TRPV1基因的shRNA逆转录病毒载体的构建和鉴定

目的构建针对人瞬态电压感受器阳离子通道,子类V,成员1(TRPV1)基因的p SUPERretro-puro TRPV1逆转录病毒表达载体,感染U87-MG细胞,观测其沉默TRPV1基因的效果。方法利用Ambion在线设计软件设计针对人TRPV1基因的sh RNA干扰序列,克隆到p SUPERretro-puro载体上。对重组质粒进行DNA序列测定和酶切分析。用磷酸钙法制备p SUPERretro-puro TRPV1逆转录病毒,将其感染到U87-MG细胞,经嘌呤酶素筛选阳性克隆后采用荧光定量PCR及Western blotting法检测TRPV1表达。结果经DNA测序和酶切鉴定表明,p SUPERretro-puro TRPV1表达载体构建成功。p SUPERretropuro TRPV1逆转录病毒感染U87-MG细胞后,细胞中TRPV1表达量明显降低。结论成功构建了针对人TRPV1基因的p SUPERretro-puro TRPV1表达载体。p SUPERretro-puro TRPV1能有效下调TRPV1基因在U87-MG细胞中的表达,为将来应用其研究TRPV1在利多卡因致细胞损伤中的作用奠定了基础。

Objective To construct a recombinant retroviral vector expressing shRNA targeting human TRPV1 gene and observe its down-regulation effect in the U87-MG cell line. Methods The oligonucleotides designed by Ambion on-line CAD software targeting to TRPV1 were cloned into the pSUPERretro RNAi plasmid. The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis. The pSUPERretro-puro TRPV1 shRNA retrovirus was prepared by calcium phosphate method and transfected into U87-MG cells. After the screening by purinase, the expression levels of TRPV1 mRNA and protein were detected by lfuorescent quantitation PCR and Western blotting. Results The expression vector pSUPERretro-puro TRPV1 shRNA was successfully constructed, which was conifrmed by the DNA sequencing and the enzyme digestion analysis. The pSUPERretro-puro TRPV1 shRNA retrovirus can down-regulate expression of TRPV1 effectively after transfection in U87-MG cells. Conclusion The pSUPERretro-puro TRPV1 shRNA expression vector was successfully constructed. The expression of TRPV1 gene was down-regulated effectively in U87-MG cells transfacted with pSUPERretro-puro TRPV1 shRNA, which laid a basis for its application in the research of cell injury induced by lidocaine.