摘要
目的构建在哺乳动物细胞中表达的Omi/HtrA2短发夹RNA(shRNA)的表达质粒,观察其对大鼠肾小管上皮细胞NRK-52E缺氧/复氧模型中Omi/HtrA2表达的影响。方法根据Genbank中Omi/HtrA2 mRNA分别设计2对多聚核苷酸序列,退火形成互补双链DNA,分别克隆至经双酶切后的载体Pgenesil-1上,构建Omi/HtrA2特异性的shRNA表达载体PGP1和PGP2,进行酶切和测序鉴定。制作NRK-52E缺氧/复氧模型,将表达质粒通过脂质体介导转染NRK-52E,Western blot检测Omi/HtrA2蛋白质表达。结果将合成的DNA片段退火后克隆至Pgenesil-1载体上,构建Omi/HtrA2特异shRNA表达载体PGP1和PGP2;经酶切和测序证实构建成功,无碱基突变发生。将表达质粒介导转染NRK-52E缺氧/复氧模型后,细胞Omi/HtrA2蛋白质表达显著降低。结论成功构建针对大鼠Omi/HtrA2基因的shRNA表达载体PGP1和PGP2,它们能有效抑制NRK-52E缺氧/复氧模型中Omi/HtrA2表达。
Objective To construct and identify two Omi/HtrA2 specific small hairpin RNA(shRNA)expression plasmids,and to explore its influence on Omi/HtrA2 expression in a hypoxia-reoxygenation model.Methods Two pairs of Omi/HtrA2 specific oligonucleotides were designed and synthesized.These oligonucleotides were annealed to form double strand DNA fragments,and then these fragments were cloned into Pgenesil-1 plasmid,respectively.The recombinant Omi/HtrA2-shRNA expression constructs were confirmed by restriction enzymes digestion and by sequencing.Rat renal epithelial cell NRK-52E hypoxia-reoxygenation model was established.The recombined plasmids were transected into NRK-52E with liposome-mediated method.Expression of Omi/HtrA2 in transferred cells was detected by Western blot.Results The Omi/HtrA2-shRNA expression plasmids,named PGP1 and PGP2,were successfully constructed and identified by restriction enzyme and sequence analysis.Expression of Omi/HtrA2 in cells transfected with Omi/HtrA2-shRNA was much lower than that transfected with Pgenesil-1 plasmid.Conclusion Two Omi/HtrA2-shRNA expression plasmids are successfully constructed and they could effectively inhibit Omi/HtrA2 expression in epithelial cells of hypoxia-reoxygenation rats.
出处
《山西医科大学学报》
CAS
2008年第3期214-217,共4页
Journal of Shanxi Medical University