黃洪超(Huang Hongchao，Dept Spine Surg Tianjin Orthop Hosp，Tianjin 300211)…∥Chin J Orthop.－ 2010，30(12).－1228～1234

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

黃洪超(Huang Hongchao，Dept Spine Surg Tianjin Orthop Hosp，Tianjin 300211)…∥Chin J Orthop.－ 2010，30(12).－1228～1234

ObjectiveTo construct green fluorescent protein(GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.MethodsThe encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase chain reaction(PCR).hBMP2 gene was inserted into pTA2-T-easy and pSELECT-GFPzeo-MCS eukaryotic expression vector，and then transferred into competence DHSα cells.After screening，pSELEC-GFPzeo-hBMP2 was obtained and identified by sequence analysis.The recombinant vector pSELECT-GFP zeo-rhBMP2 was transfected into CHO cells.The successful trasfection was verified by fluorescence microscope in 48 －72 hours.The RT-PCR and immunofluorescence was used to confirm the hBMP2 expression.Western Blotting was used to detect the secretion of hBMP2.ResultsA 1216 bp fragment was obtained by PCR，the same as expectant fragment.The recombined pSE-LECT-GFPzeo-hBMP2 eukaryotic expression vector was identified by restriction mapping and sequence analysis.The results were identical with that of reported hBMP2 sequence(Genebank NM-001200).The successful transfection was verified by fluorescence microscope in 48 － 72 hours.The stable expression in eukaryotic cells was confirmed by immunofluorescence and RT-PCR which showed an obvious band between 1000－2000 bp.Western Blotting identified the immunogenicity of recombinant human BMP2 with the molecular weight of about 17 ×103.ConclusionThe pSELECT-GFPzeohBMP2 eukaryotic expression vector was constructed successfully.21 refs，9 figs.

(Authors)