Construction of the enhanced yellow fluorescent protein expression vector carrying IFN-gamma gene.
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Lan
Y
Y
Sun Yat-Sen Memorial Hospital, Sun Yat-sen University of Medical Science, Guangzhou 510120, China.
Ge
J
J
Zhuo
Y
Y
Wang
J
J
Chen
H
H
Liu
H
H
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D007371:Interferon-gamma