Purpose: To construct the enhanced yellow fluorescent protein (EYFP) vector carrying interferon-gamma gene (ifn-gamma) in order to provide an ideal reporter in the expression of ifn-gamma and location of protein in vitro and in vivo.
Method: According to the nucleotide sequence of ifn-gamma gene, a pair of oligonucleotides was designed as primer whose two end contained nucleotide sequence of EcoR V and Not I restriction endonuclease respectively. The gene encoding for inf-gamma was amplified using PCR technqiue. After the PCR product was retrieved and purified, it was digested with EcoR V and Not I restriction endonuclease, and then cloned into the plasmid pIRES-EYFP. The recombinant plasmid pIRES-EYFPIFN-gamma was identified by restriction endonuclease enzyme analysis and DNA sequence analysis.
Results: The ifn-gamma was successfully amplified and verified by partial DNA sequence analysis. The recombinant plasmid was correctly screened.
Conclusion: The EYFP expression vector carrying ifn-gamma gene was successfully established. This research work has formed a base for monitoring the ifn-gamma gene expression and protein position in living cells.