CONSTRUCTION OF RECOMBINANT RETROVIRAL VECTOR CARRYING RAT IGF-1 GENE

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.