Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector
10.3760/cma.j.issn.0253-2352.2010.12.012
- VernacularTitle:可体内、外示踪的BMP2真核表达载体的构建和表达
- Author:
Hongchao HUANG
;
Jun MIAO
;
Chunrong LIU
;
Yongcheng HU
;
Qun XIA
;
Kesong SHI
- Publication Type:Journal Article
- Keywords:
Bone morphogenetic proteins;
Protein engineering;
CHO cells;
Fluorescent antibody technique,indirect;
Liposomes
- From:
Chinese Journal of Orthopaedics
2010;30(12):1228-1234
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector. Methods The 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.Results A 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. Conclusion The pSELECT-GFPzeo-hBMP2 eukaryotic expression vector was constructed successfully.
