大鼠蛋白激酶Cγ基因小发卡RNA慢病毒载体的构建及其干扰效率的鉴定  

Construction of shRNA lentivirus vector targeting rat PKCγ gene and identification of its interference efficiency

作　　者：肖兴鹏[1] 田学愎[1] 曹菲[1] 陈益[1] 高峰[1] 田玉科[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉科教研室,湖北武汉430030

出　　处：《医学研究生学报》2009年第10期1012-1015,I0001,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30672027);高等学校博士学科点专项科研基金资助项目(批准号:20060487046)

摘　　要：目的:研究指出蛋白激酶Cγ(PKCγ)在脊髓水平上参与了伤害性信号的传递,文中拟构建大鼠PKCγ基因的小发卡RNA(shRNA)慢病毒载体,并在细胞水平鉴定其干扰效率。方法:根据PKCγ-mRNA序列,选择3条19nt的靶序列,设计并合成包含正、反义靶序列的互补DNA链,退火后插入到pLVTHM载体的H1启动子后获得重组质粒,同时构建一个非特异性对照质粒。将所构建的质粒与pMDLg-pRRE、pR sv-REV、pMD2G共转染293T细胞,包装产生慢病毒后,分别感染C6细胞,经免疫印迹技术(W estern b lot)检测PKCγ基因的蛋白表达水平以评价慢病毒载体的抑制效率。结果:测序证实成功构建了3个大鼠PKCγ基因shRNA慢病毒载体,分别感染C6细胞后pLV-PKC2和pLV-PKC3可明显降低PKCγ蛋白的表达,其中以pLV-PKC2(靶向位点:+1913^+1931)的慢病毒抑制效率最高。结论:成功构建了大鼠PKCγ基因shRNA慢病毒载体,且慢病毒载体pLV-PKC2能特异、高效地抑制PKCγ基因的表达。0bjective： Protein kinase C（PKC）,especially PKCγ,is reportedly involved in the nociceptive processing at the spinal level.This study was designed to construct the shRNA lentivirus vectors of the rat PKCγ gene and identify their interfering efficiency at the cell level.Methods： Three target sequences（19nt） were selected according to the PKCγ-mRNA sequence and the sense and antisense oligo-nucleotides were designed and synthesized.After annealing,these double DNA strands were cloned to the pLVTHM vector that contained the H1 promoter and green fluorescent protein（GFP）.Meanwhile,an unrelated sequence was used as the negative control.All virus stocks were produced by transfection of 293T cells with the vector plasmid,pMDLg-pRRE,Rsv-REV and pMD2G.Then the lentivirus was transduced to the C6 cells and the inhibition efficiency of the recombinant shRNA lentivirus vector was identified by Western blotting.Results： The results of DNA sequencing confirmed the successful construction of the 3 shR...更多NA lentivirus vectors,among which pLV-PKC2（site： ＋1913-＋1931） possessed the highest inhibition efficiency.Conclusion： The shRNA lentivirus vectors of pLV-PKC1-3 were constructed successfully and the shRNA lentivirus vector of pLV-PKC2 could specifically and highly efficiently down-regulate the PKCγ gene expression.

关 键 词：蛋白激酶CΓ 慢病毒 RNA干扰 

分 类 号：Q782[生物学—分子生物学]