用于转化生长因子β/Smad信号转导通路抑制剂筛选的慢病毒报告载体的构建

Construction of lentiviral reporter vectors for screening inhibitors of transforming growth factor-β/Smad signal transduction pathway

目的构建由转化生长因子β(TGF-β)诱导的含有Smad响应元件(SmadRE)和红色荧光蛋白mCherry或荧光素酶报告基因序列的慢病毒报告载体,为筛选TGF-β/Smad信号转导通路抑制剂提供有效工具。方法合成UAS-SmadRE-Pmin-Gal4VP64-mCherry和UAS-SmadRE-Pmin-Gal4VP64-lucifer⁃ase报告基因序列,分别将其插入经EcoRⅠ和BamHⅠ双酶切后的pCDH-GFP-Puro-noCMV慢病毒质粒载体,获得响应TGF-β的含有SmadRE和mCherry或荧光素酶报告基因序列的慢病毒报告载体,分别命名为pCDH-USPG-mCherry和pCDH-USPG-luciferase。利用慢病毒三质粒包装系统在293FT细胞中分别包装出慢病毒并感染A549细胞(分别命名为USPG-mCherryA549和USPG-luciferaseA549细胞),在倒置荧光显微镜下观察细胞中绿色荧光蛋白(GFP)的表达,鉴定A549细胞是否被感染成功。用TGF-β(10μg·L^(-1))及其受体拮抗剂LY2109761(5μmol·L^(-1))和SB431542(10μmol·L^(-1))分别处理USPG-mCherry A549和USPG-luciferaseA549细胞24 h,倒置荧光显微镜下观察USPG-mCherryA549细胞mCherry的表达,用荧光素酶报告基因检测试剂盒检测USPG-luciferaseA549细胞荧光素酶的表达,验证所构建慢病毒报告载体用于筛选TGF-β/Smad信号转导通路抑制剂的可行性。用TGF-β及其受体拮抗剂LY2109761和SB431542处理A549细胞24 h,Western印迹法检测A549细胞磷酸化Smad2/3蛋白表达,验证LY2109761和SB431542对TGF-β/Smad信号转导通路的抑制作用。结果经限制性内切酶酶切鉴定及测序分析,成功构建pCDH-USPG-mCherry和pCDH-USPG-luciferase慢病毒重组质粒;分别转染293FT细胞包装出慢病毒并感染A549细胞,倒置荧光显微镜下观察到被感染的A549细胞表达GFP,表明USPGmCherryA549和USPG-luciferaseA549细胞构建成功。加入TGF-β处理24 h后,USPG-mCherryA549细胞表达mCherry,USPG-luciferaseA549细胞表达荧光素酶;而加入LY2109761和SB431542后,USPG-mCherryA549细胞几乎检测不到mCherry的表达,USPG-luciferaseA549细胞检测不到荧光素酶的表达。Western印迹法结果表明,TGF-β刺激后A549细胞中有磷酸化Smad2/3蛋白表达,加入LY2109761和SB431542后未检测到磷酸化Smad2/3蛋白表达。结论成功构建由TGF-β诱导的受Smad调控表达mCherry和荧光素酶的2个慢病毒报告载体,可有效感染目的细胞。该慢病毒报告载体可用于筛选TGF-β/Smad信号转导通路抑制剂。

OBJECTIVE To construct lentiviral vectors containing Smad response elements(Smad RE)and red fluorescent protein mCherry or luciferase sequences induced by transforming growth factor-β(TGF-β).METHODS UAS-Smad RE-Pmin-Gal4VP64-mCherry and UAS-Smad RE-Pmin-Gal4VP64-luciferase reporter gene sequences were designed,constructed and inserted into the pCDH-GFP-Puro-noCMV lentiviral plasmid vector digested by enzyme EcoRⅠand BamHⅠ,respectively.The TGF-βresponded recombinant lentiviral vectors containing Smad RE and mCherry or luciferase sequences were named pCDH-USPG-mCherry and pCDH-USPG-luciferase,respectively.pCDH-USPG-mCherry and pCDH-USPG-luciferase were packaged in 293FT cells by using the lentivirus three-plasmid pack⁃aging system and infected A549 cells(named USPG-mCherry A549 and USPG-luciferase A549 cells,respectively),and the expression of green fluorescent protein(GFP)was observed under a fluores⁃cence microscope to find out whether A549 cells were infected.USPG-mCherry A549 and USPG-lucif⁃erase A549 cells were treated with TGF-β(10μg·L^(-1))and its receptor antagonists LY2109761(5μmol·L^(-1))and SB431542(10μmol·L^(-1))for 24 h.The expression of mCherry in USPG-mCherry A549 cells was detected by a fluorescence microscope,while the expression of luciferase was detected by luciferase reporter gene detection kit to verify the feasibility of the constructed lentiviral reporter vector for screening TGF-β/Smad signaling pathway inhibitors.A549 cells was treated with LY2109761 and SB431542 for 24 h,and the phosphorylation of Smad2/3 proteins in A549 cells were detected by Western blotting to verify the inhibitory effect of LY2109761 and SB431542 on TGF-β/Smad signaling pathway.RESULTS The pCDH-USPG-mCherry and pCDH-USPG-luciferase recombinant lentiviral plasmid vectors were constructed and verified by enzyme digestion and sequence.A549 cells were infected by pCDH-USPG-mCherry or pCDH-USPG-luciferase lentivirus packaged in 293FT,respectively.The infected A549 cells were able to express GFP as was observed under a fluorescence microscope,indicating that USPG-mCherry A549 and USPG-luciferase A549 cells were constructed.After 24 h of TGF-βtreatment,USPG-mCherry A549 cells expressed mCherry,and USPG-luciferase A549 cells expressed luciferase.However,after LY2109761 and SB431542 were added,the mCherry expression could hardly be detected in USPG-mCherry A549 cells,and the luciferase expression was not detected in USPG-luciferase A549 cells.Western blotting results showed that phosphorylated Smad2/3 protein was expressed in A549 cells after stimulation with TGF-β,but after LY2109761 and SB431542 were added,phosphorylated Smad2/3 protein was not detected in A549 cells.CONCLUSION Lentiviral reporter vectors have been constructed,which can effectively infect target cells and serve as an effective tool for screening inhibi⁃tors of TGF-β/Smad signaling pathway.