Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector / 中华骨科杂志

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.

Constructing bicistronic eukaryotic expression plasmid containing human bone morphogenetic protein-2 labeled with green fluorescent protein / 中国组织工程研究

BACKGROUND: Bone morphogenetic protein-2(BMP-2) production of targeted cells is promoted by transfection of adenoviral vectors containing gene, but there are some immune responses. Transfection with plasmid as vector holds promise.OBJECTIVE: To explore the feasibility to construct human bone morphogenetic protein-2 eukaryotic expression vector labeled with green fluorescent protein (GFP).DESIGN: Single sample observation.SETTING: Tianjin Hospital.MATERIALS: The experiment was performed at the Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2(R)-T Easy by Dingguo, China; restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.METHODS: With pcDNA3.1/CT-hBMP2 as template, hBMP2 target fragment was subcloned by PCR binding with designed specific primers. The fragment was bound with pTA2-T-easy and pSELECT-GFPzeo-MCS, separately, and transfected into DH5 α cells. pSELECT-GFPzeo-hBMP2 containing GFP was obtained after screening.MAIN OUTCOME MEASURES: hBMP2 sequence was identified by PCR; whether hBMP2 was cloned into pTA2-hBMP2 and pSELECT-GFPzeo-MCS was identified by digestion and sequencing.RESULTS: A target fragment of 1 216 bp was obtained by PCR amplification, and cloned into pTA2-T-easy and pSELECT-GFPZeo-MCS. The screening and sequencing results showed that the target fragment was 100% matched with BMP2cDNA sequence (NM-001200) from GenBank.CONCLUSION: hBMP2 eukaryotic expression vector labeled with green fluorescent protein is successfully constructed.