竞争性RT-PCR检测铜对Ctr1 mRNA表达的影响  

Determination of Effect of Copper on the Expression of Ctr1 mRNA in PK15 Cell by Competitive Polymerase Chain Reaction

作　　者：付世新[1] 郑鑫[2] 梁海涛[2] 朱世成[1] 王哲[1] 张艳宇[1] 

机构地区：[1]解放军军需大学军事兽医系 [2]吉林农业大学动物科技学院,吉林长春130118

出　　处：《中国兽医学报》2004年第2期186-189,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目 (3 9970 5 67)

摘　　要：为了应用竞争 RT- PCR法检测铜对猪传代肾细胞 (PK15 )中特异性铜转运蛋白 Ctr1基因 m RNA表达水平的影响 ,经 2次克隆 ,将筛选出一段 4 0 0 bp的核苷酸序列 ,插入 RT- PCR扩增出的 5 30 bp Ctr1目的片段中 ,构建了插入突变型 Ctr1- c DNA竞争模板。再将竞争模板与各试验组 c DNA同时进行 PCR扩增。结果 ,猪肾 PK15细胞 Ctr1基因m RNA表达量在 7.8～ 31.2 μmol/ L Cu范围内随铜添加浓度的升高减少 ,当铜添加浓度达到 6 2 .5 μmol/ L 时 ,其表达量又有所回升 ,呈双相反应。To quantitatively determine the effect of copper on Ctr1 mRNA in PK15 cell by competitive polymerase chain reaction,a 400 bp fragment was inserted into the 530 bp fragment with two primers and a new recombinant plasmid was constructed as the internal DNA competitive template.Competitive template was amplified with cDNA in the experimental groups.The expression of copper transporter(Ctr1) gene mRNA showed a double-way response to copper ionic concentration in culture media,decreasing with enhancing copper ionic concentration from 7.8 to 31.2 μmol/L Cu and increasing with enhancing copper ionic concentration to 62.5 μmol/L Cu in culture media.

关 键 词：铜 PKl5细胞 CtrI基因 克隆 竞争RT-PCR 猪 传代肾细胞 

分 类 号：S816.72[农业科学—饲料科学]