Construction And Identification Of ShRNA Expression Vector For Gene IRAK1

ObjectiveTo induce HTLV1 cells IRAK1 gene transcriptional silencing, using RNA interference, for future research of IRAKI gene in vitro or in vivo of the central nervous system autoimmune functional recovery after injury to provide an effective method.MethodspGPU6/GFP/Neo start in the expression of siRNA H1 promoter sequence requirements on the use of Ambion's online siRNA Tatget finder software (http://www.ambion.com/techlib/misc/siRNA finder.html) design and synthesis shRNA sequence. PCR apparatus in accordance with the following procedures shRNA template annealing:95℃5 min,85℃5 min,75℃5 min,70℃5 min,4℃preservation. The enzyme treatment, pGPU6/GFP/Neo linear vector and its construction. From plasmids with BamHⅠ, PstⅠrestriction enzyme digestion, respectively.ResultsWe successfully constructed the eukaryotic expression vector of shRNA mode. pGPU6/GFP/Neo were also contain Neomycin resistance selection marker protein can be expressed GFP RNAi vector, Kanamycin/Neo cards that represent the resistance and neomycin resistance. pGPU6/GFP/Neo plasmid with BamHⅠ, PstⅠenzyme digestion, electrophoresis. The results showed that all plasmids are recombinant vectors, which are designed shRNA interference fragment has indeed inserted into the RNAi vector. Each select two clones were sequenced (Biotechnology Co., Ltd. ShanghaiYingJun), DNA sequencing plasmids encoding sequence into the correct position, no gene mutation, consistent with the design.ConclusionThe recombinant plasmid was successfully constructed for further study in vivo treatment of autoimmune injury and lay a foundation.