GM-CSF gene-modified ovarian cancer NuTu-19-GM-CSF Construction.docVIP

 PAGE \* MERGEFORMAT 18 GM-CSF gene-modified ovarian cancer NuTu-19/GM-CSF Construction Of: Ling Li Qi, Wang Ying, Wang Yunping, Cai-Xia Feng, high wind [Abstract] Objective To construct secreted expression of granulocyte - monocyte colony stimulating factor (GM-CSF) in ovarian cancer NuTu-19/GM-CSF. Methods Th GM-CSF plasmid was transformed into E. coli in resistant colonies were selected monoclonal sent for sequencing. amplified by PCR, GM-CSF gene fragment PCR products were connected to the pGEM-T easy vector, transformed into E. coli was amplified by SDS alkaline lysis method Teasy- GM-CSF plasmid, restriction enzyme digestion with Teasy-GM-CSF recombinant vector, connected by the same enzyme digestion of pLXSN retroviral vector. transfected using calcium phosphate precipitation method for virus packaging cell line PA317 packaging, the supernatant containing the virus infection NuTu-19 ovarian cancer cells by G418 infection success NuTu-19/GM-CSF pressure screening cells with immunocytochemistry and ELISA assay NuTu-19/GM-CSF secretion GM-CSF protein expression. Results Th GM-CSF plasmid DNA sequencing results and GenBank Accession #: NM_000758 determined after comparing the gene sequence for human GM-CSF gene coding sequence, PCR products were digested electrophoresis in Mark 400-500 bp in one band, in line with GM-CSF gene sequence length, recombinant Teasy-GM-CSF plasmid with EcoR , BamH restriction enzyme digestion, the electrophoresis identified one between 400-500 bp in the target band, recombinant retroviral vector pLGM-CSFSN enzyme electrophoresis purposeful band, G418 selection pressure after infection NuTu-19 cells, 14 d after the formation of monoclonal continue to grow slowly, the infection was detected by immunocytochemistry successful NuTu-19 cell surface significantly brown particles precipitation, ELISA assay of cell culture supernatant of GM-CSF concentration was 150 pg / mL. Conclusion We successfully constructed to secrete human GM