rAQP4-M1真核表达载体的构建及在CHO细胞中的表达

Construction of eukaryotic expression vector of rat AQP4-M1 and its expression in CHO cells

目的构建rAQP4-M1基因真核表达载体,建立稳定转染AQP4-M1的CHO细胞系。方法应用RT-PCR从大鼠脑组织中扩增rAQP4-M1,并将PCR产物双酶切后插入到pcDNA3.1(+)真核表达载体中,经过菌落PCR、双酶切及测序验证的pcDNA3.1(+)/rAQP4-M1质粒通过脂质体转染CHO细胞。G418筛选建立稳定转染rAQP4-M1的CHO细胞系。采用细胞免疫荧光和免疫印迹技术检测rAQP4-M1在该细胞系中的表达。结果成功构建了pcDNA3.1(+)/rAQP4-M1表达质粒,建立了稳定转染的CHO细胞系。免疫荧光检测结果显示,rAQP4-M1在CHO细胞膜上稳定表达;免疫印迹检测可见34×103处的阳性条带,表明rAQP4-M1在该细胞系中成功表达。蓝绿温和胶电泳技术证实了rAQP4-M23而不是rAQP4-M1参与了正交排列颗粒(orthogonalarraysofparticles,OAPs)的形成。结论 rAQP4-M1在CHO细胞系中稳定表达成功,rAQP4-M1未参与OAPs的形成。

Objective To construct an eukaryotic expression vector of rat aquaporins （AQP4）-M1 and to establish its stable transfectants in CHO cells.Methods The fragment of rAQP4-M1 was amplified from rat brain by RT-PCR,and then the PCR products were cloned into pcDNA3.1（＋） after NdeⅠ and XhoⅠ digestion.The eukaryotic expression vector pcDNA3.1（＋）/rAQP4-M1 was sequenced and transfected into CHO cells by Lipofectamine 2000.After screening by G418,stably transfected CHO cell line was established.The expression of rAQP4-M1 was identified by immunocytochemistry and immunoblotting.Blue-native polyacrylamide gel electrophoresis （BN-PAGE） was used to detect the formation of orthogonal arrays of particles （OAPs）.Results The eukaryotic expression plasmid of pcDNA3.1（＋）/rAQP4-M1 was successfully constructed and its stable transfectants in CHO cells was established.Immunofluorescence image revealed that rAQP4-M1 was expressed successfully on the membrane of CHO cells.Immunoblotting results following SDS-PAGE indicated the positive band was at 34×103,which manifesting that rAQP4-M1 was successfully and stab1y expressed in CHO cells.Results of BN-PAGE indicated that it was rAQP4-M23 not rAQP4-M1 that played a role in the formation of OAPs.Conclusion The recombinant eukaryotic expression vector of pcDNA3.1（＋）/rAQP4-M1 is constructed successfully and stab1y expressed in CHO cells,which provides a foundation for further studies on the function of AQP4 isoforms of M1 and M23.