S6K1 shRNA基因重组腺病毒的构建及对基因沉默效果的研究

Construction and gene silencing evaluation of S6K1 shRNA gene recombinant adenovirus

目的 构建S6K1 shRNA基因重组腺病毒（S6K1Ax）并在细胞及小鼠肝脏验证对S6K1基因的沉默效果.方法 设计3种S6K1 shRNA序列,通过在pcPUR质粒与pcDNA3.1质粒、cosmid质粒之间的拼接将S6K1 shRNA转染进腺病毒,筛选沉默效果最佳的S6K1Ax并在293细胞扩增纯化得到高效价的S6K1Ax.感染来源于小鼠的肝脏、肌肉、脂肪细胞系,在Western blot水平评价其对S6K1蛋白表达的抑制效果.将S6K1Ax注射进C57BL/6J小鼠尾静脉,6 d后处死小鼠取肝脏,逆转录-聚合酶链反应和Western blot观察小鼠肝脏S6K1在mRNA和蛋白水平的表达.检测注射S6K1Ax前后小鼠血清丙氨酸氨基转移酶（ALT）变化.结果 Western blot证实制备的S6K1 Ax可抑制来源于小鼠的肝脏、肌肉、脂肪3种细胞系和小鼠C57BL/6J肝脏S6K1的蛋白表达.小鼠肝脏S6K1 mRNA结果显示：对照组为1.39±0.21,实验组为0.63±0.09,t=6.132,P〈0.01,差异有统计学意义.S6K1Ax注射小鼠,ALT水平注射前为（15.15±4.43）U/L,注射后为（17.32±4.22）U/L,t=1.451,P〉0.05,差异没有统计学意义.结论 构建的S6K1Ax可将来源于小鼠的细胞系及C57BL/6J小鼠肝脏S6K1基因沉默,为研究S6K1的基因功能提供了适合的实验工具.

Objective To construct S6K1 shRNA gene recombinant adenovirus（S6K1 Ax）and evaluate its gene silencing effects on mouse cell lines and C57 BL/6J mice level.Methods Three S6K1 shRNA gene sequences were designed and spliced from pcPUR plasmid,pcDNA3.1 plasmid to cosmid plasmid and transfected into adenovirus.S6K1 Ax which has best gene silencing effect was selected and proliferated in 293 cell.Silencing effect of S6K1Ax was checked on mice AML12,C2C12,3T3-L1 cell lines.C57BL/6J liver was obtained after S6K1 Ax was injected into mice tail vein six days later.S6K1 was evaluated by qRT-PCR and Western blot.Alanine transferanse（ALT）was examined before and after S6K1 Ax injected.Results S6K1Ax can silence S6K1 expression of mouse AML12,C2C12 and 3T3-L1 cell lines and liver of C57 BL/6J mice on Western blot.S6K1 mRNA expression of C57BL/6J liver were control group 1.39±0.21 vs S6K1Ax group 0.63±0.09,t=6.132.P〈0.01.ALT of mice hepatic function did not change after S6K1Ax injected：before（15.15±4.43）U/L,after（17.32±4.22）U/L,t=1.451,P〉0.05.Conclusion Construction of shS6K1 Ax can knockdown S6K1 gene on mice cell lines and C57BL/6J mice liver,it provides a good tool to study the function of S6K1.