[Construction of a eukaryotic expression vector containing enhanced green fluorescence protein and recombinant human bone morphogenetic protein-2].
OBJECTIVE
To construct recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP containing enhanced green fluorescence protein (EGFP) and recombinant human bone morphogenetic protein-2 (rhBMP-2).


METHODS
A pair of primers specific for amplifying the DNA fragment encoding rhBMP-2 were designed and synthesized. The targeted DNA fragment was amplified from pIRES-rhBMP2 by PCR.rhBMP-2 and EGFP were inserted to the proper sites of vector pIRES, and internal ribozyme entry site (IRES) sequence was between the genes coding for rhBMP-2 and EGFP. The recombinant plasmid pIRES-rhBMP2-EGFP was first propagated in E.coli DH5alpha, and then was confirmed to contain hBMP2 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis.


RESULTS
The construction of the recombinant eukaryotic dual-expression plasmid pIRES-rhBMP2-EGFP and the open reading frame were confirmed through restriction enzyme mapping analysis and DNA sequencing.


CONCLUSIONS
By PCR, T/A cloning, the cDNA fragment encoding rhBMP2 and EGFP fragment can be cloned into pIRES to construct the recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP.
Liu
Yu
Y
Stomatological Hospital, Medical College of Nanjing University, Nanjing 210008, Jiangsu Province, China.
Sun
Qin-feng
QF
Yang
Pi-shan
PS
Du
Fang
F
D055396:Bone Morphogenetic Protein 2 / Q000096:biosynthesis*; D018076:DNA, Complementary; D004926:Escherichia coli; D005453:Fluorescence; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006801:Humans; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000096:biosynthesis*
D055396:Bone Morphogenetic Protein 2; D018076:DNA, Complementary; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C485184:enhanced green fluorescent protein; C499469:recombinant human bone morphogenetic protein-2; D049452:Green Fluorescent Proteins