摘要
目的 构建 TGF- βR / Fc融合基因的真核表达载体 p Ig- TR ,使其在中国仓鼠肾细胞 (CHO)中呈分泌性表达 ,为肺间质纤维化基因治疗的研究奠定基础 .方法 采用RT- PCR方法从正常人肺组织中扩增出目的基因 TGF- βR c DNA的胞膜外部分 ,再将 TGF- βR c DNA片段亚克隆到p Ig 真核表达载体 ,该载体在其多克隆位点下游含有人Ig G1 Fc基因组片段 ,由此可形成 TGF-βR / Fc融合基因 ;采用脂质体转染技术转染 CHO细胞使其瞬时表达融合蛋白 ;应用细胞免疫荧光技术检测融合蛋白的表达 ;采用免疫沉淀的方法检测 CHO细胞培养上清内融合蛋白的表达 .结果 DNA测序结果在 Blast上作同源性比较证明插入片段就是TGF-βR c DNA的胞膜外部分 ,细胞免疫荧光染色呈阳性反应 ,CHO细胞培养上清内检测到融合蛋白的表达 .结论 p Ig- TR 真核表达载体构建成功 ,并能够在 CHO细胞中分泌性表达有活性的融合蛋白 TGF-βR /
AIM To construct the pIg TRⅡ eukaryotic expression vector of the cDNA sequence encoding bioactive extracellular domain of human transforming growth factor beta type Ⅱreceptor (TGF βRⅡ) to form the fusion gene TGF βRⅡ/Fc and to express the chimeric protein TGF βRⅡ/Fc in CHO cells. This work would contribute to the foundation of gene therapy of pulmonary fibrosis. METHODS Total RNA was extracted from human normal lung tissue and subjected to reverse transcription, and then the human TGF βRⅡ cDNA gene was amplified by RT PCR. The PCR product was cloned into pMD18 Tvector and the sequence was confirmed by restriction enzyme digestion and sequencing.Then the specific TGF βRⅡ encoding region fragment was subcloned into pIg plasmid to form the pIg TRⅡ eukaryotic expression vector. The recombinant plasmid was transfected into CHO cells with liposome transfection reagent for transient expression. The expression of the chimeric protein TGF βRⅡ/Fc was identified by immunofluorescent assay and immunoprecipitation technology. RESULTS Restriction enzyme digestion and DNA sequence analysis revealed that the insert fragment was identical to the published TGF βRⅡcDNA sequence. The chimeric protein was detected by cellular immunofluorescence and immunoprecipitation technology. CONCLUSION Construction of the pIg TRⅡ eukaryotic expression vector of the fusion gene TGF βRⅡ/Fc and its expression in CHO cells are both successful.
出处
《第四军医大学学报》
北大核心
2002年第4期371-375,共5页
Journal of the Fourth Military Medical University
关键词
间质性肺疾病
肺纤维化
受体
转化生长因子Β
克隆
真核细胞
lung diseases, interstitial
pulmonary fibrosis
receptors, transforming growth factor beta
cloning, molecular
eukaryotic cells
