小鼠神经介素B及其受体基因过表达质粒的构建及表达

Construction and expression of mouse neuromedin B and neuromedin B receptor gene overexpression vectors

目的构建小鼠神经介素B(neuromedin B,NMB)及其受体(neuromedin B receptor,NMBR)基因慢病毒过表达质粒,并于RAW264.7细胞中表达,以期为小鼠NMB和NMBR基因过表达对RAW264.7细胞增殖及凋亡影响的深入研究奠定基础。方法经RT-PCR法扩增获得小鼠NMB和NMBR基因的编码序列(coding sequence,CDS),插入载体pCD513B-1,构建重组质粒pCD513B-1-NMB和pCD513B-1-NMBR;于HEK-293T细胞包装获得小鼠NMB和NMBR基因过表达慢病毒,通过倍比稀释法检测病毒效价。用慢病毒感染RAW264.7细胞48 h后,qPCR法检测小鼠NMB和NMBR基因mRNA的表达情况,以未感染细胞为对照。结果经双酶切鉴定证明重组质粒构建正确。小鼠NMB和NMBR基因过表达慢病毒滴度分别为6×10~6和8×10~6TU/mL。2种慢病毒转染RAW264.7细胞中小鼠NMB和NMBR基因mRNA表达量显著高于对照组(t分别为24.158和14.958,P均<0.01)。结论成功构建了小鼠NMB和NMBR基因过表达质粒,能够显著提高RAW264.7细胞NMB和NMBR基因的表达量。

Objective To construct the lentiviral overexpression vectors of mouse neuromedin B(NMB)and neuromedin B receptor(NMBR)genes and express them in RAW264.7 cells,so as to lay a foundation for further study on the effects of mouse NMB and NMBR gene overexpression on the proliferation and apoptosis of RAW264.7 cells.Methods The coding sequences(CDSs)of mouse NMB and NMBR genes were amplified by RT-PCR and inserted into vector pCD513B-1 to construct recombinant plasmids pCD513B-1-NMB and pCD513B-1-NMBR.Mouse NMB and NMBR gene overexpression lentiviruses were obtained through packaging by HEK-293T cells,and the virus titers were detected by double dilution method.After infection with lentivirus for 48 h,RAW264.7 cells were detected for the expression of NMB and NMBR mRNA by qPCR using the uninfected cells as control.Results The recombinant plasmids were constructed correctly as identified by double enzyme digestion.The virus titers of mouse NMB and NMBR gene overexpression lentiviruses were 6×106and 8×106TU/mL,respectively.The mRNA expression levels of mouse NMB and NMBR genes in RAW264.7 cells transfected with two lentiviruses were significantly higher than those in the control group(t=24.158 and 14.958,respectively,each P<0.01).Conclusion Mouse NMB and NMBR gene overexpression vectors were successfully constructed,which can significantly increase the expression of NMB and NMBR genes in RAW264.7 cells.