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目的:构建大鼠过氧化物酶体增生因子激活受体γ(peroxisomeproliferator-activatedreceptorgamma)基因慢病毒表达载体,获得可供转染的滴度,为进一步研究该基因在肝星状细胞活化(Hepaticstellatecells,HSC)及肝纤维化中的作用机制提供物质基础。方法:大鼠PPAR-γ基因序列进行PCR扩增,与经AgeI酶切后的pGC-FU-3FLAG载体连接产生慢病毒载体表达质粒pGC-fu-3flag-PPARG,转化DH5α,PCR筛选阳性克隆,测序并转入293T细胞Western blot鉴定,而后将pGC-fu-3flag-PPARG,pHelper 1.0,pHelper 2.0三质粒共转染293T细胞,包装成慢病毒,收集上清浓缩病毒测定病毒滴度。结果:DNA测序及Westernblot鉴定证实构建的大鼠PPAR-γ基因慢病毒表达载体pGC-fu-3flag-PPARG正确,浓缩慢病毒悬液的滴度为2×108TU/ml。结论:成功构建携带大鼠PPAR-γ基因的重组慢病毒表达载体。
OBJECTIVE: To construct a lentiviral vector expressing rat peroxisomeproliferator-activated receptor β (gamma) gene and obtain a titer for transfection. To further investigate the role of this gene in hepatic stellate cell activation HSC) and the mechanism of action in liver fibrosis provide the material basis. Methods: The rat PPAR-γ gene sequence was amplified by PCR and ligated with pGC-FU-3FLAG vector after AgeI digestion to generate lentiviral vector pGC-fu-3flag-PPARG. The recombinant plasmid was transformed into DH5α, The 293T cells were sequenced and transfected into 293T cells. The 293T cells were cotransfected into 293T cells with pGC-fu-3flag-PPARG, pHelper 1.0 and pHelper 2.0 and packaged into lentivirus. The supernatant concentrated virus was collected and the virus titer was determined. Results: DNA sequencing and Western blot confirmed that the constructed rat PPAR-γ lentiviral vector pGC-fu-3flag-PPARG was correct and the titer of the lentivirus suspension was 2 × 108TU / ml. Conclusion: The recombinant lentiviral vector carrying rat PPAR-γ gene was successfully constructed.