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RNAi抑制PrP表达载体的构建及其在PrP功能研究中的初步应用

The Establishment and Application of Inhibited Expression Vector of Prion Protein by RNA Interference
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摘要 本文旨在构建有效抑制朊蛋白(Prion protein,PrP)表达的重组质粒,并以此为工具控制PrP表达,从而探讨PrP对细胞SOD活性的影响。设计并化学合成1对含有发夹结构的寡核苷酸片段(shPrP),退火后与表达载体pG-super(Hairpin siRNA expressing vector)定向连接,构建重组质粒pG-super-shPrP。对重组子进行PCR鉴定,测序正确后,脂质体法转染C6细胞,采用实时荧光定量RT-PCR检测PrP mRNA的表达水平,以验证pG-super-shPrP的抑制效率;结果表明:重组质粒pG-super-shPrP构建成功,且显著降低C6细胞PrP mRNA表达(P<0.05),抑制效率为34.2%。利用pG-super、pG-super-shPrP分别转染C6细胞,并检测细胞SOD总活性及SOD表达水平,探讨PrP对细胞SOD活性的影响及其作用机制,结果表明PrP促进细胞SOD的活性(P<0.01),但对细胞SOD的表达量无影响,即PrP对SOD活性的促进作用与SOD1的表达量无关。本研究在成功构建了PrP的RNA干扰表达质粒的基础上,利用此质粒,在细胞水平上揭示了PrP对细胞SOD活性的促进作用。 In present research,we intend to construct pG-super-siPrP expression plasmid and explore its function in C6 cells, shPrP was subcloned into pG-super. Recombinant plasmid pG-super-shPrP was transformed into Topl0 E. coli, and the ampicillin resistant clones were identified by PCR and DNA sequencing. C6 cells were transfected with the identified pG-super-shPrP and pG-super （the control group） by LipofectamineTM 2000. PrP mRNA was quantified by real-time RT-PCR. The results showed that the expression of PrP mRNA in pG-super-shPrP group decreased by 34.2% compared with the control group （P〈0. 05）. The detection results of the total activity of superoxide dismutase indicated that PrP promoted the SOD activity. In conclusion, pG- super-shPrP expression plasmid was constructed effectively and took effect in C6 cells, which helped to further study about PrP biochemical and physiological role in animal and offer a attractive therapeutic approach to fight against prion disease.
作者 李玉荣 周向梅 尹晓敏 杨建民 乔俊文 赵德明
机构地区 中国农业大学动物医学院国家动物传染性海绵状脑病实验室
出处 《畜牧兽医学报》 CAS CSCD 北大核心 2008年第3期337-342,共6页 ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金 国家自然基金(30500371) 国家科技支撑计划(2006BAD06A14) 科技部基金(2005KDA21205-7)
关键词 RNAI C6细胞 朊蛋白 表达 超氧化物歧化酶 RNAi C6 cells prion protein（PrP） expression superoxide dismutase （SOD）
分类号 S852.659.7 [农业科学—基础兽医学]
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参考文献13

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畜牧兽医学报
2008年 第3期

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