【摘要】 目的:利用pGenesil-1质粒构建针对热休克蛋白HSP70-2的短发夹RNA(shRNA)表达载体。方法:设计针对HSP70-2表达shRNA结构的互补DNA序列,经退火成双链,分别克隆至带有U6启动子的质粒载体pGenesil-1中,构建重组质粒,转化大肠杆菌DH5α菌株,扩增,提取质粒,酶切鉴定后测序分析。利用质粒转染大鼠精原细胞,用RT-PCR和Western blotting法检测转染后HSP70-2 mRNA和蛋白表达变化。结果:成功构建靶向HSP70-2的3个shRNA重组质粒载体pGenesil-1-HSP70-21、pGenesil-1-HSP70-22和pGenesil-1-HSP70-23。酶切鉴定和测序分析重组质粒,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功。质粒筛选实验提示pGenesil-1-HSP70-23质粒对精原细胞HSP70-2基因的抑制作用最强,而pGenesil-1-HSP70-21的抑制作用最弱。结论:成功构建表达靶向HSP70-2的shRNA重组质粒载体。更多还原

【Abstract】 Objective: To construct the recombinant plasmid expressing HSP70-2-targeted short hairpin RNA(shRNA) by pGenesil-1 plasmids vector.Methods: Three pairs of DNA sequences were designed,and then synthesized into complementary chains by annealing,respectively.Then the obtained products containing short hairpin structure were inserted into plasmid vector pGenesil-1 with U6 promoter.The recombinant plasmids were transformed into Escherichia coli strain DH5α for screening and amplifying.The sequence analysis of the plasmids was identified by restriction enzyme.Results: The three HSP70-2-targeted shRNAs were successfully inserted into the plasmid vector pGenesil-1,and the coding sequences of the obtained shRNAs were consistent with the designed fragments.pGenesil-1-HSP70-23 exerted the most significant inhibition effect on HSP70-2 gene.Conclusion: The recombinant plasmid series of HSP70-2-targeted shRNAs is successfully constructed in pGenesil1 plasmid.更多还原