pIRES2-EGFP-gAd质粒载体构建及在大鼠肾脏的表达

Construction of eukaryotic expression plasmid pIRES2-EGFP-gAd and its expression in rat kidneys

背景:已有研究证实经腹腔注射基因转染肾脏是一种简便有效的实验方法。目的:构建表达球形脂联素(gAd)的pIRES2-EGFP-gAd质粒,观察其在大鼠肾脏的表达。方法:以pET/gAd质粒为模板,PCR扩增目的片段,转化大肠杆菌,用EcoRⅠBamHⅠ双酶切后,与pIRES2-EGFP荧光质粒连接,重组构建pIRES2-EGFP-gAd质粒。将构建成功的pIRES2-EGFP-gAd质粒采用脂质体介导经腹腔注射正常大鼠体内,于注射24,48,96h和7d留取肾脏标本进行冰冻切片,荧光显微镜下观察绿色荧光蛋白在肾组织中的表达,同时采用Western bot检测肾组织绿色荧光蛋白的表达。结果与结论:重组构建的pIRES2-EGFP-gAd载体经双酶切电泳分析及DNA测序证实无碱基突变。在腹腔注射脂质体/pIRES2-EGFP-gAd转染复合物24h,即可在肾小球肾间质检测到绿色荧光,注射48h,荧光进一步增强,之后逐渐减弱,至注射7d时,荧光强度明显减弱。Western bot检测的绿色荧光蛋白的表达结果与冰冻组织切片结果一致。说明脂质体能够介导pIRES2-EGFP-gAd真核载体在大鼠肾脏表达。

BACKGROUND：Previous studies have demonstrated that intraperitoneal injection of gene transfected kidney is an effective and simple experimental method.OBJECTIVE：To construct the eukaryotic expression plasmid pIRES2-EGFP-gAd,and to observe the expression of globular adiponectin（gAd） in rat kidney.METHODES：The gAd cDNA fragments were obtained by PCR from pET/gAd.Then,the PCR product was translated into JM109.It was digested by two restrictive endonucleases,and then the gAd cDNA was collected and recombined with eukaryotic expression vector pIRES2-EGFP by using gene recombination technique.The recombined plasmid pIRES2-EGFP-gAd was transfected into normal rat kidney with Lipofectamine Transfection Reagent by intraperitoneal injection.The kidney tissues were collected at different time points（24,48,96 hours,7 days） after injection,and the gAd/GFP green fluorescence protein expression was determined by fluorescence microscopy and Western Blot respectively.RESULT AND CONCLUSION：The pIRES2-EGFP-gAd expression plasmid was constructed successfully.The gAd/GFP green fluorescent protein was detected at the glomeruli and tubular at 24 hours after injection and the fluorescence intensity became stronger at 48 hours.The level of fluorescence protein expression became gradually weakened at 7 days.In Western bot test,same results were observed.The pIRES2-EGFP-gAd gene was expressed in rat kidney successfully by Lipofectamine Reagent with intraperitoneal injection.