MEK5基因siRNA重组腺病毒表达载体的构建及其对MEK5的表达抑制

Construction of MEK5 siRNA Recombinant Adenovirus Vector and Its Inhibitory Effect on MEK5 Expression

目的 构建人MEK5基因的小干扰RNA（siRNA）重组腺病毒表达载体,观察其在胃腺癌SGC7901细胞中对MEK5的表达抑制.方法 设计并合成针对人MEK5基因3个不同部位siRNA靶点的模板DNA序列,以MluⅠ及XhoⅠ克隆入pRNAT-H1.1/Adeno穿梭载体中,将得到的重组穿梭质粒用PmeⅠ线性化后在大肠埃希菌BJ5183中与腺病毒骨架质粒pAdEasy-1进行同源重组.将PacⅠ酶切鉴定正确的重组腺病毒质粒经乙醇沉淀后转染293A细胞,包装得到具有感染能力的pAd-MEK5-siRNA重组腺病毒.病毒体外转导人胃腺癌SGC7901细胞,Western印迹法检测其对MEK5表达的抑制.结果 经酶切和测序鉴定均证实pAd-MEK5-siRNA重组腺病毒载体构建成功,其插入序列正确无误.Western印迹检测结果显示pAd-MEK5-siRNA2可抑制MEK5基因的表达,其有效抑制率达75.5 %.结论本研究成功地构建了针对人MEK5基因的siRNA重组腺病毒载体,为进一步深入研究MEK5基因在人胃腺癌细胞中的作用和功能奠定了基础.

Objective To construct a small interfering RNA （siRNA） adenovirus expression vector targeting MEK5 and to study its inhibitive effect on MEK5 expression in gastric adenocarcinomas SGC7901 ceils. Methods Three different siRNA template DNA sequences of MEK5 gene were designed by Genscript siRNA design software. The corresponding DNA vitro, annealed and then cloned into the pRNAT-H1.1/Adeno shuttle fragments were synthesized in vector. The recombinant shuttle vectors were confirmed by DNA sequencing, then transformed into Escherichia coli B J5183 carry- ing backbone plasmid PAdeasy-1 to obtain adenovirus plasmid through homologous recombina- tion. The adenovirus plasmid was transfected into 293A cells to form adenovirus particle. Then, the adenovirus particles were transduced into SGC7901 cells. Western blot was carried out to analyze the suppression effect of MEK5 siRNA vectors in SGC7901 cells and to screen the best vector that had the highest effect on inhibition of MEK5 expression. Results DNA sequencing and western blot confirmed the effective silence effect on MEK5 by one MEK5 siRNA adenovirus expression vector pRNATH1.1/Adeno-MEK5 siRNA2, with a suppression ratio up to 75.5 %. Conclusion The recombinant pAd-MEKS-siRNA expression vector effectively targeting MEK5 provides a tool for further investigation of MEK5 function in gastric adenocarcinomas cells.