CAJ | 학술논문

目的构建磷脂酰肌醇激酶-3 beta(PI3Kcb)基因小干涉RNA (siRNA)的重组35型腺病毒载体( Ad5/F35).方法在PI3Kcb mRNA序列中选择1个特异性靶序列,体外合成对应发卡样DNA片段,经退火后,将其定向克隆人siRNA载体pDC316-EGFP-U6,获得重组质粒pDC316-PI3Kcb siRNA-EGFP.骨架质粒pBHG-fiber5/35和穿梭质粒pDC316-PI3 Kcb siRNA-EGFP共转染293细胞,同源重组产生Ad5/F35-PI3 Kcb-siRNA.经聚合酶链反应(PCR)鉴定目的基因的表达.结果 PCR表明Ad5/F35-PI3Kcb-siRNA质粒构建正确.结论获得的Ad5/F35-PI3Kcb-siRNA可以用于转基因疼痛治疗的实验研究.
목적 구건린지선기순격매-3 beta(PI3Kcb)기인소간섭RNA (siRNA)적중조35형선병독재체( Ad5/F35).방법 재PI3Kcb mRNA서렬중선택1개특이성파서렬,체외합성대응발잡양DNA편단,경퇴화후,장기정향극륭인siRNA재체pDC316-EGFP-U6,획득중조질립pDC316-PI3Kcb siRNA-EGFP.골가질립pBHG-fiber5/35화천사질립pDC316-PI3 Kcb siRNA-EGFP공전염293세포,동원중조산생Ad5/F35-PI3 Kcb-siRNA.경취합매련반응(PCR)감정목적기인적표체.결과 PCR표명Ad5/F35-PI3Kcb-siRNA질립구건정학.결론 획득적Ad5/F35-PI3Kcb-siRNA가이용우전기인동통치료적실험연구.
Objective To construct the recombinant adenovirus' s containing fibers derived from B group serotype 35 (rAd5/F35) vector with phosphatidylinositol kinase-3 catalytic beta polypeptide-siRNA (PI3Kcb-siRNA).Methods A target-specific sequence from PI3Kcb mRNA was used to synthesize the corresponding hairpin DNA fragments in vitro.After annealing,the DNA products were cloned into siRNA vector pDC316-EGFP-U6 to obtain the recombinant siRNA vector plasmid pDC316-PI3Kcb siRNA-EGFP.The 293 cells were cotransfected by the skeleton plasmid of pBHG-fiber5/35 and the shuttle plasmid of pDC316-PDP,and the recombinant plasmid of Ad5/F35-PI3Kcb-siRNA was obtained.The expression of the transfected genes was evaluated by polymerase chain reaction (PCR) and immunocytochemical stain.Results The identification of PCR and immunocytochemical stain showed that the construction of the recombinant Ad5/F35-PI3Kcb-siRNA plasmid could be confirmed.Conclusion The Ad5/F35-PI3Kcb-siRNA vector can be used in empirical study of transgenic pain therapy.