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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 a shRNA targeting B7-H1, construct a lentiviral expression vector, and detect its silencing effect on B7-H1 gene in U251 cells after packaging into viral particles. METHODS: Three pairs of shRNA targeting B7-H1 mRNA were designed, the sense and antisense strands were chemically synthesized, and annealed to ligate the pLKO.1 vector. After sequencing, the virus was packaged and infected into U251 cells. The interference effect was detected by qRT-PCR and Western blot respectively. Results The results of qRT-PCR and Western blot confirmed that two of the three designed RNA interference sequences effectively silenced the expression of B7-H1 in U251 cells. Conclusion The prepared shR-NA lentivirus against B7-H1 can specifically silence B7-H1.