真核表达载体pIRES2-EGFP-NT3的构建及在豚鼠耳蜗成纤维细胞中的表达

目的 构建真核表达载体pIRES2-EGFP-NT3，并观察其在豚鼠耳蜗成纤维细胞中的表达及分布。方法 用RT－PCR法，从人肝脏组织中克隆NT3-cDNA基因，构建真核表达载体pIRES2-EGFP-NT3并经PCR及EcoR I/Bam H I双酶切鉴定。用脂质体介导的方法，将用真核表达载体IRES2-EGF-NT3瞬时转染豚鼠原代耳蜗成纤维细胞。转染48h后，分别在荧光显微镜下及用免疫组化法观察转染细胞中NT－3的表达。结果 人NT－3 cDNA的PCR产物约为744bp序列测定结果用BLAST软件进行同源性比较，同源性为100％。经Eco R I/Bam H I双酶切证实，NT－3 cDNA已成功地克隆到真核表达载体IRES2-EGFP中。用脂质体介导法，将pIRES2-EGFP-NT3瞬时转染豚鼠原代耳蜗成纤维细胞，在荧光显微镜下观察到呈绿色荧光的成纤维细胞。NT－3免疫组化染色结果显示，转染NT－3基因的成纤维细胞胞浆呈棕黄色着色；而转染空载体的成纤维细胞免疫组化染色呈阴性。结论 成功地构建真核表达载体pIRES2-EGFP-NT3并在豚鼠耳蜗原代成纤维细胞中表达，为NT－3基因转染治疗致聋豚鼠耳试验奠定了基础。

Construction of the eukaryotic expression vector pIRES2-EGFP-NT3 and expression in guinea pig cochlea fibroblast

Aim To construct the eukaryotic expression vector pIRES2 EGFP NT3 and observe its expression in guinea pig cochlea fibroblast. Methods The NT 3 cDNA was cloned by RT PCR from human liver tissue and was sequenced. The eukaryotic expression vector (pIRES2 EGFP NT3) was constructed and identified by PCR and with Eco R I and Bam H I digestion. The pIRES2 EGFP NT3 was transfected into guinea pig cochlea fibroblast by using lipofectamine. After transfection of 48 h, the NT 3 expression in transfected fibroblasts were observed and detected under fluorescence microscope and by immunor histochemical staining respectively. Results The size of PCR product of NT 3 gene was about 744 bp. Sequencing result revealed the sequence of amplified NT 3 gene was identical with that in GenBank. Restrictive enzyme( Eco R I/ Bam H I) digestion analysis showed that recombinant expression vector pIRES2 EGFP NT3 had been constructed successfully. The transfected fibroblasts displaying green fluorescence were observed under fluorescence microscope. The immunohistochemical staining showed that positive reactant of brown yellow could be seen in the cytoplasm of fibroblasts transfected by pIRES2 EGFP NT3, while the no NT 3 expression was observed in the fibroblasts transfected by empty vector. Conclusion The recombinant expression vector pIRES2 EGFP NT3 is constructed successfullu and can be expressed in the fibroblasts. The present study lay the foundation for the furhter test of treating deafened guinea pig by NT 3 gene transfection.