Construction of lentiviral vector for late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 interference and its role on inflammatory factor secretion of macrophages / 中华传染病杂志

ABSTRACT

Objective To construct lentiviral vector of late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 ( lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.Methods Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage.There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control.The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction ( RT-qPCR ) and Western blot.The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-αsecreted by the cells were detected by RT-qPCR.T test was used for comparison between groups.Results The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully.The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000 ±0.000, 0.596 ±0.125 and 0.120 ±0.080, respectively.The expression of lamtor2 in the sh2 group was significantly lower than that in the sh 1 group (t＝3.399, P＝0.015), and they were both significantly lower than the control group ( t ＝3.333 and 9.734, respectively, both P ＜0.05).After infection with Klebsiella pneumoniae, expression levels of IL-1β( t ＝15.20), IL-6 (t＝43.30) and TNF-α(t＝12.67) were significantly higher than those in the control group (all P＜0.01).Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism , which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.