摘要
目的:探索对人PTTG1基因mRNA序列有较高干扰效率的位点,构建对PTTG1基因的表达有较高抑制效率的重组质粒。方法:筛选、合成1对互补DNA单链,退火为双链后与双酶切后的质粒载体连接,构建了表达短发夹RNA的1种重组质粒plk0.1-puro/PTTG1。重组质粒以不同浓度转染,并于转染后不同时间收集细胞,以β-actin为内参,采用RT-PCR和Western blot技术分别检测PTTG1基因的mRNA、蛋白质的相对表达水平。结果:与空白对照组相比,6孔板中每孔转染质粒plk0.1-puro/PTTG 11000ng以上、转染超过12d时,PTTG1基因mRNA、蛋白质的相对表达水平下降70%以上。结论:重组质粒plk0.1-puro/PTTG1,在6孔细胞培养板中以1000ng/孔转染12d以上时,有效抑制了人PTTG1基因的表达,在关于PTTG1基因的研究中具有较好的应用价值。
Objective To explore the interference sites with high efficiency for the mRNA of human PTTG1 gene, and build on the recombinant plasmids which can restrain the expression of PTTG1gene. Methods Two pairs of single-stranded DNA sequences were selected and synthesised, and double-stranded DNA molecules were obtained after annealing. The two molecules were linked respectively to the vector plk0.1-puro plasmid which was digested, then two kinds of recombinant plasmids named plk0.1-puro/PTTG1 was successfully constructed. The two kinds of plasmids were transfected into Hep-2 ceils with different concentrations, and the cells were collected at different times after transfection RT-PCR and Western blot techniques were used to detect the relative expression level of PTTG1 gene＇s mRNA and protein, while 13-actin was used as internal control. Results Compared with the control, in 6-well cell culture plates, when the recombinant plasmid plk0.1-puro/PTTG1 were transfected above 1000ng in each well, and more than 12 d, the relative expression level of PTTG1 gene＇s mRNA and protein reduced at less 70%. Conclusions The recombinant plasmid plk0.1-puro/PTTG1 can effectively inhibit the expression of human PTTG1 gene, it has a better value in studies on PTTG1 gene.
出处
《实用医学杂志》
CAS
北大核心
2011年第16期2913-2917,共5页
The Journal of Practical Medicine
基金
河南省杰出人才创新基金(编号:084200410016)
