氧浓度非敏感性低氧诱导因子-1α表达载体的构建及其在PC12细胞系中的表达  

Construction of recombinant eukaryotic expression vector pEGFPC1-HIF-1α and its expression in eukaryotic cells PC12

作　　者：赵宁辉[2] 李冉[1] 黄晓玮[2] 

机构地区：[1]昆明医学院第二附属医院神经外科,650101 [2]云南大学生物资源保护与利用实验室

出　　处：《中华创伤杂志》2010年第2期160-164,共5页Chinese Journal of Trauma

基　　金：基金项目：国家自然科学基金资助项目（30660190）;云南省社会发展科技计划省科技厅一昆医联合专项资助项目（2008CD021）;云南省中青年学术技术带头人后备人才资助项目（2009C1034）;昆明医学院第二附属医院博士启动基金资助项目（2008）

摘　　要：目的构建大鼠非氧浓度敏感性低氧诱导因子1α（HIF-1α）真核表达载体并观察其在PCI2细胞系中的表达。方法应用RT—PCR从脊髓损伤区域的细胞总RNA中扩增HIF—1αcDNA，采用重叠延伸PCR的方法克隆获得缺失氧敏感性降解结构域（ODD）的HIF-1α突变体基因克隆，采用质粒pEGFP—C1构建重组真核融合表达载体，将其转入培养的PC12神经细胞系中，应用所融合的绿色荧光蛋白的表达和Western blot鉴定其在细胞内的表达。结果通过重叠延伸PCR的方法成功扩增得到非氧浓度敏感性的HIF1α突变体基因（HIF1αAODD）序列，并构建了过量表达缺失ODD的转录调控因子HIF1α突变体的重组表达质粒（pEGFPCI一HIF1αAODD）。将其转染PC12细胞系后，Westernblot和绿色荧光蛋白结果均表明HIF—letAODD蛋白能在PC12神经细胞系中正确表达。结论成功构建了pEGFPCI一HIF1αAODD真核表达载体并在PC12细胞系中观察到融合蛋白表达。Objective To construct the eukaryotic expression vector with the mutant of hypoxia inducible factor 1α （HIF-1α） that could over express nonhypoxic dependent HIF-1α and investigate its expression in eukaryotic cells PC12. Methods The ceding sequence of HIF-1α was amplified from to- tal RNA from the injured spinal cord of rats by means of RT-PCR. Then, overlapping extension PCR was employed to obtain one complete sequence encoding HIF-1α mutant without oxygen-sensitive degradation domain （ODD）. By using gene recombination technique, the gene HIF-1α AODD was inserted into eu- karyotic expression vector pEGFPCI that contained a reporter gene of enhanced green fluorescent protein. The recombinant plasmid was successfully constructed and transfected into the neural cell line PC12. The expression of HIF-1α AODD was analyzed by fluorescent observation and Western blot. Results A 2kb gene fragment encoding HIF-1αAODD was successfully obtained by overlapping extension PCR. On this basis, the recombinant plasmid pEGFPC1-HIF-1αAODD was constructed and testified with restriction en- zyme digestion and DNA sequencing. After this recombinant plasmid was transfeeted into the neural cell line PC12, both fluorescent observation and Western blot demonstrated thai HIF-1αAODD was over ex- pressed under nonhypoxia conditions. Conclusion The eukaryotic expression vector pEGFPC1-HIF-1αAODD is successfully constructed and HIF-1αAODD over expresses in transfeeted PC12 cell lines.

关 键 词：低氧诱导因子1Α 质粒 转染 PCI2细胞系 

分 类 号：R346[医药卫生—基础医学]