RECONSTRUCTION AND EXPRESSION OF RETROVIRUS VECTOR WITH HUMAN D_2 RECEPTOR GENE

Objective To construct the eukaryotic expression vector of human full-length D -2 receptor gene for further study on role in PD. Methods cDNA fragment of D -2 receptor was subcloned into retrovirus vector pLNCX2 which was used to transfected into the PT67 cell line.Cell clones were obtained through G418 selection with passage.D -2R gene expression was detected with in situ hybridization,immunocytochemistry and Western blot assay.The packaging cell medium containing virus particles was collected and infected bone marrow stromal cells(MSCs) and the efficiency was measured with immunofluorescence. Results The retrovirus vector pLNCX2-D -2R was constructed successfully.More than 80% PT67 cells expressed the target gene.Porteins were detected in plasm and cell membrane was lysed and proteins were harvested for Western blot assays.A fragment of 48.84kD length was manifested with Western blot.Virus particles were secreted from PT-67 cells and were used to further infect MSCs.Conclusion The cloned human D -2 receptor gene was expressed in PT67 cells and MSCs was efficiently infected by the recombinant retrovirus.pLNCX2-D -2R could be used to delivery vehicle for gene therapy of PD.