摘要
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.
出处
《山东大学学报(医学版)》
CAS
北大核心
2011年第7期61-64,共4页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金资助项目(30970992)
关键词
硫氧还蛋白
抗菌肽PR39
融合基因
重组腺相关病毒
Thioredoxin
Antimicrobial peptide PR39
Fusion gene
Recombinant adeno-associated virus