摘要
目的利用干扰RNA技术构建线粒体加工肽酶(mitochondrial processing peptidases,MPPs)干扰RNA真核表达载体,探讨MPPs在神经细胞中的作用,为帕金森病的研究提供有价值的资料。方法(1)根据Gen-Bank提供的大鼠MPPsα亚基(peptidase mitochondrial processing alpha,Pmpca)序列,设计并合成4对MPPs干扰RNA单链寡核苷酸:MPPs-SR-1113,MPPs-SR-1425,MPPs-SR-561,MPPs-SR-903。(2)使互补的单链寡核苷酸模板退火,再转化为能表达其小发卡结构RNA的DNA序列。(3)使PGPU6/GFP/Neo载体的线性化。(4)shRNA与载体的连接。(5)酶切鉴定并测序鉴定结果。结果(1)酶切结果表明,所有质粒均为阳性重组载体。(2)经鉴定证实,构建的siRNAs序列与基因库中序列完全相同,并且未发现有突变、缺失、插入等异常存在。结论利用干扰RNA技术可成功构建MPPs干扰RNA真核表达载体。
Objective Eukaryotic expression vector of RNA interference specific for mitochondrial processing peptidases（MPPs） was constructed. It is critical to research the role that MPPs play in the nerve cells and provide valuable theoretical basis for the parkinsonism. Methods （ 1 ） Four pairs of siRNAs of MPPs gene were designed according to the principle of RNA interference ： MPPs-SR-1113, MPPs- SR-1425, MPPs- SR-561, MPPs- SR-903. （ 2 ） Complementary single and strand DNA oligos was annealed and convertred into cDNA coding expression of small hairpin RNAs of siRNA. （3） Vectors named PGPU6/GFP/Neo were linearized. （4） shRNAs and vectors were connected together separately. （5） Four eukaryotic expression vectors of RNA interference specific for MPPs were identified by the restriction map and the sequence anlaysis. Results （1）According to the restriction map, four recombinant plasmid vectors were positive. （2）Construction of siRNA sequences was as same as gene banks sequence by characterization and there were no mutations,deletion and in- sertion. Conclusion Eukaryotic expression vectors of RNA interference specific for MPPs were constructed.
出处
《中风与神经疾病杂志》
CAS
CSCD
北大核心
2010年第1期32-34,共3页
Journal of Apoplexy and Nervous Diseases
基金
内蒙古自治区自然基金资助项目(20080404MS1114)
