大鼠PPARγ shRNA慢病毒载体的构建与鉴定  

Construction and identification of PPARγ shRNA lentiviral vector in rats

作　　者：叶宽萍[1] 王少华[1] 孙子林[1] 黄艳[1] 

机构地区：[1]东南大学附属中大医院内分泌病科,南京市210009

出　　处：《江苏医药》2011年第13期1528-1531,共4页Jiangsu Medical Journal

摘　　要：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 and identify a lentiviral vector for RNA interference of the peroxisome proliferators-activated receptor γ（PPARγ） gene.Methods A pair of complementary small hairpin RNA（shRNA） oligonucleotides targeting PPARγ was designed and inserted into pLentiLox3.7（pLL3.7） vector by gene recombinant technique,which was then identied by double digestion with XbaⅠ/XhoⅠ and DNA sequencing.The pLL3.7 empty vector（group NC）,pLL-PPARγ-sh1RNA（group S1） and pLL-PPARγ-sh2RNA（group S2） were respectively co-transfected with packaging plasmids into human embryonic kidney 293T cells,and the virus titer was then determined.After rat pheochromocytoma PC12 cells and hippocampus neural stem cells were infected with virus,the expressions of PPARγ mRNA and protein were detected by RT-PCR and Western blot,respectively.Results The shRNA sequence was successfully inserted into the lentiviral vector.The recombinant lentiviral vector was successfully packaged in 293T cells,and the virus titer was 5×106IU/ml.Compared with group NC,the levels of PPARγ mRNA were decreased in groups of S1 and S2（P0.05）,and the expression of PPARγ protein was also significantly declined in group S1（P0.05）.Conclusion A lentiviral vector for RNA interference targeting the PPARγ gene is successfully constructed.

关 键 词：过氧化物酶体增殖激活受体Γ RNA干扰 慢病毒载体 

分 类 号：R587[医药卫生—内分泌]