目的 :构建可在真核细胞中表达碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)的真核表达载体 ,为进一步开展bFGF基因治疗缺血性脑血管疾病奠定基础。方法 :①采用线栓法建立SD大鼠大脑中动脉局灶性脑缺血模型 ,取缺血灶周围脑组织经逆转录聚合酶链反应 (RT PCR)扩增bFGF基因 ;②将bFGFDNA克隆到pGEM TEasy质粒 ,经菌落PCR和HindⅢ和EcoRI双酶切鉴定 ;③然后将bFGFDNA亚克隆到pEGFP N3质粒 ;通过抗性基因筛选阳性克隆 ,经菌落PCR、酶切和测序鉴定。结果 :菌落PCR、酶切和DNA序列鉴定均证实插入片段与GenBank报道的bFGF基因序列一致。结论 :经RT PCR反应得到了特异的bFGF基因片段并成功构建了bFGF荧光真核表达载体 OBJECTIVE: To construct an eukaryotic expression vector capable of expressing basic fibroblast growth factor (bFGF) in eukaryotic cells, and lay the foundation for the further development of bFGF gene therapy for ischemic cerebrovascular diseases. Methods: ① The middle cerebral artery occlusion (MCAO) model of SD rats was established by thread occlusion method. The bFGF gene was amplified by reverse transcription polymerase chain reaction (RT PCR) from the brain tissue around the ischemic area. ② The bFGF cDNA was cloned into pGEM TEasy plasmid was digested with Hind Ⅲ and EcoRI. The bFGFDNA was then subcloned into pEGFP N3 plasmid. Positive clones were screened by resistance gene and identified by colony PCR, restriction enzyme digestion and sequencing. Results: The results of colony PCR, enzyme digestion and DNA sequencing confirmed that the inserted fragment was consistent with the bFGF gene sequence reported by GenBank. Conclusion: The specific bFGF gene fragment was obtained by RT-PCR and the bFGF fluorescent eukaryotic expression vector was successfully constructed