人心肌营养素-1重组腺病毒载体的构建、纯化及表达

Construction and purification of human cardiotrophin-1 adenovirus vector and its expression in vitro and in vivo

目的 构建人心肌营养素 1重组腺病毒载体 ,以期用于神经系统退行性疾病及损伤的在体研究。方法 构建穿梭质粒pDC3 16 huCT1和pDC3 16 eGFP ,CsCl梯度离心制备高纯度的病毒基因组质粒pBHloxdeltaE1,3Cre、pHG14 0。培养2 93细胞 ,CaCl2 法质粒共转染 2 93细胞构建并筛选腺病毒AdCMV huCT1和AdCMV eGFP。病毒原液转染 2 93细胞及NIH3T3细胞 ,PCR、RT PCR及免疫组化鉴定。CsCl梯度离心纯化病毒 ,空斑试验检测病毒滴度。纯化病毒注入大鼠颈脊髓 ,RT PCR及免疫组化检验AdCMA huCT的在体表达。结果 采用双质粒共转染 2 93细胞Cre loxP位点同源重组方法构建了E1和E3缺失的含MCMV启动子、外源基因和SV40PloyA的AdCMV huCT1和AdCMV eGFP载体。经过PCR、RT PCR和免疫组化证实腺病毒载体构建成功。病毒经过CsCl梯度离心纯化后 ,AdCMV huCT1滴度达到 3 .0× 10 1 0 pfu。RT PCR及免疫组化显示AdCMV huCT1在大鼠脊髓中特异性表达。结论 构建并纯化了体外及体内高效表达的AdCMV huCT1。

Objective To construct human cardiotrophin 1(huCT 1) adenovirus vector for central nervous system(CNS) gene therapy in vivo . Methods The huCT 1 and eGFP genes were cloned into shuttle plasmid pDC316 to construct pDC316 huCT 1 and pDC316 eGFP. Virus genome plasmids pBHGloxdeltaE1,3Cre and pHG140 were purified by CsCl banding certification. Recombinant replication defective adenovirus vectors AdCMV huCT1 and AdCMV eGFP were rescued in 293 packaging cells by co transfection and Cre mediated recombination of both plasmids pDC316 huCT1 and pBHGloxdelta1,3Cre. The insert gene and its expression were identified by PCR, RT PCR and immunohistochemistry after recombinant adenovirus transfected 293 cells and NIH 3T3 cells. Recombinant adenovirus vectors were purified by CsCl banding and titrated by plaque forming test. AdCMV huCT1 expression in vivo was analyzed by RT PCR and immunohistochemistry after transfection of the cervical spinal cord in adult rats. Results We have constructed two recombinant adenoviral vectors: AdCMV huCT1 and AdCMV eGFP, containing MCMV promoter, foreign DNA and SV40 PolyA with deletions of E1 and E3 regions. The positive huCT 1 mRNA and protein were identified in AdCMV huCT1 transfected NIH 3T3 cells and rat cervical spinal cord. The titer of virus stocks was generally up to 3.0×10 10 plaque forming units(pfu) per milliliter. Conclusion Recombinant purified AdCMV huCT1 vectors can be highly expressed in vitro and in vivo and is suitable for CNS gene therapy in vivo .