中文摘要：

目的构建携带人PAPS合成酶1（hpapss1）全长cDNA的重组腺病毒,并感染巨噬细胞使PAPSS1过表达,为研究其在胆固醇代谢中的作用提供工具。方法PCR扩增hpapss1全长cDNA,继而构建了真核表达载体pCDNA3.0-hpapss1,再将hpapss1片段亚克隆入腺病毒穿梭质粒pshuttle-CMV,得到转移质粒pshuttle-CMV-hpapss1,并将其转化含腺病毒基因组质粒pAdEasy-1的BJ5183感受态菌（AdEasier-1cells）。筛选并鉴定重组正确的腺病毒质粒pAdEasy-1-hpapss1,在阳离子脂质体介导下,转染腺病毒包装细胞（293细胞）进行包装和扩增。携带hpapss1全长cDNA的重组腺病毒感染THP-1来源的巨噬细胞48h后,用Western blot方法测定蛋白水平来确定PAPSS1的过表达。结果成功制备携带hpapss1全长cDNA的重组腺病毒,滴度为5.3×10^8pfu/mL,实现了hpapss1基因在巨噬细胞中的过表达。结论携带hpapss1全长cDNA的重组腺病毒的成功制备为进一步探讨PAPSS1及氧化固醇硫酸化在巨噬细胞胆固醇代谢平衡中的作用及机制建立了很好的模型和工具。

英文摘要：

Objective To construct the recombinant adenovirus carrying hpapssl 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-hpapssl 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 hpapssl was constructed successfully. The viral titer was 5.3 × 10^8 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.