重组HIF—1α及NIS慢病毒表达载体的构建及功能鉴定  

Construction of recombinant HIF-la and NIS lentiviral expression plasmid and its functional identi fication

作　　者：石朔[1] 郭睿[1] 王利华[1] 张敏[1] 张淼[1] 苗莹[1] 李彪[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院核医学科,200025

出　　处：《中华核医学与分子影像杂志》2014年第2期130-135,共6页Chinese Journal of Nuclear Medicine and Molecular Imaging

基　　金：国家自然科学基金（30900375）;上海交通大学“医工（理）交叉研究基金”（YG2010MS22）

摘　　要：目的构建肌凝蛋白轻链-2（MLC-2v）启动下HIF-1α及人NIS慢病毒真核表达载体，探讨外源基因在心肌细胞中的特异性表达和NIS作为报告基因的可行性。方法应用基因重组技术构建慢病毒（Lv）-延长因子（EF）1-HIF-1α-内部核糖体进入位点（IRES）-NIS及Lv-MLC-HIF-1α-IRES-NIS慢病毒表达载体。对重组质粒进行酶切、测序鉴定后，采用脂质体转染法将重组质粒转入Hela细胞。细胞免疫荧光及Westernb1a分别验证HIF-1α及NIS蛋白在细胞中的定位及定量表达；制备重组病毒，分别以不同的感染复数（MOI）感染H9C2大鼠心肌细胞、NIH-3T3小鼠胚胎成纤维细胞和L6大鼠骨骼肌成肌细胞，Westernb1a确定最佳MOI并验证MLC·2v启动子的特异性；分别行H9C2细胞动态摄碘和NaClO4抑制实验。采用两样本t检验分析数据。结果成功构建Lv-EFl-HIF-1a-IRES-NIS及Lv-MLC-HIF-1α-IRES-NIS慢病毒表达载体。2种质粒分别转染Hela细胞，免疫荧光显示HIF-1α蛋白表达于胞质，NIS蛋白表达于胞膜；Westernb1a显示EFl启动下2种蛋白质的表达量多于MLC-2v启动下相应表达。Westernb1a蛋白检测后测定蛋白灰度值，阳性对照组NIS及HIF-1α灰度值分别为69-8和71-9，EF1启动下分别为109-4和92-7，MLC-2v启动下分别为141-9和132-4。Lv-MLC-HIF-1α-IRES-NIS病毒感染H9C2细胞，MOI为20时NIS蛋白表达最高，为最佳MOI。2种病毒以MOI为20分别感染H9C2、NIH-3T3及L6细胞，Westernb1a显示EF1启动下NIS蛋白在3种细胞中均有表达，灰度值分别为23.4、29.8和28.6，相差较小。MLC-2v启动下仅H9C2细胞中有大量NIS蛋白表达，NIS蛋白在H9C2、L6及NIH-3T3细胞的灰度值分别为157.9、178.8和2173。H9C2细胞的摄碘高峰时间为40min，峰值为4287.2计数·min-1，是阴性对照组（254.g计数·min-1）的16.85倍（t=5.34，P〈0.01）。摄碘可被NaC国O4抑制，抑制率达85.5％（t=21.3，P〈0.01）。结论MLC-2v可启动治疗基因HIFObjective To construct a recombinant lentivirus vector containing the human NIS gene and HIF-la with the myosin light chain-2v（MLC-2v） as a promoter and to investigate the specific expres sion and feasibility of NIS as a reporter gene in cardiomyocytes. Methods The target gene HIF-la and NIS were subcloned into the lentivirus （Lv）-elongation factor （EF） 1-HIF-la-internal ribosome entry site （IRES）-NIS and Lv-MLC-HIF-lcMRES-NIS lentivirus vectors. The recombinated vectors were transfected into Hela ceils by lipofectamine 2000. The expression of HIF-la and NIS in the transfected Hela cells was detected by indirect immunofluorescence and Western blot. The H9C2 cells were exposed to different multi- plicities of infection （MOI; 5, 10, 20, 40） with packaged virus particles. The infection efficiency was de-tected by Western blot. MOI 20 was used for H9C2, NIH-3T3 and L6 cell lines and the specificity of the MLC-2v promoter was detected by the count of NIS protein in the 3 different cell lines with Western blot. The function and features of NIS protein were evaluated by dynamic iodine uptake and NaC104 iodine uptake inhibition tests in vitro. Two-sample t test was used to analyze the data. Results The two recombinant lenti- virus vectors were constructed successfully. The HIF-Iot protein was expressed in the cytoplasm and the NIS protein was expressed on the cell membrane in Hela cells. The grey levels of NIS and HIF-1a proteins in the positive control were 69.8 and 71.9, respectively, which were 109.4 and 92.7 after being prompted by EF1, and 141.9 and 132.4 by MLC-2v. The expression of these proteins was much higher by EF1 promoter than that by MLC-2v promoter. The optimal MOI for the Lv-MLC-HIF-la-IRES-NIS virus to infect H9C2 ceils was 20. With the MOI of 20, the grey levels of NIS protein promoted by EF1 were 23.4, 29.8 and 28.6 for H9C2, NIH-3T3 and L6 cells infected with Lv-EF1-HIF-loMRES-NIS virus, respectively. The expression of NIS protein promoted by MLC-2v was much higher in H9C2 cells th

关 键 词：缺氧诱导因子1 Α亚基 DNA 重组 质粒 转染 钠 碘转运体 

分 类 号：R373[医药卫生—病原生物学] Q78[医药卫生—基础医学]