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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
