurately inserted into plasmid, and PCR and sequencing results showed that the genes encoding the three proteins were correct. (2) Establishment and selection of the cell strain stably expressi-ng Ga15/T1R2/T1R3. At the first, the antibiotic sensitivity test were performed to determine the optimal concentration of G418and blasticidin to screen stable expressing Ga15and Ga15/T1R2/T13HEK293cell strain respectively. The optimal concentration of G418and blasticidin are5p,g/mL and4μg/mL.Considering it's difficult to transfect HEK293cell line with three genes at the same time, so in the first the stable expressing Ga15cell strain was established. Then T1R2/T1R3genes were cotransfected into HEK293-Ga15cells. Finally, the stable HEK293cell strain expressing Ga15/T1R2/T1R3was primal established by the recombinant tag(GFP, RFPand YFP).(3) Identification of Ga15/T1R2/T1R3protein. The stable cell strain HEK293-Ga15/T1R2/T1R3was further validated by PCR and western blot results, which showed that Ga15/T1R2/T1R3protein expressed in the HEK293cells.