CHO-hGPR91工程细胞株的构建及鉴定  

Construction and identification of engineering cells CHO-hGPR91

作　　者：陈曦[1] 韩春光[2] 胡明[3] 刘超[2] 刘永学[2] 

机构地区：[1]安徽医科大学研究生学院,合肥230032 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]海军总医院海战伤救治研究中心,北京100048

出　　处：《国际药学研究杂志》2012年第6期507-511,共5页Journal of International Pharmaceutical Research

摘　　要：目的构建携带hGPR91蛋白表达序列的pcDNA3.1(+)-hGPR91表达载体,并将其转入中国仓鼠卵巢细胞(CHO细胞)中,经筛选获得CHO-hGPR91工程细胞株,并对工程细胞株表达受体功能进行初步鉴定。方法采用PCR法扩增hGPR91全长开放阅读框,通过T4连接酶将其连接到载体pcDNA3.1(+)上,再通过Lipofectamine 2000将重组载体转染入CHO细胞中,并以抗生素G418加压筛选,RT-PCR及Western印迹法检测工程细胞株中hGPR91 mRNA及蛋白的表达情况,以Fluo4-AM荧光染料检测琥珀酸对细胞内Ca2+浓度([Ca2+]i)的影响。结果所构建CHO-hGPR91工程细胞株能够成功表达hGPR91的mRNA及蛋白,在琥珀酸的刺激下能够明显引起工程细胞株[Ca2+]i的变化;而空载体pcDNA3.1(+)转染CHO细胞所得的CHO-pcDNA3.1(+)细胞则无上述效应。结论成功构建了CHO-hGPR91工程细胞株,琥珀酸作为GPR91的特异性配体,能够明显改变工程细胞株的[Ca2+]i,为进一步研究GPR91的功能及针对以GPR91为靶点的药物研发奠定了基础。Objective To construct pcDNA3.1（＋）-hGPR91 expression vector containing protein sequence of hGPR91, transfer it into Chinese hamster ovary（CHO） cells,and preliminarily identify the function of the engineering cells CHO-hGPR91 after isolation.Methods The open reading frames of the hGPR91 were obtained by PCR,and subcloned into the pcDNA3.1（＋） vector with T4 DNA ligase.The recombinant vectors were transfected into CHO cells by Lipofectamine 2000.The mRNA and protein expression of hGPR91 were detected by RT-PCR and Western-blot,respectively.After stimulation with succinate,the changes of intracellular Ca 2 ＋ concentration in the engineering cells were measured using Fluo 4-AM fluorescent indicator.Results The mRNA and protein of hGPR91 were successfully expressed in CHO-hGPR91 engineering cells and succinate stimulation could increase the intracellular Ca 2 ＋ concentration of the CHO-hGPR91,but no such effects were observed with CHO-pcDNA3.1（＋） which were transfected with parent vector pcDNA3.1（＋）.Conclusion The CHO-hGPR91 engineering cells expressing GPR91 were successfully constructed and succinate could change the intracellular Ca 2 ＋ concentration in the engineering cells,which may provide basis for the research on the function of GPR91 and development of drugs targeting GPR91.

关 键 词：G蛋白偶联受体 GPR91 工程细胞株 

分 类 号：TQ920.6[轻工技术与工程—发酵工程]