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

作　　者：李玉荣[1] 周向梅[1] 尹晓敏[1] 杨建民[1] 乔俊文[1] 赵德明[1] 

机构地区：[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

出　　处：《畜牧兽医学报》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)

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.

关 键 词：RNAI C6细胞 朊蛋白 表达 超氧化物歧化酶 

分 类 号：S852.659.7[农业科学—基础兽医学]