稳定表达烟酸受体GPR109A的中国仓鼠卵巢细胞株的建立

Establishment of a Chinese Hamster Ovary Cell Line Stably Expressing Nicotinic Acid Receptor GPR109A

目的将GPR109A基因插入pEGFP-N3载体中构建重组质粒pEGFP-GPR109A,并建立稳定表达烟酸受体GPR109A的中国仓鼠卵巢(Ch inese ham ster ovary,CHO)细胞株。方法构建重组质粒pEGFP-GPR109A,重组质粒转化大肠杆菌DH5 a,重组质粒经PCR、酶切、测序验证正确后,应用脂质体转染技术将该质粒导入CHO细胞,用抗生素G418筛选稳定表达的细胞。倒置荧光显微镜观察克隆细胞株荧光信号,RT-PCR检测GPR109A基因mRNA表达,W estern B lotting检测绿色荧光蛋白与烟酸受体GPR109A的融合蛋白(GFP-GPR109A)的表达,激光共聚焦显微镜观察融合蛋白的细胞定位。结果 pEGFP-GPR109A真核表达质粒构建正确,融合蛋白GFP-GPR109A在CHO细胞中稳定表达,表达的融合蛋白主要定位于细胞膜。结论成功构建pEGFP-GPR109A真核表达载体并建立其稳定表达的CHO细胞株;该细胞株的建立有助于GPR109A的更多生理病理功能研究,也为下一步抗动脉粥样硬化的药物筛选奠定了基础。

Aim To construct pEGFP-GPR109A recombinant plasmid by inserting GPR109A gene into pEGFP-N3 vector and establish a Chinese hamster ovary（CHO）cell line stably expressing nicotinic acid receptor GPR109A. Methods The pEGFP-GPR109A recombinant plasmid was constructed,which was subsequently transformed into DH5α E coli.After identification by PCR,digestion with restriction endonuclease and sequencing,the recombinant plasmid was transfected into CHO cells via lipofectamine 2000.The stable transfectants were screened by antibiotic G418.The fluorescent signal of cloned cell lines were detected by fluorescent microscope.The GPR109A gene mRNA expression was analyzed by RT-PCR and the fusion protein expression of green fluorescence protein and nicotinic acid receptor GPR109A（GFP-GPR109A） was detected by Western Blotting.The cell localization of fusion protein was detected by laser scanning confocal microscope. Results The results of PCR,restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pEGFP-GPR109A.Western Blotting showed that the fusion protein GFP-GPR109A was expressed stably in CHO cells.The fusion protein was mainly localized on cell membrane detected by laser scanning confocal microscope. Conclusion The eukaryotic expression vector pEGFP-GPR109A has been successfully constructed and a CHO cell line stably expressing fusion protein GFP-GPR109A was established,which facilitated the physiological and pathological function research of GPR109A and also provided important foundation for following up drug screening for atherosclerosis treatment.