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目的构建过氧化物酶体增殖激活受体γ(PPARγ)基因RNA干扰慢病毒载体并鉴定。方法设计针对PPARγ的小发夹RNA(shRNA)序列,应用基因重组技术插入到pLentiLox3.7(pLL3.7)慢病毒载体中,XbaⅠ和XhoⅠ双酶切和DNA测序鉴定重组克隆。将pLL3.7空载体(NC组)、pLL-PPAR-γsh1RNA(S1组)、pLL-PPAR-γsh2RNA(S2组)三组质粒分别与辅助包装质粒共转染人胚肾293T细胞,并测定病毒滴度;病毒感染大鼠肾上腺嗜铬细胞瘤PC12细胞和大鼠海马神经干细胞后,分别用RT-PCR和Western blot检测PPARγmRNA和蛋白表达。结果 PPARγshRNA成功插入慢病毒载体。慢病毒载体经293T细胞包装成功,测定病毒的滴度为5×106IU/ml。与NC组相比,S1、S2组PPARγmRNA水平下降(P<0.05),且S1组PPARγ蛋白表达亦明显下降(P<0.05)。结论成功构建PPARγ基因RNA干扰慢病毒载体。
Objective To construct the RNA interference lentivirus vector of peroxisome proliferator - activated receptor γ (PPARγ) gene and identify it. Methods A small hairpin RNA (shRNA) sequence targeting PPARγ was designed and inserted into pLentiLox3.7 (pLL3.7) lentiviral vector using gene recombination technique. The recombinant clones were identified by double digestion with Xba Ⅰ and Xho Ⅰ and DNA sequencing. Three plasmids of pLL3.7 empty vector (NC group), pLL-PPAR-γsh1RNA (S1 group) and pLL-PPAR-γsh2RNA (S2 group) were cotransfected into 293T cells of human embryonic kidney separately with the helper packaging plasmid, The expression of PPARγmRNA and protein was detected by RT-PCR and Western blot respectively after infection of rat adrenal pheochromocytoma PC12 cells and rat hippocampal neural stem cells. Results PPARγ shRNA was successfully inserted into the lentiviral vector. The lentiviral vector was successfully packaged in 293T cells and the titer of the virus was determined to be 5 × 10 6 IU / ml. Compared with NC group, PPARγmRNA level in S1, S2 group decreased (P <0.05), and PPARγ protein expression in S1 group also decreased significantly (P <0.05). Conclusion The PPARγ gene RNAi lentiviral vector was successfully constructed.