hTRX-PR39重组腺相关病毒载体的构建

Construction of a hTRX-PR39 recombinant adeno-associated virus vector

目的探讨hTRX-PR39融合基因在脑缺血疾病中的治疗作用。方法将已经构建好的hTRX-PR39融合基因插入到具有相应酶切位点的pSSCMV病毒载体质粒中,得到重组腺相关病毒载体质粒,酶切电泳鉴定。将已构建的重组腺相关病毒载体质粒、腺病毒辅助质粒PFG140和包装质粒pAAV/Ad,三质粒磷酸钙共沉淀法转染293细胞系,通过同源重组获得hTRX-PR39重组腺相关病毒载体,收集病毒,斑点杂交(Dot blot)法测定病毒滴度。结果成功构建重组腺相关病毒质粒pSSCMV/hTRX-PR39。包装、回收病毒后,Dot blot法测定重组病毒滴度为3.46×(1012～1013)PFU/mL。结论成功构建pSSCMV/hTRX-PR39重组腺相关病毒载体,并包装较高浓度的重组病毒。

Objective To construct a vector of recombinant adeno-associated virus（AAV） containing the fusion gene hTRX-PR39,and explore the role of fusion gene hTRX-PR39 as a target gene in the treatment of cerebral ischemic disease.Methods The constructed fusion gene hTRX-PR39 was inserted into the EcoRI-BamHI site of vector plasmid pSSCMV,and the vector of hTRX-PR39 recombinant AAV was constructed.The recombinant AAV stock was packaged.Renal embryo 293 cells were co-transfected with the recombinant AAV vector of plasmid pSSCMV/hTRX-PR39,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140.Recombinant AAV was produced by homologous recombination of the 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization.Results The vector of hTRX-PR39 recombinant AAV was successfully constructed.High titer of recombinant AAV was obtained by homologous recombination in renal embryo 293 cells（3.46×1012-3.46×1013PFU/mL）.Conclusion The recombinant AAV vector encoding the gene hTRX-PR39 was successfully constructed in this study by molecular cloning and in vitro recombination techniques,laying the foundation for further research into genetic therapy of cerebral ischemic disease.