携带血红素加氧酶1基因的重组腺相关病毒的制备

目的:制备携带大鼠血红素加氧酶1(rHO-1)基因的重组腺相关病毒(AAV)。方法:通过RT-PCR的方法,从大鼠脾脏组织中扩增rHO-1基因,克隆入通用型AAV载体质粒pSNAV中构建重组质粒pSNAV-rHO-1,通过酶切和测序鉴定。利用Lipofectamine将pSNAV-rHO-1转染BHK-21细胞,G418筛选得到表达外源基因的细胞系BHK/rHO-1。用具有能表达Rap和Cap及包装重组AAV功能的一种重组单纯疱疹病毒HSV1-rc/△UL2分别感染这株细胞系后收获并纯化病毒,DNA斑点杂交检测重组病毒滴度。结果:扩增了rHO-1基因,测序证实与GenBank中的一致,病毒滴度为1.5×1015v.g/L。结论:成功获得了高滴度、高纯度的重组AAV-rHO-1,这为以后心血管系统疾病的基因治疗研究奠定了基础。

Preparation of Recombinant Adeno-associated Virus Carrying Heme Oxygenase-1 Gene

Objective: To construct the recombinant adeno-associated virus(AAV) vector carrying rat heme oxygenase-1(rHO-1) gene.Methods: The gene sequence coding rHO-1 was amplified from the rat spleen by RT-PCR.The fragment was cloned into a general AAV vector plasmid pSNAV to construct the recombinant vector pSNAV-rHO-1,which was verified by restriction digestion and DNA sequencing,then the recombinant pSNAV-rHO-1 was transfected into BHK-21 packaging cells by lipofectine-mediated transfection.AAV vector cells-BHK-21 carrying rHO-1 gene were then screened using G418 and called BHK/rHO-1 cells.BHK/rHO-1 cells were subsequently infected with the recombinant virus HSV1-rc/△UL2,which could express Rep and Cap,and was able to package and recombine AAV.The infected BHK-21 cells were collected and purified.The titer of the recombinant virus AAV-rHO-1 was detected by DNA dot blot.Results: The gene sequence coding rHO-1 was amplified from the rat spleen,which was identical with that of GenBank by DNA sequencing.The titer of AAV-rHO-1 was approximately 1.5×1015 v.g/L.Conclusion: The high-titer,purified viral stock of AAV-rHO-1 was successfully obtained,which can serve as the further experimental study of gene therapy of cardiovascular diseases.