HIV-1辅受体的配体在HeLa细胞的表达及其对合胞体形成的抑制

Expression of the bicistronic vector with RANTES and SDF-1 genes and inhibition of syncytium formation

目的 观察HIV 1辅受体的配体、趋化因子RANTES和SDF 1的双顺反子表达载体pCMV R K S K在HeLa细胞系表达 ,并对其抗HIV 1感染作用进行初步观察。方法 应用PCR扩增RANTES KDEL基因 ,鉴定后与真核表达质粒 pCMV S/K连接 ,构建RANTES和SDF 1双顺反子表达载体 pCMV R K S K ,酶切鉴定并测序。脂质体介导转染HeLa细胞 ,间接免疫荧光及放射免疫沉淀法检测RANTES和SDF 1表达。合胞体形成实验初步检测其抗HIV 1感染的作用。结果 酶切鉴定和测序证明成功构建了 pCMV R K S K双顺反子表达载体 ,间接免疫荧光及放射免疫沉淀法证实RANTES和SDF 1可以表达于HeLa细胞。pCMV R K S K转染能够抑制M和T嗜性HIV 1膜蛋白诱导的合胞体形成。结论 双顺反子表达载体 pCMV R K S K转染的HeLa细胞可以表达HIV 1辅受体的配体RANTES和SDF 1,并能抵抗HIV 1感染。

Objective To identify the expression of bicistronic vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors and its inhibition of HIV-1 infection. Methods In order to construct the bicistronic expression vector pCMV-R-K-S-K,RANTES-KDEL, which was amplified from pCMV-R-K by PCR, was ligased with eukaryotic expression vector pCMV-S/K. Transfection into HeLa cells with the new recombinant vector was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Syncytium formation detection was carried out to observe the effect of HIV-1 infection. Results The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were showed expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. The HeLa cells expressing RANTES and SDF-1 were found to be able to inhibite M and T tropic HIV-1 envelop-mediated syncytium formation. Conclusions pCMV-R-K-S-K was constructed and expressed in cell line HeLa successfully. The chemokines expressed in target cells can bind new synthesized HIV-1 coreceptors, CCR5 and CXCR4 and prevent their transport to the cell surface.