HTLV-1 Tax基因真核表达载体的构建及对RPE细胞的影响  

Construction of HTLV-1 Tax gene eukaryotic expression vector and its effect on RPE cells

作　　者：邓江云[1] 聂玉红 崔冬梅[1] 梁静文[1] 吴开力[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广东省广州市510060

出　　处：《眼科新进展》2008年第12期881-885,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30572002;30630064)~~

摘　　要：目的构建人T淋巴细胞白血病病毒-1(human T-cell leukemia virus type1,HTLV-1)Tax基因真核表达载体,在人视网膜色素上皮(retinal pigment epithelium,RPE)细胞表达Tax蛋白,观察其对细胞的影响。方法应用基因重组技术构建重组载体PIRES2-EGFP-Tax(PT),鉴定后将重组载体转染入RPE细胞,应用RT-PCR、Western-blot和免疫荧光法检测细胞内Tax基因和蛋白的表达;通过MTT和流式细胞仪检测质粒(空白载体、PT和Green-Tax)转染RPE细胞24h和48h后对RPE细胞生长活力及细胞周期的影响。结果经双酶切鉴定和测序分析证实成功地构建了HTLV-1Tax真核表达载体;通过RT-PCR、Western-blot和免疫荧光法检测到转染Tax重组载体的RPE细胞中有Tax基因和蛋白的表达。质粒转染RPE细胞后,细胞生长活力受到抑制,G1期减少而S期增加。但转染Tax质粒和空白载体,对RPE细胞的生长活力和细胞周期产生相似影响。结论成功构建了HTLV-1Tax基因真核表达载体,并在RPE细胞中表达Tax蛋白;Tax质粒对RPE细胞的影响不排除是转染试剂所致。Objective To construct a eukaryotic expression vector of human T-cell leukemia virus type 1（HTLV-1） Tax gene expressing Tax protein in human retinal pigment epithelial cell（RPEC） and study the influence of Tax gene and protein to RPECs.Methods The vector PIRES2-EGFP-Tax（PT） was reconstructed by gene recombinant technique and was confirmed by sequencing analysis.Then the transfection of vector was taken into RPECs.Tax gene and protein expression in RPECs were examined by RT-PCR,Western-blot and immunofluorescence.The viability of RPECs was evaluated by MTT method and the cell cycle of RPECs was evaluated by flow cytometry assay after transfection for 24 hours and 48 hours.Results It was confirmed that the eukaryotic expression vector PT was constructed successfully by endonuclease digestion and sequencing analysis.Tax gene and protein expression were detected in RPECs with transfection of vector by RT-PCR,Western-blot and immunofluorescence analysis.The transfection of the vectors could inhibit the viability of cells and interfered the cell cycle,including a decrease of the G1 stage and an increase of the S stage.However,these cellular changes occurred in RPECs with transfection of Tax vector or transfection of control vectors.Conclusions A eukaryotic expression vector PT is constructed successfully,and express Tax protein in RPECs.The effect of vector on RPECs may be caused by transfection reagent.

关 键 词：人T淋巴细胞白血病病毒-1 Tax基因 TAX蛋白 视网膜色素上皮细胞 

分 类 号：R774.1[医药卫生—眼科]