MEKK2基因siRNA重组腺病毒表达载体的构建及其对MEKK2的表达抑制

Construction of MEKK2 siRNA Recombinant Adenovirus Vector and Its Effect on the Inhibition of MEKK2 Expression

设计并合成针对人MEKK2基因3个不同部位siRNA靶点的模板DNA序列,将合成的互补片段退火后克隆入pRNAT-H1.1/Adeno穿梭载体中,并使其在大肠杆菌BJ5183中与腺病毒骨架质粒pAdEasy-1进行同源重组。将经鉴定正确的重组腺病毒质粒转染293A细胞,包装得到具有感染能力的pAd-MEKK2-siRNA重组腺病毒。病毒体外转导人胃腺癌AGS细胞,Western blot印迹法检测其对MEKK2表达的抑制。经酶切和测序鉴定均证实pAd-MEKK2-siRNA重组腺病毒载体构建成功,其插入序列正确无误。Western blot印迹检测结果显示,重组腺病毒表达载体可抑制MEKK2基因的表达,以pAd-MEKK2-siRNA2(针对MEKK2 cDNA 992-1 010的片段)抑制效果为最佳,pAd-MEKK2-siRNA1(针对MEKK2 cDNA 1 456-1 474的片段)和pAd-MEKK2-siRNA3(针对MEKK2 cDNA 1 351-1 369的片段)则未见明显的抑制效果。DNA Ladder和细胞存活测定结果表明,敲减MEKK2的表达后,AGS细胞接受H2O2刺激后的凋亡受到较强抑制、细胞存活数增加,明显高于野生型细胞和转导siRNA阴性对照腺病毒细胞接受H2O2刺激后的,差异具有统计学意义(P<0.05),而后两者之间差异无统计学意义(P>0.05)。该研究成功地构建了针对MEKK2基因的siRNA重组腺病毒载体,为进一步深入研究MEKK2基因在人胃腺癌细胞中的作用和功能奠定了基础。

Three different siRNA template DNA sequences of MEKK2 gene were designed by Genscript siRNA design software.The corresponding DNA fragments were synthesized in vitro,annealed and then cloned into the pRNAT-H1.1/Adeno shuttle vector.The recombinant shuttle vectors were confirmed by DNA sequencing,then transformed into Escherichia coli BJ5183 carrying backbone plasmid pAdEasy-1 to obtain adenovirus plasmid through homologous recombination.The adenovirus plasmid was transfected into 293A cells to form adenovirus particle.Followed,the adenovirus particles were transduced into AGS cells.Western blot was carried out to analyze the suppression effect of MEKK2 adenovirus expression vectors in AGS cells and to screen the best vector that had the highest effect on inhibition of MEKK2 expression.DNA sequencing confirmed that the MEKK2 siRNA adenovirus expression vector were successfully constructed and the results of Western blot showed that one of the three MEKK2 siRNA adenovirus vectors（pAdeno-MEKK2 siRNA2,targeting MEKK2 cDNA sequences of 992-1 010） could effectively silence MEKK2 expression in AGS cells.No obvious inhabition effect was found in pAdenoMEKK2 siRNA1（targeting MEKK2 cDNA sequences of 1 456-1 474） and pAdeno-MEKK2 siRNA3（targeting MEKK2 cDNA sequences of 1 351-1 369）.In conclusion,the recombinant pAd-MEKK2-siRNA expression vector targeted on MEKK2 was successfully constructed,which can effectively suppress MEKK2 expression in AGS cells.The apoptosis induced by H2O2 was significantly lower,whereas cell survival number was significantly higher in MEKK2 knocked down AGS cells than those in transduced siRNA negative control cells or wild type cells（P0.05）.This study will provide a foundation for further investigation on the function of MEKK2 gene in gastric adenocarcinomas cells.