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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 investigate the therapeutic effect of hTRX-PR39 fusion gene in cerebral ischemia. Methods The constructed hTRX-PR39 fusion gene was inserted into the plasmid pSSCMV vector with the corresponding restriction sites to obtain the recombinant adeno-associated virus vector plasmid, which was identified by restriction enzyme digestion. The constructed adeno-associated virus vector plasmid, adenovirus helper plasmid PFG140 and packaging plasmid pAAV / Ad, three plasmid calcium phosphate coprecipitation transfected 293 cell lines, obtained by homologous recombination hTRX-PR39 recombinant adeno-associated virus vector, The virus was collected and Dot blot was used to determine the virus titer. Results The recombinant adeno-associated virus plasmid pSSCMV / hTRX-PR39 was successfully constructed. After packaging and recovering the virus, the recombinant virus titer was determined by Dot blot method as 3.46 × (1012 ~ 1013) PFU / mL. Conclusion The recombinant adeno-associated virus vector pSSCMV / hTRX-PR39 was successfully constructed and packaged with a high concentration of recombinant virus.