WDR5真核表达载体的构建及其在LNCaP细胞中的稳定表达

目的 构建人源WDR5全长结构基因真核表达载体, 建立稳定表达WDR5的LNCaP细胞株.方法PCR扩增WDR5基因, 将WDR5插入pCI-neo载体中, 酶切及测序鉴定重组质粒.利用脂质体转染法将重组质粒转染LNCaP细胞, Real Time-PCR检测转染细胞WDR5的m RNA表达水平.结果 经酶切及测序证实真核表达载体pCI-neo-WDR5构建正确.重组质粒转染到LNCaP细胞后, 用G418筛选获得稳定表达的细胞株, Real Time-PCR方法检测到WDR5基因在转录水平的表达.结论 PCI-neo-WDR5成功转染LNCaP细胞株中并稳定表达, 为下一步WDR5的深入研究及应用奠定了基础.

Construction of WDR5 Eukaryotic Expression Vector and Its Stable Expression in LNCaP Cells

Objective To construct a recombinant vector containing human WDR5 and establish LNCaP cell strain with stable expression of WDR5. Me thods WDR5 gene was amplified by PCR and was cloned into eukaryotic expression vector pCI-neo. The resulted pCI-neo-WDR5 plasmid was verified by restriction enzyme digestion and DNA sequencing. LNCaP cells were transfected with pCI-neo-WDR5 by liposome. The expression of WDR5 in the cells was examined by Real-time PCR. Re s ults Restriction enzyme digestion and sequencing analysis showed that WDR5 c DNA was correctly cloned into pCI-neo vector. After transfection of LNCaP cells with the recombinant plasmids, the monoclonal cell strain of stably expressed were obtained by G418 screening, which showed expressions of WDR5 m RNAs detected by Real Time-PCR. Conclus ion The recombinant of PCI-neo-WDR5 stably express in the transfected LNCaP cells, which laid a foundation for further study on WDR5.