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Omi/HtrA2 shRNA重组真核表达质粒的构建及其在大鼠肾小管上皮细胞缺氧/复氧模型中的作用 被引量:1

Construction of Omi/HtrA2 specific small hairpin RNA expression plasmid and its role in a hypoxia-reoxygenation model
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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
关键词 OMI/HTRA2 SHRNA 重组质粒 Omi/HtrA2 shRNA recombinant plasmid
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同被引文献14

  • 1王晓樑,王瑾,吕小萍,王澎,刘慧荣.线粒体丝氨酸蛋白酶Omi/HtrA2与细胞凋亡[J].生理科学进展,2006,37(3):285-288. 被引量:7
  • 2徐宗全,陈孝平,李常海,李高鹏,张志发.线粒体促凋亡因子Omi/HtrA2在肝癌组织中表达的研究[J].中国组织化学与细胞化学杂志,2006,15(6):617-622. 被引量:7
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