【摘要】 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荧光真核表达载体更多还原

【Abstract】 Aim: To construct basic fibroblast growth factor (bFGF) gene fluorescent eukaryotic expression plasmid for gene therapy of ischemic cerebral vascular diseases. Methods:The focal cerebral ischemia model of mature Sprague Dawley rat was established, then the coding sequence of bFGF was amplified by reverse transcriptase PCR(RT PCR).The bFGF gene was cloned into pGEM T Easy vector and identified by PCR, restriction enzyme analysis.The bFGF gene was determined subcloned into pEGFP N3 plasmid, then the recombinant vector was selected and identified by PCR, restriction enzyme analysis, and nucleotide sequence determination. Results: Correct construction of pEGFP N3 bFGF was identified by methods of PCR amplication, restriction enzyme analysis, and nucleotide sequence determination. Conclusion: An eukaryotic expression plasmid pEGFP N3 bFGF has been constructed successfully.更多还原