含B7-H1 shRNA基因的慢病毒表达载体的制备及其抑制效果鉴定

Preparation and knockdown efficiency evaluation of shRNA expressing lentiviral vector targeting B7-H1

目的设计针对B7-H1的shRNA序列,构建慢病毒表达载体,包装成病毒颗粒后检测其对U251细胞中B7-H1基因的沉默效果。方法设计3对针对B7-H1 mRNA的shRNA,化学合成正义链和反义链,退火后与酶切后的pLKO.1载体进行连接。测序正确后包装成病毒并感染U251细胞,分别用qRT-PCR及Western blot法检测干扰效果。结果 qRT-PCR及Westernblot结果证实设计的3条RNA干扰序列中有2条可有效沉默U251细胞中B7-H1的表达。结论所制备的针对B7-H1的shR-NA慢病毒能特异性沉默B7-H1。

Objective To design and package shRNA expressing lentiviral particles targeting BT-H1, and evaluate their inhibitory effect on BT-H1 expression in U251 cells. Methods Three shRNAs targeting B？-H] was designed and the sense and antisense primers were produced by chemical synthesis. After annealing, they were linked into restriction enzyme diges- ted 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 BT-H1 mRNA and protein levels respectively. Results qRT-PCR and Western blotting showed that two of the three shRNAs effectively knocked-down B？-HI expression in U251 cells. Conclusion The packaged lentiviral particles can specifically inhibit BT-H1 expression, which will be helpful for further functional study on B7-H1.