Nogo受体特异性RNA干扰载体合成

目的构建表达Nogo受体(NgR)特异性siRNA199的重组质粒。方法按照Elbashir等设计原则和siRNA表达载体的要求,在合成、筛选出基因沉默效率较高的siRNA199基础上,设计带有BamH I、HindⅢ酶切黏性末靖、终止识别序列和LOOP环的shRNA,并将其克隆入载体pRNAT-U6．1/Neo,构建成NgR特异siRNA199重组质粒,然后进行酶切鉴定及基因测序。结果设计的shRNA成功克隆人载体pRNAT-U6．1/Neo,构建成NgR特异siRNA199重组质粒,酶切鉴定及基因测序表明设计序列完全相符,目的基因序列准确无误。结论重组质粒构建成功,为构建病毒载体及观察重组质粒抑制大鼠NgR基因的表达对脊髓损伤的修复作用奠定了基础。

The generation of the vector of RNA interference targeting Nogo receptor

Objective To construct a recombinant plasmid expressing siRNA199 cognate to NgR gene.Methods Based on SiRNA199 having greater effect on NgR mRNA suppressing and according to the Elbashir's criteria and the rules of siRNA expression vectors, we designed a short hairpin RNA with BamHI, Hind III sites and a loop structure, cloned it into the pRNAT-U6.1/Neo vector, and confirmed the clone of siRNA199 into the vector by restriction enzyme analysis and gene sequencing.Results Restriction enzyme analysis and gene sequencing showed the molecular weight and sequence of the vector was same with the designed sequence. It may suggest the short hairpin RNA targeting siRNA199 was cloned in pRNAT-U6.1/Neo vector.Conclusion The recombinant plasmid vector targeting NgR-spccific siRNA199 was successfully created. It will be useful in generating viral vector and observing the repair effect on spinal cord injury after suppressing NgR mRNA of rats by vector based RNA interference.