TRPV1基因过表达逆转录病毒载体的构建和鉴定

Construction and Identification of Retrovirus Vector Over-Expressing TRPV1 Gene

目的 TRPV1基因过表达逆转录病毒载体的构建和鉴定研究。方法 PCR法扩增TRPV1基因的编码序列,克隆到pBaBe-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pBaBe-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用荧光定量PCR和Western blot法检测TRPV1表达。结果经DNA测序和酶切鉴定表明,pBaBe-puro-TRPV1表达载体构建成功。pBaBe-puroTRPV1逆转录病毒感染U87-MG细胞后,细胞中TRPV1表达量明显增高。结论成功构建了人TRPV1基因的pBaBe-puro-TRPV1表达载体。pBaBe-puro-TRPV1能有效上调TRPV1基因在U87-MG细胞中的表达,为应用其研究TRPV1在利多卡因致细胞损伤中的作用奠定了基础。

Objective Construction and identification of recombinant retroviral vector expressing TRPV1 gene. Methods Human TRPV1 gene coding sequence was amplified and cloned into the pBaBe - puro vector. The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis. The pBaBe - puro - TRPV1 retrovirus was prepared by calcium phosphate method and transformed into U87 - MG cells. After the screening by purinase, the expression levels of TRPV1 mRNA and protein were detected by fluorescent quantitation PCR and Western blot. Results The expression vector pBaBe -puro -TRPV1 was successfully constructed, which was confirmed by the DNA sequencing and the enzyme digestion analysis. The pBaBe - puro - TRPV1 retrovirus can up - regulate expression of TRPV1 effectively after transfection in U87 - MG cells. Conclusion The pBaBe - puro - TRPV1 expression vector was successfully constructed. The protein expression of TRPV1 gene was up - regulated effectively in U87 - MG cells transfacted with pBaBe - puro - TRPV1, which laid a basis for its application in the research of cell injury induced by lidocaine.