Recombinant Adenoviruses Driving Hydroxysteroid-delta 24 Reductase Expression In Neuron

Two recombinant adenoviruses driving DHCR24 expression specifically in neuron were constructed to explore to explore the correlation between antioxidant or A?-cleaving activity of DHCR24 and the neurodegenerative disease.A about 500bp DNA fragment of SYN1 promoter was obtained from the human or rat genome by PCR,respectively.The PCR product was subcloned into pGEMT easy,followed by the picking up and sequencing.The plasmid obtained from the positive clone was digested and inserted into the upstream of DHCR24 cDNA of recombinant pcDNA3.1A-DHCR24-myc.The fragment SYN1-DHCR24-myc was digested with KpnI and PmeI and then inserted into pShuttle vector.A fragment containing SYN1-DHCR24-myc was inserted into replication-deficient adenoV virus DNA bone by the recombinantion.Finally the linearised recombinant adenovirus DNA fragment was transfected into 293 cells using the lipofectamine to package the entire adenovirus.After amplification and purification,the titers of obtained of recombinant adenovirus Ad-DHCR24s were about 1×109pfu/? L and 3.5×109pfu/?L respectively.The neuron-specific expression of DHCR24 driving by obtained ad-DHCR24s were investigated in 293,N2A neuroblastoma cell and MIN6 pancreatic cell infected with recombinant adenovirus by Western blot analysis.The results showed that an about 56kDa band was present in the 293 and N2A cells,respectively,but not in the MIN6 cells,demonstrating that the recombinant adenoviruses have been successfully constructed.This result lays the foundation for further study on function of DHCR24 and its correlation with degenerative neuropathy.