靶向WNT5A基因的shRNA逆转录病毒表达载体的构建与鉴定

Construction and identif ication of recombinant retroviral vector expressing shRNA targeting human WNT5A gene

目的:构建人WNT5A shRNA逆转录病毒表达载体。方法:根据人WNT5A基因mRNA序列设计并合成两条互补的DNA单链寡核苷酸,将退火后形成的双链连接于pSUPER Retro RNAi质粒,构建WNT5A shRNA逆转录病毒表达载体,经脂质体介导入GPG293细胞,包装成逆转录病毒。用该逆转录病毒感染人鼻咽癌细胞,Western blot法和RT-PCR检测细胞WNT5A的表达。结果:目的序列成功连接于载体并包装成逆转录病毒,免疫印迹检测和RT-PCR检测结果表明构建的WNT5A shRNA逆转录病毒表达载体能显著抑制鼻咽癌细胞WNT5A的表达。结论:成功构建人WNT5A shRNA逆转录病毒表达载体。

Objective:To construct the recombinant retroviral vector expressing shRNA targeting human WNT5A gene. Methods :According to WNT5A mRNA sequence in the Genbank, two pairs of oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSUPER Retro RNAi plasmid. The recombinants were finally sequenced and identified by sequencing. The recombinant plasmids were transfected into GPG293 cells by lipofectamine reagent. Nasopharyngeal cancer cells were infected with the viral supernatant from the GPG293 cells. The alteration of WNT5A expression was examined by Western blotting and RT-PCR assays. Results:pSUPER RetroRNAi/ WNT5A shRNA expression vector were successfully constructed. Sequence analysis revealed that inserted fragment was identical to the synthesized siRNA oligonucleotides. The results of western blotting and RT-PCR showed that transfection of pSUPER RetroRNAi/WNT5A shRNA significantly down-regulated the WNT5A expression. Conclusion:The recombinant plasmid expressing the shRNA targeting WNT5A gene has been successfully constructed, which laying a foundation for further study on WNT5A signaling pathway