p300RNA干扰慢病毒载体的构建及其在CNE-2细胞中的表达

Construction of a Lentivirus Interfering Vector Carrying p300Gene with RNA Interfering and Its Expression in the CNE-2 Cells

目的构建p300基因RNA干扰慢病毒载体获得稳定感染的人鼻咽癌CNE-2细胞株,以观察CNE-2细胞中p300的表达及其功能。方法设计p300基因特异性RNA干扰靶序列,与经Hpa I和Xho I酶切后的载体连接,产生重组慢病毒质粒p LL-GFP-Puro-shp300,筛选阳性克隆,测序鉴定,与慢病毒包装载体共转染293T细胞,包装产生慢病毒,对病毒滴度和感染效率进行检测。结果 p LL-GFP-Puro-shp300慢病毒RNA干扰载体经酶切和测序鉴定证实准确连接测序正确,且包装出来的病毒纯化后滴度达107TU·m L-1。RT-q PCR、Western blot结果显示感染后p300 mRNA及蛋白水平显著下降,证明重组慢病毒对CNE-2细胞p300基因表达有明显沉默作用。结论成功构建靶向p300基因RNA干扰慢病毒载体,为进一步研究p300在鼻咽癌发生过程中的作用奠定基础。

Objective To construct a lentiviral vector carrying p300 gene with RNA interfering, and to establish human nasopharyngeal carcinoma cells with stably inhibited p300. Methods Two pairs of small hairpin RNA specific for p300 were designed, synthesized and cloned into the pLL3.7-GFP-Puro vector. Then, the lentivirual plasmid was transfected with the other two packaging plasmids into 293T cells to product and amplify lentivirus. After the infection of lentivirus mediated siRNA-p300, the mRNA and protein expression of p300 were observed. Results The lentivirus vector of siRNA-p300 was constructed successfully. The virus titres were above 10^7 TU ·mL^-1. pLL-GFP-Puro-shp300 was effective to inhibit p300 expression in mRNA and protein levels of CNE-2 cells. Conclusion A lentiviral vector carrying p300 gene with RNA interfering is successfully constructed. This study lays a foundation for further study of mechanisms of p300 in the nasopharyngeal carcinoma.