人氧化固醇结合蛋白8（ORP8)重组腺病毒载体的构建与鉴定及病毒滴度测定

Previous study showed that ORP8 participated in cholesterol transport and was involved in liver cell apoptosis and proliferation, but the mechanism involved of ORP8 remained unknown. To better study the cellular functions of ORP8 in mammalian cells ,we constructed a recombinant adenovirus vector containing ORP8 gene that could express ORP8 by using gateway recombinant cloning technology. First, we cloned full-length ORP8 cDNA by PCR amplification and inserted it into the skeleton vector pDown-mcs-IRES/EGFP by BP recombination reaction, the recombinant plasmid pDown-ORP8/IRES/eGFP then used LR recombination reaction to co-recombinant pDown-ORP8/IRES/eGFP and destination vector pAV.Des1d into the expression vector pAV.EX1d-ORP8/IRES/eGFP. We transfected the expression vector into 293A cells, packaged the recombined adenovirus expressing ORP8.The detection by Western blot and fluorescent confirmed that ORP8 expressed in 293A cells successfully. Finally, we collected high-titer adenovirus by purification and concentration, and determined of the virus titer. The study provided a very useful tool for further research on the cellular functions for ORP8.