靶向B7-H1的短发夹RNA表达慢病毒载体的制备及敲低效率评估
[Preparation and knockdown efficiency evaluation of shRNA expressing lentiviral vector targeting B7-H1].
作者信息
Huangfu Luokai, Wang Xi, Guo Zhangyan, Xi Wenjin, Shi Shengjia, Yang Fan, Chen Xiaoyan, Yang Angang, Zhang Jianning, Wen Weihong
机构信息
Department of Neurosurgery, Fourth Military Medical University, Xi'an, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2013 Mar;29(3):242-5.
OBJECTIVE
To design and package shRNA expressing lentiviral particles targeting B7-H1, and evaluate their inhibitory effect on B7-H1 expression in U251 cells.
METHODS
Three shRNAs targeting B7-H1 was designed and the sense and antisense primers were produced by chemical synthesis. After annealing, they were linked into restriction enzyme digested pLKO.1 vector. Confirmed by DNA sequencing, the lentiviral particles were packaged and applied to infect U251 cells. qRT-PCR and Western blotting were used to detect the B7-H1 mRNA and protein levels respectively.
RESULTS
qRT-PCR and Western blotting showed that two of the three shRNAs effectively knocked-down B7-H1 expression in U251 cells.
CONCLUSION
The packaged lentiviral particles can specifically inhibit B7-H1 expression, which will be helpful for further functional study on B7-H1.
目的
设计并包装靶向B7-H1的表达短发夹RNA（shRNA）的慢病毒颗粒，评估其对U251细胞中B7-H1表达的抑制作用。
方法
设计3条靶向B7-H1的shRNA，通过化学合成制备 sense和antisense引物。退火后，将它们连接到经限制性内切酶消化的pLKO.1载体中。经DNA测序确认后，包装慢病毒颗粒并用于感染U251细胞。分别采用qRT-PCR和蛋白质免疫印迹法检测B7-H1的mRNA和蛋白质水平。
结果
qRT-PCR和蛋白质免疫印迹法显示，3条shRNA中有2条能有效下调U251细胞中B7-H1的表达。
结论
包装的慢病毒颗粒可特异性抑制B7-H1的表达，这将有助于对B7-H1进行进一步的功能研究。
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