【摘要】 目的构建胰岛素样生长因子-1(IGF-1)基因重组逆转录病毒,为将IGF-1基因应用于神经系统疾病的治疗打下基础。方法质粒pcDNA3.1-IGF-1经EcoR I和Xho I双酶切后亚克隆至逆转录病毒载体pLXSN,构建成重组逆转录病毒表达载体pLXSN-IGF-1,采用酶切及测序对重组体进行鉴定。而后脂质体转染pLXSN-IGF-1至包装细胞pA317,检测培养上清病毒滴度。结果重组真核基因表达载体pLXSN-IGF-1经EcoR I和Xho I双酶切后,得到了400 bp和6.0 kb两条带;以重组体为模板进行PCR检测,400 bp的目的基因片段呈阳性;重组体测序结果与预期结果完全一致;建立了细胞系PA317-IGF-1,其培养上清平均病毒滴度为6.5×105CFU/ml。结论成功构建了IGF-1基因重组逆转录病毒。更多还原

【Abstract】 Objective To establish a recombinant retroviral vector containing insulin-like growth factor-1(IGF-1) gene and to provide the basis for the application of IGF-1 in treating nervous system diseases such as stroke.Methods The plasmid pcDNA3.1-IGF-1 was cut by EcoR I/Xho I,and subcloned to retroviral vector pLXSN,resulting in the recombinant plasmid(pLXSN-IGF-1.)The recombinant IGF-1 expression vector was evaluated by using enzyme cutting and sequencing.By the Lipofectamine 2000,pLXSN-IGF-1 was transferred to packaging cell line-pA317.Culture supernatant of these cells was detected for titration of the recombinant virus.Results The two fragments from recombined IGF-1 eukaryotic expression vector by EcoR I and Xho I represented 400?bp and 6.0?kb by agarose electrophoresis,and PCR showed positive fragment which was about 400?bp long,and sequence analysis showed the same sequence as expected.The cell line pA317-IGF-1 was established,and average virus titer of the recombinant virus in the culture supernatant was about 6.5×10~5 CFU/ml.Conclusion A recombinant retroviral containing IGF-1 gene was successfully constructed.更多还原