Construction and purification of human cardiotrophin-1 adenovirus vector and its expression in vitro and in vivo / 第三军医大学学报

ABSTRACT

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 .