Construction and identification of recombinant retroviral vector pLNCX2-human calcitonin gene-related peptide α

Objective To construct and identify the recombinant retroviral vector encoding human calcitonin gene-related peptideα (hCGRPα) gene.Methods Using genetic engineering techniques,the fragment of hCGRPα cDNA and the fragment of pLNCX2 carrier,respectively 438 bp and 6.1 kb,were recovered after double digestion,then mixed according to molar ratio of 7∶1 after purification.The hCGRPα gene was cloned into the retroviral vector pLNCY2.High titer viral particles of pLNCX2- hCGRPα were obtained after transfection of PT67 cells by Lipofectine 2000.Then the recombinant retrovirus was transfected into NIH3T3 cells and the titer was measured.Results Restriction,and sequencing analysis confirmed that the hCGRPα cDNA was successfully inserted into the retroviral vector.The titer of recombinant retrovirus with hCGRPα gene was 1.7 × 106 pfu/ml and the retrovirus was effectively transfected into NIH3T3 cells.Conclusion The recombinant retroviral vector pLNCX-hCGRPα had been constructed successfully,which can be used for further investigation on gene therapy. Key words: Retrovirus; Calcitonin gene-related peptide; Gene cloning; Osteonecrosis of the femoral head