畜牧兽医学报 ›› 2008, Vol. 39 ›› Issue (3): 337-342.doi:

• 预防兽医 • 上一篇    下一篇

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

李玉荣;周向梅;尹晓敏;杨建民;乔俊文;赵德明   

  1. 1．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，北京 100094；2．河北农业大学动物科技学院，保定 071000
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2008-03-24 发布日期:2008-03-24

The Establishment and Application of Inhibited Expression Vector of Prion Protein by RNA Interference

LI Yu-rong;ZHOU Xiang-mei;YIN Xiao-min;YANG Jian-min;QIAO Jun-wen;ZHAO De-ming   

  1. 1. National Animal Transmissible Spongiform Encephalopathies Laboratory，College of Veterinary Medicine, China Agricultural University,Beijing 100094,China；2.College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000，China
  • Received:1900-01-01 Revised:1900-01-01 Online:2008-03-24 Published:2008-03-24

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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。

Abstract: 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 Top10 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 LipofectamineTM2000．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.

ISSN 0366-6964
CN 11-1985/S
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