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Published on: January 12, 2015
[Construction of a eukaryotic expression vector containing enhanced green fluorescence protein and recombinant human
Yu Liu1, Qin-feng Sun, Pi-shan Yang
1Stomatological Hospital, Medical College of Nanjing University, Nanjing 210008, Jiangsu Province, China.
Purpose:
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.
Conclusion:
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.
