RARE介导的视黄酸可调控的IFN-α表达载体的构建与鉴定

Construction and identification of an IFN-α gene eukaryotic expression vector containing retinoic acid response element

目的构建一个含视黄酸反应元件 ( RARE)的 IFN- α表达载体 ,使其在真核细胞内的表达受视黄酸 ( RA)的诱导。方法运用 DNA重组技术构建含有 4个 RARE重复序列的 IFN- α真核表达载体 ( p RARE4 - IFN- α) ,经酶切、PCR鉴定和测序确认后 ,用脂质体转染法使其转染 HL- 60细胞 (人早幼粒白血病细胞系 )、Bcap- 3 7和 MCF- 7细胞 (人乳腺癌细胞系 ) ,RA处理后 ,RT- PCR分析 IFN- α m RNA水平 ,EL ISA检测培养细胞上清中 IFN- α含量。结果 p RARE4 - IFN- α转染的 HL- 60、Bcap- 3 7和 MCF- 7细胞 ,IFN- α分泌量为 16～ 2 4 ng/ ( m L· 10 6 )细胞 ,而经 RA处理 2 4 h,IFN- α表达水平可增加 8～ 13倍 ,但经 RA处理 2 4 h的细胞 ,换无 RA的新鲜培养基继续培养 2 4 h后 ,IFN- α表达水平又下降到 2 5～ 3 4 ng/ ( m L· 10 6 )细胞。结论带有 RARE的 IFN- α表达载体转染细胞后 ,外源性 IFN- α基因的表达受 RA的诱导。

ObjectiveTo investigate the possibility of the expression of exogenous IFN-α gene mediated by retinoic acid(RA)through retinoic acid response element (RARE). Methods An IFN-α gene eukaryotic expression vector containing four copies of RARE(pRARE4-IFN-α)was constructed with recombinant DNA technique, identificated with restriction endonuc- lease digest and PCR and DNA sequence analysis, then transfected into the cells of HL-60,Bcap-37 and MCF-7 with liposome DOTAP. The expression of IFN-α gene was analyzed with RT-PCR and ELISA. Results ELISA showed that the pRARE4-IFN-α transfected HL-60, Bcap-37 and MCF-7 cells treated with RA for 24 hours produced 184,211 and 241 ng/(mL·10 6) cells of IFN-α compared with 21,16 and 24 ng/(mL·10 6) cells in untreated transfected cells respectively, as compared with 15 ng/(mL·10 6) cells in nontransfected HL-60 cells treated with RA for 24 hours.ConclusionOur study suggest that RA is able to induce the expression of exogenous IFN-α gene in cells transfected with pRARE4-IFN-α.