Construction of the Enhanced Yellow Fluorescent Protein Expression Vector Carrying IFN-γ Gene

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

Expression and characterization of Mac-1-FP fusion protein in CHO cells

Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes.