Construction of recombinant adenovirus carrying hpapss1 cDNA

Objective To construct the recombinant adenovirus carrying hpapss1 cDNA and overexpress PAPSS1 in macrophages. Methods The full length of hpapss1 cDNA was amplified and cloned into eukaryotic expression vector pCDNA3.0 (pCDNA3.0-hpapss1). The segment of hpapss1 was subcloned into the shuttle plasmid pshuttle-CMV (pshuttle-CMV-hpapss1). The homologous recombination of pshuttle-CMV-hpapss1 and the backbone plasmid pAdEasy-1 took place in the E.coli BJ5183,and then the recombinant adenoviral plasmid (pAdEasy-1-hpapss1) was generated. The identified adenovirus vector was transfected into 293 cells to pack and amplify the adenovirus. The viral titer was checked by adenovirus plaque assay. Overxepression of PAPSS1 in THP-1 derived macrophages infected with Ad-hpapss1 for 48h was determined by Western blot. Results The recombinant adenovirus carrying hpapss1 was constructed successfully. The viral titer was 5.3×108 pfu/mL. The recombinant adenovirus could introduce hpapss1 into macrophages. Conclusions The recombinant adenovirus carrying hpapss1 would be a good model to study the effect of PAPSS1 and oxysterol sulfation on cholesterol metabolism.