胰岛素样生长因子-1基因真核表达载体的构建

CONSTRUCTION OF RECOMBINANT RETROVIRAL VECTOR CARRYING RAT IGF-1 GENE

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。

Objective To establish a recombinant retreviral vector containing insulin-like growth factor-1 （IGF-1） gone 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 subcloncd to retroviral vector pI2CSN, resulting in the recombinant plasmid pLXSN-IGF-1. The recombinant IGF-1 expression vector was evaluated by using enzyme cutting and sequencing. By the Lipofcctaminc 2000, pLXSN-IGF-1 was transferred to packaging cell linc-pA317. Culture supernatant of these cells was detected for titration of the recombinant virus.Results The two fragments from recombincd IGF-1 cukaryotic expression vector by EcoR I and Xho I represented 400 bp and 6.0 kb by agarosc clcctrophorcsis, 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.