HIV-1辅受体配体的融合表达载体的构建及表达

Construction and Expression of the Bicistronic Expression Vector pCMV-R-K-S-K

应用PCR扩增RANTES-KDEL基因,鉴定后与真核表达载体pCMV—S/K连接,构建成HIV-1辅受体的配体、趋化因子RANTES和SDF-1的融合表达载体pCMV-R—K-S—K,酶切鉴定和测序证明成功构建了pCMV—R—K—S—K融合表达载体。脂质体介导pCMV—R—K-S—K转染HeLa细胞,间接免疫荧光证实RANTES和SDF-1可高效表达于HeLa细胞。结果表明构建的pCMV—R—K—S—K融合表达载体能在HeLa细胞中高效表达,可用于下一步的HIV-1感染实验。

In order to construct bicistronic expression vector of RANTES and SDF-l,the ligands of HIV-1 principal coreceptors,RANTES-KDEL were amplified from pCMV-R-K by PCR and ligased with eukaryotic expression vector pCMV-S/K. The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. Then pCMV-R-K-S-K transfection into HeLa cells was carried out by lipofectin. RAN TES and SDF-1 were showed expressed in HeLa cells by indirect immumofluo-rescence. The results show that pCMV-R-K-S-K was constructed and expressed in cell line HeLa successfully, which will contribute to gene therapy of HIV-1.