简论shRNA表达载体pWH的构建及用于HIF基因的沉默的论文.docVIP

简论shRNA?表达载体pWH?1的构建及用于?HIF1基因的?沉默的论文 简?论shRNA表?达载体pWH1?的构建及用于H?IF1基因的沉?默的论文 ?【摘要】 ? 目的: ?构建用于rna?i的shrna?（small ?hairpin? rna）表达?载体及检测其对?低氧诱导因子?1（hypox?iaindu?cible f?actor1?, hif1）?基因的沉默效果?。方法: 从人?血基因组中pc?r扩增出h1基?因启动子, 克?隆入酶切处理后?的pegfp?c1载体片段中?, 此载体命名?为pwh1。以?人hif1 c?dna基因为靶?标设计引物, ?退火后克隆入p?wh1。新的载?体转染sgc7?901细胞, ?然后用rtp?cr和west?ern blo?t检测hif1?基因的表达改变?。结果: 构建?的pwh1载体?能很好地表达针?对hif1基因?的shrna,? rtpcr?和wester?n blot的?结果显示hif?1基因的mrn?a和蛋白表达水?平均明显下降。?结论: 成功构?建了shrna?表达载体pwh?1, 这对于基?因的功能研究具?有重要的意义。? ? 【关键词】 ? ?shrna 表?达载体 rna?干涉 hif1?基因 [abs?tract]a?im: to ?constru?ct the ?express?ion vec?tor of ?small h?airpin ?rna（shr?na）and ?to test? its ef?ficacy ?in sile?ncing t?he hypo?xiaind?ucible ?factor?1 (hif1?)gene. ?methods?: the h?1 gene ?promote?r was a?mplifie?d from ?the gen?ome of ?the hum?an bloo?d cells? by pcr?. then ?the pro?moter w?as clon?ed into? the pe?gfpc1 ?vector ?digeste?d with ?the res?trictio?n enzym?e. the ?constru?cted ve?ctor wa?s named? pwh ? 1. the? primer? was de?signed ?to targ?et the ?human h?if1 cdn?a gene.? the an?nealed ?primer ?fragmen?t was c?loned i?nto pwh? 1. ?the new? constr?ucted p?lasmid ?was tra?nsfecte?d into ?the sgc?7901 ce?ll line?. then ?the exp?ression? level ?of hif1? gene w?as assa?yed by ?rtpcr ?and wes?tern bl?ot. res?ults: t?he newl?y const?ructed ?plasmid? expres?sed shr?na to t?arget t?he hif1? gene.t?he resu?lts of ?rtpcr ?and wes?tern bl?ot show?ed the ?express?ion of ?hif1 ge?ne was ?reduced? dramat?icaly i?n mrna ?and at ?protein? level.? conclu?sion: t?he succ?essful ?constru?ction o?f shrna? expres?sion ve?ctor (p?wh1) pr?ovides ?a tool ?for fur?ther re?search ?into th?e funct?ion of ?a novel? gene. ?[keywor?ds]shrn?a; expr?ession ?vector;? rna in?terfere?nce; hi?f1 gene? rna干涉(?rna int?erferen?ce, rna?i)是将双链r?na（dsrn?a）导入细胞引?起特异基因mr?na降解的一种?细胞反应过程。?wwW..CO?m它是转录后基?因沉寂（ptg?s）的一种。1?998年, f?ire等[1]?在利用反义核酸?技术来抑制线虫?基因表达时意外?地发现, 由正?义和反义rna?退火形成的ds?rna引起的基?因表达抑制要比?单独应用正义或?反义rna强1?0倍以上。ds?rna引起的基?